Paulo Pires

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Scholarly Contributions

Chapters

• Pires, P., Bernas, M. J., & Witte, M. H. (2023). Lymphatic pathophysiology. In Rutherford's Vascular Surgery and Endovascular Therapy(pp 108-126). Chapter 10: Elsevier.
• Witte, M. H., Bernas, M., Pires, P., Sidaway, A. N., & Perler, B. A. (2022). Lymphatic pathophysiology, Chapter 10. In Rutherford’s Vascular Surgery, Tenth Edition(pp 108-126). Philadelphia: Elsevier.
• Witte, M. H., Bernas, M., Pires, P., Sidaway, A. N., & Perler, B. A. (2021). Lymphatic pathophysiology, Chapter 10. In Rutherford’s Vascular Surgery, Tenth Edition. Philadelphia: Elsevier.

Journals/Publications

• Dennis, M. R., Pires, P. W., & Banek, C. T. (2023). Vascular Dysfunction in Polycystic Kidney Disease: A Mini-Review. Journal of vascular research, 60(3), 125-136.
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  Polycystic kidney disease (PKD) is one of the most common hereditary kidney diseases, which is characterized by progressive cyst growth and secondary hypertension. In addition to cystogenesis and renal abnormalities, patients with PKD can develop vascular abnormalities and cardiovascular complications. Progressive cyst growth substantially alters renal structure and culminates into end-stage renal disease. There remains no cure beyond renal transplantation, and treatment options remain largely limited to chronic renal replacement therapy. In addition to end-stage renal disease, patients with PKD also present with hypertension and cardiovascular disease, yet the timing and interactions between the cardiovascular and renal effects of PKD progression are understudied. Here, we review the vascular dysfunction found in clinical and preclinical models of PKD, including the clinical manifestations and relationship to hypertension, stroke, and related cardiovascular diseases. Finally, our discussion also highlights the critical questions and emerging areas in vascular research in PKD.
• Hoyer-Kimura, C., Hay, M., Konhilas, J. P., Morrison, H. W., Methajit, M., Strom, J., Polt, R., Salcedo, V., Fricks, J. P., Kalya, A., & Pires, P. W. (2023). PNA5, A Novel Mas Receptor Agonist, Improves Neurovascular and Blood-Brain-Barrier Function in a Mouse Model of Vascular Cognitive Impairment and Dementia. Aging and disease, Online ahead of print.
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  It is well established that decreased brain blood flow, increased reactive oxygen species production (ROS), and pro-inflammatory mechanisms accelerate neurodegenerative disease progressions, including vascular cognitive impairment and dementia (VCID). Previous studies in our laboratory have shown that our novel glycosylated Angiotensin-(1-7) Mas receptor agonist PNA5 reverses cognitive deficits, decreases ROS production, and inhibits inflammatory cytokine production in our preclinical mouse model of VCID that is induced by chronic heart failure (VCID-HF). In the present study, the effects of VCID-HF and treatment with PNA5 on microglia activation, blood-brain-barrier (BBB) integrity, and neurovascular coupling were assessed in our mouse model of VCID-HF. Three-month-old male C57BL/6J mice were subjected to myocardial infarction (MI) to induce heart failure for four weeks and then treated with subcutaneous injections of extended-release PNA5. Microglia activation, BBB permeability, cerebral perfusion, and neurovascular coupling were assessed. Results show that in our VCID-HF model, there was an increase in microglial activation and recruitment within the CA1 and CA3 regions of the hippocampus, a disruption in BBB integrity, and a decrease in neurovascular coupling. Treatment with PNA5 reversed these neuropathological effects of VCID-HF, suggesting that PNA5 may be an effective disease-modifying therapy to treat and prevent VCID. This study identifies potential mechanisms by which heart failure may induce VCID and highlights the possible mechanisms by which treatment with our novel glycosylated Angiotensin-(1-7) Mas receptor agonist, PNA5, may protect cognitive function in our model of VCID.
• Kamali, A., Dieckhaus, L. A., Peters, E. C., Preszler, C. A., Witte, R., Pires, P. W., Hutchinson, E., & Laksari, K. (2023). Ultrasound, photoacoustic, and magnetic resonance imaging to study hyperacute pathophysiology of traumatic and vascular brain injury. Journal of neuroimaging : official journal of the American Society of Neuroimaging, 33(4), 534-546.
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  Cerebrovascular dynamics and pathomechanisms that evolve in the minutes and hours following traumatic vascular injury in the brain remain largely unknown. We investigated the pathophysiology evolution in mice within the first 3 hours after closed-head traumatic brain injury (TBI) and subarachnoid hemorrhage (SAH), two significant traumatic vascular injuries.
• Polk, F. D., Hakim, M. A., Silva, J. F., Behringer, E. J., & Pires, P. W. (2023). Endothelial K2 channel dysfunction in aged cerebral parenchymal arterioles. American journal of physiology. Heart and circulatory physiology, Online ahead of print.
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  Aging is associated with cognitive decline incompletely understood mechanisms. Cerebral microvascular dysfunction occurs in aging, particularly impaired endothelium-mediated dilation. Parenchymal arterioles are bottlenecks of the cerebral microcirculation, and dysfunction causes a mismatch in nutrient demand and delivery, leaving neurons at risk. Extracellular nucleotides elicit parenchymal arteriole dilation by activating endothelial purinergic receptors (P2Y), leading to opening of K channels, including inwardly-rectifying K channels (K2). These channels amplify hyperpolarizing signals, resulting in dilation. However, it remains unknown if endothelial P2Y and K2 signaling are altered in brain parenchymal arterioles during aging. We hypothesized that aging impairs endothelial P2Y and K2 function in parenchymal arterioles. We observed reduced dilation to the purinergic agonist 2-methyl-S-ADP (1 µM) in arterioles from Aged (>24-month-old) mice when compared to Young (4-6 months of age) despite similar hyperpolarization in endothelial cells tubes. No differences were observed in vasodilation or endothelial cell hyperpolarization to activation of small- and intermediate-conductance Ca-activated K channels (K2.3 / K3.1) by NS309. Hyperpolarization to 15 mM [K] was smaller in Aged than Young mice, despite a paradoxical increased dilation in Aged arterioles to 15 mM [K] that was unchanged by endothelium removal. K2 Inhibition attenuated vasodilatory responses to 15 mM [K] and 1 µM 2-me-S-ADP in both Young and Aged arterioles. Further, we observed a significant increase in myogenic tone in Aged parenchymal arterioles, which was not enhanced by endothelium removal. We conclude that aging impairs endothelial K2 channel function in the cerebral microcirculation with possible compensation by smooth muscle cells.
• Blackwell, J. A., Silva, J. F., Louis, E. M., Savu, A., Largent-Milnes, T. M., Brooks, H. L., & Pires, P. W. (2022). Cerebral arteriolar and neurovascular dysfunction after chemically induced menopause in mice. American journal of physiology. Heart and circulatory physiology, 323(5), H845-H860.
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  Cognitive decline is linked to decreased cerebral blood flow, particularly in women after menopause. Impaired cerebrovascular function precedes the onset of dementia, possibly because of reduced functional dilation in parenchymal arterioles. These vessels are bottlenecks of the cerebral microcirculation, and dysfunction can limit functional hyperemia in the brain. Large-conductance Ca-activated K channels (BK) are the final effectors of several pathways responsible for functional hyperemia, and their expression is modulated by estrogen. However, it remains unknown whether BK function is altered in cerebral parenchymal arterioles after menopause. Using a chemically induced model of menopause, the 4-vinylcyclohexene diepoxide (VCD) model, which depletes follicles while maintaining intact ovaries, we hypothesized that menopause would be associated with reduced functional vasodilatory responses in cerebral parenchymal arterioles of wild-type mice via reduced BK function. Using pressure myography of isolated parenchymal arterioles, we observed that menopause (Meno) induced a significant increase in spontaneous myogenic tone. Endothelial function, assessed as nitric oxide production and dilation after cholinergic stimulation or endothelium-dependent hyperpolarization pathways, was unaffected by Meno. BK function was significantly impaired in Meno compared with control, without changes in voltage-gated K channel activity. Cerebral functional hyperemia, measured by laser-speckle contrast imaging during whisker stimulation, was significantly blunted in Meno mice, without detectable changes in basal perfusion. However, behavioral testing identified no change in cognition. These findings suggest that menopause induces cerebral microvascular and neurovascular deficits. Cerebral parenchymal arterioles from menopause mice showed increased myogenic tone. We identified an impairment in smooth muscle cell BK channel activity, without a reduction in endothelium-dependent dilation or nitric oxide production. Microvascular dysfunction was associated with a reduction in neurovascular responses after somatosensory stimulation. Despite the neurovascular impairment, cognitive abilities were maintained in menopausal mice.
• Hakim, M., Pires, P., & Behringer, E. (2022). Isolation and functional resolution of arteriolar endothelium of mouse brain parenchyma. Journal of Visualized Experiments, 181, 10.3791/63463. doi:10.3791/63463
• Park, S., Cho, J. M., Mookherjee, S., Pires, P., & Symons, J. D. (2022). Recent Insights Concerning Autophagy and Endothelial Cell Nitric Oxide Generation. Current Opinion in Physiology, 30, 100614. doi:https://doi.org/10.1016/j.cophys.2022.100614
• Peters, E. C., Gee, M. T., Polk, F. D., Kath, A. M., Pawlowski, L. N., Lessa Sacoman, J., & Pires, P. (2022). Amyloid-β Disrupts Unitary Calcium Entry Through Endothelial NMDA Receptors in Mouse Cerebral Arteries. Journal of Cerebral Blood Flow and Metabolism, 42(1), 145-161. doi:10.1177/0271678X211039592
• Mustacich, D. M., Kylathu, R., Bernas, M. J., Myles, R. J., Jones, J. A., Kanady, J. D., Simon, A., Georgieva, T. G., Witte, M. H., Erickson, R. P., & Pires, P. (2021). Abnormal lymphatic phenotype in a CRISPR mouse model of the human lymphedema-causing Connexin 47 R260C point mutation. Lymphology, 54(1), 78-91.
• Pires, P., Erickson, R. P., Witte, M. H., Georgieva, T. G., Simon, A., Kanady, J. D., Jones, J. A., Myles, R. J., Bernas, M. J., Kylathu, R., & Mustacich, D. M. (2021). Abnormal lymphatic phenotype in a CRISPR mouse model of the human lymphedema-causing Connexin47 R260C point mutation.. Lymphology, 54(2), 78-91.
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  Connexin proteins form gap junctions controlling exchange of ions and small molecules between cells and play an important role in movement of lymph within lymphatic vessels. Connexin47 (CX47) is highly expressed in lymphatic endothelial cells and CX47 missense mutations, i.e., R260C, cosegregate with primary lymphedema in humans. However, studies utilizing CX47 knockout mice have failed to demonstrate any lymphatic anomalies. To unravel the lymphatic consequences of expressing a mutant CX47 protein, we used CRISPR technology to create a mouse carrying a Cx47 missense mutation (Cx47R259C) equivalent to the human CX47R260C missense mutation associated with human primary lymphedema. Intradermal Evans Blue dye injection identified a 2-fold increase in regional lymph nodes in homozygous Cx47R259C mice compared to wildtype, particularly in the jugular region (4.8 ± 0.4 and 2.0 ± 0.0, respectively, p
• Pires, P. (2018). Cannabinoids during ischemic strokes: friends or foes?. American Journal of Physiology-Heart and Circulatory Physiology.
• Pires, P. (2018). Nanoscale remodeling of ryanodine receptor cluster size underlies cerebral microvascular dysfunction in Duchenne muscular dystrophy. Proceedings of the National Academy of Sciences.
• Pires, P. (2018). Neuroprotective effects of TRPA1 channels in the cerebral endothelium following ischemic stroke. eLife.
• Pires, P. W., McClain, J. L., Hayoz, S. F., & Dorrance, A. M. (2018). Mineralocorticoid receptor antagonism prevents obesity-induced cerebral artery remodeling and reduces white matter injury in rats. Microcirculation (New York, N.Y. : 1994), 25(5), e12460.
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  Midlife obesity is a risk factor for dementia development. Obesity has also been linked to hyperaldosteronism, and this can be modeled in rats by high fat (HF) feeding from weaning. Aldosterone, or activation of the mineralocorticoid receptor (MR) causes cerebrovascular injury in lean hypertensive rats. We hypothesized that rats fed a HF diet would show inward middle cerebral artery (MCA) remodeling that could be prevented by MR antagonism. We further proposed that the cerebral artery remodeling would be associated with white mater injury.
• Pires, P. (2017). Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility. Science Signaling.
• Pires, P. (2017). The angiotensin II receptor type 1b is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cellS. The Journal of Physiology.
• Pires, P. W., & Earley, S. (2017). Redox regulation of transient receptor potential channels in the endothelium. Microcirculation (New York, N.Y. : 1994), 24(3).
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  ROS and RNS are important mediators of signaling pathways in the endothelium. Specific members of the TRP superfamily of cation channels act as important Ca influx pathways in endothelial cells and are involved in endothelium-dependent vasodilation, regulation of barrier permeability, and angiogenesis. ROS and RNS can modulate the activity of certain TRP channels mainly by modifying specific cysteine residues or by stimulating the production of second messengers. In this review, we highlight the recent literature describing redox regulation of TRP channel activity in endothelial cells as well as the physiological importance of these pathways and implication for cardiovascular diseases.
• Matin, N., Pires, P. W., Garver, H., Jackson, W. F., & Dorrance, A. M. (2016). DOCA-salt hypertension impairs artery function in rat middle cerebral artery and parenchymal arterioles. Microcirculation (New York, N.Y. : 1994), 23(7), 571-579.
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  Chronic hypertension induces detrimental changes in the structure and function of surface cerebral arteries. Very little is known about PAs, which perfuse distinct neuronal populations in the cortex and may play a role in cerebrovascular disorders. We investigated the effect of DOCA-salt induced hypertension on endothelial function and artery structure in PAs and MCAs.
• Pires, P. W., & Earley, S. (2016). A TRPC3 signalling complex promotes cerebral artery remodelling during hypertension. Cardiovascular research, 109(1), 4-5.
• Pires, P. W., & Earley, S. (2016). No Static at All: Tuning Into the Complexities of Ca2+ Signaling in the Endothelium. Circulation research, 118(7), 1042-4.
• Pires, P. W., Dabertrand, F., & Earley, S. (2016). Isolation and Cannulation of Cerebral Parenchymal Arterioles. Journal of visualized experiments : JoVE.
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  Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves - innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.
• Pires, P. (2015). TRPs in the kidney - location, location, location. Acta Physiologica.
• Pires, P. (2015). Unitary TRPV3 channel Ca2+ influx events elicit endothelium-dependent dilation of cerebral parenchymal arterioles. American Journal of Physiology-Heart and Circulatory Physiology.
• Pires, P. (2015). Vascular biology: Localized TRPA1 channel Ca²⁺ signals stimulated by reactive oxygen species promote cerebral artery dilation. Science Signaling.
• Pires, P. (2014). The effects of obesity on the cerebral vasculature. Current Vascular Pharmacology.
• Pires, P. (2014). Tumor necrosis factor-α inhibition attenuates middle cerebral artery remodeling but increases cerebral ischemic damage in hypertensive rats. American Journal of Physiology - Heart and Circulatory Physiology.
• Pires, P. (2013). Improvement in middle cerebral artery structure and endothelial function in stroke-prone spontaneously hypertensive rats after macrophage depletion. Microcirculation.
• Pires, P. (2013). The effects of hypertension on the cerebral circulation. American Journal of Physiology - Heart and Circulatory Physiology.
• Pires, P. (2012). Direct regulation of blood pressure by smooth muscle cell mineralocorticoid receptors. Nature Medicine.
• Pires, P. (2012). The development of hypertension and hyperaldosteronism in a rodent model of life-long obesity. Endocrinology.
• Pires, P. (2011). Doxycycline, a matrix metalloprotease inhibitor, reduces vascular remodeling and damage after cerebral ischemia in stroke-prone spontaneously hypertensive rats. American Journal of Physiology - Heart and Circulatory Physiology.
• Pires, P. (2010). Effects of gestational and lactational fenvalerate exposure on immune and reproductive systems of male rats. Journal of Toxicology and Environmental Health - Part A: Current Issues.
• Pires, P. (2010). Tempol, a superoxide dismutase mimetic, prevents cerebral vessel remodeling in hypertensive rats. Microvascular Research.
• Pires, P. (2009). Metalloproteinases 2 and -9 activity during promotion and progression stages of rat liver carcinogenesis. Journal of Molecular Histology.
• Pires, P. (2008). Chronic ethanol intake promotes double gluthatione S-transferase/transforming growth factor-α-positive hepatocellular lesions in male Wistar rats. Cancer Science.
• Pires, P. (2007). Liver lesions produced by aflatoxins in Rana catesbeiana (bullfrog). Ecotoxicology and Environmental Safety.

Proceedings Publications

• Pires, P., Laksari, K., Hutchinson, E. B., Peters, E., Dieckhaus, L. A., & Kamali, A. (2021, Oct.). The Golden Hour: Early outcomes of traumatic brain injury and stroke. In Biomedical Engineering Society.

Presentations

• Vance, C., Kath, A. M., Williams, C., Witte, M. H., & Pires, P. W. (2021, September). Amyloid-β reduces contraction frequency of superficial cervical lymphatic in mice. 28th World Congress of Lymphology. Athens, Greece: International Society of Lymphology.
• Witte, R. S., Pires, P., & Hutchinson, E. B. (2021, Feb.). Mouse Brain Photoacoustic Imaging to Detect Hyper-Acute Pathophysiology Following Traumatic Brain Injury, Ischemia and Hemorrhage.. VisualSonics Live Imaging Workshop: “From Brain to Belly”VisualSonics.

Poster Presentations

• Peters, E. C., Ayon, R. J., & Pires, P. W. (2020, April). Amyloid-β(1-42) Inhibits Ca2+ Transients Induced by N-Methyl-D-Aspartate Receptor in The Cerebral Vasculature. Experimental Biology. San Diego, CA: American Physiological Society.
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  Endothelium-dependent dilation of cerebral arteries, a process dependent on transient increases in intracellular calcium (Ca2+), contributes to neurovascular coupling (NVC), which is altered in cerebral amyloid angiopathy (CAA).The N-methyl-D-aspartate receptor (NMDAR), a nonselective cation channel with high Ca2+ permeability, has been shown to mediate endothelium-dependent dilation in cerebral arteries. NMDAR activity is reduced by amyloid-β, which accumulates around the cerebral vasculature during CAA. We hypothesized that amyloid-β impairs NMDAR-induced Ca2+ transients in endothelial cells of cerebral arteries, which impairs endothelium-mediated dilation in mice. All animal experiments were approved by the University of Arizona IACUC. Data are means ± SEM, both in male and female mice (no sex differences were observed). Cerebral arteries isolated from mice expressing the genetically-encoded calcium indicator GCaMP8 in endothelial cells (cdh5:Gcamp8) were prepared en face for time-lapse imaging of endothelial Ca2+ transients induced by NMDAR activation. In fields of view that displayed Ca2+ transients, we found that the NMDAR agonist NMDA (1 µM) increased the frequency of endothelial Ca2+ transients compared to baseline (0.22 ± 0.06 vs 0.58 ± 0.15 Hz, baseline vs NMDA, n = 10 and 13 fields of view from at least 3 mice, p < 0.05, one way ANOVA). Pre-incubation of preparations with the NMDAR antagonist D-AP5 (10 µM) prevented NMDA induction of endothelial cell Ca2+ transients (frequency: 0.14 ± 0.05, n = 10 fields of view, p < 0.05 vs NMDA, one-way ANOVA). These data suggest that endothelial NMDAR Ca2+ transients can be stimulated in cerebral arteries via NMDA. We then tested whether the peptide amyloid-β(1-42), commonly found in CAA, blunted NMDAR-elicited Ca2+ transients. Cerebral artery preparations were incubated for 30 minutes with 5 µM amyloid-β(1-42), then exposed to NMDA. Our preliminary data suggests that pre-incubation of preparations with amyloid-β blunts NMDA-dependent induction of endothelial cell Ca2+ transients (0.21 ± 0.04, n = 10 fields of view, p < 0.05 vs NMDA, one-way ANOVA). In order to evaluate the effects of amyloid-β on dilation of cerebral arterioles, we then performed ex vivo pressure myography experiments with cerebral parenchymal arterioles from a mouse model of familial Alzheimer’s disease without aging (5x-FAD) or wildtype littermates. Our preliminary results suggest that NMDA-elicited dilation of parenchymal arterioles may be impaired in 5x-FAD mice (vasodilation (%): 11.06 ± 0.78 vs 6.21 ± 2.09, wildtype vs 5x-FAD, n = 3 arterioles from 3 mice, p = 0.067, two-tailed Student’s t-test). These preliminary data suggest that NMDA receptors in the cerebrovascular endothelium of wildtype mice mediate arteriolar dilation via an increase in Ca2+ transients. Further, amyloid-β may impair the activity of endothelial NMDA receptors and thus contribute to neurovascular dysfunction via impaired arteriolar dilation in individuals with CAA.