Tatiana Kalin

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• Bian, F., Goda, C., Wang, G., Lan, Y. W., Deng, Z., Gao, W., Acharya, A., Reza, A. A., Gomez-Arroyo, J., Merjaneh, N., Ren, X., Goveia, J., Carmeliet, P., Kalinichenko, V. V., & Kalin, T. V. (2024). FOXF1 promotes tumor vessel normalization and prevents lung cancer progression through FZD4. EMBO molecular medicine, 16(5), 1063-1090.
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  Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control the reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific overexpression of FOXF1 normalized tumor vessels and inhibited the progression of lung cancer. FOXF1 deficiency decreased Wnt/β-catenin signaling in TECs through direct transcriptional activation of Fzd4. Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/β-catenin signaling in TECs, normalized tumor vessels and inhibited the progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/β-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has promise for future therapies in NSCLC.
• Deng, Z., Gao, W., Kohram, F., Li, E., Kalin, T. V., Shi, D., & Kalinichenko, V. V. (2024). Fluorinated amphiphilic Poly(β-Amino ester) nanoparticle for highly efficient and specific delivery of nucleic acids to the Lung capillary endothelium. Bioactive materials, 31, 1-17.
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  Endothelial cell dysfunction occurs in a variety of acute and chronic pulmonary diseases including pulmonary hypertension, viral and bacterial pneumonia, bronchopulmonary dysplasia, and congenital lung diseases such as alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). To correct endothelial dysfunction, there is a critical need for the development of nanoparticle systems that can deliver drugs and nucleic acids to endothelial cells with high efficiency and precision. While several nanoparticle delivery systems targeting endothelial cells have been recently developed, none of them are specific to lung endothelial cells without targeting other organs in the body. In the present study, we successfully solved this problem by developing non-toxic poly(β-amino) ester (PBAE) nanoparticles with specific structure design and fluorinated modification for high efficiency and specific delivery of nucleic acids to the pulmonary endothelial cells. After intravenous administration, the PBAE nanoparticles were capable of delivering non-integrating DNA plasmids to lung microvascular endothelial cells but not to other lung cell types. IVIS whole body imaging and flow cytometry demonstrated that DNA plasmid were functional in the lung endothelial cells but not in endothelial cells of other organs. Fluorination of PBAE was required for lung endothelial cell-specific targeting. Hematologic analysis and liver and kidney metabolic panels demonstrated the lack of toxicity in experimental mice. Thus, fluorinated PBAE nanoparticles can be an ideal vehicle for gene therapy targeting lung microvascular endothelium in pulmonary vascular disorders.
• Li, E., Wen, B., Gao, D., Kalin, T. R., Wang, G., Kalin, T. V., & Kalinichenko, V. V. (2024). The bone marrow of mouse-rat chimeras contains progenitors of multiple pulmonary cell lineages. Frontiers in cell and developmental biology, 12, 1394098.
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  Radiation-induced lung injury (RILI) is a common complication of anti-cancer treatments for thoracic and hematologic malignancies. Bone marrow (BM) transplantation restores hematopoietic cell lineages in cancer patients. However, it is ineffective in improving lung repair after RILI due to the paucity of respiratory progenitors in BM transplants. In the present study, we used blastocyst injection to create mouse-rat chimeras, these are artificial animals in which BM is enriched with mouse-derived progenitor cells. FACS-sorted mouse BM cells from mouse-rat chimeras were transplanted into lethally irradiated syngeneic mice, and the contribution of donor cells to the lung tissue was examined using immunostaining and flow cytometry. Donor BM cells provided long-term contributions to all lung-resident hematopoietic cells which includes alveolar macrophages and dendritic cells. Surprisingly, donor BM cells also contributed up to 8% in pulmonary endothelial cells and stromal cells after RILI. To identify respiratory progenitors in donor BM, we performed single-cell RNA sequencing (scRNAseq). Compared to normal mouse BM, increased numbers of hematopoietic progenitors were found in the BM of mouse-rat chimeras. We also identified unique populations of hemangioblast-like progenitor cells expressing , and along with mesenchymal stromal cells expressing , and that were absent or ultra-rare in the normal mouse BM. In summary, by using rats as "bioreactors", we created a unique mouse BM cell transplant that contributes to multiple respiratory cell types after RILI. Interspecies chimeras have promise for future generations of BM transplants enriched in respiratory progenitor cells.
• Merjaneh, N., Hajjar, M., Lan, Y. W., Kalinichenko, V. V., & Kalin, T. V. (2024). The Promise of Combination Therapies with FOXM1 Inhibitors for Cancer Treatment. Cancers, 16(4).
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  Forkhead box M1 (FOXM1) is a transcription factor in the forkhead (FOX) family, which is required for cellular proliferation in normal and neoplastic cells. FOXM1 is highly expressed in many different cancers, and its expression is associated with a higher tumor stage and worse patient-related outcomes. Abnormally high expression of FOXM1 in cancers compared to normal tissue makes FOXM1 an attractive target for pharmacological inhibition. FOXM1-inhibiting agents and specific FOXM1-targeted small-molecule inhibitors have been developed in the lab and some of them have shown promising efficacy and safety profiles in mouse models. While the future goal is to translate FOXM1 inhibitors to clinical trials, potential synergistic drug combinations can maximize anti-tumor efficacy while minimizing off-target side effects. Hence, we discuss the rationale and efficacy of all previously studied drug combinations with FOXM1 inhibitors for cancer therapies.
• Mohammed, A. N., Kohram, F., Lan, Y. W., Li, E., Kolesnichenko, O. A., Kalin, T. V., & Kalinichenko, V. V. (2024). Transplantation of alveolar macrophages improves the efficacy of endothelial progenitor cell therapy in mouse model of bronchopulmonary dysplasia. American journal of physiology. Lung cellular and molecular physiology, 327(1), L114-L125.
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  Bronchopulmonary dysplasia (BPD) is a severe complication of preterm births, which develops due to exposure to supplemental oxygen and mechanical ventilation. Published studies demonstrated that the number of endothelial progenitor cells (EPC) is decreased in mouse and human BPD lungs and that adoptive transfer of EPC is an effective approach in reversing the hyperoxia-induced lung damage in mouse model of BPD. Recent advancements in macrophage biology identified the specific subtypes of circulating and resident macrophages mediating the developmental and regenerative functions in the lungs. Several studies reported the successful application of macrophage therapy in accelerating the regenerative capacity of damaged tissues and enhancing the therapeutic efficacy of other transplantable progenitor cells. In the present study, we explored the efficacy of combined cell therapy with EPC and resident alveolar macrophages (rAM) in hyperoxia-induced BPD mouse model. rAM and EPC were purified from neonatal mouse lungs and were used for adoptive transfer to the recipient neonatal mice exposed to hyperoxia. Adoptive transfer of rAM alone did not result in engraftment of donor rAM into the lung tissue but increased the mRNA level and protein concentration of proangiogenic CXCL12 chemokine in recipient mouse lungs. Depletion of rAM by chlodronate-liposomes decreased the retention of donor EPC after their transplantation into hyperoxia-injured lungs. Adoptive transfer of rAM in combination with EPC enhanced the therapeutic efficacy of EPC as evidenced by increased retention of EPC, increased capillary density, improved arterial oxygenation, and alveolarization in hyperoxia-injured lungs. Dual therapy with EPC and rAM has promise in human BPD. Recent studies demonstrated that transplantation of lung-resident endothelial progenitor cells (EPC) is an effective therapy in mouse model of bronchopulmonary dysplasia (BPD). However, key factors regulating the efficacy of EPC are unknown. Herein, we demonstrate that transplantation of tissue-resident alveolar macrophages (rAM) increases CXCL12 expression in neonatal mouse lungs. rAM are required for retention of donor EPC in hyperoxia-injured lungs. Co-transplantation of rAM and EPC improves the efficacy of EPC therapy in mouse BPD model.
• Wang, G., Wen, B., Guo, M., Li, E., Zhang, Y., Whitsett, J. A., Kalin, T. V., & Kalinichenko, V. V. (2024). Identification of endothelial and mesenchymal FOXF1 enhancers involved in alveolar capillary dysplasia. Nature communications, 15(1), 5233.
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  Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, a lethal lung disease affecting newborns and infants. Identification of new FOXF1 upstream regulatory elements is critical to explain why frequent non-coding FOXF1 deletions are linked to the disease. Herein, we use multiome single-nuclei RNA and ATAC sequencing of mouse and human patient lungs to identify four conserved endothelial and mesenchymal FOXF1 enhancers. We demonstrate that endothelial FOXF1 enhancers are autoactivated, whereas mesenchymal FOXF1 enhancers are regulated by EBF1 and GLI1. The cell-specificity of FOXF1 enhancers is validated by disrupting these enhancers in mouse embryonic stem cells using CRISPR/Cpf1 genome editing followed by lineage-tracing of mutant embryonic stem cells in mouse embryos using blastocyst complementation. This study resolves an important clinical question why frequent non-coding FOXF1 deletions that interfere with endothelial and mesenchymal enhancers can lead to the disease.
• Wen, B., Li, E., Wang, G., Kalin, T. R., Gao, D., Lu, P., Kalin, T. V., & Kalinichenko, V. V. (2024). CRISPR-Cas9 Genome Editing Allows Generation of the Mouse Lung in a Rat. American journal of respiratory and critical care medicine, 210(2), 167-177.
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  Recent efforts in bioengineering and embryonic stem cell (ESC) technology allowed the generation of ESC-derived mouse lung tissues in transgenic mice that were missing critical morphogenetic genes. Epithelial cell lineages were efficiently generated from ESC, but other cell types were mosaic. A complete contribution of donor ESCs to lung tissue has never been achieved. The mouse lung has never been generated in a rat. We sought to generate the mouse lung in a rat. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing was used to disrupt the gene in rat one-cell zygotes. Interspecies mouse-rat chimeras were produced by injection of wild-type mouse ESCs into -deficient rat embryos with lung agenesis. The contribution of mouse ESCs to the lung tissue was examined by immunostaining, flow cytometry, and single-cell RNA sequencing. Peripheral pulmonary and thyroid tissues were absent in rat embryos after CRISPR-Cas9-mediated disruption of the gene. Complementation of rat blastocysts with mouse ESCs restored pulmonary and thyroid structures in mouse-rat chimeras, leading to a near-99% contribution of ESCs to all respiratory cell lineages. Epithelial, endothelial, hematopoietic, and stromal cells in ESC-derived lungs were highly differentiated and exhibited lineage-specific gene signatures similar to those of respiratory cells from the normal mouse lung. Analysis of receptor-ligand interactions revealed normal signaling networks between mouse ESC-derived respiratory cells differentiated in a rat. A combination of CRISPR-Cas9 genome editing and blastocyst complementation was used to produce mouse lungs in rats, making an important step toward future generations of human lungs using large animals as "bioreactors."
• Acharya, A., Bian, F., Gomez-Arroyo, J., Wagner, K. A., Kalinichenko, V. V., & Kalin, T. V. (2023). Hypoxia represses FOXF1 in lung endothelial cells through HIF-1α. Frontiers in physiology, 14, 1309155.
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  Forkhead Box F1 (FOXF1) transcription factor plays a critical role in lung angiogenesis during embryonic development and lung repair after injury. FOXF1 expression is decreased in endothelial cells after lung injury; however, molecular mechanisms responsible for the FOXF1 transcript changes in injured lung endothelium remain unknown. We used immunostaining of injured mouse lung tissues, FACS-sorted lung endothelial cells from hypoxia-treated mice, and data from patients diagnosed with hypoxemic respiratory failure to demonstrate that hypoxia is associated with decreased FOXF1 expression. Endothelial cell cultures were used to induce hypoxia and identify the upstream molecular mechanism through which hypoxia inhibits FOXF1 gene expression. Bleomycin-induced lung injury induced hypoxia in the mouse lung tissue which was associated with decreased expression. Human mRNA was decreased in the lungs of patients diagnosed with hypoxemic respiratory failure. Mice exposed to hypoxia exhibited reduced expression in the lung tissue and FACS-sorted lung endothelial cells. , hypoxia (1% of O) or treatment with cobalt (II) chloride increased HIF-1α protein levels but inhibited expression in three endothelial cell lines. Overexpression of HIF-1α in cultured endothelial cells was sufficient to inhibit expression. siRNA-mediated depletion of HIF-1α prevented the downregulation of gene expression after hypoxia or cobalt (II) chloride treatment. Hypoxia inhibits FOXF1 expression in endothelial cells in a HIF-1α dependent manner. Our data suggest that endothelial cell-specific inhibition of HIF-1α via gene therapy can be considered to restore FOXF1 and improve lung repair in patients with severe lung injury.
• Bian, F., Lan, Y. W., Zhao, S., Deng, Z., Shukla, S., Acharya, A., Donovan, J., Le, T., Milewski, D., Bacchetta, M., Hozain, A. E., Tipograf, Y., Chen, Y. W., Xu, Y., Shi, D., Kalinichenko, V. V., & Kalin, T. V. (2023). Lung endothelial cells regulate pulmonary fibrosis through FOXF1/R-Ras signaling. Nature communications, 14(1), 2560.
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  Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF.
• Donovan, J., Deng, Z., Bian, F., Shukla, S., Gomez-Arroyo, J., Shi, D., Kalinichenko, V. V., & Kalin, T. V. (2023). Corrigendum: Improving anti-tumor efficacy of low-dose Vincristine in rhabdomyosarcoma the combination therapy with FOXM1 inhibitor RCM1. Frontiers in oncology, 13, 1163510.
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  [This corrects the article DOI: 10.3389/fonc.2023.1112859.].
• Donovan, J., Deng, Z., Bian, F., Shukla, S., Gomez-Arroyo, J., Shi, D., Kalinichenko, V. V., & Kalin, T. V. (2023). Improving anti-tumor efficacy of low-dose Vincristine in rhabdomyosarcoma the combination therapy with FOXM1 inhibitor RCM1. Frontiers in oncology, 13, 1112859.
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  Rhabdomyosarcoma (RMS) is a highly metastatic soft-tissue sarcoma that often develops resistance to current therapies, including vincristine. Since the existing treatments have not significantly improved survival, there is a critical need for new therapeutic approaches for RMS patients. FOXM1, a known oncogene, is highly expressed in RMS, and is associated with the worst prognosis in RMS patients. In the present study, we found that the combination treatment with specific FOXM1 inhibitor RCM1 and low doses of vincristine is more effective in increasing apoptosis and decreasing RMS cell proliferation compared to single drugs alone. Since RCM1 is highly hydrophobic, we developed innovative nanoparticle delivery system containing poly-beta-amino-esters and folic acid (NP), which efficiently delivers RCM1 to mouse RMS tumors . The combination of low doses of vincristine together with intravenous administration of NP nanoparticles containing RCM1 effectively reduced RMS tumor volumes, increased tumor cell death and decreased tumor cell proliferation in RMS tumors compared to RCM1 or vincristine alone. The combination therapy was non-toxic as demonstrated by liver metabolic panels using peripheral blood serum. Using RNA-seq of dissected RMS tumors, we identified as a uniquely downregulated gene after the combination treatment. Knockdown of in RMS cells recapitulated the effects of the combination therapy. Altogether, combination treatment with low doses of vincristine and nanoparticle delivery of FOXM1 inhibitor RCM1 in a pre-clinical model of RMS has superior anti-tumor effects and decreases CHAC1 while reducing vincristine toxicity.
• Kolesnichenko, O. A., Flood, H. M., Zhang, Y., Ustiyan, V., Cuervo Jimenez, H. K., Kalin, T. V., & Kalinichenko, V. V. (2023). Endothelial progenitor cells derived from embryonic stem cells prevent alveolar simplification in a murine model of bronchopulmonary dysplasia. Frontiers in cell and developmental biology, 11, 1209518.
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  Vascular remodeling and compromised alveolar development are hallmarks of chronic pulmonary diseases such as bronchopulmonary dysplasia (BPD). Despite advances in neonatal healthcare the number of BPD cases worldwide continues to increase. One approach to overcoming the premature arrest in lung development seen in BPD is to stimulate neonatal angiogenesis via delivery and engraftment of endothelial progenitor cells (EPCs). One such population is resident to the pulmonary microvasculature and expresses both FOXF1 and c-KIT. Previous studies have shown that c-KITFOXF1 EPCs are highly sensitive to elevated levels of oxygen (hyperoxia) and are decreased in premature infants with BPD and hyperoxia-induced BPD mouse models. We hypothesize that restoring EPCs through transplantation of c-KITFOXF1 EPCs derived from pluripotent embryonic stem cells (ESCs), will stimulate neonatal angiogenesis and alveolarization in mice with hyperoxia-induced lung injury. Utilizing a novel ESC line with a FOXF1:GFP reporter, we generated ESC-derived c-KITFOXF1 EPCs . Using a second ESC line which contains FOXF1:GFP and tdTomato transgenes, we differentiated ESCs towards c-KITFOXF1 EPCs and tracked them after injection into the neonatal circulation of hyperoxia-injured mice. After a recovery period in room air conditions, we analyzed c-KITFOXF1 EPC engraftment and quantified the number of resident and circulating endothelial cells, the size of alveolar spaces, and the capillary density after EPC transplantations. Herein, we demonstrate that addition of BMP9 to the directed endothelial differentiation protocol results in very efficient generation of c-KITFOXF1 EPCs from pluripotent ESCs. ESC-derived c-KITFOXF1 EPCs effectively engraft into the pulmonary microvasculature of hyperoxia-injured mice, promote vascular remodeling in alveoli, increase the number of resident and circulating endothelial cells, and improve alveolarization. Altogether, these results provide a proof-of-principle that cell therapy with ESC-derived c-KITFOXF1 EPCs can prevent alveolar simplification in a hyperoxia-induced BPD mouse model.
• Pradhan, A., Che, L., Ustiyan, V., Reza, A. A., Pek, N. M., Zhang, Y., Alber, A. B., Kalin, T. R., Wambach, J. A., Gu, M., Kotton, D. N., Siefert, M. E., Ziady, A. G., Kalin, T. V., & Kalinichenko, V. V. (2023). Novel FOXF1-Stabilizing Compound TanFe Stimulates Lung Angiogenesis in Alveolar Capillary Dysplasia. American journal of respiratory and critical care medicine, 207(8), 1042-1054.
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  Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is linked to heterozygous mutations in the (Forkhead Box F1) gene, a key transcriptional regulator of pulmonary vascular development. There are no effective treatments for ACDMPV other than lung transplant, and new pharmacological agents activating FOXF1 signaling are urgently needed. Identify-small molecule compounds that stimulate FOXF1 signaling. We used mass spectrometry, immunoprecipitation, and the ubiquitination assay to identify TanFe (transcellular activator of nuclear FOXF1 expression), a small-molecule compound from the nitrile group, which stabilizes the FOXF1 protein in the cell. The efficacy of TanFe was tested in mouse models of ACDMPV and acute lung injury and in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV. We identified HECTD1 as an E3 ubiquitin ligase involved in ubiquitination and degradation of the FOXF1 protein. The TanFe compound disrupted FOXF1-HECTD1 protein-protein interactions and decreased ubiquitination of the FOXF1 protein in pulmonary endothelial cells . TanFe increased protein concentrations of FOXF1 and its target genes , , and in LPS-injured mouse lungs, decreasing endothelial permeability and inhibiting lung inflammation. Treatment of pregnant mice with TanFe increased FOXF1 protein concentrations in lungs of embryos, stimulated neonatal lung angiogenesis, and completely prevented the mortality of mice after birth. TanFe increased angiogenesis in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV with deletion. TanFe is a novel activator of FOXF1, providing a new therapeutic candidate for treatment of ACDMPV and other neonatal pulmonary vascular diseases.
• Shukla, S., Saha, T., Rama, N., Acharya, A., Le, T., Bian, F., Donovan, J., Tan, L. A., Vatner, R., Kalinichenko, V., Mascia, A., Perentesis, J. P., & Kalin, T. V. (2023). Ultra-high dose-rate proton FLASH improves tumor control. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology, 186, 109741.
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  Proton radiotherapy (PRT) offers potential benefits over other radiation modalities, including photon and electron radiotherapy. Increasing the rate at which proton radiation is delivered may provide a therapeutic advantage. Here, we compared the efficacy of conventional proton therapy (CONV) to ultrahigh dose-rate proton therapy, FLASH, in a mouse model of non-small cell lung cancers (NSCLC).