Background: Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca ²⁺ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca ²⁺ 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 ²⁺ 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 ²⁺ 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.

Significance: Pulmonary hypertension (PH) is a serious disorder with a mortality rate of 40% over 5 y after diagnosis. Endothelial dysfunction is a major contributor to increased pulmonary arterial pressure (PAP) in PH. Here, we provide evidence that endothelial cell caveolin-1–TRPV4 channel signaling lowers resting PAP. Moreover, an impairment of endothelial caveolin-1–TRPV4 channel signaling elevates PAP in PH. In PH, there is an up-regulation of NOX1 and iNOS enzymes in endothelial cells, which increases peroxynitrite levels. Peroxynitrite, in turn, targets endothelial caveolin-1 and lowers endothelial caveolin-1–TRPV4 channel signaling. Finally, decreasing peroxynitrite levels rescues endothelial caveolin-1–TRPV4 channel signaling and lowers PAP in PH.

Background: Recent studies demonstrate that spatially restricted, local Ca ²⁺ signals are key regulators of endothelium‐dependent vasodilation in systemic circulation. There are drastic functional differences between pulmonary arteries ( PA s) and systemic arteries, but the local Ca ²⁺ signals that control endothelium‐dependent vasodilation of PA s are not known. Localized, unitary Ca ²⁺ influx events through transient receptor potential vanilloid 4 ( TRPV 4) channels, termed TRPV 4 sparklets, regulate endothelium‐dependent vasodilation in resistance‐sized mesenteric arteries via activation of Ca ²⁺ ‐dependent K ⁺ channels. The objective of this study was to determine the unique functional roles, signaling targets, and endogenous regulators of TRPV 4 sparklets in resistance‐sized PA s. Methods and Results: Using confocal imaging, custom image analysis, and pressure myography in fourth‐order PA s in conjunction with knockout mouse models, we report a novel Ca ²⁺ signaling mechanism that regulates endothelium‐dependent vasodilation in resistance‐sized PA s. TRPV 4 sparklets exhibit distinct spatial localization in PA s when compared with mesenteric arteries, and preferentially activate endothelial nitric oxide synthase ( eNOS ). Nitric oxide released by TRPV 4‐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 TRPV 4 channels, thus limiting sparklet activity. Moreover, we discovered that adenosine triphosphate dilates PA s through a P2 purinergic receptor‐dependent activation of TRPV 4 sparklets. Conclusions: Our results reveal a spatially distinct TRPV 4‐endothelial nitric oxide synthase signaling mechanism and its novel endogenous regulators in resistance‐sized PA s.

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 Ca²⁺ entry pathways in multiple cell types. Here, we provide direct evidence supporting Ca²⁺ 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. Significance Primary Open Angle Glaucoma (POAG) is the most common form of glaucoma that leads to irreversible vision loss and blindness worldwide. POAG is often associated with elevated intraocular pressure (IOP). The trabecular meshwork (TM), a molecular sieve-like structure, tightly controls IOP by constantly adjusting the resistance to aqueous humor (AH) outflow. In POAG, there is increased resistance to AH outflow, elevating IOP. Here, we show that mechanically activated TRPV4 channels in TM play a critical role in IOP regulation via calcium-mediated activation of eNOS signaling and production of nitric oxide (NO). Importantly, we show that impaired TRPV4 channel activity results in reduced NO bioavailability and elevated IOP in glaucoma. Our findings support impaired TRPV4-eNOS signaling to the pathogenesis of POAG.

Background: Ca ²⁺ signals in smooth muscle cells (SMCs) contribute to vascular resistance and control blood pressure. Increased vascular resistance in hypertension has been attributed to impaired SMC Ca ²⁺ signaling mechanisms. In this regard, transient receptor potential vanilloid 4 (TRPV4 _SMC ) ion channels are a crucial Ca ²⁺ entry pathway in SMCs. However, their role in blood pressure regulation has not been identified. Methods: We used SMC-specific TRPV4 ^−/− (TRPV4 _SMC ^−/− ) mice to assess the role of TRPV4 _SMC channels in blood pressure regulation. We determined the contribution of TRPV4 _SMC channels to the constrictor effect of α1 adrenergic receptor (α1AR) stimulation and elevated intraluminal pressure: 2 main physiologic stimuli that constrict resistance-sized arteries. The contribution of spatially separated TRPV4 _SMC channel subpopulations to elevated blood pressure in hypertension was evaluated in angiotensin II–infused mice and patients with hypertension. Results: We provide first evidence that TRPV4 _SMC channel activity elevates resting blood pressure in normal mice. α1AR stimulation activated TRPV4 _SMC channels through PKCα (protein kinase Cα) signaling, which contributed significantly to vasoconstriction and blood pressure elevation. Intraluminal pressure–induced TRPV4 _SMC channel activity opposed vasoconstriction through activation of Ca ²⁺ -sensitive K ⁺ (BK) channels, indicating functionally opposite pools of TRPV4 _SMC channels. Superresolution imaging of SMCs revealed spatially separated α1AR:TRPV4 and TRPV4:BK nanodomains in SMCs. These data suggest that spatially separated α1AR–TRPV4 _SMC and intraluminal pressure–TRPV4 _SMC –BK channel signaling have opposite effects on blood pressure, with α1AR–TRPV4 _SMC signaling dominating under resting conditions. Furthermore, in patients with hypertension and a mouse model of hypertension, constrictor α1AR–PKCα–TRPV4 signaling was upregulated, whereas dilator pressure–TRPV4–BK channel signaling was disrupted, thereby increasing vasoconstriction and elevating blood pressure. Conclusions: Our data identify novel smooth muscle Ca ²⁺ -signaling nanodomains that regulate blood pressure and demonstrate their impairment in hypertension.