Ronald L Heimark

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Chapters

• Heimark, R. L. (2009). Regulation of N-cadherin in Epithelial-Mesenchymal Transition. In Molecular and Functional Diversities of Cadherin/Protocadherin. SignPost Publishing.
• Heimark, R. L., & Alexander, N. R. (2006). REGULATION OF CADHERINS DURING PROSTATE CANCER PROGRESSION. In Cancer Metastasis- Biology and Treatment. Springer, Dordrecht. doi:10.1007/978-1-4020-5129-6_3
• Heimark, R. L., & Alexander, N. R. (2006). Regulation of Cadherins During Prostate Cancer Progression. In Cell Adhesion and Cytoskeletal Molecules in Metastasis(pp 47-68). Springer.
• Runyan, R. B., Heimark, R. L., Camenisch, T. D., & Barnett, J. (2005). Epithelial-mesenchymal transformation. In Rise and Fall of Epithelial Phenotype:Concepts of Epithelial-Mesenchymal Transition(pp 40-65). Molecular Biology Intelligence Unit.
• Heimark, R. L. (1993). Cell-cell Adhesion molecules of the blood brain barrier. In The Blood Brain Barrier: Cellular and Molecular Biology(pp 87-106).
• Heimark, R. L., & Schwartz, S. M. (1988). Endothelial Morphogenesis. In Endothelial Cell Biology in Health & Disease(pp 127-143). Springer US. doi:10.1007/978-1-4613-0937-6_6
• Heimark, R. L., & Schwartz, S. M. (1988). The role of cell-cell interaction in the regulation of Endothelial Cell Growth. In The Molecular and Cellular Biology of Wound Repair(pp 359-369). Plenum Press.
• Heimark, R. L., & Davie, E. W. (1981). [14] Bovine and human plasma prekallikrein. In Methods in Enzymology(pp 157-172). doi:10.1016/s0076-6879(81)80016-x
• Davie, E. W., Fujikawa, K., Kisiel, W., Kurachi, K., & Heimark, R. L. (1978). THE EARLY PHASE OF BLOOD COAGULATION. In Molecular Basis of Biological Degradation Processes. doi:10.1016/B978-0-12-092150-8.50009-6
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  Factor XII (Hageman factor) is a plasma protein that participates in the early phase of the intrinsic and extrinsic pathways of blood coagulation. Bovine factor XII is a single-chain molecule which is converted to factor XII a by minor proteolysis. Factor XII a is composed of a heavy and a light chain held together by a disulfide bond(s), The active site in this protein is in the light chain of the molecule. In the intrinsic pathway, factor XII a converts factor XI to factor XI a in the presence of high molecular weight kininogen. In the extrinsic pathway, factor XII a converts factor VII to factor VII a .
• Traut, R. R., Heimark, R. L., & Sun, T. T. (1974). Protein Topography of Ribosomal Subunits from Escherichia Coli. In Ribosomes(pp 271-308). Cold Spring Harbor.

Journals/Publications

• Batai, K., Chen, Y., Rheinheimer, B. A., Arora, A., Pandey, R., Heimark, R. L., Bracamonte, E. R., Ellis, N. A., & Lee, B. R. (2023). Clear cell renal cell carcinoma molecular variations in non-Hispanic White and Hispanic patients. Cancer medicine, 12(11), 12792-12801.
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  The United States is becoming increasingly diverse, but few molecular studies have assessed the progression of clear cell renal cell carcinoma (ccRCC) in diverse patient populations. This study examined ccRCC molecular variations in non-Hispanic White (NHW) and Hispanic patients and their effect on the association of gene expression with high-grade (Grade 3 or 4) ccRCC and overall mortality.
• Estrada-Mendizabal, R. J., Dhaliwal, A. S., Bertolo, A. J., Batai, K., Heimark, R., Recio-Boiles, A., & Chipollini, J. (2023). Prostate Cancer Disparities in Metastatic and Treatment Status for Hispanic Americans Based on Country of Origin Compared to Non-Hispanic Whites Using the National Cancer Database. Clinical genitourinary cancer.
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  Among Hispanic-American (HA) men, prostatic cancer (PCa) accounts for nearly one-quarter of the total cancer burden. We sought to identify differences in PCa presentation and treatment status for HA subgroups based on country/region of origin.
• Heimark, R., Recio-Boiles, A., Batai, K., Cheng, C., & Chipollini, J. (2022). Disparities in prostate cancer: An ethnicity comparative focus among Hispanic Americans versus non-Hispanic whites.. Journal of Clinical Oncology, 40(6_suppl), 23-23. doi:10.1200/jco.2022.40.6_suppl.023
• Rubio, N. E., Heimark, R., & Jie, T. (2016). Role of Myeloid Derived Suppressor Cells in the Inflammatory Response of Pancreatitis and Pancreatic Cancer. ANNALS OF SURGICAL ONCOLOGY, 23(Suppleemnt 1), S155.
• Futscher, B. W., Heimark, R. L., Vrba, L., & Rheinheimer, B. A. (2020). Identification and transcriptional regulation of the mir-218-1 alternative promoter. bioRxiv. doi:10.1101/2020.09.29.319202
• Futscher, B. W., Heimark, R. L., Vrba, L., & Rheinheimer, B. A. (2020). Transcriptional regulation of SLIT2 expression in pancreatic cancer cell lines. bioRxiv. doi:10.1101/2020.09.29.319129
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  Abstract Background SLIT2 has been shown to serve as a tumor suppressor in breast, lung, colon, and liver cancers. Additionally, expression of SLIT2 has been shown to be epigenetically regulated in prostate cancer. Therefore, we sought to determine transcriptional regulation of SLIT2 in pancreatic ductal adenocarcinoma. Methods RNA expression of SLIT2, SLIT3, and ROBO1 was examined in a panel of pancreatic ductal adenocarcinoma cell lines while protein expression of ROBO1 and SLIT2 was examined in tumor tissue. Methylation of the SLIT2 promoter was determined using Sequenom while histone modifications were queried by chromatin immunoprecipitation. Reexpression of SLIT2 was tested by treatment with 5-aza-2’deoxycytidine and Trichostatin A. Results Pancreatic cancer cell lines fall into three distinct groups based on SLIT2 and ROBO1 expression. The SLIT2 promoter is methylated in pancreatic ductal adenocarcinoma and SLIT2 expression is dependent on the level of methylation at specific CpG sites. Treatment with 5-aza-2’deoxycytidine (but not Trichostatin A) led to SLIT2 reexpression. The SLIT2 promoter is bivalent in pancreatic ductal adenocarcinoma and histone marks around the transcriptional start site are responsible for transcription. Conclusions Loss of SLIT2 expression modulated by epigenetic silencing may play a role in pancreatic ductal adenocarcinoma progression.
• Heimark, R. L., & Rheinheimer, B. A. (2020). mir-218 modulates ARF6 expression and inhibits pancreatic ductal adenocarcinoma invasion. bioRxiv. doi:10.1101/2020.09.29.319236
• Heimark, R. L., Jie, T., Ong, E., Camacho, L., Cardenas, A., & Rheinheimer, B. A. (2020). Inhibition of Sphingosine-1-Phosphate signaling in pancreatic ductal adenocarcinoma leads to downregulation of growth and invasion. bioRxiv. doi:10.1101/2020.09.29.318964
• Heimark, R. L., Jie, T., Padi, M., Camacho, L., Grant, A. D., & Rheinheimer, B. A. (2020). Cell intrinsic signaling in MEN1 mutant pancreatic neuroendocrine tumors unveils novel signaling pathways associated with de-differentiation. bioRxiv, 2020.09. 29.318873. doi:10.1101/2020.09.29.318873
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  Abstract Objectives Preliminary genomic analysis of primary pancreatic neuroendocrine tumors revealed a complex mutational landscape with four common oncogenic events; however, critical activation pathways responsible for pancreatic neuroendocrine tumor progression and metastasis have yet to be elucidated. Here, we analyzed six primary pancreatic neuroendocrine tumors to determine which pathways are deregulated and responsible for progression. Methods Selected genomic profiling of six primary pancreatic neuroendocrine tumors was performed using the Ion Torrent Comprehensive Cancer Panel with matched transcriptomes analyzed by Affymetrix Clariom D arrays. Validation of gene expression changes were measured by quantitative PCR using TaqMan assays and immunohistochemistry on tumor specimens. Results MEN1 was mutated in half (50%) of our sequenced tumors while FGFR3 was mutated in 2/6 (33%). Transcriptome analysis revealed that ITGA2 and EZH2 were overexpressed in MEN1 mutant tumors whereas ALK and VEGFA were overexpressed in FGFR3 mutant tumors. Immunohistochemistry revealed increased nuclear ITGA2 and EZH2 staining along with increased VE-Cadherin staining and loss of membranous E-cadherin localization in MEN1 and FGFR3 mutant tumors. Conclusions Our results suggest that pancreatic neuroendocrine tumors containing MEN1 and FGFR3 mutations are more aggressive and de-differentiated than their wild-type counterparts. Additionally, we provide novel chemotherapeutic target FGFR3 for patients with this disease.
• Heimark, R. L., Jie, T., Riall, T. S., & Rheinheimer, B. A. (2019). Pancreatic mixed acinar cell carcinoma: genomic analysis and characterization of a patient-derived organoid culture. Hpb, 21(S1), S98eS188. doi:10.1016/j.hpb.2019.03.260
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  Despite national guidelines recommending the receipt of adjuvant therapy for patients undergoing resection for pancreatic adenocarcinoma, a significant proportion of patients do not receive it. Efforts to improve post-operative outcomes should be pursued to improve the proportion of patients who receive adjuvant therapy following resection for pancreatic adenocarcinoma. Given that a significant proportion of patients do not receive adjuvant therapy, the importance of neoadjuvant therapy should be consider
• Cheung, L. S., Zheng, X., Wang, L., Baygents, J. C., Guzman, R., Schroeder, J. A., Heimark, R. L., & Zohar, Y. (2016). Adhesion dynamics of circulating tumor cells under shear flow in a bio-functionalized microchannel. JOURNAL OF MICROMECHANICS AND MICROENGINEERING, 21(5).
• Heimark, R. L., Galbraith, D. W., Doetschman, T., Weng, N., & Samadder, P. (2016). Flow cytometry and single nucleus sorting for Cre-based analysis of changes in transcriptional states. Cytometry Part A, 89(5), 430-442. doi:10.1002/cyto.a.22847
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  The organs of eukaryotic organisms comprise complex interspersions of cell types, whose different molecular activities, and corresponding cellular states, cooperate during development to produce the final, functional organ. Dysfunction of organs in disease, particularly oncogenesis, initiates with changes of state of a minor subset of cells. It therefore is hard to detect early molecular indicators of disease within an overwhelming background of normal cells. Flow cytometry and sorting provides a convenient way to purify minority subpopulations, if a specific fluorophore can be unambiguously and exclusively associated with this subpopulation. We have generated a number of transgenic mouse lines expressing a nuclear-localized version of the Green Fluorescent Protein (GFP), within which the production of a chimeric histone 2B-GFP protein occurs under the control of a constitutively-active, actin-derived promoter, separated by a Floxed-STOP sequence. In the presence of Cre recombinase, within F1 progeny of these mouse lines, excision of the STOP sequence activates transcription which results in the emergence of cells containing green fluorescent nuclei. We describe the characterization of these lines using a combination of microscopic imaging, flow cytometry and sorting, and Reverse-Transcription polymerase chain reaction of transcripts within single sorted nuclei isolated from tissue homogenates. These lines should be particularly useful for analysis of transcriptional changes in oncogenesis. © 2016 International Society for Advancement of Cytometry.
• Cieza-Rubio, N., Jie, T., & Heimark, R. L. (2015). Mechanistic Studies in the Inflammatory Response of Pancreatitis and Pancreatic Cancer: Role of Myeloid Derived Suppressor Cells. PANCREAS, 44(8), 1367.
• Rheinheimer, B., Vrba, L., Futscher, B., & Heimark, R. L. (2013). Epigenetic silencing alters the SLIT2/ROBO1/miR-218-1 signaling axis in pancreatic cancer. CANCER RESEARCH, 72(13 Supplenent), B13.
• Chandramouli, A., Onyeagucha, B. C., Mercado-Pimentel, M. E., Stankova, L., Shahin, N. A., LaFleur, B. J., Heimark, R. L., Bhattacharyya, A. K., & Nelson, M. A. (2012). MicroRNA-101 (miR-101) post-transcriptionally regulates the expression of EP4 receptor in colon cancers. Cancer biology & therapy, 13(3), 175-83.
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  Expression of the PGE2 receptor, EP4, is up-regulated during colorectal carcinogenesis. However the mechanism leading to deregulation of the EP4 receptor is not known. The present study was conducted to investigate the regulation of EP4 receptor by miRNAs.
• Doetschman, T., Barnett, J. V., Runyan, R. B., Camenisch, T. D., Heimark, R. L., Granzier, H. L., Conway, S. J., & Azhar, M. (2012). Transforming Growth Factor beta signaling in adult cardiovascular diseases and repair. Cell and Tissue Research, 347(1), 203-223.
• Cheung, L. S., Zheng, X., Wang, L., Guzman, R., Schroeder, J. A., Heimark, R. L., Baygents, J. C., & Zohar, Y. (2010). Kinematics of Specifically Captured Circulating Tumor Cells in Bio-Functionalized Microchannels. JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, 19(4), 752-763.
• Vrba, L., Jensen, T. J., Garbe, J. C., Heimark, R. L., Cress, A. E., Dickinson, S., Stampfer, M. R., & Futscher, B. W. (2010). Role for DNA methylation in the regulation of miR-200c and miR-141 expression in normal and cancer cells. PloS one, 5(1), e8697.
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  The microRNA-200 family participates in the maintenance of an epithelial phenotype and loss of its expression can result in epithelial to mesenchymal transition (EMT). Furthermore, the loss of expression of miR-200 family members is linked to an aggressive cancer phenotype. Regulation of the miR-200 family expression in normal and cancer cells is not fully understood.
• Heimark, R. L., Chueng, L. S., Zheng, X., Stopa, A., Heimark, R. L., & Zohar, Y. (2009). Detachment of Captured cancer cells under flow accleration in a bio-functionalized microchannel. LAB ON A CHIP, 9(12), 1721-1731.
• Moran, C. M., Garriock, R. J., Miller, M. K., Heimark, R. L., Gregorio, C. C., & Krieg, P. A. (2008). Expression of the fast twitch troponin complex, fTnT, fTnI and fTnC, in vascular smooth muscle. Cell motility and the cytoskeleton, 65(8), 652-61.
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  It is generally believed that proteins of the troponin complex are not expressed in smooth muscle. We have directly assayed for expression of troponin transcripts in mouse vascular smooth muscle and found that troponin sequences normally associated with fast twitch skeletal muscle (fTnT, fTnI, fTnC) were present at significant levels in the thoracic aorta. In situ hybridization experiments demonstrated that fTnT, fTnI and fTnC transcripts were expressed in the smooth muscle layer of mouse blood vessels of all sizes. Protein blot analysis using rat tissue showed that at least two members of the troponin complex, Troponin T and Troponin I, were translated in vascular smooth muscle of the aorta. Finally, immuno-fluorescence microscopy of rat aortic smooth muscle revealed that TnT and TnI are localized in a unique pattern, coincident with the distribution of tropomyosin. It seems likely therefore, that a complete troponin complex is expressed in vascular smooth muscle and is associated with the contractile machinery of the cell. These observations raise the possibility that troponins play a role in regulation of smooth muscle function.
• Zohar, Y., Baygents, J. C., Heimark, R. L., Schroeder, J. A., & Guzman, R. (2008). Innovative Microsystems: Novel Nanostructures to Capture Circulating Breast Cancer Cells. Annual rept. 23 Apr 2008-22 Apr 2009. doi:10.21236/ada486651
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  Abstract : The goal of this project is to develop a microsystem for sorting metastatic breast cancer cells from a heterogeneous suspension of cells circulating in the blood stream. Conceptually, the technique requires the transformation of a distinguishing biochemical characteristic of the target cells, such as up-regulated cadherin phenotype, into a mechanical or electrical that makes it possible to selectively manipulate the cells on the microscale. The project includes developments of a model system of cells to evaluate cadherin-mediated cell sorting and an integrated bio-functional microfluidic system to capture target cells from heterogeneous suspensions of cells. We have succeeded in the transfection of MDA-MB-231 cells with an N-cadherin expression vector deriving a homogeneous population. An anti-N-cad functionalized surface has been shown to capture N-cad expressing prostate cancer cells (PC3N) with high degree of selectivity. An assay to characterize and a technique to control the amount of immobilized anti-N-cad antibodies on surfaces have been developed to maximize the cell capture efficiency. Microchannels with anti-N-cad functionalized surfaces have been fabricated. Under flow conditions, the capture rate is poor; however, after 15min of incubation time, the capture rate is high. Once captured, the cell/surface adhesion bond is strong enough to sustain high flow-induced shears stress.
• Cherukuri, D. P., Chen, X. B., Goulet, A., Young, R. N., Han, Y., Heimark, R. L., Regan, J. W., Meuillet, E., & Nelson, M. A. (2007). The EP4 receptor antagonist, L-161,982, blocks prostaglandin E2-induced signal transduction and cell proliferation in HCA-7 colon cancer cells. Experimental cell research, 313(14), 2969-79.
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  Accumulating evidence indicates that elevated levels of prostaglandin E(2) (PGE(2)) can increase intestinal epithelial cell proliferation, and thus play a role in colorectal tumorigenesis. PGE(2) exerts its effects through four G-protein-coupled PGE receptor (EP) subtypes, named the EP1, EP2, EP3, and EP4. Increased phosphorylation of extracellular regulated kinases (ERK1/2) is required for PGE(2) to stimulate cell proliferation of human colon cancer cells. However, the EP receptor(s) that are involved in this process remain unknown. We provide evidence that L-161,982, a selective EP4 receptor antagonist, completely blocks PGE(2)-induced ERK phosphorylation and cell proliferation of HCA-7 cells. In order to identify downstream target genes of ERK1/2 signaling, we found that PGE(2) induces expression of early growth response gene-1 (EGR-1) downstream of ERK1/2 and regulates its expression at the level of transcription. PGE(2) treatment induces phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 residue and CRE-mediated luciferase activity in HCA-7 cells. Studies with dominant-negative CREB mutant (ACREB) provide clear evidence for the involvement of CREB in PGE(2) driven egr-1 transcription in HCA-7 cells. In conclusion, this study reveals that egr-1 is a target gene of PGE(2) in HCA-7 cells and is regulated via the newly identified EP4/ERK/CREB pathway. Finally our results support the notion that antagonizing EP4 receptors may provide a novel therapeutic approach to the treatment of colon cancer.
• Heimark, R. L., Meuillet, E. J., Nelson, M. A., Regan, J. W., Han, Y., Young, R. N., Goulet, A. C., Chen, X. B., & Cherukuri, D. P. (2007). The EP4 receptor antagonist, L-161,982, blocks prostaglandin E2-induced signal transduction and cell proliferation in HCA-7 colon cancer cells. Experimental Cell Research. doi:10.1016/j.yexcr.2007.06.004
• Stamer, W. D., Heimark, R. L., Perry, A., & Zanello, S. B. (2007). Regulation of Junctional Proteins in Schlemm’s Canal Cells. Investigative Ophthalmology & Visual Science.
• Cheung, L. S., Zheng, X., Stopa, A., Baygents, J. C., Guzman, R., Schroeder, J. A., Heimark, R. L., & Zohar, Y. (2006). Detachment of captured cancer cells under flow acceleration in a bio-functionalized microchannel. LAB ON A CHIP, 9(12), 1721-1731.
• Cox, C. M., D'Agostino, S. L., Miller, M. K., Heimark, R. L., & Krieg, P. A. (2006). Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo. Developmental biology, 296(1), 177-89.
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  The peptide growth factor apelin is the high affinity ligand for the G-protein-coupled receptor APJ. During embryonic development of mouse and frog, APJ receptor is expressed at high levels in endothelial precursor cells and in nascent vascular structures. Characterization of Xenopus apelin shows that the sequence of the bioactive region of the peptide is perfectly conserved between frogs and mammals. Embryonic expression studies indicate that apelin is expressed in, or immediately adjacent to, a subset of the developing vascular structures, particularly the intersegmental vessels. Experimental inhibition of either apelin or APJ expression, using antisense morpholino oligos, results in elimination or disruption of intersegmental vessels in a majority of embryos. In gain of function experiments, apelin peptide is a potent angiogenic factor when tested using two in vivo angiogenesis assays, the frog embryo and the chicken chorioallantoic membrane. Furthermore, studies using the mouse brain microvascular cell line bEnd.3 show that apelin acts as a mitogenic, chemotactic and anti-apoptotic agent for endothelial cells in culture. Finally, we show that, similar to a number of other angiogenic factors, expression of the apelin gene is increased under conditions of hypoxia. Taken together, these studies indicate that apelin is required for normal vascular development in the frog embryo and has properties consistent with a role during normal and pathological angiogenesis.
• Heimark, R. L., Glackin, C. A., Summers, C., Rekapally, H., Tran, N. V., & Alexander, N. R. (2006). N-cadherin Gene Expression in Prostate Carcinoma Is Modulated by Integrin-Dependent Nuclear Translocation of Twist1. Cancer Research, 66(7), 3365-3369. doi:10.1158/0008-5472.can-05-3401
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  The gain of N-cadherin expression in carcinomas has been shown to be important in the regulation of cell migration, invasion, and survival. Here, we show that N-cadherin mRNA expression in PC-3 prostate carcinoma cells is dependent on β1 integrin–mediated cell adhesion to fibronectin and the basic helix-loop-helix transcription factor Twist1. Depletion of Twist1 mRNA by small interfering RNA resulted in decreased expression of both Twist1 and N-cadherin and the inhibition of cell migration. Whereas Twist1 gene expression was independent of β1 integrin–mediated adhesion, Twist1 protein failed to accumulate in the nuclei of cells cultured in anchorage-independent conditions. The increased nuclear accumulation of Twist1 following cell attachment was suppressed by treatment with an inhibitor of Rho kinase or a β1 integrin neutralizing antibody. The effect of Twist1 on induction of N-cadherin mRNA …
• Heimark, R. L., Krieg, P. A., Miller, M. K., D'Agostino, S. L., & Cox, C. M. (2006). Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo. Developmental Biology, 177-1898. doi:10.1016/j.ydbio.2006.04.452
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  The peptide growth factor apelin is the high affinity ligand for the G-protein-coupled receptor APJ. During embryonic development of mouse and frog, APJ receptor is expressed at high levels in endothelial precursor cells and in nascent vascular structures. Characterization of Xenopus apelin shows that the sequence of the bioactive region of the peptide is perfectly conserved between frogs and mammals. Embryonic expression studies indicate that apelin is expressed in, or immediately adjacent to, a subset of the developing vascular structures, particularly the intersegmental vessels. Experimental inhibition of either apelin or APJ expression, using antisense morpholino oligos, results in elimination or disruption of intersegmental vessels in a majority of embryos. In gain of function experiments, apelin peptide is a potent angiogenic factor when tested using two in vivo angiogenesis assays, the frog embryo and the chicken chorioallantoic membrane. Furthermore, studies using the mouse brain microvascular cell line bEnd.3 show that apelin acts as a mitogenic, chemotactic and anti-apoptotic agent for endothelial cells in culture. Finally, we show that, similar to a number of other angiogenic factors, expression of the apelin gene is increased under conditions of hypoxia. Taken together, these studies indicate that apelin is required for normal vascular development in the frog embryo and has properties consistent with a role during normal and pathological angiogenesis.
• Heimark, R., Alexander, N. R., Tran, N. L., Rekapally, H., Summers, C. E., Glackin, C., & Heimark, R. L. (2006). N-cadherin gene expression in prostate carcinoma is modulated by integrin-dependent nuclear translocation of Twist1. Cancer research, 66(7).
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  The gain of N-cadherin expression in carcinomas has been shown to be important in the regulation of cell migration, invasion, and survival. Here, we show that N-cadherin mRNA expression in PC-3 prostate carcinoma cells is dependent on beta(1) integrin-mediated cell adhesion to fibronectin and the basic helix-loop-helix transcription factor Twist1. Depletion of Twist1 mRNA by small interfering RNA resulted in decreased expression of both Twist1 and N-cadherin and the inhibition of cell migration. Whereas Twist1 gene expression was independent of beta(1) integrin-mediated adhesion, Twist1 protein failed to accumulate in the nuclei of cells cultured in anchorage-independent conditions. The increased nuclear accumulation of Twist1 following cell attachment was suppressed by treatment with an inhibitor of Rho kinase or a beta(1) integrin neutralizing antibody. The effect of Twist1 on induction of N-cadherin mRNA required an E-box cis-element located within the first intron (+2,627) of the N-cadherin gene. These data raise the possibility that integrin-mediated adhesion to interstitial matrix proteins during metastasis differentially regulates the nuclear/cytoplasmic translocation and DNA binding of Twist1, activating N-cadherin transcription.
• Lee, L. M., Heimark, R. L., Baygents, J. C., & Zohar, Y. (2006). Self-aligned immobilization of proteins utilizing PEG patterns. Nanotechnology, 17(4), S29-33.
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  A novel self-aligned method to selectively immobilize proteins on a silicon dioxide surface is developed in conjunction with a standard lift-off patterning technique of a PEG layer. The approach is designed to photolithographically pattern regions that specifically bind target proteins and particles, surrounded by regions that suppress non-specific attachment of bio-species. The physical and biological properties of the derivatized surfaces at the end of the fabrication process are characterized.
• Lee, L. M., Heimark, R. L., Guzman, R., Baygents, J. C., & Zohar, Y. (2006). Low melting point agarose as a protection layer in photolithographic patterning of aligned binary proteins. LAB ON A CHIP, 6(8), 1080-1085.
• Lee, L. M., Heimark, R. L., Guzman, R., Baygents, J. C., & Zohar, Y. (2006). Low melting point agarose as a protection layer in photolithographic patterning of aligned binary proteins. Lab on a chip, 6(8), 1080-5.
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  A novel photolithography method to build aligned patterns of two different proteins is presented. Chessboard patterns of 125 microm x 125 microm squares are constructed on a silicon dioxide substrate, using standard photoresist chemistries in combination with low-temperature oxygen plasma etching. Low-melting-point agarose (LMPA) is used to protect underlying protein layers and, at the appropriate stage, the digestive enzyme GELase (EPICENTRE) is used to selectively remove the prophylactic LMPA layers. Two antibodies, mouse-IgG and human-IgG, were immobilized and patterned by this procedure. The patterned antibodies maintained the specificity of their antigen-antibody binding, as demonstrated by fluorescence microscopy. In addition, normalized fluorescence intensity profiles illustrate that the patterned proteins layers are uniform (standard deviations below 0.05). Finally, a trypsin activity test was conducted to probe the effect of the patterning protocol on immobilized enzymes; the results imply that this photolithographic process using LMPA as a protection layer preserves 70% of immobilized enzyme activity.
• Derbyshire, Z. E., Haltfer, U. M., Heimark, R. L., Vaillancourt, R. R., & Sy, T. H. (2005). Angiotensin II stimulated transcription of cyclooxygenase II is regulated by a novel kinase cascade involving Pyk2, MEKK4 and annexin II. Molecular and Cellular Biochemistry, 271, 77-90. doi:doi: 10.1007/s11010-005-5386-9
• Lee, L. M., Heimark, R. L., Baygents, J. C., & Zohar, Y. (2005). Self-aligned immobilization of proteins utilizing PEG patterns. NANOTECHNOLOGY, 17(4), S29-S33.
• Vokes, S. A., Yatskievych, T. A., Heimark, R. L., Antin, P., & Krieg, P. (2004). Hedgehog Signaling is Essential for Endothelial Tube formation during Vasculogenesis. Development.
• Wessells, H., King, S. H., Schmelz, M., Nagle, R. B., & Heimark, R. L. (2004). Immunohistochemical comparison of vascular and sinusoidal adherens junctions in cavernosal endothelium. Urology, 63(1), 201-6.
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  To characterize endothelial cell-to-cell junctions in the sinusoids and microvasculature of the corpus cavernosum.
• Alexander, N. R., Tran, N. L., & Heimark, R. L. (2002). Co-expression of N-cadherin and E-cadherin in prostate carcinoma cells promotes a migratory phenotype. Proceedings of the American Association for Cancer Research, 43, 368.
• Heimark, R. L., Koachar, S., & Stamer, W. D. (2002). Human Schlem'scancal cells express the endothelial adherens junction proteins VE-cadherin and PECAM-1. Current Eye Research, 25(6), 299-308.
• Heimark, R. L., Vaillancourt, R. R., Adams, D. G., & Tran, N. L. (2002). Signal Transduction from N-cadherin Increases Bcl-2. Journal of Biological Chemistry, 277(36), 32905-32914. doi:10.1074/jbc.m200300200
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  Associated with the metastatic progression of epithelial tumors is the dynamic regulation of cadherins. Whereas E-cadherin is expressed in most epithelium and carcinomas, recent studies suggest that the up-regulation of other cadherin subtypes in carcinomas, such as N-cadherin, may function in cancer progression. We demonstrate that a signal transduction cascade links the N-cadherin·catenin adhesion complex to up-regulation of the anti-apoptotic protein Bcl-2. In suspension, aggregates of DU-145 cells, an E-cadherin expressing human prostate carcinoma line, survive loss of integrin-dependent adhesion by a different anti-apoptotic signaling pathway than the N-cadherin expressing lines PC3 and PC3N. N-cadherin intercellular adhesion mediates a 3.5-fold increase in Bcl-2 protein expression, whereas the level of the proapoptotic protein Bax remains constant. Only N-cadherin ligation in PC3 cells, which express both N-cadherin and E-cadherin, is sufficient to induce activation of Akt/protein kinase B. N-cadherin homophilic ligation initiates phosphatidylinositol 3-kinase-dependent activation of Akt resulting in Akt phosphorylation of Bad on serine 136. Following N-cadherin homophilic adhesion phosphatidylinositol 3-kinase was identified in immunoprecipitates of the N-cadherin·catenin complex. The recruitment of phosphatidylinositol 3-kinase to the adhesion complex is dependent on ligation of N-cadherin and an organized actin cytoskeleton because cytochalasin D blocks the recruitment. We propose that N-cadherin homophilic adhesion can initiate anti-apoptotic signaling, which enhances the Akt cell survival pathway in metastatic cancer.
• Heimark, R., Tran, N. L., Adams, D. G., Vaillancourt, R. R., & Heimark, R. L. (2002). Signal transduction from N-cadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization. The Journal of biological chemistry, 277(36).
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  Associated with the metastatic progression of epithelial tumors is the dynamic regulation of cadherins. Whereas E-cadherin is expressed in most epithelium and carcinomas, recent studies suggest that the up-regulation of other cadherin subtypes in carcinomas, such as N-cadherin, may function in cancer progression. We demonstrate that a signal transduction cascade links the N-cadherin.catenin adhesion complex to up-regulation of the anti-apoptotic protein Bcl-2. In suspension, aggregates of DU-145 cells, an E-cadherin expressing human prostate carcinoma line, survive loss of integrin-dependent adhesion by a different anti-apoptotic signaling pathway than the N-cadherin expressing lines PC3 and PC3N. N-cadherin intercellular adhesion mediates a 3.5-fold increase in Bcl-2 protein expression, whereas the level of the proapoptotic protein Bax remains constant. Only N-cadherin ligation in PC3 cells, which express both N-cadherin and E-cadherin, is sufficient to induce activation of Akt/protein kinase B. N-cadherin homophilic ligation initiates phosphatidylinositol 3-kinase-dependent activation of Akt resulting in Akt phosphorylation of Bad on serine 136. Following N-cadherin homophilic adhesion phosphatidylinositol 3-kinase was identified in immunoprecipitates of the N-cadherin.catenin complex. The recruitment of phosphatidylinositol 3-kinase to the adhesion complex is dependent on ligation of N-cadherin and an organized actin cytoskeleton because cytochalasin D blocks the recruitment. We propose that N-cadherin homophilic adhesion can initiate anti-apoptotic signaling, which enhances the Akt cell survival pathway in metastatic cancer.
• Bair, E. L., Massey, C. P., Tran, N. L., Borchers, A. H., Heimark, R. L., Cress, A. E., & Bowden, G. T. (2001). Integrin- and cadherin-mediated induction of the matrix metalloprotease matrilysin in cocultures of malignant oral squamous cell carcinoma cells and dermal fibroblasts. Experimental cell research, 270(2), 259-67.
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  Matrilysin is a matrix metalloprotease (MMP) overexpressed in a number of cancers including skin, head and neck squamous cell carcinomas, and prostate and colon adenocarcinomas. Matrilysin has been shown to play a role in the degradation of the basement membrane that separates epithelium from stroma allowing tumor cells to intravasate into the bloodstream and metastasize. Here, we show that an oral squamous cell carcinoma cell line (SCC-25) expresses low levels of promatrilysin when cultured alone. However, when SCC-25 cells are cocultured with human foreskin fibroblasts (HFF), there is a 40-fold induction of promatrilysin expression. We tested whether this induction of promatrilysin expression was due to the release of paracrine factors, cell-cell interactions, or cell-matrix interactions. Our results indicate induced promatrilysin expression is the result of both cell-cell and cell-matrix interactions. We demonstrate that beta1 integrins as well as cadherins, specifically N-cadherin and E-cadherin, are involved in the induction of promatrilysin expression. Our results are of general interest in relation to the regulation of MMP expression through cell surface receptor regulation. Further investigation may lead to the identification of novel targets for suppression of invasion and metastasis in oral tumors.
• Westerband, A., Crouse, D., Richter, L. C., Aguirre, M. L., Wixon, C. C., James, D. C., Mills, J. L., Hunter, G. C., & Heimark, R. L. (2001). Vein adaptation to arterialization in an experimental model. Journal of vascular surgery, 33(3), 561-9.
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  The events preceding myointimal thickening in vein grafts after vascular reconstructions are not well characterized. Indeed, the injury response associated with vein graft arterialization may be different than that observed in the balloon angioplasty model. Therefore, we used a rat model to study the early cellular response after arterialization of vein grafts.
• Heimark, R. L., Vincent, P. A., Minnear, F. L., Goldberg, P. L., & Hurst, V. (1999). Rearrangement of adherens junctions by transforming growth factor-β1: role of contraction. American Journal of Physiology-lung Cellular and Molecular Physiology, 276(4), L582-L595. doi:10.1152/ajplung.1999.276.4.l582
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  The signal transduction pathways that lead to disruption of pulmonary endothelial monolayer integrity by transforming growth factor-β1 (TGF-β1) have not been elucidated. The purpose of this investigation was to determine whether disassembly of the adherens junction is temporally associated with the TGF-β1-induced decrease in pulmonary endothelial monolayer integrity. Measurement of albumin clearance and electrical resistance showed that monolayer integrity started to decrease between 1 and 2 h post-TGF-β1 treatment and continued to slowly decrease over the next 6 h. Immunofluorescence microscopy of monolayers between 2 and 3 h post-TGF-β1 showed that β-catenin, plakoglobin, α-catenin, and cadherin-5 were colocalized both at the cell periphery and in newly formed bands that are perpendicular to the cell-cell border. At 4 h post-TGF-β1, cells began separating; however, β- and α-catenin, plakoglobin, and cadherin-5 could still be found at the cell periphery at areas of cell separation and in strands between separated cells. By 8 h, these junctional proteins were no longer present at the cell periphery at areas of cell separation. The myosin light chain kinase inhibitor KT-5926 prevented the TGF-β1-induced change in integrity but did not inhibit the formation of actin stress fibers or the formation of bands containing adherens junction proteins that were perpendicular to the cell-cell junction. Overall, these results suggest that adherens junction disassembly occurs after cell separation during TGF-β1-induced decreases in pulmonary endothelial monolayer integrity and that the loss of integrity may be due to the activation of a myosin light chain kinase-dependent signaling cascade.
• Hurst V, I. V., Goldberg, P. L., Minnear, F. L., Heimark, R. L., & Vincent, P. A. (1999). Rearrangement of adherens junctions by transforming growth factor-beta1: role of contraction. The American journal of physiology, 276(4 Pt 1), L582-95.
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  The signal transduction pathways that lead to disruption of pulmonary endothelial monolayer integrity by transforming growth factor-beta1 (TGF-beta1) have not been elucidated. The purpose of this investigation was to determine whether disassembly of the adherens junction is temporally associated with the TGF-beta1-induced decrease in pulmonary endothelial monolayer integrity. Measurement of albumin clearance and electrical resistance showed that monolayer integrity started to decrease between 1 and 2 h post-TGF-beta1 treatment and continued to slowly decrease over the next 6 h. Immunofluorescence microscopy of monolayers between 2 and 3 h post-TGF-beta1 showed that beta-catenin, plakoglobin, alpha-catenin, and cadherin-5 were colocalized both at the cell periphery and in newly formed bands that are perpendicular to the cell-cell border. At 4 h post-TGF-beta1, cells began separating; however, beta- and alpha-catenin, plakoglobin, and cadherin-5 could still be found at the cell periphery at areas of cell separation and in strands between separated cells. By 8 h, these junctional proteins were no longer present at the cell periphery at areas of cell separation. The myosin light chain kinase inhibitor KT-5926 prevented the TGF-beta1-induced change in integrity but did not inhibit the formation of actin stress fibers or the formation of bands containing adherens junction proteins that were perpendicular to the cell-cell junction. Overall, these results suggest that adherens junction disassembly occurs after cell separation during TGF-beta1-induced decreases in pulmonary endothelial monolayer integrity and that the loss of integrity may be due to the activation of a myosin light chain kinase-dependent signaling cascade.
• Tran, N. L., Nagle, R. B., Cress, A. E., & Heimark, R. L. (1999). N-cadherin expression in human prostate carcinoma cell lines- An Epithelial-mesenchymal transformation mediating adhesion with stromal cells. American Journal of Pathology, 155(3), 787-798.
• Wong, R. K., Baldwin, A. L., & Heimark, R. L. (1999). Cadherin-5 redistribution at sites of TNF-α and IFN-γ-induced permeability in mesenteric venules. American Journal of Physiology-heart and Circulatory Physiology, H736-748. doi:10.1152/ajpheart.1999.276.2.h736
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  The response of the endothelial permeability barrier in microvascular networks of the rat mesentery to perfused immune inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was examined. TNF-α (12.5 U/ml) treatment did not change albumin permeability, but in combination with IFN-γ (20 U/ml), there was a marked increase in the number of sites of extravascular albumin in postcapillary venules. Endothelial integrity was characterized by cadherin-5 immunoreactivity, which was localized to the continuous intercellular junctions of endothelium in arterioles, capillaries, and venules. Perfusion with the combined cytokines showed that the increased albumin permeability was dose dependent and correlated with the focal disorganization of cadherin-5 at intercellular junctions of venular endothelium. No correlation was found between the increase in albumin permeability and the localization of intravascular leukocytes or extravascular mast cells. These results show that the combination of TNF-α and IFN-γ induces an endothelial phenotype with focal loss of cadherin-5 intercellular adhesion, which, in part, facilitates passage of blood macromolecules and cells to the interstitium.
• Heimark, R. L., Ihnat, D., Gentile, A. T., Hunter, G. C., Mills, J. L., & Westerband, A. (1998). Topography of cell replication in human vein graft stenoses.. Circulation, II-325.
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  Analysis of the cellular composition of human autogenous vein graft lesions at the time of revision provides an opportunity to identify the cellular processes leading to the development of stenosis in humans after vascular reconstruction.Human vein graft-threatening stenotic lesions were identified by duplex scanning within 3 to 18 months after infrainguinal bypass and surgically removed. They were serially studied by immunocytochemistry for expression of the proliferating cell nuclear antigen (PCNA) in different cell types: alpha-actin-positive smooth muscle cells (SMCs), endothelial cells (ECs), monocytes, and macrophages. Proliferation indexes were separately obtained for each layer of the vessel wall by determining the mean percentage of PCNA-positive nuclei among the total number of nuclei present within the intima, the media, and the adventitia, respectively. The percentage distribution of the replicating cell types was also determined. We report that in autogenous vein graft (n = 14) the intima of the lesion displayed fewer PCNA + nuclei (1.03 +/- 0.88) than the underlying media (3.14 +/- 0.74) or the adventitia (3.01 +/- 0.74). Replicating SMCs were predominantly in the medial layer (68% of PCNA + cells) of stenotic vein grafts. In the adventitia, the proliferation was most intense in the endothelium of microvessels (65% of PCNA + nuclei).Our findings reveal a 3-fold greater proliferative activity in the media and the adventitia as compared with the intima of autogenous vein graft lesions, in contrast to cellular proliferation identified in recurrent coronary stenotic plaques. Moreover, there are distinctive patterns of distribution of the different cell populations among the 3 layers. The results indicate a proliferative response of the media and the adventitia of autogenous vein grafts transplanted into the arterial circulation, in addition to the cellular proliferation observed in the intima of the lesion.
• Westerband, A., Mills, J. L., Hunter, G. C., Gentile, A. T., Ihnat, D., & Heimark, R. L. (1998). Topography of cell replication in human vein graft stenoses. Circulation, 98(19 Suppl), II325-9; discussion II329-30.
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  Analysis of the cellular composition of human autogenous vein graft lesions at the time of revision provides an opportunity to identify the cellular processes leading to the development of stenosis in humans after vascular reconstruction.
• Haselton, F. R., & Heimark, R. L. (1997). Role of cadherins 5 and 13 in the aortic endothelial barrier. Journal of cellular physiology, 171(3), 243-51.
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  We investigated the role of the cadherins 5 and 13 in the solute barrier formed by aortic endothelial cells in vitro. In confluent monolayers of bovine aortic endothelial cells, immunofluorescence with antibodies to the external domain of cadherin 5 (Mab 9H7) or to cadherin 13 (Mab Ec6C10) found staining for both cadherins at endothelial cell borders. Western blotting with an antibody to the characteristic cadherin cytoplasmic tail or with an antibody to the extracellular domain of cadherin 5 revealed a single 125 kD protein band. A second larger band was found at 130 kD with the anti-cadherin 13 Mab which was not recognized by an antibody to the cadherin cytoplasmic tail. A calcium switch strategy was used to investigate the involvement of these cadherins in the endothelial barrier. Changes in the permeability of small solutes in an endothelial cell column produced by a decrease in calcium concentration followed by a return to normal calcium, with or without antibody, were recorded. We found that anti-cadherin 5 IgG (10 micrograms/ml) interfered with the reforming of interendothelial junctions after restoration of calcium at every time point tested for a total of 45 min after restoration of calcium. The anti-cadherin 13 IgG (10 micrograms/ml) did not block reforming of the endothelial barrier in a similar manner. The presence of this antibody delayed only by 15 min the restoration of the normal barrier. Without calcium switch, addition of either monoclonal antibody (10 micrograms/ml) to the endothelial cell column had no effect on solute permeability. These results suggest that cadherin 5 in bovine aortic endothelial cells has a major functional role in forming the calcium-sensitive endothelial junction in vitro and may play an important role in the normal structure and function of the in vivo barrier.
• Heimark, R. L., & Haselton, F. R. (1997). Role of cadherins 5 and 13 in the aortic endothelial barrier. Journal of Cellular Physiology, 171(3), 243-251. doi:10.1002/(sici)1097-4652(199706)171:3<243::aid-jcp2>3.0.co;2-o
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  We investigated the role of the cadherins 5 and 13 in the solute barrier formed by aortic endothelial cells in vitro. In confluent monolayers of bovine aortic endothelial cells, immunofluorescence with antibodies to the external domain of cadherin 5 (Mab 9H7) or to cadherin 13 (Mab Ec6C10) found staining for both cadherins at endothelial cell borders. Western blotting with an antibody to the characteristic cadherin cytoplasmic tail or with an antibody to the extracellular domain of cadherin 5 revealed a single 125 kD protein band. A second larger band was found at 130 kD with the anti-cadherin 13 Mab which was not recognized by an antibody to the cadherin cytoplasmic tail. A calcium switch strategy was used to investigate the involvement of these cadherins in the endothelial barrier. Changes in the permeability of small solutes in an endothelial cell column produced by a decrease in calcium concentration followed by a return to normal calcium, with or without antibody, were recorded. We found that anti-cadherin 5 IgG (10 micrograms/ml) interfered with the reforming of interendothelial junctions after restoration of calcium at every time point tested for a total of 45 min after restoration of calcium. The anti-cadherin 13 IgG (10 micrograms/ml) did not block reforming of the endothelial barrier in a similar manner. The presence of this antibody delayed only by 15 min the restoration of the normal barrier. Without calcium switch, addition of either monoclonal antibody (10 micrograms/ml) to the endothelial cell column had no effect on solute permeability. These results suggest that cadherin 5 in bovine aortic endothelial cells has a major functional role in forming the calcium-sensitive endothelial junction in vitro and may play an important role in the normal structure and function of the in vivo barrier.
• Schram, C. A., Runyan, R. R., & Heimark, R. L. (1997). Regulation of cadherin 5 during epithelial mesenchymal transformation of atrioventricular endocardium. Molecular Biology of the Cell, 8, 1899.
• Westerband, A., Mills, J. L., Marek, J. M., Heimark, R. L., Hunter, G. C., & Williams, S. K. (1997). Immunocytochemical determination of cell type and proliferation rate in human vein graft stenoses. Journal of vascular surgery, 25(1), 64-73.
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  Vascular reconstructions are prone to fail as a result of the development of stenotic lesions, which have historically been attributed to myointimal hyperplasia. In animal models, these lesions are associated with marked proliferative smooth muscle cell (SMC) response to vascular injury. However, recent studies using sensitive immunocytochemical techniques in human lesions have generally failed to detect significant cellular proliferation. To clarify the role of cellular proliferation in humans, we characterized the cellular composition and proliferative index of 14 early infrainguinal vein graft stenoses.
• SCHRAMM, C., & HEIMARK, R. (1995). CADHERIN-5 EXPRESSION IN ENDOTHELIAL DIFFERENTIATION AND BLOOD-VESSEL GROWTH DURING EMBRYOGENESIS. DEVELOPMENTAL BIOLOGY, 170(2), 753.
• Heimark, R. L., Suzuki, S., John, T. S., Sano, K., & Tanihara, H. (1994). Cloning of Five Human Cadherins Clarifies Characteristic Features of Cadherin Extracellular Domain and Provides Further Evidence for Two Structurally Different Types of Cadherin. Cell Adhesion and Communication, 15-26. doi:10.3109/15419069409014199
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  The entire coding sequences for five possible human cadherins, named cadherin-4, -8, -11, -12 and -13, were determined. The deduced amino acid sequences of cadherin-4 and cadherin-13 showed high homology with those of chicken R-cadherin or chicken T-cadherin, suggesting that cadherin-4 and cadherin-13 are mammalian homologues of the chicken R-cadherin or T-cadherin. Comparison of the extracellular domain of these proteins with those of other cadherins and cadherin-related proteins clarifies characteristic structural features of this domain. The domain is subdivided into five subdomains, each of which contains a cadherin-specific motif characterized by well-conserved amino acid residues and short amino acid sequences. Moreover, each subdomain has unique features of its own. The comparison also provides additional evidence for two structurally different types of cadherins: the first type includes B-, E-, EP-, M, N-, P- and R-cadherins and cadherin-4; the second type includes cadherin-5 through cadherin-12. Cadherin-13 lacks the sequence corresponding to the cytoplasmic domain of typical cadherins, but the extracellular domain shares most of the features common to the extracellular domain of cadherins, especially those of the first type of cadherins, suggesting that cadherin-13 is a special type of cadherin. These results, and those of other recent cloning studies, indicate that many cadherins with different properties are expressed in various tissues of different organisms.
• Tanihara, H., Kido, M., Obata, S., Heimark, R. L., Davidson, M., St John, T., & Suzuki, S. (1994). Characterization of cadherin-4 and cadherin-5 reveals new aspects of cadherins. Journal of cell science, 107 ( Pt 6), 1697-704.
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  Several properties of cadherin-4 and cadherin-5 were characterized by using the cDNA transfection approach. The proteins of both cadherins had a relative molecular mass of about 130 kDa and were present at the cell periphery, especially at cell-cell contact sites. These cadherins were easily digested with trypsin, and Ca2+ protected cadherin-4, but not cadherin-5, from the digestion. In immunoprecipitation, cadherin-4 co-precipitated with two major proteins of 105 kDa and 95 kDa, respectively. The 105 kDa and the 95 kDa proteins are likely to correspond to alpha- and beta-catenins. Cadherin-5 co-precipitated with only one major protein of 95 kDa, but seems to associate with the 105 kDa protein. On the other hand, plakoglobin or gamma-catenin did not co-precipitate well with either cadherin-4 or cadherin-5 in immunoprecipitation, but plakoglobin also appears to associated weakly with these cadherins. Cadherin-4 transfectants aggregated within 30 minutes in a cell aggregation assay, but cadherin-5 transfectants did not aggregate under the same conditions. Furthermore, the transfectants of chimeric cadherin-4 with cadherin-5 cytoplasmic domain showed cell aggregation activity comparable to that of wild-type cadherin-4 transfectants, whereas the transfectants of chimeric cadherin-5 with cadherin-4 cytoplasmic domain did not show appreciable cell aggregation, suggesting that the extracellular domains of cadherins, in conjunction with their cytoplasmic domains, play an important role in cell aggregation activity. These results show that cadherin-4 is very similar to the classical cadherins, whereas cadherin-5 is functionally as well as structurally distinct from classical cadherins.
• Tanihara, H., Sano, K., Heimark, R. L., St John, T., & Suzuki, S. (1994). Characteristic Features of Cadherin Extracellular Domain and provides further Evidence for 2 Structurall Different types of Cadherin. Cell Adhesion and Communication, 2(1), 15-26.
• Tanihara, H., Sano, K., Heimark, R. L., St John, T., & Suzuki, S. (1994). Cloning of five human cadherins clarifies characteristic features of cadherin extracellular domain and provides further evidence for two structurally different types of cadherin. Cell adhesion and communication, 2(1), 15-26.
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  The entire coding sequences for five possible human cadherins, named cadherin-4, -8, -11, -12 and -13, were determined. The deduced amino acid sequences of cadherin-4 and cadherin-13 showed high homology with those of chicken R-cadherin or chicken T-cadherin, suggesting that cadherin-4 and cadherin-13 are mammalian homologues of the chicken R-cadherin or T-cadherin. Comparison of the extracellular domain of these proteins with those of other cadherins and cadherin-related proteins clarifies characteristic structural features of this domain. The domain is subdivided into five subdomains, each of which contains a cadherin-specific motif characterized by well-conserved amino acid residues and short amino acid sequences. Moreover, each subdomain has unique features of its own. The comparison also provides additional evidence for two structurally different types of cadherins: the first type includes B-, E-, EP-, M, N-, P- and R-cadherins and cadherin-4; the second type includes cadherin-5 through cadherin-12. Cadherin-13 lacks the sequence corresponding to the cytoplasmic domain of typical cadherins, but the extracellular domain shares most of the features common to the extracellular domain of cadherins, especially those of the first type of cadherins, suggesting that cadherin-13 is a special type of cadherin. These results, and those of other recent cloning studies, indicate that many cadherins with different properties are expressed in various tissues of different organisms.
• Sano, K., Tanihara, H., Heimark, R. L., Obata, S., Davidson, M., St John, T., Taketani, S., & Suzuki, S. (1993). Protocadherins: a large family of cadherin-related molecules in central nervous system. The EMBO journal, 12(6), 2249-56.
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  Using the polymerase chain reaction, we have isolated numerous rat and human cDNAs of which the deduced amino acid sequences are highly homologous to the sequences of the extracellular domain of cadherins. The entire putative coding sequences for two human proteins defined by two of these cDNAs have been determined. The overall structure of these molecules is very similar to that of classic cadherins, but they have some unique features. The extracellular domains are composed of six or seven subdomains that are very similar to those of cadherins, but have characteristic properties. The cytoplasmic domains, on the other hand, have no significant homology with those of classic cadherins. Since various cDNAs with almost identical features were obtained also from Xenopus, Drosophila and Caenorhabditis elegans, it appears that similar molecules are expressed in a variety of organisms. We have tentatively named these proteins protocadherins. They are highly expressed in brain and their expression appears to be developmentally regulated. The proteins expressed from the two full-length cDNAs in L cells were approximately 170 or 150 kDa in size, and were localized mainly at cell-cell contact sites. Moreover, the transfectants showed cell adhesion activity.
• Coffin, J. D., Harrison, J., Schwartz, S., & Heimark, R. (1991). Angioblast differentiation and morphogenesis of the vascular endothelium in the mouse embryo. Developmental biology, 148(1), 51-62.
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  Bandeiraea simplicifolia B4 isolectin (BSLB4) and polyclonal antisera against von Willebrand factor (VWF) were used to study the origin of endothelial cells and their organization into blood vessels in the postimplantation mouse embryo. Examination of BSLB4-stained whole mounted and sectioned embryos revealed intense staining of the endothelium, highlighting large vessels, capillaries, and many individual cells. Dorsal aorta formation was first obvious at E7 when many lectin-positive cells appeared in paraxial and lateral plate mesoderm. As development proceeded to E8, BSLB4-positive cells became organized into craniocaudal lines destined to become the aorta proper. At E9, BSLB4 stained all vessels of the embryo including the dorsal aorta, the intersomitic arteries, and the endocardium. VWF expression was not detected until E8 when BSLB4/VWF double-stained sections revealed the dorsal aortae as the first VWF-positive vessels, while other endothelium visible with BSLB4 remained negative for VWF immunostaining. By E12 many other vessels became VWF-positive, including the aortic arches, the intersomitic arteries, and the cardinal veins. However, many angioblasts and capillaries remained VWF-negative, reflecting the heterogeneous expression of VWF among endothelium that has been reported in adults of other species. The histochemical data reported here support the conclusions of earlier avian studies by showing distinct vascular patterns in the initial formation of vessels from isolated angioblasts (vasculogenesis), followed by the extension and organization of the initial vascular structures (angiogenesis). Moreover, our data suggest that the endothelium arises from distinct VWF-positive sources associated with the dorsal aorta, as well as VWF-negative sources associated with other vessels in the embryo.
• Heimark, R. L. (1991). Calcium-Dependent and Calcium-Independent Cell Adhesion Molecules in the Endothelium. Annals of the New York Academy of Sciences, 614, 229-239.
• Bavisotto, L. M., Schwartz, S. M., & Heimark, R. L. (1990). Modulation of Ca2(+)-dependent intercellular adhesion in bovine aortic and human umbilical vein endothelial cells by heparin-binding growth factors. Journal of cellular physiology, 143(1), 39-51.
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  Cultured endothelial cells have been shown to possess two mechanisms of intercellular adhesion: Ca2(+)-dependent and Ca2(+)-independent. We report here that growth of bovine aortic endothelial cells (BAEC) in complete medium containing purified basic fibroblast growth factor (bFGF, 6 ng/ml) results in loss of Ca2(+)-dependent intercellular adhesion. In the presence of heparin (90 micrograms/ml), this effect is reproduced upon treatment with acidic fibroblast growth factor (aFGF, 6 ng/ml) or endothelial cell growth supplement (ECGS, 100 micrograms/ml), in both human umbilical vein endothelial cells (HUVEC) and BAEC. Treatment at these doses with aFGF in the absence of heparin or with heparin alone is without significant effect. Loss of Ca2(+)-dependent adhesion following treatment of cells with heparin-binding growth factors (HBGFs) is prevented by pre-treatment of cell layers with cycloheximide. The Ca2(+)-independent adhesion mechanism is unaffected by HBGF treatment. Exposure of endothelial cells to HBGFs, moreover, prevents the eventual establishment of quiescence in growing cultures and restimulates replication in confluent cultures that have reached a final density-inhibited state. Addition of bFGF alone or aFGF + heparin at these doses results in a 4-fold increase in DNA synthesis over untreated control cultures at saturation density as reflected by thymidine index. A single addition of bFGF (6 ng/ml) to untreated quiescent confluent BAEC monolayers results in an increase in 3H-TdR incorporation reaching a peak at 22 hours with a parallel loss of Ca2(+)-dependent adhesiveness. Fluorescent staining with rhodamine-phalloidin demonstrates an altered distribution of polymerized F-actin in the bFGF-treated monolayers, marked by disruption of the dense peripheral microfilament bands retained by untreated confluent monolayers. Together, these results indicate that the mitogenic effect of HBGFs in cultured endothelial cells is associated with a "morphogenic" set of responses, perhaps dependent on breakdown of calcium-dependent cell-cell contacts.
• Heimark, R. L., Schwartz, S. M., & Bavisotto, L. M. (1990). Modulation of Ca2+-dependent intercellular adhesion in bovine aortic and human umbilical vein endothelial cells by heparin-binding growth factors. Journal of Cellular Physiology, 143(1), 39-51. doi:10.1002/jcp.1041430106
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  Cultured endothelial cells have been shown to possess two mechanisms of intercellular adhesion: Ca2(+)-dependent and Ca2(+)-independent. We report here that growth of bovine aortic endothelial cells (BAEC) in complete medium containing purified basic fibroblast growth factor (bFGF, 6 ng/ml) results in loss of Ca2(+)-dependent intercellular adhesion. In the presence of heparin (90 micrograms/ml), this effect is reproduced upon treatment with acidic fibroblast growth factor (aFGF, 6 ng/ml) or endothelial cell growth supplement (ECGS, 100 micrograms/ml), in both human umbilical vein endothelial cells (HUVEC) and BAEC. Treatment at these doses with aFGF in the absence of heparin or with heparin alone is without significant effect. Loss of Ca2(+)-dependent adhesion following treatment of cells with heparin-binding growth factors (HBGFs) is prevented by pre-treatment of cell layers with cycloheximide. The Ca2(+)-independent adhesion mechanism is unaffected by HBGF treatment. Exposure of endothelial cells to HBGFs, moreover, prevents the eventual establishment of quiescence in growing cultures and restimulates replication in confluent cultures that have reached a final density-inhibited state. Addition of bFGF alone or aFGF + heparin at these doses results in a 4-fold increase in DNA synthesis over untreated control cultures at saturation density as reflected by thymidine index. A single addition of bFGF (6 ng/ml) to untreated quiescent confluent BAEC monolayers results in an increase in 3H-TdR incorporation reaching a peak at 22 hours with a parallel loss of Ca2(+)-dependent adhesiveness. Fluorescent staining with rhodamine-phalloidin demonstrates an altered distribution of polymerized F-actin in the bFGF-treated monolayers, marked by disruption of the dense peripheral microfilament bands retained by untreated confluent monolayers. Together, these results indicate that the mitogenic effect of HBGFs in cultured endothelial cells is associated with a "morphogenic" set of responses, perhaps dependent on breakdown of calcium-dependent cell-cell contacts.
• Heimark, R. L., Schwartz, S. M., & Majesky, M. W. (1990). Developmental Mechanisms Underlying Pathology of Arteries. Physiological Reviews, 70(4), 1177-1209.
• Schwartz, S. M., Heimark, R. L., & Degner, M. (1990). Identification of a Ca2(+)-dependent cell-cell adhesion molecule in endothelial cells.. The Journal of cell biology, 110(5), 1745-56. doi:10.1083/jcb.110.5.1745
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  Confluent cultures of aortic endothelial cells contain two different cell-cell adhesion mechanisms distinguished by their requirement for calcium during trypsinization and adhesion. A hybridoma clone was isolated producing a monoclonal antibody Ec6C10, which inhibits Ca2(+)-dependent adhesion of endothelial cells. There was no inhibition of Ca2(+)-independent adhesion of endothelial cells and only a minor effect on Ca2(+)-dependent adhesion of smooth muscle cells. Immunoblotting analysis shows that the antibody Ec6C10 recognizes a protein in endothelial but not epithelial cells with an apparent molecular weight of 135,000 in reducing conditions and 130,000 in non-reducing conditions. Monoclonal antibody Ec6C10 reacts with an antigen at the cell surface as shown by indirect immunofluorescence of confluent endothelial cells in a junctional pattern outlining the cobblestone morphology of the monolayer. Removal of extracellular calcium increased the susceptibility of the antigen recognized by antibody Ec6C10 to proteolysis by trypsin. The role of the Ca2(+)-dependent cell adhesion molecule in organization of the dense peripheral microfilament band in confluent endothelial cells was examined by adjusting the level of extracellular calcium to modulate cell-cell contact. Addition of the monoclonal antibody Ec6C10 at the time of the calcium switch inhibited the extent of formation of the peripheral F-actin band. These results suggest an association between cell-cell contact and the peripheral F-actin band potentially through the Ca2(+)-dependent CAM.
• Schwartz, S. M., Heimark, R. L., & Majesky, M. W. (1990). Developmental mechanisms underlying pathology of arteries. Physiological Reviews, 4, 1177-1209. doi:10.1152/physrev.1990.70.4.1177
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  This review tries to provide a general, and very speculative, view of growth control mechanisms that may be common to the development of blood vessels and to pathological processes including cell proliferation. From a developmental point of view, vascular growth is most likely to include local autocrine or paracrine mechanisms that permit the two cells of the vessel wall to grow, organize into the characteristic tubular and layered structures of the vessel wall, and eventually achieve a return to quiescence. The "real" mechanisms controlling growth in vivo are difficult to ascertain from studies in culture. For example, a large list of angiogenesis molecules must be able to generate endothelial replication, but in culture many of these molecules are inhibitory for each endothelial replication. Similarly, in culture, we have a long list of smooth muscle mitogens, but none of these have as of yet been proven to control smooth muscle growth in vivo. Endothelial growth control has been attributed to the presence of membrane molecules able to inhibit endothelial replication and to the actions of soluble growth factors and their receptors. Unfortunately for the former hypothesis we still lack specific molecules with the properties of contact inhibition of replication. The data discussed here, however, suggest that modulation of expression or function of cell-cell adhesive molecules could be critical both to morphogenic changes and to mitogenesis by release of cells from cell-cell contact. Moreover, our data and data from other laboratories suggest that angiogenic factors, including the HBGFs and TGF-beta, may function in angiogenesis by altering cell-cell and cell-cell substrate interactions rather than via a primary effect on cell replication. This view of angiogenesis is consistent with the absence of a mitogenic effect of some angiogenic factors. Although endothelial cell replication is obviously necessary to angiogenesis, the lack of mitogenic effect of some factors suggests a need for a more general explanation of the actions of angiogenic factors. Endothelial injury may be interrelated with smooth muscle growth. The simplest possibility is that a failure of the endothelial cell barrier function, due either to denudation or an increase in adhesivity for leukocytes, would permit access of platelets or leukocytes to the vessel wall. These extrinsic cells, in turn, would stimulate smooth muscle cell replication by release of growth factors. The second possibility is that the endothelial cell may itself release growth factors into the vessel wall.(ABSTRACT TRUNCATED AT 400 WORDS)
• Chen, C. S., Thiagarajan, P., Schwartz, S. M., Harlan, J. M., & Heimark, R. L. (1987). The platelet glycoprotein IIb/IIIa-like protein in human endothelial cells promotes adhesion but not initial attachment to extracellular matrix. The Journal of cell biology, 105(4), 1885-92.
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  On platelets the membrane glycoprotein IIb/IIIa complex (GPIIb/IIIa) functions in adhesive interactions with fibrinogen, von Willebrand factor, and fibronectin. However, the function of GPIIb/IIIa-like proteins on endothelial cells, as well as the ligand(s) the complex binds, is unknown. Using a highly specific polyclonal antibody we have explored the function of GPIIb/IIIa-like proteins on human umbilical vein endothelial cells (HUVE). Analysis by immunoblotting shows that this antiserum recognizes the endothelial GPIIIa-like protein of the complex. The IgG fraction of the polyclonal antiserum and its Fab' fragments detach confluent and subconfluent HUVE from extracellular substrata. The effect of the anti-GPIIb/IIIa IgG is not toxic as the detached cells maintain their viability after trypsinization and replating. Anti-GPIIb/IIIa IgG does not inhibit HUVE binding to extracellular matrix or purified fibronectin in an attachment assay despite the presence of intact GPIIb/IIIa on HUVE detached from substrate by various methods. Apparently, the GPIIb/IIIa-like protein on HUVE is important in normal HUVE adhesion to the extracellular matrix, but it is not required in the initial attachment of HUVE to extracellular matrix.
• Funakoshi, T., Heimark, R. L., Hendrickson, L. E., McMullen, B. A., & Fujikawa, K. (1987). Human Placental Anticoagulant Protein Isolation and Characterization. Biochemistry, 26(17), 5572-5578.
• Heimark, R. L., Fujikawa, K., McMullen, B. A., Hendrickson, L. E., & Funakoshi, T. (1987). Human placental anticoagulant protein: isolation and characterization. Biochemistry, 26(17), 5572-5578. doi:10.1021/bi00391a053
• Heimark, R. L., Twardzik, D. R., & Schwartz, S. M. (1986). Inhibition of Endothelial Regeneration by Type Beta Transforming Growth Factor from Platelets. Science, 233(4768), 1078-1080.
• Heimark, R. L., & Schwartz, S. M. (1985). The Role of Membrane: Membraine Inteactions in the Regulation of Endothelial Cell Growth. Journal of Cell Biology, 100(6), 1934-1940.
• Gabbiani, G., Gabbiani, F., Heimark, R. L., & Schwartz, S. M. (1984). Organization of actin cytoskeleton during early endothelial regeneration in vitro. Journal of cell science, 66, 39-50.
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  The pattern of early cell movement after an experimental 'wound' and the organization of actin in stationary and moving cultured endothelial cells have been studied by means of: time-lapse photography; indirect immunofluorescence using anti-actin antibodies with and without pretreatment with the actin destabilizing factor present in human plasma; and differential centrifugation and densitometric analysis of stained sodium dodecylsulphate/polyacrylamide gels in order to evaluate the total and relative amounts of G and F-actin. Up to 5 h after a single scratch, movement consists of a coordinate spreading and translocation of a band of about 10 cells from the wound edge. Compared to stationary cells, moving endothelial cells show: no significant changes in the intensity and distribution of immunofluorescent staining with anti-actin antibodies, but an increased sensitivity of cytoplasmic actin, including stress fibres, to the actin-destabilizing factor purified from human plasma; and no significant change in the total amount of actin, but a decreased relative amount of F-actin and a corresponding increased relative amount of G-actin. We conclude that endothelial cell movement in vitro is accompanied by a rapid change in the state of actin organization characterized by an overall decrease in cytoplasmic F-actin.
• Heimark, R. L., & Schwartz, S. M. (1984). Binding of Coagulation Factor IX and Factor X to the Endothelial Cell Surface. Biochemical and Biophysical Research Communication, 111(2), 723-731.
• Heimark, R. L., Davie, E. W., Fujikawa, K., Ohkubo, I., & Kurachi, K. (1983). Initiation of intrinsic blood coagulation.. Advances in Experimental Medicine and Biology.
• Kurachi, K., Ohkubo, I., Heimark, R. L., Fujikawa, K., & Davie, E. W. (1983). Initiation of Intrinsic Blood Coagulation. Advances In Experimental Medicine and Biolofy, 156, 39-44.
• Kurachi, K., Ohkubo, I., Heimark, R. L., Fujikawa, K., & Davie, E. W. (1983). Initiation of intrinsic blood coagulation. Advances in experimental medicine and biology, 156, 39-44.
• Schwartz, S. M., & Heimark, R. L. (1983). Binding of coagulation factors IX and X to the endothelial cell surface.. Biochemical and biophysical research communications, 111(2), 723-31. doi:10.1016/0006-291x(83)90365-0
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  Bovine coagulation factors IX and X bind to independent sites on bovine aortic endothelial cells. Binding studies with cells maintained serum-free showed that there are at least two classes of binding sites for factor IX and factor X with a dissociation constant of 4.9 x 10(-9) M and 2.1 x 10(-8) M for the respective high affinity sites. Ca+2 was required for specific binding and was reversed by addition of EDTA or EGTA. Competition experiments showed that factor IX and factor IXa bind to the same sites, which are different from the factor X binding sites. Neither binding of factor IX or factor X is inhibited by addition of prothrombin or protein C. Indirect immunofluorescence of factor IX indicated that binding was diffuse on the cell surface.
• Heimark, R. L., & Davie, E. W. (1981). Bovine and Human Plasma Prekallikrein. Methods In Enzymology, 80(Part C), 157-172.
• McRae, B. J., Kurachi, K., Heimark, R. L., Fujikawa, K., Davie, E. W., & Powers, J. C. (1981). Mapping the active sites of bovine thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, plasma kallikrein, and trypsin with amino acid and peptide thioesters: development of new sensitive substrates. Biochemistry, 20(25), 7196-206.
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  The subsite specificities of bovine factor IXa, factor Xa, factor XIa, factor XIIa, thrombin, plasma kallikrein, and trypsin were mapped with amino acid, dipeptide, and longer peptide thioester substrates. Each substrate contained a P1 Arg residue. The P1' residues included thiol residues which are analogues of valine, leucine, and isoleucine, respectively, and the P2 residue included 12 representative amino acid residues. Longer substrates with the sequence at the antithrombin III reactive site and at the zymogen activation site of various coagulation factors were also studied. The enzymatic hydrolysis of the thioesters was measured in the presence of 4,4'-dithiodipyridine which provides a very sensitive assay for the free thiol. The thioesters were excellent substrates for the coagulation factors studied, and the kcat/Km values for the best thioester substrates were higher than those previously reported for most of these enzymes. Thrombin and plasma kallikrein were the most active of the coagulation factors toward the thioester substrates. The best substrate for thrombin was Z-Gly-Arg-SCH2C6H5, although substrates containing proline in the P2 position were also quite effective. Some of the better substrates for plasma kallikrein had a P2 Phe or Trp residue. Factor IXa was the least reactive of the coagulation factors and hydrolyzed only four of the dipeptide thioesters. Substrates with bulky hydrophobic groups such as Phe or Trp in the P2 position were the most reactive with factor IXa. Factor Xa hydrolyzed all the thioester substrates tested, the most reactive being Z-Gly-Arg-SCH2C6H5. This is consistent with the fact that glycine and arginine are present in the P2 and P1 positions, respectively, of the factor Xa sensitive bonds in prothrombin which is the physiological substrate for factor Xa. Bovine factor XIa showed the least amount of specificity of the various coagulation factors and was quite reactive toward all of the thioester substrates. The most sensitive substrate for this enzyme was also Z-Gly-Arg-SCH2C6H5. Factor XIIa preferred the dipeptide with a P2 Phe, although the simpler thioester Z-Arg-SCH2CH(CH3)2 was more reactive. Trypsin hydrolyzed all of the thioester substrates at a high rate and showed little substrate specificity. With all enzymes studied, extension of the thioester substrate beyond P2 or the P1' thiol leaving group did not lead to an improvement in hydrolysis. Due to their high kcat/Km values and the ease of detecting the thiol leaving group, thioester substrates should be extremely useful for future studies of coagulation proteases.
• Fujikawa, K., Heimark, R. L., Kurachi, K., & Davie, E. W. (1980). Activation of bovine factor XII (Hageman factor) by plasma kallikrein. Biochemistry, 19(7), 1322-30.
• Heimark, R. L., Davie, E. W., Fujikawa, K., & Kurachi, K. (1980). Surface activation of blood coagulation, fibrinolysis and kinin formation. Nature, 5772, 456-460. doi:10.1038/286456a0
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  The activation of plasma prekallikrein by single-chain factor XII has been studied in the presence of high molecular weight kininogen and kaolin. The data indicate that factor XII can initiate blood coagulation, fibrinolysis or kinin generation in the presence of kaolin and does so by converting prekallikrein to kallikrein. An enzyme cascade is then generated leading to the formation of fibrin, plasmin or bradykinin in three closely related physiological events
• Heimark, R. L., Kurachi, K., Fujikawa, K., & Davie, E. W. (1980). Surface Activation of Blood- Coagulation Fibrinolysis and Kinin Formation. Nature, 286(5772), 456-460.
• Heimark, R. L., & Davie, E. W. (1979). Isolation and Characterization of Bovine Plasma Prekallikrein (Fletcher Factor). Biochemistry, 18, 5743-5750.
• Heimark, R. L., Hershey, J. W., & Traut, R. R. (1976). Cross-linking of Initiation Factor IF2 to Proteins L7-L12 in the 70S Ribosomal Subunit. Journal of Biological Chemistry, 251(24), 7779-7784.
• Heimark, R. L., Kahan, L., Johnston, K., Traut, R. R., & Hershey, J. W. (1976). Cross-linking of Initiation Factor IF3 to Proteins of Escherichia coli 30S Ribosomal Subunit. Journal of Molecular Biology, 105(2), 219-230.
• Bollen, A., Heimark, R. L., Cozzone, A., Traut, R. R., & Hershey, J. W. (1975). Cross-linking of initiation factor IF-2 to Escherichia coli 30 S ribosomal proteins with dimethylsuberimidate. The Journal of biological chemistry, 250(11), 4310-4.
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  The 30 S ribosomal proteins near the binding site for initiation factor IF-2 in Escherichia coli were identified by allowing complexes of 30 S subunits, [32P]phosphoryl initiation factor IF-2 and nonradioactive initiation factors IF-1 and IF-3, to react with the protein cross-linking reagent dimethylsuberimidate. Noncross-linked initiation factors were removed by centrifugation of the complexes in buffer containing a high salt concentration; the protein was extracted from the pelleted particles; and cross-linked species containing initiation factor IF-2 and ribosomal proteins were partially purified by column chromatography on Sephadex G-75. The mixture of cross-linked products was analyzed by radioimmunodiffusion with antisera prepared against 20 individual 30 S ribosomal proteins S1, S2, S11, S12, S13, S14, and S19 was interpreted to mean that initiation factor IF-2 was present in covalent cross-linked complexes containing those proteins. The results imply that these 30 S ribosomal proteins are near the binding site for initiation factor IF-2.
• SUN, T., HEIMARK, R., & TRAUT, R. (1975). PROTEIN TOPOGRAPHY OF ESCHERICHIA-COLI 30S RIBOSOMAL-SUBUNIT - PRELIMINARY MODEL E-COLI-30S SUBUNIT-RIBOSOME-SPATIAL ARRANGEMENT OF PROTEINS. Molecular and Cellular Biochemistry, 6(1), 33-41.
• Traut, R. R., Sun, T. T., R, R. T., & Heimark, R. L. (1975). The protein topography of the E. coli 30S ribosomal subunit: a preliminary model E. coli/30S subunit/ribosome/spatial arrangement of proteins.. Molecular and cellular biochemistry, 6(1), 33-41. doi:10.1007/bf01731864
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  A three dimensional model is presented which shows the apatial arrangement of 20 of the 21 proteins of the 30S ribosomal subunit of Escherichia coli. The model fulfills several purposes: (a) It summarizes currently available structural and functional data on ribosomal proteins; (b) It suggests an interesting correlation between stoichiometry and function. Functional proteins are both clustered and fractional; (c) It can be evaluated in relationships or point out critical experiments by which it can be tested.

Proceedings Publications

• Futscher, B. W., Futscher, B. W., Heimark, R. L., Heimark, R. L., Vrba, L., Vrba, L., Rheinheimer, B. A., & Rheinheimer, B. A. (2014). Abstract 127: Alternative transcription of the SLIT2/mir-218-1 signaling axis mediates pancreatic cancer invasion through the regulation of invadopodia. In American Association of Cancer Research, 74, 127-127.
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  Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Guidance molecules from the Netrin, Slit, Ephrin, and Semaphorin gene families were originally described as cues for the directional guidance of axons in the developing nervous system. More recently, members of these families have been found to have critical roles in tumor invasion. The SLIT2/mir-218-1 signaling axis has properties of a tumor suppressor pathway via the inhibition of directional migration and is epigenetically silenced in many cancers. We propose that mir-218-1 expression and function is independent of SLIT2 signaling in pancreatic ductal adenocarcinoma and inhibits tumor dissemination following the conversion of precursor lesions to invasive carcinoma. We determined that KRAS-dependent pancreatic cancer cell lines express SLIT2 mRNA while KRAS-independent lines show silenced SLIT2 expression. Using chromatin immunoprecipitation (ChIP) and Sequenom, we confirmed that loss of SLIT2 in KRAS-independent lines was due to enrichment of silencing histone mark H3K27me3 and DNA hypermethylation at the SLIT2 promoter, respectively. Activating marks H3K4me2 and H4ac are found at the SLIT2 promoter in KRAS-dependent lines suggesting that SLIT2 is lost during pancreatic cancer progression due to epigenetic silencing. mir-218-1 is an intronic microRNA found within intron 15 of the SLIT2 gene. Many intronic microRNAs are transcribed from the host gene promoter; however, mir-218-1 expression in pancreatic cancer cell lines does not correlate with SLIT2 gene expression. Therefore, we examined whether mir-218-1 had an alternative promoter independent of the SLIT2 promoter. Using ChIP for H3K4me3, a marker for promoter regions, and H4ac, a marker of permissive chromatin, we discovered a putative alternative promoter of mir-218-1 in all pancreatic cancer cell lines regardless of KRAS dependence. The putative alternative mir-218-1 promoter was also found to have basal transcription via enhanced luciferase reporter activity. This suggests that mir-218-1 expression is important and may contain its own tumor suppressor properties independent of SLIT2 signaling. Since pancreatic cancer is an invasive disease we examined the role of miR-218 target genes in specialized cellular structures, termed invadopodia. These structures have been found to facilitate the invasive phenotype of many cancer types and are dynamic actin-rich cellular projections that localize proteases to degrade extracellular matrix. Transfection of a miR-218 antagomir enhanced invasion in a modified Boyden chamber while addition of mir-218 reduced cell invasion. Our results further demonstrate that mir-218 functions in the suppression of invadopodia maturation and metalloproteinase matrix degradation. Overall, our data establishes that the SLIT2/mir-218-1 signaling axis is an important mediator of pancreatic cancer invasion through the extracellular matrix. Citation Format: Brenna A. Rheinheimer, Lukas Vrba, Bernard W. Futscher, Ronald L. Heimark. Alternative transcription of the SLIT2/mir-218-1 signaling axis mediates pancreatic cancer invasion through the regulation of invadopodia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 127. doi:10.1158/1538-7445.AM2014-127
• Raza, O., Heimark, R. L., Grewal, G. S., Bharara, M., Armstrong, D., & Anghel, E. L. (2014). Thermography for in-vivo wound healing models. In Summer Sim'14: Proceedings of the Summer Simulation Multiconference, 46.
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  The objective of this study was to create a wound healing model based on longitudinal thermal images of wounds to assess the pro-angiogenic properties of Eu(OH)3 nanorods using an in vivo animal model. Eu(OH)3 nanorods are bioactive nanoparticles and induce angiogenesis which results in relatively faster skin wound healing in diabetics. We tracked wound area of full thickness cutaneous wounds for 18 days using digital photographs and wound temperature using infrared imaging as a surrogate indictor for inflammation. Cutaneous wound area decreased faster for mice treated with Eu(OH)3 than those treated with control vehicle dressing [62% (Europium) Vs 43% (Control) wound area reduction at Day 18]. This study provides a novel methodology to assess wound healing for in-vivo models using thermography.
• Heimark, R. L., & Rheinheimer, B. A. (2012). Abstract A72: Epigenetic loss of SLIT2 leads to an autocrine-to-paracrine switch of the SLIT2/ ROBO1 signaling network in pancreatic cancer.. In The American Association for Cancer Research, 72, A72.
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  University of Arizona Student Showcase 2012 President’s Award, University of Arizona Student Showcase 2012 1st Place Biological Sciences, AACR Special Meeting Pancreatic Cancer: Progress and Challenges Scholar-in-Training Award
• Zheng, X. J., Cheung, L. S., Jiang, L., Schroeder, J. A., Heimark, R. L., Baygents, J. C., Guzman, R., Zohar, Y., & , . (2011, 2011). DYNAMIC STATES OF ADHERING CANCER CELLS UNDER SHEAR FLOW IN AN ANTIBODY-FUNCTIONALIZED MICROCHANNEL. In 2011 IEEE 24TH INTERNATIONAL CONFERENCE ON MICRO ELECTRO MECHANICAL SYSTEMS (MEMS), 849-852.
• Zheng, X. J., Cheung, L. S., Wang, L., Schroeder, J., Heimark, R. L., Baygents, J. C., Guzman, R., Zohar, Y., & , . (2011, 2010). QUANTITATIVE SPECIFIC BINDING OF BREAST CANCER CELLS IN AN ANTIBODY-FUNCTIONALIZED MICROCHAMBER ARRAY. In MEMS 2010: 23RD IEEE INTERNATIONAL CONFERENCE ON MICRO ELECTRO MECHANICAL SYSTEMS, TECHNICAL DIGEST, 939-942.
• Zheng, X., Cheung, S. L., Wang, L., Schroeder, J. A., Heimark, R. L., Baygents, J. C., Guzman, R., Zohar, Y., & , . (2004, 2010). SPECIFIC BINDING OF CANCER CELLS USING A MICROCHAMBER ARRAY FUNCTIONALIZED WITH ANTIBODIES. In IMECE 2009: PROCEEDINGS OF THE ASME INTERNATIONAL MECHANICAL ENGINEERING CONGRESS AND EXPOSITION, VOL 12, PTS A AND B, 821-827.
• Cheung, L. S., Zheng, X. J., Stopa, A., Schroeder, J., Heimark, R. L., Baygents, J. C., Guzman, R., Zohar, Y., & , . (1997, 2009). FLOW ACCELERATION EFFECT ON CANCER CELL DEFORMATION AND DETACHMENT. In IEEE 22ND INTERNATIONAL CONFERENCE ON MICRO ELECTRO MECHANICAL SYSTEMS (MEMS 2009), 431-434.
• Tait, J. F., Heimark, R. L., Funakoshi, T., & Fujikawa, K. (1987). PLACENTAL ANTICOAGULANT PROTEIN. In XIth International Congress on Thrombosis and Haemostasis.
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  An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and mono S (Pharmacia). Approximately 30 mg of the protein was purified from one placenta. The purified protein gave a single band on SDS polyacrylamide gel and had a molecular weight of 36,500. This protein inhibited both kaolin and thromboplastin induced clotting times of normal human plasma. It also inhibited the clotting time of platelet-rich plasma induced by factor Xa, but did not affect thrombin activity of fibrinogen-fibrin conversion. The protein neither bound factor Xa nor inhibited the amidase activity of factor Xa. This protein specifically bound to phospholipid vesicles prepared from a mixture of phosphatidylserine and phosphatidylcholine (20 to 80 weight ratio) in the presence of calcium ions. The purified protein was digested with cyanogen bromide and the resulting fragments were separated by FPLC. Partial amino acid sequences of the cyanogen bromide fragments showed that this protein was composed of at least three repeats that were homologous to the four repeats found in lipocortin I and II. Lipocortins are known to inhibit the phospholipase A2 activity, probably by binding to the phospholipid substrate. These results indicate that the placental anticoagulant protein is a member of the family of lipocortins and probably inhibits coagulation by binding to phospholipid vesicles. Supported in part by grants HL 16919 and HL 18645 from National Institute of Health.
• Scipio, R. G., Lindquist, P., Kurachi, K., Heimark, R. L., Fujikawa, K., & Davie, E. W. (1977). The Role of Factor IX (Christmas Factor) in Blood Coagulation. In VIth International Congress on Thrombosis and Haemostasis.
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  Factor IX participates in the middle phase of the intrinsic pathway of blood coagulation. The reactions leading to the activation of factor IX involve prekallikrein, high molecular weight kininogen, and factor XII. These proteins interact in the presence of a surface such as kaolin and give rise to the activation of factor XI. Factor XIa then converts factor IX to factor IXa in the presence of calcium ions. In this reaction, factor IX (a single-chain glycoprotein of mol. wt.-~55,000) is converted to factor IXa in a two-step reaction. In the first step, an internal peptide bond is cleaved leading to the formation of an intermediate lacking enzymatic activity. This intermediate contains two polypeptide chains held together by a disulfide bond(s). In the second step, an activation peptide is split from the heavy chain of the intermediate giving rise to factor IXa (mol. wt. ~45,000). Factor IXa is composed of a heavy chain (mol. wt.~27,000) and a light chain (mol. wt. ~16,000) held together by a disulfide bond(s). The activation mechanism is essentially identical for human and bovine factor IX. Factor IXa is a serine protease with esterase activity and is sensitive to protease inhibitors such as antithrombin III. Factor IX is also activated by the protease from Russell’s viper venom, but this reaction involves only a single cleavage in the precursor molecule. The critical step in the activation of factor IX by factor XIa or the protease from Russell’s viper venom is the cleavage of the same internal Arg-Val peptide bond and the formation of a new amino-terminal sequence of Val-Val-Gly-Gly- in the heavy chain of the enzyme.

Presentations

• Batai, K., Chen, Y., Rheinheimer, B., Heimark, R. L., Ellis, N., & Lee, B. R. (2022, April). Clear cell renal cell carcinoma molecular characteristics in Hispanic Americans compared to European Americans. . American Association for Cancer Research Annual Meeting. New Orleans LA: American Association for Cancer Research.
• Batai, K., Chen, Y., Rheinheimer, B., Heimark, R., Ellis, N., & Lee, B. R. (2022). Clear cell renal cell carcinoma molecular characteristics in Hispanic Americans compared to European Americans. AACR Annual Meeting. New Orleans.
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  Population Sciences Cancer Health Disparities Research
• Jie, T., Rheinheimer, B. A., & Heimark, R. L. (2019, February). Functional Genomics of Pancreatic Neuroendocrine Tumor Metastases to the Liver. 2019 Academic Surgical Congress.
• Rheinheimer, B. A., Riall, T. S., Heimark, R. L., & Jie, T. (2019, March). Pancreatic Mixed Acinar Cell Carcinoma: Genomic Analysis and Characterization of a Patient-derived Organoid Culture. 2019 The Americas Hepato-Pancreato-Biliary Association.
• Rico-Boiles, A., Batai, K., Cheng, C., Heimark, R. L., & Chipollini, J. (2022). Disparities in Prostate Cancer: An Ethnicity Comparative Focus Among Hispanic Americans versus Non-Hispanic Whites.. GU ASCO. San Francisco: ASCO.
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  Abstract #357084
• Wilder, J. A., Ware, V., Sokoloff, M., Rheinheimer, B., Patnode, J. M., Kelsey, G., Hernandez, K., Heimark, R. L., & Dias, R. (2020). Abstract C049: A common microRNA-flanking variant (rs13136737) inhsa-miR-302/367affects risk of prostate cancer progression in young men. Cancer Epidemiology, Biomarkers & Prevention.
• Rheinheimer, B., Heimark, R. L., & Jie, T. (2019, January). Deregulated growth factor signaling in pancreatic neuroendocrine liver metastasis. ASCO Gastrointestinal Cancers Symposium.
• Heimark, R. L., & Rubio, N. C. (2017, February). Myeloid Derived Suppressor Cells in the inflammatory Response of Pancreatitis and Pancreatic Cancer. American College of Surgeons. Las Vegas, NV.
• Heimark, R. L., Vrba, L., Rheinheimer, B. A., Heimark, R. L., & Futscher, B. W. (2015). Abstract A79: Post-transcriptional regulatory networks of pancreatic tumor invasion. Cancer Research.
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  Guidance molecules from the Slit gene family were originally described as cues for the directional guidance of axons in the developing nervous system. More recently, members of these families have been found to play critical roles in epithelial development, angiogenesis and tumorigenesis. SLIT2 has properties of a potential tumor suppressor, is mutated at a low frequency in pancreatic tumors and is epigenetically silenced in many cancers. mir-218-1 is an intronic microRNA found within intron 15 of the SLIT2 gene. We propose that cell intrinsic mir-218-1 expression and function is independent of SLIT2 signaling in pancreatic ductal adenocarcinoma. This dynamic expression of mir-218-1 can inhibit tumor dissemination following the conversion of precursor lesions to invasive carcinoma. In normal pancreatic tissue, SLIT2 is highly expressed in the ductal compartment and expressed at a reduced level in the majority of primary pancreatic ductal adenocarcinomas (PDACs). We determined that KRAS-dependent pancreatic cancer cell lines express SLIT2 while KRAS-independent cell lines show silenced SLIT2 expression. Sequenom and chromatin immunoprecipitation (ChIP) confirmed that loss of SLIT2 in KRAS-independent lines was due to DNA hypermethylation and enrichment of silencing histone marks at the SLIT2 promoter and transcriptional start site with an inverse correlation between SLIT2 and EZH2 expression. Activating histone marks are found at both the SLIT2 promoter and transcriptional start site in KRAS-dependent lines suggesting that SLIT2 is lost during pancreatic cancer progression due to epigenetic silencing. Many intronic microRNAs are transcribed from the host gene promoter; however, mir-218-1 expression is variable in pancreatic cancer cell lines and does not correlate with SLIT2 gene expression. Therefore, we examined whether mir-218-1 had an alternative promoter independent of the SLIT2 promoter. Using ChIP for H3K4me3, a marker for promoter regions, we discovered an alternative promoter of mir-218-1 . ChIP for H4ac and Sequenom showed that the chromatin structure within this putative alternative promoter is acetylated, unmethylated, and, thus, permissive for transcription. The putative alternative mir-218-1 promoter was also found to have basal transcription via enhanced luciferase reporter activity and enrichment of RNA polymerase II at the H3K4me3/H4ac peak. In silico analysis of the mir-218-1 alternative promoter region indicated that DNA binding sites for transcriptional repressors MYC/MAX and ZEB1 are present. In silico analysis also showed the presence of DNA binding sites for transcriptional activators CDX1, LEF1/TCF1 and NF-κB. The transcription factor NF-κB and oncogene KRAS frequently function cooperatively to promote tumor progression; however, maintenance of ductal architecture is also important in pancreatic cancer invasion and metastasis. Site-directed mutagenesis of the NF-κB binding site decreased luciferase reporter activity suggesting that NF-κB is an activator of mir-218-1 expression at this alternative promoter. This suggests that mir-218-1 expression is uncoupled from SLIT2 expression and may contain its own tumor suppressor properties independent of SLIT2 signaling. Matrix metalloproteinases (MMPs) are associated with tumor initiation and progression by targeting extracellular matrix (ECM) components. Invadopodia are structures that have been found to facilitate the invasive phenotype of many cancer types and are dynamic actin-rich cellular projections that localize proteases such as MMP2, MMP9, and MMP14 to degrade the ECM. In addition to ROBO1 , we identified additional novel mir-218-1 target genes that control PDAC cell invasion. Our results further demonstrate that mir-218-1 functions in the suppression of invadopodia maturation and metalloproteinase matrix degradation. Overall, our data establishes that mir-218-1 is a key independent mediator of pancreatic cancer invasion through the extracellular matrix. Citation Format: Brenna A. Rheinheimer, Lukas Vrba, Bernard W. Futscher, Ronald L. Heimark. Post-transcriptional regulatory networks of pancreatic tumor invasion. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A79.
• Jie, T., Rheinheimer, B. A., Riall, T. S., & Heimark, R. L. (2019, February). Genomic Analysis of a Patient-Derived Organoid Model of Mixed Acinar/Neuroendocrine Cell Carcinoma. 2019 Academic Surgical Congress.
• Rheinheimer, B. A., Heimark, R. L., & Jie, T. (2019, March). Deregulated growth factor signaling in pancreatic neuroendocrine liver metastasis. 2019 The Americas Hepato-Pancreato-Biliary Association.
• Rheinheimer, B. A., Jie, T., Rheinheimer, B. A., Heimark, R. L., Heimark, R. L., & Jie, T. (2019, February). Functional Genomics of Pancreatic Neuroendocrine Tumor Metastases to the Liver. 2019 Academic Surgical Congress.
• Rheinheimer, B., Vrba, L., Futcher, B. W., & Heimark, R. L. (2014, APR). Alternative transcription of the SLIT2/ROBO1/miR-218-1 signaliong axis in pancreatic cancer. Cancer Research.
• Abrams, A., Jie, T., Ong, E., Heimark, R. L., Bhattacharyya, A. K., & Patel, C. (2013, Feb/Spring). Characterization of Pancreatic Adenocarcinoma of Primary and Peritoneal Metastasis a Pilot Study Using Biomarkers for Metastasis (EMT). 2013 Americas Hepato-Pancreato-Biliary Association Meeting. Miami, FL.
• Heimark, R. L., Vrba, L., Rheinheimer, B., Heimark, R. L., & Futscher, B. W. (2013). Abstract B13: Epigenetic silencing alters the SLIT2/ROBO1/miR-218-1 signaling axis in pancreatic cancer. Cancer Research.
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  Pancreatic ductal adenocarcinoma (PDAC) is characterized by early tumor dissemination from pancreatic intraepithelial neoplasia (PanIN) precursor lesions through tumor budding and perineural invasion. Therefore, it is necessary to elucidate the mechanism of transcriptional regulation of genes and signaling pathways that lead to early spread of this disease. Neuronal guidance molecules are secreted membrane-bound ligands that act as signaling cues in morphogenesis, depending on the presence of their cognate receptors. Four major classes of guidance molecules include the Ephrins and Ephrin receptors, Slits and Robo receptors, Netrins and Unc5H receptors, and Semaphorins and Plexin receptors. Mammals encode three SLIT genes (SLIT1-3). The SLIT2/miR-218-1 signaling axis has properties of a tumor suppressor pathway via the inhibition of epithelial cell growth, directional migration, ductal morphogenesis, and is epigenetically silenced in lung, colon, prostate and breast cancers. SLIT2 is also a target for polycomb repressor complex 2 protein EZH2 which enriches H3K27me3 at the SLIT2 promoter. We proposed that changes in DNA methylation patterns and chromatin dynamics lead to differences in SLIT2 and miR-218-1 expression in PDAC and may mediate ductal expansion and early spread following the conversion of PanIN precursor lesions to invasive carcinoma. The SLIT2 promoter contains a large CpG island (290 CpG sites) that extends 2,021 base pairs (bp) upstream of the transcriptional start site (TSS) through the first exon and ends 1,251 bp into the first intron (-2,021 to +1,251). In our studies, we determined that KRAS-dependent pancreatic cancer cell lines express SLIT2 and ROBO1 in a cell autonomous manner while KRAS-independent cell lines have silenced SLIT2 expression and upregulated ROBO1 expression. We confirmed that loss of SLIT2 in KRAS-independent cell lines was due to DNA hypermethylation of its promoter using Sequenom. We cloned 1.4 kb of the SLIT2 promoter upstream of the firefly luciferase gene to evaluate activity of this region in transcriptional control. We transfected the SLIT2 reporter into pancreatic cancer cell lines. The cell lines showed relative promoter activity similar to the level of SLIT2 transcripts. Chromatin immunoprecipitation (ChIP) shows that silencing histone mark H3K27me3 is found at the TSS of the SLIT2 gene in KRAS-independent lines while activating mark H3K4me2 is found at the TSS of the SLIT2 gene in KRAS-dependent lines. Treatment with demethylating agents, but not histone deacetylase inhibitors, reactivates SLIT2 expression suggesting that DNA promoter methylation is the dominant epigenetic mark in silencing SLIT2 expression. miR-218-1 is an intronic microRNA found within intron 15 of the SLIT2 gene. Most intronic miRNAs are transcribed from the host gene promoter; however, miR-218 expression is variable in pancreatic cancer cell lines and does not correlate with SLIT2 expression. Therefore, we proposed that miR-218-1 has an alternative TSS other than the SLIT2 promoter. Using ChIP for H3K4me3, which defines gene promoters, we discovered a putative alternative TSS of miR-218-1. ChIP analysis using activating mark H4ac shows that this TSS is active in all pancreatic cancer cell lines regardless of KRAS dependence suggesting that the tumor suppressive function of SLIT2 may be mediated through miR-218-1 instead. Overall, our data establishes that the SLIT2/ROBO1/miR-218-1 signaling axis is epigenetically regulated in pancreatic cancer leading to tumor expansion and progression along intrapancreatic neurons that express the SLIT2 ligand. Citation Format: Brenna Rheinheimer, Lukas Vrba, Bernard Futscher, Ronald L. Heimark. Epigenetic silencing alters the SLIT2/ROBO1/miR-218-1 signaling axis in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B13.
• Rheinheimer, B. A., & Heimark, R. L. (2012). Abstract A72: Epigenetic loss of SLIT2 leads to an autocrine-to-paracrine switch of the SLIT2/ ROBO1 signaling network in pancreatic cancer.. Cancer Research.
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  Guidance molecules from the Netrin, Slit, Ephrin, and Semaphorin gene families were originally described as cues for the directional guidance of axons in the developing nervous system. More recently, members of these families have been found to have critical roles in epithelial development, angiogenesis and cancer. The SLIT2/ROBO1 signaling axis has properties of a potential tumor suppressor pathway via the inhibition of epithelial cell growth, directional migration, ductal morphogenesis, and is epigenetically silenced in lung, colon and breast cancers. We proposed that changes in SLIT2 and ROBO1 expression in pancreatic ductal adenocarcinoma may mediate ductal expansion following the conversion of PanIN precursor lesions to invasive carcinoma. The SLIT2 receptor, ROBO1 , is a member of the immunoglobulin (Ig) superfamily and is required for lung and mammary development in mammals. ROBO1 has an alternative splice variant, DUTT1 , and these two variants have different initial exons and initiating codons which may suggest the two proteins have distinct functions. In our studies, we found that all pancreatic cancer cell lines and primary pancreatic cancer specimens express only the DUTT1 isoform. We also determined that as pancreatic cancer cell lines become KRAS-independent, ROBO1 expression increases. Furthermore, using immunohistochemistry (IHC), we found that ROBO1 protein expression in primary pancreatic cancer tissue specimens is localized to the ductal compartment with no stromal staining seen. In normal pancreas, ROBO1 expression is weak while its ligand SLIT2 is strongly expressed in both the acinar and ductal compartments in vitro and in vivo. Mammals encode three SLIT genes ( SLIT1 3 ). The secreted SLIT2 protein is not diffusible, but has a cleavage site within its EGF-like repeats creating two fragments which allow it to act either as a short or long range guidance cue with each fragment appearing to have its own cell-association characteristics. The 5’ promoter of SLIT2 has been shown to be methylated resulting in gene silencing in early stages of several epithelial cancers suggesting a possible tumor suppressor role. miR-218-1 is an intronic microRNA found between exons 15 and 16 of the SLIT2 gene and targets a complimentary sequence in the ROBO1 3’ untranslated region (UTR) indicative of a potential regulation of receptor availability in the presence of ligand. In our studies, we determined that the KRAS-dependent pancreatic cancer cell lines express SLIT2 and ROBO1 in a cell autonomous manner. The KRAS-independent cell lines, however, have silenced SLIT2 and miR 218-1 expression. Using IHC we found that high levels of SLIT2 are seen in normal pancreas localized to the acinar and ductal compartments. Reduced SLIT2 expression is seen in primary pancreatic cancer tissue specimens. We confirmed that loss of SLIT2 mRNA in KRAS-independent lines was due to DNA hypermethylation shown by methylation specific PCR and Sequenom analysis. Chromatin immunoprecipitation analysis shows that silencing histone marks are found in the 5’ promoter of the SLIT2 gene in KRAS-independent lines. Treatment with demethylating agents reactivate SLIT2 and miR-218-1 expression suggesting that epigenetic mechanisms controlling the SLIT2 promoter also regulate miR-218-1 expression. Overall, our data establishes that the SLIT2/ ROBO1 signaling axis is a dynamic pathway in pancreatic cancer that can act in the tumor expansion and progression along intrapancreatic neurons that express the SLIT2 ligand. Citation Format: Brenna A. Rheinheimer, Ronald L. Heimark. Epigenetic loss of SLIT2 leads to an autocrine-to-paracrine switch of the SLIT2/ ROBO1 signaling network in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A72.

Poster Presentations

• Chipollini, J., Heimark, R. L., Recio Boiles, A., Zeng, J., & Garcia, K. (2022, Fall). Prostate cancer presentation in Hispanic Americans: a nationwide comparative analysis of Hispanic Americans versus non-Hispanic whites. Western Section American Urological Association.
• Daw, J. N., Diermeier, S., Chung, S., Montfort, W. R., & Heimark, R. L. (2021). Induction of NOS2 by Pto-inflammatory Cytokines and Regulation by the lncRNA MaTAR20 in Triple Negative Breast Cancer. UACC Retreat. Tucson.
• Wong, R. K., & Heimark, R. L. (1998, November). Proinflammatory cytokines TNF-alpha and IFN-gamma alter permeability and the cadherin-5/catenin complex via beta-catenin cleavage.. Cell Biology. San Francisco: The American Society for Cell Biology.
• Tatsuguchi, M., Rekapally, H., Summers, C., & Heimark, R. (1995, OCT 26). Id2 (Inhibitor of DNA binding) functions as a regulator of AV canal cushion mesenchyme in the morphogenesis of cardiac valves. CIRCULATION.
• Coffin, J. D., Harrison, J., Schwartz, S. M., & Heimark, R. L. (1989, DEC). Expression of Von Willebrand Factor in Endothelium of Post-Implantation Mouse Embryos. 29th Annual Meeting of American Society for Cell Biology. Houston, TX.

Others

• Heimark, R. L., Riall, T. S., Rheinheimer, B. A., & Jie, T. (2019, February). Genomic Analysis of a Patient-Derived Organoid Model of Mixed Acinar/Neuroendocrine Cell Carcinoma. 2019 Academic Surgical Congress.
• Jie, T., Heimark, R. L., & Rheinheimer, B. A. (2019, February). Functional Genomics of Pancreatic Neuroendocrine Tumor Metastases to the Liver. 2019 Academic Surgical Congress.
• Jie, T., Heimark, R. L., & Rheinheimer, B. A. (2019, March). Deregulated growth factor signaling in pancreatic neuroendocrine liver metastasis. 2019 The Americas Hepato-Pancreato-Biliary Association.
• Jie, T., Heimark, R. L., Riall, T. S., & Rheinheimer, B. A. (2019, March). Pancreatic Mixed Acinar Cell Carcinoma: Genomic Analysis and Characterization of a Patient-derived Organoid Culture. 2019 The Americas Hepato-Pancreato-Biliary Association.
• Jie, T., Heimark, R. L., & Rheinheimer, B. (2019, January). Deregulated growth factor signaling in pancreatic neuroendocrine liver metastasis. ASCO Gastrointestinal Cancers Symposium.
• Schramm, C., Runyan, R., & Heimark, R. (1986, NOV). Regulation of cadherin 5 during epithelial-mesenchymal transformation of atrioventricular endocardium.. MOLECULAR BIOLOGY OF THE CELL.