Publications by authors named "Swapnil K Sonkusare"

33 Publications

Guidelines for the measurement of vascular function and structure in isolated arteries and veins.

Am J Physiol Heart Circ Physiol 2021 May 14. Epub 2021 May 14.

Cardiovascular Translational Research Center and Department of Cell Biology and Anatomy, grid.254567.7University of South Carolina, Columbia, South Carolina, United States.

The measurement of vascular function in isolated vessels has revealed important insights into the structural, functional, and biomechanical features of the normal and diseased cardiovascular system, and has provided a molecular understanding of the cells that constitutes arteries and veins and their interaction. Further, this approach has allowed the discovery of vital pharmacological treatments for cardiovascular diseases. However, the expansion of the vascular physiology field has also brought new concerns over scientific rigor and reproducibility. Therefore, it is appropriate to set guidelines for the best practices of evaluating vascular function in isolated vessels. These guidelines are a comprehensive document detailing the best practices and pitfalls for the assessment of function in large and small arteries and veins. Herein, we bring together experts in the field of vascular physiology with the purpose of developing guidelines for evaluating ex vivo vascular function. By utilizing this document, vascular physiologists will have consistency amongst methodological approaches, producing more reliable and reproducible results.
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http://dx.doi.org/10.1152/ajpheart.01021.2020DOI Listing
May 2021

Endothelial Transient Receptor Potential V4 Channels Mediate Lung Ischemia-Reperfusion Injury.

Ann Thorac Surg 2021 May 4. Epub 2021 May 4.

Department of Surgery, University of Virginia School of Medicine, Charlottesville, VA. Electronic address:

Background: Lung ischemia-reperfusion injury (IRI), involving severe inflammation and edema, is a major cause of primary graft dysfunction following transplant. Activation of transient receptor potential vanilloid 4 (TRPV4) channels modulates vascular permeability. Thus, this study tests the hypothesis that endothelial TRPV4 channels mediate lung IRI.

Methods: C57BL/6 wild-type (WT), TRPV4, tamoxifen-inducible endothelial TRPV4 knockout (TRPV4), and tamoxifen-treated control (TRPV4) mice underwent lung IR using a left lung hilar-ligation model (n≥6 mice/group). WT mice were also treated with a TRPV4-specific inhibitor (GSK2193874; 1mg/kg) (WT+GSK219). Partial pressure of oxygen (PaO), edema (wet-to-dry weight ratio), compliance, neutrophil infiltration, and cytokine concentrations in bronchioalveolar lavage fluid were assessed. Pulmonary microvascular endothelial cells (PMVECs) were characterized in vitro following exposure to hypoxia-reoxygenation.

Results: Compared to WT, PaO following IR was significantly improved in TRPV4 mice (133.1±43.9 vs 427.8±83.1 mmHg, p<0.001) and WT+GSK219 mice (133.1±43.9 vs 447.0±67.6 mmHg, p<0.001). Pulmonary edema and neutrophil infiltration were also significantly reduced after IR in TRPV4 and WT+GSK219 mice versus WT. TRPV4 mice following IR demonstrated significantly improved oxygenation versus control (109.2±21.6 vs 405.3±41.4 mmHg, p<0.001) as well as significantly improved compliance, and significantly less edema, neutrophil infiltration and proinflammatory cytokine production (TNF-α, CXCL1, IL-17, IFN-γ). Hypoxia-reoxygenation-induced permeability and CXCL1 expression by PMVECs was significantly attenuated by TRPV4 inhibitors.

Conclusions: Endothelial TRPV4 plays a key role in vascular permeability and lung inflammation following IR. TRPV4 channels may be a promising therapeutic target to mitigate lung IRI and decrease the incidence of primary graft dysfunction following transplant. (Word Count: 249/250).
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http://dx.doi.org/10.1016/j.athoracsur.2021.04.052DOI Listing
May 2021

Caveolar peroxynitrite formation impairs endothelial TRPV4 channels and elevates pulmonary arterial pressure in pulmonary hypertension.

Proc Natl Acad Sci U S A 2021 Apr;118(17)

Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908;

Recent studies have focused on the contribution of capillary endothelial TRPV4 channels to pulmonary pathologies, including lung edema and lung injury. However, in pulmonary hypertension (PH), small pulmonary arteries are the focus of the pathology, and endothelial TRPV4 channels in this crucial anatomy remain unexplored in PH. Here, we provide evidence that TRPV4 channels in endothelial cell caveolae maintain a low pulmonary arterial pressure under normal conditions. Moreover, the activity of caveolar TRPV4 channels is impaired in pulmonary arteries from mouse models of PH and PH patients. In PH, up-regulation of iNOS and NOX1 enzymes at endothelial cell caveolae results in the formation of the oxidant molecule peroxynitrite. Peroxynitrite, in turn, targets the structural protein caveolin-1 to reduce the activity of TRPV4 channels. These results suggest that endothelial caveolin-1-TRPV4 channel signaling lowers pulmonary arterial pressure, and impairment of endothelial caveolin-1-TRPV4 channel signaling contributes to elevated pulmonary arterial pressure in PH. Thus, inhibiting NOX1 or iNOS activity, or lowering endothelial peroxynitrite levels, may represent strategies for restoring vasodilation and pulmonary arterial pressure in PH.
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http://dx.doi.org/10.1073/pnas.2023130118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092599PMC
April 2021

Impaired TRPV4-eNOS signaling in trabecular meshwork elevates intraocular pressure in glaucoma.

Proc Natl Acad Sci U S A 2021 Apr;118(16)

Department of Pharmacology and Neuroscience, North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107;

Primary Open Angle Glaucoma (POAG) is the most common form of glaucoma that leads to irreversible vision loss. Dysfunction of trabecular meshwork (TM) tissue, a major regulator of aqueous humor (AH) outflow resistance, is associated with intraocular pressure (IOP) elevation in POAG. However, the underlying pathological mechanisms of TM dysfunction in POAG remain elusive. In this regard, transient receptor potential vanilloid 4 (TRPV4) cation channels are known to be important Ca entry pathways in multiple cell types. Here, we provide direct evidence supporting Ca entry through TRPV4 channels in human TM cells and show that TRPV4 channels in TM cells can be activated by increased fluid flow/shear stress. TM-specific TRPV4 channel knockout in mice elevated IOP, supporting a crucial role for TRPV4 channels in IOP regulation. Pharmacological activation of TRPV4 channels in mouse eyes also improved AH outflow facility and lowered IOP. Importantly, TRPV4 channels activated endothelial nitric oxide synthase (eNOS) in TM cells, and loss of eNOS abrogated TRPV4-induced lowering of IOP. Remarkably, TRPV4-eNOS signaling was significantly more pronounced in TM cells compared to Schlemm's canal cells. Furthermore, glaucomatous human TM cells show impaired activity of TRPV4 channels and disrupted TRPV4-eNOS signaling. Flow/shear stress activation of TRPV4 channels and subsequent NO release were also impaired in glaucomatous primary human TM cells. Together, our studies demonstrate a central role for TRPV4-eNOS signaling in IOP regulation. Our results also provide evidence that impaired TRPV4 channel activity in TM cells contributes to TM dysfunction and elevated IOP in glaucoma.
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http://dx.doi.org/10.1073/pnas.2022461118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072326PMC
April 2021

Extra-Nuclear Functions of the Transcription Factor Grainyhead-Like 3 in the Endothelium-Interaction with Endothelial Nitric Oxide Synthase.

Antioxidants (Basel) 2021 Mar 11;10(3). Epub 2021 Mar 11.

IUF-Leibniz Research Institute for Environmental Medicine, 40225 Düsseldorf, Germany.

We previously demonstrated that the transcription factor Grainyhead-like 3 (GRHL3) has essential functions in endothelial cells by inhibiting apoptosis and promoting migration as well as activation of endothelial nitric oxide synthase (eNOS). We now show that a large portion of the protein is localized to myo-endothelial projections of murine arteries suggesting extra-nuclear functions. Therefore, we generated various deletion mutants to identify the nuclear localization signal (NLS) of GRHL3 and assessed potential extra-nuclear functions. Several large-scale deletion mutants were incapable of activating a GRHL3-dependent reporter construct, which could either be due to deficiencies in transcriptional activation or to impaired nuclear import. One of these mutants encompassed a predicted bipartite NLS whose deletion led to the retention of GRHL3 outside the nucleus. Interestingly, this mutant retained functions of the full-length protein as it could still inhibit pathways inducing endothelial cell apoptosis. As apoptosis protection by GRHL3 depends on NO-production, we examined whether GRHL3 could interact with eNOS and showed a direct interaction, which was enhanced with the extra-nuclear GRHL3 variant. The observation that endogenous GRHL3 also interacts with eNOS in intact murine arteries corroborated these findings and substantiated the notion that GRHL3 has important extra-nuclear functions in the endothelium.
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http://dx.doi.org/10.3390/antiox10030428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000391PMC
March 2021

The Calcium Signaling Mechanisms in Arterial Smooth Muscle and Endothelial Cells.

Compr Physiol 2021 Apr 1;11(2):1831-1869. Epub 2021 Apr 1.

Department of Pharmacology, University of Virginia, Charlottesville, Virginia, USA.

The contractile state of resistance arteries and arterioles is a crucial determinant of blood pressure and blood flow. Physiological regulation of arterial contractility requires constant communication between endothelial and smooth muscle cells. Various Ca signals and Ca -sensitive targets ensure dynamic control of intercellular communications in the vascular wall. The functional effect of a Ca signal on arterial contractility depends on the type of Ca -sensitive target engaged by that signal. Recent studies using advanced imaging methods have identified the spatiotemporal signatures of individual Ca signals that control arterial and arteriolar contractility. Broadly speaking, intracellular Ca is increased by ion channels and transporters on the plasma membrane and endoplasmic reticular membrane. Physiological roles for many vascular Ca signals have already been confirmed, while further investigation is needed for other Ca signals. This article focuses on endothelial and smooth muscle Ca signaling mechanisms in resistance arteries and arterioles. We discuss the Ca entry pathways at the plasma membrane, Ca release signals from the intracellular stores, the functional and physiological relevance of Ca signals, and their regulatory mechanisms. Finally, we describe the contribution of abnormal endothelial and smooth muscle Ca signals to the pathogenesis of vascular disorders. © 2021 American Physiological Society. Compr Physiol 11:1831-1869, 2021.
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http://dx.doi.org/10.1002/cphy.c200030DOI Listing
April 2021

Calcium Signal Profiles in Vascular Endothelium from Cdh5-GCaMP8 and Cx40-GCaMP2 Mice.

J Vasc Res 2021 11;58(3):159-171. Epub 2021 Mar 11.

Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia, USA,

Introduction: Studies in Cx40-GCaMP2 mice, which express calcium biosensor GCaMP2 in the endothelium under connexin 40 promoter, have identified the unique properties of endothelial calcium signals. However, Cx40-GCaMP2 mouse is associated with a narrow dynamic range and lack of signal in the venous endothelium. Recent studies have proposed many GCaMPs (GCaMP5/6/7/8) with improved properties although their performance in endothelium-specific calcium studies is not known.

Methods: We characterized a newly developed mouse line that constitutively expresses GCaMP8 in the endothelium under the VE-cadherin (Cdh5-GCaMP8) promoter. Calcium signals through endothelial IP3 receptors and TRP vanilloid 4 (TRPV4) ion channels were recorded in mesenteric arteries (MAs) and veins from Cdh5-GCaMP8 and Cx40-GCaMP2 mice.

Results: Cdh5-GCaMP8 mice showed lower baseline fluorescence intensity, higher dynamic range, and higher amplitudes of individual calcium signals than Cx40-GCaMP2 mice. Importantly, Cdh5-GCaMP8 mice enabled the first recordings of discrete calcium signals in the intact venous endothelium and revealed striking differences in IP3 receptor and TRPV4 channel calcium signals between MAs and mesenteric veins.

Conclusion: Our findings suggest that Cdh5-GCaMP8 mice represent significant improvements in dynamic range, sensitivity for low-intensity signals, and the ability to record calcium signals in venous endothelium.
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http://dx.doi.org/10.1159/000514210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102377PMC
March 2021

A venous-specific purinergic signaling cascade initiated by Pannexin 1 regulates TNFα-induced increases in endothelial permeability.

Sci Signal 2021 Mar 2;14(672). Epub 2021 Mar 2.

Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

The endothelial cell barrier regulates the passage of fluid between the bloodstream and underlying tissues, and barrier function impairment exacerbates the severity of inflammatory insults. To understand how inflammation alters vessel permeability, we studied the effects of the proinflammatory cytokine TNFα on transendothelial permeability and electrophysiology in ex vivo murine veins and arteries. We found that TNFα specifically decreased the barrier function of venous endothelium without affecting that of arterial endothelium. On the basis of RNA expression profiling and protein analysis, we found that claudin-11 (CLDN11) was the predominant claudin in venous endothelial cells and that there was little, if any, CLDN11 in arterial endothelial cells. Consistent with a difference in claudin composition, TNFα increased the permselectivity of Cl over Na in venous but not arterial endothelium. The vein-specific effects of TNFα also required the activation of Pannexin 1 (Panx1) channels and the CD39-mediated hydrolysis of ATP to adenosine, which subsequently stimulated A adenosine receptors. Moreover, the increase in vein permeability required the activation of the Ca channel TRPV4 downstream of Panx1 activation. Panx1-deficient mice resisted the pathologic effects of sepsis induced by cecal ligation and puncture on life span and lung vascular permeability. These data provide a targetable pathway with the potential to promote vein barrier function and prevent the deleterious effects of vascular leak in response to inflammation.
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http://dx.doi.org/10.1126/scisignal.aba2940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011850PMC
March 2021

Role of the purinergic signaling network in lung ischemia-reperfusion injury.

Curr Opin Organ Transplant 2021 Apr;26(2):250-257

Department of Surgery.

Purpose Of Review: Primary graft dysfunction (PGD) is the leading cause of early mortality following lung transplantation and is typically caused by lung ischemia-reperfusion injury (IRI). Current management of PGD is largely supportive and there are no approved therapies to prevent lung IRI after transplantation. The purinergic signaling network plays an important role in this sterile inflammatory process, and pharmacologic manipulation of said network is a promising therapeutic strategy. This review will summarize recent findings in this area.

Recent Findings: In the past 18 months, our understanding of lung IRI has improved, and it is becoming clear that the purinergic signaling network plays a vital role. Recent works have identified critical components of the purinergic signaling network (Pannexin-1 channels, ectonucleotidases, purinergic P1 and P2 receptors) involved in inflammation in a number of pathologic states including lung IRI. In addition, a functionally-related calcium channel, the transient receptor potential vanilloid type 4 (TRPV4) channel, has recently been linked to purinergic signaling and has also been shown to mediate lung IRI.

Summary: Agents targeting components of the purinergic signaling network are promising potential therapeutics to limit inflammation associated with lung IRI and thus decrease the risk of developing PGD.
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http://dx.doi.org/10.1097/MOT.0000000000000854DOI Listing
April 2021

Technical brief: Direct, real-time electrochemical measurement of nitric oxide in ex vivo cultured human corneoscleral segments.

Mol Vis 2020 5;26:434-444. Epub 2020 Jun 5.

Department of Pharmacology and Neuroscience, North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth, TX.

Chronic elevation of intraocular pressure (IOP) is a major risk factor associated with primary open angle glaucoma (POAG), a common form of progressive optic neuropathy that can lead to debilitating loss of vision. Recent studies have identified the role of nitric oxide (NO) in the regulation of IOP, and as a result, several therapeutic ventures are currently targeting enhancement of NO signaling in the eye. Although a low level of NO is important for ocular physiology, excess exogenous NO can be detrimental. Therefore, the ability to directly measure NO in real time is essential for determining the role of NO signaling in glaucomatous pathophysiology. Historically, NO activity in human tissues has been determined by indirect methods that measure levels of NO metabolites (nitrate/nitrite) or downstream components of the NO signaling pathway (cGMP). In this proof-of-concept work, we assess the feasibility of direct, real-time measurement of NO in ex vivo cultured human corneoscleral segments using electrochemistry. A NO-selective electrode (ISO-NOPF200) paired to a free radical analyzer (TBR1025) was placed on the trabecular meshwork (TM) rim for real-time measurement of NO released from cells. Exogenous NO produced within cells was measured after treatment of corneoscleral segments with esterase-dependent NO-donor O2-acetoxymethylated diazeniumdiolate (DETA-NONOate/AM; 20 μM) and latanoprostene bunod (5-20 μM). A fluorescent NO-binding dye DAF-FM (4-Amino-5-methylamino- 2',7'-difluorofluorescein diacetate) was used for validation. A linear relationship was observed between the electric currents measured by the NO-sensing electrode and the NO standard concentrations, establishing a robust calibration curve. Treatment of ex vivo cultured human donor corneoscleral segments with DETA-NONOate/AM and latanoprostene bunod led to a significant increase in NO production compared with vehicle-treated controls, as detected electrochemically. Furthermore, the DAF-FM fluorescence intensity was higher in outflow pathway tissues of corneoscleral segments treated with DETA-NONOate/AM and latanoprostene bunod compared with vehicle-treated controls. In conclusion, these results demonstrate that NO-sensing electrodes can be used to directly measure NO levels in real time from the tissues of the outflow pathway.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300198PMC
May 2021

Mechanisms underlying selective coupling of endothelial Ca signals with eNOS vs. IK/SK channels in systemic and pulmonary arteries.

J Physiol 2020 09 11;598(17):3577-3596. Epub 2020 Jun 11.

Robert M. Berne Cardiovascular Research Center, University of Virginia-School of Medicine, Charlottesville, VA, USA.

Key Points: Endothelial cell TRPV4 (TRPV4 ) channels exert a dilatory effect on the resting diameter of resistance mesenteric and pulmonary arteries. Functional intermediate- and small-conductance K (IK and SK) channels and endothelial nitric oxide synthase (eNOS) are present in the endothelium of mesenteric and pulmonary arteries. TRPV4 sparklets preferentially couple with IK/SK channels in mesenteric arteries and with eNOS in pulmonary arteries. TRPV4 channels co-localize with IK/SK channels in mesenteric arteries but not in pulmonary arteries, which may explain TRPV4 -IK/SK channel coupling in mesenteric arteries and its absence in pulmonary arteries. The presence of the nitric oxide-scavenging protein, haemoglobin α, limits TRPV4 -eNOS signalling in mesenteric arteries. Spatial proximity of TRPV4 channels with eNOS and the absence of haemoglobin α favour TRPV4 -eNOS signalling in pulmonary arteries.

Abstract: Spatially localized Ca signals activate Ca -sensitive intermediate- and small-conductance K (IK and SK) channels in some vascular beds and endothelial nitric oxide synthase (eNOS) in others. The present study aimed to uncover the signalling organization that determines selective Ca signal to vasodilatory target coupling in the endothelium. Resistance-sized mesenteric arteries (MAs) and pulmonary arteries (PAs) were used as prototypes for arteries with predominantly IK/SK channel- and eNOS-dependent vasodilatation, respectively. Ca influx signals through endothelial transient receptor potential vanilloid 4 (TRPV4 ) channels played an important role in controlling the baseline diameter of both MAs and PAs. TRPV4 channel activity was similar in MAs and PAs. However, the TRPV4 channel agonist GSK1016790A (10 nm) selectively activated IK/SK channels in MAs and eNOS in PAs, revealing preferential TRPV4 -IK/SK channel coupling in MAs and TRPV4 -eNOS coupling in PAs. IK/SK channels co-localized with TRPV4 channels at myoendothelial projections (MEPs) in MAs, although they lacked the spatial proximity necessary for their activation by TRPV4 channels in PAs. Additionally, the presence of the NO scavenging protein haemoglobin α (Hbα) within nanometer proximity to eNOS limits TRPV4 -eNOS signalling in MAs. By contrast, co-localization of TRPV4 channels and eNOS at MEPs, and the absence of Hbα, favour TRPV4 -eNOS coupling in PAs. Thus, our results reveal that differential spatial organization of signalling elements determines TRPV4 -IK/SK vs. TRPV4 -eNOS coupling in resistance arteries.
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http://dx.doi.org/10.1113/JP279570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484264PMC
September 2020

Endothelial TRPV4 channels and vasodilator reactivity.

Curr Top Membr 2020 12;85:89-117. Epub 2020 Feb 12.

Robert M. Berne Cardiovascular Research Center, University of Virginia-School of Medicine, Charlottesville, VA, United States; Department of Molecular Physiology and Biological Physics, University of Virginia-School of Medicine, Charlottesville, VA, United States. Electronic address:

Transient receptor potential vanilloid 4 (TRPV4) ion channels on the endothelial cell membrane are widely regarded as a crucial Ca influx pathway that promotes endothelium-dependent vasodilation. The downstream vasodilatory targets of endothelial TRPV4 channels vary among different vascular beds, potentially contributing to endothelial cell heterogeneity. Although numerous studies have examined the role of endothelial TRPV4 channels using specific pharmacological tools over the past decade, their physiological significance remains unclear, mainly due to a lack of endothelium-specific knockouts. Moreover, the loss of endothelium-dependent vasodilation is a significant contributor to vascular dysfunction in cardiovascular disease. The activity of endothelial TRPV4 channels is impaired in cardiovascular disease; therefore, strategies targeting the mechanisms that reduce endothelial TRPV4 channel activity may restore vascular function and provide therapeutic benefit. In this chapter, we discuss endothelial TRPV4 channel-dependent signaling mechanisms, the heterogeneity in endogenous activators and targets of endothelial TRPV4 channels, and the role of endothelial TRPV4 channels in the pathogenesis of cardiovascular diseases. We also discuss potentially interesting future research directions that may provide novel insights into the physiological and pathological roles of endothelial TRPV4 channels.
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http://dx.doi.org/10.1016/bs.ctm.2020.01.007DOI Listing
February 2020

Endothelial calreticulin deletion impairs endothelial function in aged mice.

Am J Physiol Heart Circ Physiol 2020 05 20;318(5):H1041-H1048. Epub 2020 Mar 20.

Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia.

Discrete calcium signals within the vascular endothelium decrease with age and contribute to impaired endothelial-dependent vasodilation. Calreticulin (Calr), a multifunctional calcium binding protein and endoplasmic reticulum (ER) chaperone, can mediate calcium signals and vascular function within the endothelial cells (ECs) of small resistance arteries. We found Calr protein expression significantly decreases with age in mesenteric arteries and examined the functional role of EC Calr in vasodilation and calcium mobilization in the context of aging. Third-order mesenteric arteries from mice with or without EC Calr knockdown were examined for calcium signals and constriction to phenylephrine (PE) or vasodilation to carbachol (CCh) after 75 wk of age. PE constriction in aged mice with or without EC Calr was unchanged. However, calcium signals and vasodilation to endothelial-dependent agonist carbachol were significantly impaired in aged EC Calr knockdown mice. Ex vivo incubation of arteries with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) significantly improved vasodilation in mice lacking EC Calr. Our data suggests diminished vascular Calr expression with age can contribute to the detrimental effects of aging on endothelial calcium regulation and vasodilation. Calreticulin (Calr) is responsible for key physiological processes in endoplasmic reticulum, especially in aging tissue. In particular, endothelial Calr is crucial to vascular function. In this study, we deleted Calr from the endothelium and aged the mice up to 75 wk to examine changes in vascular function. We found two key differences: ) calcium events in endothelium were severely diminished after muscarinic stimulation, which ) corresponded with a dramatic decrease in muscarinic vasodilation. Remarkably, we were able to rescue the effect of Calr deletion on endothelial-dependent vasodilatory function using tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum stress that is currently in clinical trials.
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http://dx.doi.org/10.1152/ajpheart.00586.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346539PMC
May 2020

Local Peroxynitrite Impairs Endothelial Transient Receptor Potential Vanilloid 4 Channels and Elevates Blood Pressure in Obesity.

Circulation 2020 04 3;141(16):1318-1333. Epub 2020 Feb 3.

Robert M. Berne Cardiovascular Research Center (M.O., K.H., E.L.C., Z.D., L.J.D., C.M., N.Y.N., S.R.J., B.E.I., S.K.S.).

Background: Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels are a major Ca influx pathway in endothelial cells, and regulatory protein AKAP150 (A-kinase anchoring protein 150) enhances the activity of TRPV4 channels.

Methods: We used endothelium-specific knockout mice and high-fat diet-fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from nonobese and obese individuals.

Results: We show that Ca influx through TRPV4 channels at myoendothelial projections to smooth muscle cells decreases resting blood pressure in nonobese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule nitric oxide attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of inducible nitric oxide synthase and NADPH oxidase 1 enzymes at myoendothelial projections in obese mice generated higher levels of nitric oxide and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at myoendothelial projections. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150 and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients.

Conclusions: These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.043385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195859PMC
April 2020

Loss of Endothelial FTO Antagonizes Obesity-Induced Metabolic and Vascular Dysfunction.

Circ Res 2020 01 5;126(2):232-242. Epub 2019 Dec 5.

From the Robert M. Berne Cardiovascular Research Center (N.K., L.A.B., M.E.G., C.A.R., A.G.W., L.J.D., S.M., E.H.M., V.S., A.K.B., N.L., S.K.S., B.E.I.), University of Virginia School of Medicine, Charlottesville.

Rationale: Increasing prevalence of obesity and its associated risk with cardiovascular diseases demands a better understanding of the contribution of different cell types within this complex disease for developing new treatment options. Previous studies could prove a fundamental role of FTO (fat mass and obesity-associated protein) within obesity; however, its functional role within different cell types is less understood.

Objectives: We identify endothelial FTO as a previously unknown central regulator of both obesity-induced metabolic and vascular alterations.

Methods And Results: We generated endothelial -deficient mice and analyzed the impact of obesity on those mice. While the loss of endothelial FTO did not influence the development of obesity and dyslipidemia, it protected mice from high-fat diet-induced glucose intolerance and insulin resistance by increasing AKT (protein kinase B) phosphorylation in endothelial cells and skeletal muscle. Furthermore, loss of endothelial FTO prevented the development of obesity-induced hypertension by preserving myogenic tone in resistance arteries. In -deficient arteries, microarray analysis identified upregulation of with significant increases in prostaglandin D levels. Blockade of prostaglandin D synthesis inhibited the myogenic tone protection in resistance arteries of endothelial -deficient mice on high-fat diet; conversely, direct addition of prostaglandin D rescued myogenic tone in high-fat diet-fed control mice. Myogenic tone was increased in obese human arteries with FTO inhibitors or prostaglandin D application.

Conclusions: These data identify endothelial FTO as a previously unknown regulator in the development of obesity-induced metabolic and vascular changes, which is independent of its known function in regulation of obesity.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007767PMC
January 2020

Novel Regulators and Targets of Redox Signaling in Pulmonary Vasculature.

Curr Opin Physiol 2019 Jun 9;9:87-93. Epub 2019 May 9.

Robert M. Berne Cardiovascular Research Center, University of Virginia-School of Medicine, Charlottesville, VA, 22908, USA.

Dysregulated redox signaling in pulmonary vasculature is central to the development of pulmonary arterial hypertension (PAH) and lung injury. Modulators of reactive oxygen species (ROS) production and downstream signaling targets are critical for mediating the physiological or pathological effects of ROS. Understanding the complex interactions between the modulators and signaling targets of ROS is essential for developing novel strategies to prevent or attenuate lung pathologies. In this review, we discuss recent studies on the modulators and targets of ROS in pulmonary endothelial and smooth muscle cells, their cellular effects, and the disease conditions associated with dysregulated redox signaling.
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http://dx.doi.org/10.1016/j.cophys.2019.04.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6690609PMC
June 2019

Calcium signals that determine vascular resistance.

Wiley Interdiscip Rev Syst Biol Med 2019 09 18;11(5):e1448. Epub 2019 Mar 18.

Robert M. Berne Cardiovascular Research Center, University of Virginia-School of Medicine, Charlottesville, Virginia.

Small arteries in the body control vascular resistance, and therefore, blood pressure and blood flow. Endothelial and smooth muscle cells in the arterial walls respond to various stimuli by altering the vascular resistance on a moment to moment basis. Smooth muscle cells can directly influence arterial diameter by contracting or relaxing, whereas endothelial cells that line the inner walls of the arteries modulate the contractile state of surrounding smooth muscle cells. Cytosolic calcium is a key driver of endothelial and smooth muscle cell functions. Cytosolic calcium can be increased either by calcium release from intracellular stores through IP3 or ryanodine receptors, or the influx of extracellular calcium through ion channels at the cell membrane. Depending on the cell type, spatial localization, source of a calcium signal, and the calcium-sensitive target activated, a particular calcium signal can dilate or constrict the arteries. Calcium signals in the vasculature can be classified into several types based on their source, kinetics, and spatial and temporal properties. The calcium signaling mechanisms in smooth muscle and endothelial cells have been extensively studied in the native or freshly isolated cells, therefore, this review is limited to the discussions of studies in native or freshly isolated cells. This article is categorized under: Biological Mechanisms > Cell Signaling Laboratory Methods and Technologies > Imaging Models of Systems Properties and Processes > Mechanistic Models.
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http://dx.doi.org/10.1002/wsbm.1448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688910PMC
September 2019

RSK2 contributes to myogenic vasoconstriction of resistance arteries by activating smooth muscle myosin and the Na/H exchanger.

Sci Signal 2018 10 30;11(554). Epub 2018 Oct 30.

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.

Smooth muscle contraction is triggered when Ca/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates the regulatory light chain of myosin (RLC). However, blood vessels from -deficient mouse embryos retain the ability to contract, suggesting the existence of additional regulatory mechanisms. We showed that the p90 ribosomal S6 kinase 2 (RSK2) also phosphorylated RLC to promote smooth muscle contractility. Active, phosphorylated RSK2 was present in mouse resistance arteries under normal basal tone, and phosphorylation of RSK2 increased with myogenic vasoconstriction or agonist stimulation. Resistance arteries from -deficient mice were dilated and showed reduced myogenic tone and RLC phosphorylation. RSK2 phosphorylated Ser in RLC in vitro. In addition, RSK2 phosphorylated an activating site in the Na/H exchanger (NHE-1), resulting in cytosolic alkalinization and an increase in intracellular Ca that promotes vasoconstriction. NHE-1 activity increased upon myogenic constriction, and the increase in intracellular pH was suppressed in -deficient mice. In pressured arteries, RSK2-dependent activation of NHE-1 was associated with increased intracellular Ca transients, which would be expected to increase MLCK activity, thereby contributing to basal tone and myogenic responses. Accordingly, -deficient mice had lower blood pressure than normal littermates. Thus, RSK2 mediates a procontractile signaling pathway that contributes to the regulation of basal vascular tone, myogenic vasoconstriction, and blood pressure and may be a potential therapeutic target in smooth muscle contractility disorders.
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http://dx.doi.org/10.1126/scisignal.aar3924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474246PMC
October 2018

TRPV4 (Transient Receptor Potential Vanilloid 4) Channel-Dependent Negative Feedback Mechanism Regulates G Protein-Coupled Receptor-Induced Vasoconstriction.

Arterioscler Thromb Vasc Biol 2018 03 4;38(3):542-554. Epub 2018 Jan 4.

From the Robert M. Berne Cardiovascular Research Center (K.H., E.L.C., L.J.D., C.M., B.E.I., S.K.S.), Department of Molecular Physiology and Biological Physics (C.M., B.E.I., S.K.S.), and Department of Pharmacology (L.J.D., S.K.S), University of Virginia-School of Medicine, Charlottesville.

Objective: Several physiological stimuli activate smooth muscle cell (SMC) GPCRs (G protein-coupled receptors) to cause vasoconstriction. As a protective mechanism against excessive vasoconstriction, SMC GPCR stimulation invokes endothelial cell vasodilatory signaling. Whether Ca influx in endothelial cells contributes to the regulation of GPCR-induced vasoconstriction remains unknown. Ca influx through TRPV4 (transient receptor potential vanilloid 4) channels is a key regulator of endothelium-dependent vasodilation. We hypothesized that SMC GPCR stimulation engages endothelial TRPV4 channels to limit vasoconstriction.

Approach And Results: Using high-speed confocal microscopy to record unitary Ca influx events through TRPV4 channels (TRPV4 sparklets), we report that activation of SMC αARs (alpha-adrenergic receptors) with phenylephrine or thromboxane A receptors with U46619 stimulated TRPV4 sparklets in the native endothelium from mesenteric arteries. Activation of endothelial TRPV4 channels did not require an increase in Ca as indicated by the lack of effect of L-type Ca channel activator or chelator of intracellular Ca EGTA-AM. However, gap junction communication between SMCs and endothelial cells was required for phenylephrine activation or U46619 activation of endothelial TRPV4 channels. Lowering inositol 1,4,5-trisphosphate levels with phospholipase C inhibitor or lithium chloride suppressed phenylephrine activation of endothelial TRPV4 sparklets. Moreover, uncaging inositol 1,4,5-trisphosphate profoundly increased TRPV4 sparklet activity. In pressurized arteries, phenylephrine-induced vasoconstriction was followed by a slow, TRPV4-dependent vasodilation, reflecting activation of negative regulatory mechanism. Consistent with these data, phenylephrine induced a significantly higher increase in blood pressure in TRPV4 mice.

Conclusions: These results demonstrate that SMC GPCR stimulation triggers inositol 1,4,5-trisphosphate-dependent activation of endothelial TRPV4 channels to limit vasoconstriction.
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http://dx.doi.org/10.1161/ATVBAHA.117.310038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823749PMC
March 2018

Nitric Oxide-Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries.

J Am Heart Assoc 2017 12 23;6(12). Epub 2017 Dec 23.

Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA

Background: Recent studies demonstrate that spatially restricted, local Ca signals are key regulators of endothelium-dependent vasodilation in systemic circulation. There are drastic functional differences between pulmonary arteries (PAs) and systemic arteries, but the local Ca signals that control endothelium-dependent vasodilation of PAs are not known. Localized, unitary Ca influx events through transient receptor potential vanilloid 4 (TRPV4) channels, termed TRPV4 sparklets, regulate endothelium-dependent vasodilation in resistance-sized mesenteric arteries via activation of Ca-dependent K channels. The objective of this study was to determine the unique functional roles, signaling targets, and endogenous regulators of TRPV4 sparklets in resistance-sized PAs.

Methods And Results: Using confocal imaging, custom image analysis, and pressure myography in fourth-order PAs in conjunction with knockout mouse models, we report a novel Ca signaling mechanism that regulates endothelium-dependent vasodilation in resistance-sized PAs. TRPV4 sparklets exhibit distinct spatial localization in PAs when compared with mesenteric arteries, and preferentially activate endothelial nitric oxide synthase (eNOS). Nitric oxide released by TRPV4-endothelial nitric oxide synthase signaling not only promotes vasodilation, but also initiates a guanylyl cyclase-protein kinase G-dependent negative feedback loop that inhibits cooperative openings of TRPV4 channels, thus limiting sparklet activity. Moreover, we discovered that adenosine triphosphate dilates PAs through a P2 purinergic receptor-dependent activation of TRPV4 sparklets.

Conclusions: Our results reveal a spatially distinct TRPV4-endothelial nitric oxide synthase signaling mechanism and its novel endogenous regulators in resistance-sized PAs.
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http://dx.doi.org/10.1161/JAHA.117.007157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5779028PMC
December 2017

Non-Endoplasmic Reticulum-Based Calr (Calreticulin) Can Coordinate Heterocellular Calcium Signaling and Vascular Function.

Arterioscler Thromb Vasc Biol 2018 01 9;38(1):120-130. Epub 2017 Nov 9.

From the Robert M. Berne Cardiovascular Research Center (L.A.B., M.E.G., K.H., R.K.P., S.K.S., B.E.I.) and Department of Molecular Physiology and Biophysics (L.A.B., S.K.S., B.E.I.), University of Virginia School of Medicine, Charlottesville; and Department of Kinesiology, College of William and Mary, Williamsburg, VA (N.A., R.L.-W.).

Objective: In resistance arteries, endothelial cell (EC) extensions can make contact with smooth muscle cells, forming myoendothelial junction at holes in the internal elastic lamina (HIEL). At these HIEL, calcium signaling is tightly regulated. Because Calr (calreticulin) can buffer ≈50% of endoplasmic reticulum calcium and is expressed throughout IEL holes in small arteries, the only place where myoendothelial junctions form, we investigated the effect of EC-specific Calr deletion on calcium signaling and vascular function.

Approach And Results: We found Calr expressed in nearly every IEL hole in third-order mesenteric arteries, but not other ER markers. Because of this, we generated an EC-specific, tamoxifen inducible, Calr knockout mouse (EC Calr Δ/Δ). Using this mouse, we tested third-order mesenteric arteries for changes in calcium events at HIEL and vascular reactivity after application of CCh (carbachol) or PE (phenylephrine). We found that arteries from EC Calr Δ/Δ mice stimulated with CCh had unchanged activity of calcium signals and vasodilation; however, the same arteries were unable to increase calcium events at HIEL in response to PE. This resulted in significantly increased vasoconstriction to PE, presumably because of inhibited negative feedback. In line with these observations, the EC Calr Δ/Δ had increased blood pressure. Comparison of ER calcium in arteries and use of an ER-specific GCaMP indicator in vitro revealed no observable difference in ER calcium with Calr knockout. Using selective detergent permeabilization of the artery and inhibition of Calr translocation, we found that the observed Calr at HIEL may not be within the ER.

Conclusions: Our data suggest that Calr specifically at HIEL may act in a non-ER dependent manner to regulate arteriolar heterocellular communication and blood pressure.
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http://dx.doi.org/10.1161/ATVBAHA.117.309886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746467PMC
January 2018

Pharmacological inhibitors of TRPV4 channels reduce cytokine production, restore endothelial function and increase survival in septic mice.

Sci Rep 2016 Sep 22;6:33841. Epub 2016 Sep 22.

Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405, USA.

Sepsis is characterized by systemic inflammation, edema formation and hypo-perfusion leading to organ dysfunction and ultimately death. Activation of the transient receptor potential vanilloid type 4 (TRPV4) channel is associated with edema formation and circulatory collapse. Here, we show that TRPV4 channels are involved in the hyper-inflammatory response and mortality associated with sepsis. Pharmacological inhibition of TRPV4 channels in mice reduced mortality in lipopolysaccharide and cecal-ligation-and-puncture models of sepsis, but not in a tumor necrosis factor-α (TNFα)-induced sepsis model. These protective effects of TRPV4 channel inhibition were attributable to prevention of the sepsis-induced surge of a broad spectrum of pro-inflammatory cytokines, including TNFα, interleukin (IL)-1 and IL-6, and subsequent preservation of endothelial cell function, including Ca signaling, integrity and endothelium-dependent vasodilation. These results suggest that TRPV4 antagonists may be of therapeutic utility in the management of sepsis.
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http://dx.doi.org/10.1038/srep33841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031985PMC
September 2016

Gain-of-function mutation in TRPV4 identified in patients with osteonecrosis of the femoral head.

J Med Genet 2016 10 21;53(10):705-9. Epub 2016 Jun 21.

Division of Hematology and Oncology, Department of Medicine, McGill University Health Centre, Montreal, Quebec, Canada.

Background: Osteonecrosis of the femoral head is a debilitating disease that involves impaired blood supply to the femoral head and leads to femoral head collapse.

Methods: We use whole-exome sequencing and Sanger sequencing to analyse a family with inherited osteonecrosis of the femoral head and fluorescent Ca(2+) imaging to functionally characterise the variant protein.

Results: We report a family with four siblings affected with inherited osteonecrosis of the femoral head and the identification of a c.2480_2483delCCCG frameshift deletion followed by a c.2486T>A substitution in one allele of the transient receptor potential vanilloid 4 (TRPV4) gene. TRPV4 encodes a Ca(2+)-permeable cation channel known to play a role in vasoregulation and osteoclast differentiation. While pathogenic TRPV4 mutations affect the skeletal or nervous systems, association with osteonecrosis of the femoral head is novel. Functional measurements of Ca(2+) influx through mutant TRPV4 channels in HEK293 cells and patient-derived dermal fibroblasts identified a TRPV4 gain of function. Analysis of channel open times, determined indirectly from measurement of TRPV4 activity within a cluster of TRPV4 channels, revealed that the TRPV4 gain of function was caused by longer channel openings.

Conclusions: These findings identify a novel TRPV4 mutation implicating TRPV4 and altered calcium homeostasis in the pathogenesis of osteonecrosis while reinforcing the importance of TRPV4 in bone diseases and vascular endothelium.
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http://dx.doi.org/10.1136/jmedgenet-2016-103829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035228PMC
October 2016

Inward rectifier potassium (Kir2.1) channels as end-stage boosters of endothelium-dependent vasodilators.

J Physiol 2016 06 4;594(12):3271-85. Epub 2016 Mar 4.

Department of Pharmacology, University of Vermont, VT, USA.

Key Points: Increase in endothelial cell (EC) calcium activates calcium-sensitive intermediate and small conductance potassium (IK and SK) channels, thereby causing hyperpolarization and endothelium-dependent vasodilatation. Endothelial cells express inward rectifier potassium (Kir) channels, but their role in endothelium-dependent vasodilatation is not clear. In the mesenteric arteries, only ECs, but not smooth muscle cells, displayed Kir currents that were predominantly mediated by the Kir2.1 isoform. Endothelium-dependent vasodilatations in response to muscarinic receptor, TRPV4 (transient receptor potential vanilloid 4) channel and IK/SK channel agonists were highly attenuated by Kir channel inhibitors and by Kir2.1 channel knockdown. These results point to EC Kir channels as amplifiers of vasodilatation in response to increases in EC calcium and IK/SK channel activation and suggest that EC Kir channels could be targeted to treat endothelial dysfunction, which is a hallmark of vascular disorders.

Abstract: Endothelium-dependent vasodilators, such as acetylcholine, increase intracellular Ca(2+) through activation of transient receptor potential vanilloid 4 (TRPV4) channels in the plasma membrane and inositol trisphosphate receptors in the endoplasmic reticulum, leading to stimulation of Ca(2+) -sensitive intermediate and small conductance K(+) (IK and SK, respectively) channels. Although strong inward rectifier K(+) (Kir) channels have been reported in the native endothelial cells (ECs) their role in EC-dependent vasodilatation is not clear. Here, we test the idea that Kir channels boost the EC-dependent vasodilatation of resistance-sized arteries. We show that ECs, but not smooth muscle cells, of small mesenteric arteries have Kir currents, which are substantially reduced in EC-specific Kir2.1 knockdown (EC-Kir2.1(-/-) ) mice. Elevation of extracellular K(+) to 14 mm caused vasodilatation of pressurized arteries, which was prevented by endothelial denudation and Kir channel inhibitors (Ba(2+) , ML-133) or in the arteries from EC-Kir2.1(-/-) mice. Potassium-induced dilatations were unaffected by inhibitors of TRPV4, IK and SK channels. The Kir channel blocker, Ba(2+) , did not affect currents through TRPV4, IK or SK channels. Endothelial cell-dependent vasodilatations in response to activation of muscarinic receptors, TRPV4 channels or IK/SK channels were reduced, but not eliminated, by Kir channel inhibitors or EC-Kir2.1(-/-) . In angiotensin II-induced hypertension, the Kir channel function was not altered, although the endothelium-dependent vasodilatation was severely impaired. Our results support the concept that EC Kir2 channels boost vasodilatory signals that are generated by Ca(2+) -dependent activation of IK and SK channels.
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http://dx.doi.org/10.1113/JP271652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908010PMC
June 2016

Traumatic brain injury disrupts cerebrovascular tone through endothelial inducible nitric oxide synthase expression and nitric oxide gain of function.

J Am Heart Assoc 2014 Dec;3(6):e001474

From the Departments of Pharmacology, University of Vermont, Burlington, VT

Background: Traumatic brain injury (TBI) has been reported to increase the concentration of nitric oxide (NO) in the brain and can lead to loss of cerebrovascular tone; however, the sources, amounts, and consequences of excess NO on the cerebral vasculature are unknown. Our objective was to elucidate the mechanism of decreased cerebral artery tone after TBI.

Methods And Results: Cerebral arteries were isolated from rats 24 hours after moderate fluid‐percussion TBI. Pressure‐induced increases in vasoconstriction (myogenic tone) and smooth muscle Ca2+ were severely blunted in cerebral arteries after TBI. However, myogenic tone and smooth muscle Ca2+ were restored by inhibition of NO synthesis or endothelium removal, suggesting that TBI increased endothelial NO levels. Live native cell NO, indexed by 4,5‐diaminofluorescein (DAF‐2 DA) fluorescence, was increased in endothelium and smooth muscle of cerebral arteries after TBI. Clamped concentrations of 20 to 30 nmol/L NO were required to simulate the loss of myogenic tone and increased (DAF‐2T) fluorescence observed following TBI. In comparison, basal NO in control arteries was estimated as 0.4 nmol/L. Consistent with TBI causing enhanced NO‐mediated vasodilation, inhibitors of guanylyl cyclase, protein kinase G, and large‐conductance Ca2+‐activated potassium (BK) channel restored function of arteries from animals with TBI. Expression of the inducible isoform of NO synthase was upregulated in cerebral arteries isolated from animals with TBI, and the inducible isoform of NO synthase inhibitor 1400W restored myogenic responses following TBI.

Conclusions: The mechanism of profound cerebral artery vasodilation after TBI is a gain of function in vascular NO production by 60‐fold over controls, resulting from upregulation of the inducible isoform of NO synthase in the endothelium.
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http://dx.doi.org/10.1161/JAHA.114.001474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338739PMC
December 2014

Vascular TRP channels: performing under pressure and going with the flow.

Physiology (Bethesda) 2014 Sep;29(5):343-60

Department of Pharmacology, University of Vermont, Burlington, Vermont

Endothelial cells and smooth muscle cells of resistance arteries mediate opposing responses to mechanical forces acting on the vasculature, promoting dilation in response to flow and constriction in response to pressure, respectively. In this review, we explore the role of TRP channels, particularly endothelial TRPV4 and smooth muscle TRPC6 and TRPM4 channels, in vascular mechanosensing circuits, placing their putative mechanosensitivity in context with other proposed upstream and downstream signaling pathways.
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http://dx.doi.org/10.1152/physiol.00009.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4214829PMC
September 2014

AKAP150-dependent cooperative TRPV4 channel gating is central to endothelium-dependent vasodilation and is disrupted in hypertension.

Sci Signal 2014 Jul 8;7(333):ra66. Epub 2014 Jul 8.

Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05403, USA. Institute of Cardiovascular Sciences, University of Manchester, Manchester M13 9NT, UK.

Endothelial cell dysfunction, characterized by a diminished response to endothelial cell-dependent vasodilators, is a hallmark of hypertension. TRPV4 channels play a major role in endothelial-dependent vasodilation, a function mediated by local Ca(2+) influx through clusters of functionally coupled TRPV4 channels rather than by a global increase in endothelial cell Ca(2+). We showed that stimulation of muscarinic acetylcholine receptors on endothelial cells of mouse arteries exclusively activated TRPV4 channels that were localized at myoendothelial projections (MEPs), specialized regions of endothelial cells that contact smooth muscle cells. Muscarinic receptor-mediated activation of TRPV4 depended on protein kinase C (PKC) and the PKC-anchoring protein AKAP150, which was concentrated at MEPs. Cooperative opening of clustered TRPV4 channels specifically amplified Ca(2+) influx at MEPs. Cooperativity of TRPV4 channels at non-MEP sites was much lower, and cooperativity at MEPs was greatly reduced by chelation of intracellular Ca(2+) or AKAP150 knockout, suggesting that Ca(2+) entering through adjacent channels underlies the AKAP150-dependent potentiation of TRPV4 activity. In a mouse model of angiotensin II-induced hypertension, MEP localization of AKAP150 was disrupted, muscarinic receptor stimulation did not activate TRPV4 channels, cooperativity among TRPV4 channels at MEPs was weaker, and vasodilation in response to muscarinic receptor stimulation was reduced. Thus, endothelial-dependent dilation of resistance arteries is enabled by MEP-localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and the coupled channel gating necessary for efficient communication from endothelial to smooth muscle cells in arteries. Disruption of this molecular assembly may contribute to altered blood flow in hypertension.
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http://dx.doi.org/10.1126/scisignal.2005052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403000PMC
July 2014

Opposing roles of smooth muscle BK channels and ryanodine receptors in the regulation of nerve-evoked constriction of mesenteric resistance arteries.

Am J Physiol Heart Circ Physiol 2014 Apr 7;306(7):H981-8. Epub 2014 Feb 7.

Department of Pharmacology, University of Vermont, Burlington, Vermont;

In depolarized smooth muscle cells of pressurized cerebral arteries, ryanodine receptors (RyRs) generate "Ca2+ sparks" that activate large-conductance, Ca2+ -, and voltage-sensitive potassium (BK) channels to oppose pressure-induced (myogenic) constriction. Here, we show that BK channels and RyRs have opposing roles in the regulation of arterial tone in response to sympathetic nerve activation by electrical field stimulation. Inhibition of BK channels with paxilline increased both myogenic and nerve-induced constrictions of pressurized, resistance-sized mesenteric arteries from mice. Inhibition of RyRs with ryanodine increased myogenic constriction, but it decreased nerve-evoked constriction along with a reduction in the amplitude of nerve-evoked increases in global intracellular Ca2+. In the presence of L-type voltage-dependent Ca2+ channel (VDCC) antagonists, nerve stimulation failed to evoke a change in arterial diameter, and BK channel and RyR inhibitors were without effect, suggesting that nerve- induced constriction is dependent on activation of VDCCs. Collectively, these results indicate that BK channels and RyRs have different roles in the regulation of myogenic versus neurogenic tone: whereas BK channels and RyRs act in concert to oppose myogenic vasoconstriction, BK channels oppose neurogenic vasoconstriction and RyRs augment it. A scheme for neurogenic vasoregulation is proposed in which RyRs act in conjunction with VDCCs to regulate nerve-evoked constriction in mesenteric resistance arteries.
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http://dx.doi.org/10.1152/ajpheart.00866.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962638PMC
April 2014

The β3 subunit contributes to vascular calcium channel upregulation and hypertension in angiotensin II-infused C57BL/6 mice.

Hypertension 2013 Jan 5;61(1):137-42. Epub 2012 Nov 5.

Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

Voltage-gated L-type Ca(2+) (Ca(v)1.2) channels in vascular smooth muscle cells are a predominant Ca(2+) influx pathway that mediates arterial tone. Channel biogenesis is accomplished when the pore-forming α(1C) subunit coassembles with regulatory Ca(v)β subunits intracellularly, and the multiprotein Ca(v)1.2 channel complex translocates to the plasma membrane to form functional Ca(2+) channels. We hypothesized that the main Ca(v)β isoform in vascular smooth muscle cells, Ca(v)β3, is required for the upregulation of arterial Ca(v)1.2 channels during the development of hypertension, an event associated with abnormal Ca(2+)-dependent tone. Ca(v)1.2 channel expression and function were compared between second-order mesenteric arteries of C57BL/6 wild-type (WT) and Ca(v)β3(-/-) mice infused with saline (control) or angiotensin II (Ang II) for 2 weeks to induce hypertension. The mesenteric arteries of Ang II-infused WT mice showed increased Ca(v)1.2 channel expression and accentuated Ca(2+)-mediated contractions compared with saline-infused WT mice. In contrast, Ca(v)1.2 channels failed to upregulate in mesenteric arteries of Ang II-infused Ca(v)β3(-/-) mice, and Ca(2+)-dependent reactivity was normal in these arteries. Basal systolic blood pressure was not significantly different between WT and Ca(v)β3(-/-) mice (98 ± 2 and 102 ± 3 mm Hg, respectively), but the Ca(v)β3(-/-) mice showed a blunted pressor response to Ang II infusion. Two weeks after the start of Ang II administration, the systolic blood pressure of Ca(v)β3(-/-) mice averaged 149 ± 4 mm Hg compared with 180 ± 5 mm Hg in WT mice. Thus, the Ca(v)β3 subunit is a critical regulatory protein required to upregulate arterial Ca(v)1.2 channels and fully develop Ang II-dependent hypertension in C57BL/6 mice.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.112.197863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748590PMC
January 2013

Elementary Ca2+ signals through endothelial TRPV4 channels regulate vascular function.

Science 2012 May;336(6081):597-601

Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405, USA.

Major features of the transcellular signaling mechanism responsible for endothelium-dependent regulation of vascular smooth muscle tone are unresolved. We identified local calcium (Ca(2+)) signals ("sparklets") in the vascular endothelium of resistance arteries that represent Ca(2+) influx through single TRPV4 cation channels. Gating of individual TRPV4 channels within a four-channel cluster was cooperative, with activation of as few as three channels per cell causing maximal dilation through activation of endothelial cell intermediate (IK)- and small (SK)-conductance, Ca(2+)-sensitive potassium (K(+)) channels. Endothelial-dependent muscarinic receptor signaling also acted largely through TRPV4 sparklet-mediated stimulation of IK and SK channels to promote vasodilation. These results support the concept that Ca(2+) influx through single TRPV4 channels is leveraged by the amplifier effect of cooperative channel gating and the high Ca(2+) sensitivity of IK and SK channels to cause vasodilation.
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http://dx.doi.org/10.1126/science.1216283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3715993PMC
May 2012