Pressure-induced Constriction Research Articles

The vasoprotective role of IGF-1 signaling in the cerebral microcirculation: prevention of cerebral microhemorrhages in aging.

Aging is closely associated with various cerebrovascular pathologies that significantly impact brain function, with cerebral small vessel disease (CSVD) being a major contributor to cognitive decline in the elderly. Consequences of CSVD include cerebral microhemorrhages (CMH), which are small intracerebral bleeds resulting from the rupture of microvessels. CMHs are prevalent in aging populations, affecting approximately 50% of individuals over 80, and are linked to increased risks of vascular cognitive impairment and dementia (VCID). Hypertension is a primary risk factor for CMHs. Vascular smooth muscle cells (VSMCs) adapt to hypertension by undergoing hypertrophy and producing extracellular matrix (ECM) components, which reinforce vessel walls. Myogenic autoregulation, which involves pressure-induced constriction, helps prevent excessive pressure from damaging the vulnerable microvasculature. However, aging impairs these adaptive mechanisms, weakening vessel walls and increasing susceptibility to damage. Insulin-like Growth Factor 1 (IGF-1) is crucial for vascular health, promoting VSMC hypertrophy, ECM production, and maintaining normal myogenic protection. IGF-1 also prevents microvascular senescence, reduces reactive oxygen species (ROS) production, and regulates matrix metalloproteinase (MMP) activity, which is vital for ECM remodeling and stabilization. IGF-1 deficiency, common in aging, compromises these protective mechanisms, increasing the risk of CMHs. This review explores the vasoprotective role of IGF-1 signaling in the cerebral microcirculation and its implications for preventing hypertension-induced CMHs in aging. Understanding and addressing the decline in IGF-1 signaling with age are crucial for maintaining cerebrovascular health and preventing hypertension-related vascular injuries in the aging population.

Uncoupling of Ca2+ sparks from BK channels in cerebral arteries underlies hypoperfusion in hypertension-induced vascular dementia

The deficit in cerebral blood flow (CBF) seen in patients with hypertension-induced vascular dementia is increasingly viewed as a therapeutic target for disease-modifying therapy. Progress is limited, however, due to uncertainty surrounding the mechanisms through which elevated blood pressure reduces CBF. To investigate this, we used the BPH/2 mouse, a polygenic model of hypertension. At 8 mo of age, hypertensive mice exhibited reduced CBF and cognitive impairment, mimicking the human presentation of vascular dementia. Small cerebral resistance arteries that run across the surface of the brain (pial arteries) showed enhanced pressure-induced constriction due to diminished activity of large-conductance Ca2+-activated K+ (BK) channels-key vasodilatory ion channels of cerebral vascular smooth muscle cells. Activation of BK channels by transient intracellular Ca2+ signals from the sarcoplasmic reticulum (SR), termed Ca2+ sparks, leads to hyperpolarization and vasodilation. Combining patch-clamp electrophysiology, high-speed confocal imaging, and proximity ligation assays, we demonstrated that this vasodilatory mechanism is uncoupled in hypertensive mice, an effect attributable to physical separation of the plasma membrane from the SR rather than altered properties of BK channels or Ca2+ sparks, which remained intact. This pathogenic mechanism is responsible for the observed increase in constriction and can now be targeted as a possible avenue for restoring healthy CBF in vascular dementia.

BK channel dysfunction underlies small vessel disease of the brain in both hypertension and Alzheimer's disease

Alzheimer's disease (AD) and hypertension-related vascular dementia share many common clinical and radiological features including cerebral small vessel disease (cSVD) of the brain, thought to be the mechanism behind the reduction in cerebral blood flow (CBF). The aim of this study was to determine if there are common mechanisms underlying a reduced CBF in dementia. CBF is determined by depolarisation induced contraction of arterial smooth muscle cells (SMCs). This contraction is buffered by the activation of the large-conductance Ca 2+ -activated K + (BK) channels on the plasma membrane (PM), by transient localised Ca 2+ release from ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR), known as Ca 2+ sparks.Hypothesis: Defects in BK channel activation leads to the cSVD phenotype in these diseases.8-month old male spontaneously hypertensive (BPH/2) mice and normotensive controls (BPN/3) were used to study hypertension-induced cSVD. To study AD related cSVD we used 18–20 month old male APP23 mice. We used a range of physiological techniques to investigate vascular ion channel function within the cerebral microvasculature. Laser Doppler flowmetry and behavioural assessments were performed on the BPH/2 and BPN/3 mice to initially determine if they are a model of hypertension-related vascular dementia.BPH/2 mice displayed reduced CBF and cognitive impairment. Pressure-induced constriction from cerebral pial arteries was significantly greater in both the BPH/2 and APP23 animals compared to controls. This was the result of reduced BK channel activity, as confirmed by reduced constriction to the BK channel blocker paxilline. Electrophysiology experiments showed a decrease in spontaneous transient outward currents, which represent BK channel activation by Ca 2+ sparks. In the APP23 model, this attenuated BK channel activation was due to reduced Ca 2+ spark frequency. However, in the BPH/2 mice, Ca 2+ spark frequency was equal between hypertensive and normotensive mice. Furthermore, there were no differences in single channel BK channel activity over a range of [Ca 2+ ]. Live cell imaging of isolated SMCs labelled with membrane dyes, showed a reduced interaction between the SR and the PM. Proximity ligation assay was used on fixed SMCs to determine if BK channel and RyR are within 40 nm of each other. SMCs from BPH/2 cerebral arteries has significantly less puncta suggesting a physical separation between Ca 2+ sparks and BK channel, rendering the [Ca 2+ ] local to the BK channel insufficient for activation.Our data indicate that vascular BK channel dysfunction is a common mechanism underpinning reduced CBF in SVD. Therapies aimed at improving vascular BK channel function could provide new therapeutic targets for both AD and hypertension-induced vascular dementia. British Heart Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intraluminal pressure elevates intracellular calcium and contracts CNS pericytes: Role of voltage-dependent calcium channels.

Arteriolar smooth muscle cells (SMCs) and capillary pericytes dynamically regulate blood flow in the central nervous system in the face of fluctuating perfusion pressures. Pressure-induced depolarization and Ca2+ elevation provide a mechanism for regulation of SMC contraction, but whether pericytes participate in pressure-induced changes in blood flow remains unknown. Here, utilizing a pressurized whole-retina preparation, we found that increases in intraluminal pressure in the physiological range induce contraction of both dynamically contractile pericytes in the arteriole-proximate transition zone and distal pericytes of the capillary bed. We found that the contractile response to pressure elevation was slower in distal pericytes than in transition zone pericytes and arteriolar SMCs. Pressure-evoked elevation of cytosolic Ca2+ and contractile responses in SMCs were dependent on voltage-dependent Ca2+ channel (VDCC) activity. In contrast, Ca2+ elevation and contractile responses were partially dependent on VDCC activity in transition zone pericytes and independent of VDCC activity in distal pericytes. In both transition zone and distal pericytes, membrane potential at low inlet pressure (20 mmHg) was approximately -40 mV and was depolarized to approximately -30 mV by an increase in pressure to 80 mmHg. The magnitude of whole-cell VDCC currents in freshly isolated pericytes was approximately half that measured in isolated SMCs. Collectively, these results indicate a loss of VDCC involvement in pressure-induced constriction along the arteriole-capillary continuum. They further suggest that alternative mechanisms and kinetics of Ca2+ elevation, contractility, and blood flow regulation exist in central nervous system capillary networks, distinguishing them from neighboring arterioles.

PS-B10-7: A VASCULAR SMOOTH MUSCLE POTASSIUM CHANNEL DYSFUNCTION UNDERLIES SMALL VESSEL DISEASE OF THE BRAIN IN BOTH HYPERTENSION AND ALZHEIMER'S DISEASE

Alzheimer's disease (AD) and hypertension-related vascular dementia share many common clinical and radiological features. Indeed, at post-mortem the majority of patients diagnosed with either disorder show extensive small vessel disease (SVD) of the brain. One phenotypic character of SVD is a reduction in cerebral blood flow (CBF). The large-conductance calcium-activated potassium (BK) channel reduces arterial contractility through hyperpolarisation of the vascular smooth muscle cell membrane. The BK channel is activated via small, localised calcium release events from ryanodine receptors (RyR), known as calcium sparks. We wished to examine whether defects in calcium spark to BK channel coupling contributes to the SVD phenotype in these diseases, which could suggest new avenues for therapy based on improvement of brain vascular health. Male spontaneously hypertensive (BPH/2) mice and normotensive controls (BPN/3) were used at 8 months of age to study hypertension-induced cSVD. To study AD we used 18–20 month old male APP23 mice that have a 7-fold increased expression of amyloid precursor protein, which results in significant amyloid-beta accumulation as is seen in human AD. During this study we used a range of physiological techniques; mainly pressure myography, patch clamp electrophysiology and high-speed spinning disc confocal microscopy, to investigate in depth, vascular ion channel function within the cerebral microvasculature of these mice. Pressure-induced constriction from cerebral pial arteries was significantly greater in both the BPH/2 and APP23 animals compared to controls. Pressure myography studies revealed that in both models there was a reduction in BK channel activity. This was confirmed with electrophysiology data that showed a decrease in spontaneous transient outward currents, which represent BK channel openings in response to calcium sparks. In the APP23 model, this reduction in BK channel function was due to a decrease in calcium spark frequency. However in the BPH/2 mice, spark frequency was equal between hypertensive individuals and controls. In this model separation of the BK channel and RyR resulted in BK channel dysfunction. Overall our data indicates that vascular BK channel dysfunction is a common mechanism underpinning reduced CBF in SVD. Therefore, therapies aimed at improving vascular BK channel function could provide new therapeutic targets for the treatment of both AD and hypertension-induced vascular dementia.

S-18-2: HYPERTENSION AS A DRIVER OF CEREBRAL SMALL VESSEL DISEASE AND DEMENTIA

Hypertension is the principal cause of vascular dementia. Unfortunately however, lowering blood pressure after the onset of cognitive decline does not slow disease progression and currently, vascular dementia is a disease with no available treatment. A principal feature of hypertension-related vascular dementia is a reduction in cerebral blood flow. Precisely how elevated blood pressure reduces cerebral blood flow is unknown and to investigate this further we used a polygenic model of hypertension, the BPH/2 mouse. Aged 8 months, BPH/2 mice exhibited cognitive impairment mimicking the human presentation of vascular dementia and in addition, cerebral blood flow was reduced. Small cerebral resistance arteries which run across the surface of the brain (pial arteries) showed enhanced pressure-induced constriction due to lack of activity of large conductance Ca2+ activated K+ channels (BK channels), key vasodilatory ion channels of cerebral vascular smooth muscle cells. BK channels are activated by transient intracellular signals from the sarcoplasmic reticulum called Ca2+ sparks, leading to hyperpolarisation and vasodilation. Combining patch clamp electrophyiology, high speed confocal imaging and proximity ligation assays we demonstrated that while the biophysical properties of BK channels and Ca2+ sparks remained intact, physical separation of the plasma membrane from the sarcoplasmic reticulum uncoupled the vasodilatory mechanism. This novel pathogenic mechanism is responsible for the increased constriction and can be now targeted as a possible avenue to restore healthy cerebral blood flow in vascular dementia.

Abstract 15590: Smooth Muscle Specific Deletion of P90 Ribosomal S6 Kinase Alters the Vasomotor Response of Resistance Arteries

Background: Smooth muscle (SM) contraction is triggered by Ca 2+ /calmodulin-dependent myosin light chain kinase (MLCK), which phosphorylates the regulatory light chain of myosin (RLC 20 ). However, additional regulatory mechanisms exist. We found that global Rsk2 KO mice had more dilated mesenteric arteries (MAs) with reduced myogenic tone and RLC 20 phosphorylation, and significantly lower blood pressure. Furthermore, RSK2-dependent activation of the Na + /H + exchanger resulted in an increase in pH i and increased Ca 2+ i transients. However, it is unclear whether our observations were due to developmental abnormalities observed in Rsk2 global KO mice. Hypothesis: RSK2 signaling in SM is a significant regulator of myogenic tone, arterial stiffness, and Ca 2+ signaling. Methods: To study the role of RSK2 signaling in vascular tone, we generated inducible SM-specific RSK2 KO mice. Detection of total and phospho proteins were determined by western blot. Pressure myography studies assessed vascular reactivity in 4 th - and 5 th -order MAs isolated from MYH11-Cre + Rsk2 fl/fl (RSK2 SM -/- ) and MYH11-Cre - Rsk2 fl/fl (RSK2 SM +/+ ) mice. We examined SM-specific Ca 2+ events in pressurized MAs by confocal fluorescent microscopy. Results: Western blot confirms knock down of RSK2 expression (~70%) in MAs and aorta. Intraluminal pressure-induced constriction of MAs is attenuated in RSK2 SM -/- . RSK2 SM -/- MAs display a delay in development of myogenic tone at 40 mmHg and decreased vascular stiffness, compared to RSK2 SM +/+ . Vasodilatory responses to Spermine NONOate (1-100 μM) in preconstricted (L-NNA; 10 μM) RSK2 SM -/- vessels were enhanced. RSK2 SM -/- MAs display fewer Ca 2+ events (97±19.6; n = 4) than RSK2 SM +/+ (400±24.5; n = 4) at 80 mmHg ( p = 0.0001). Additionally, Ca 2+ events are slower and longer-lasting in the RSK2 SM -/- . Decreased levels of p-RLC 20 are observed at 40 mmHg, but not 80 mmHg in RSK2 SM -/- vs. RSK2 SM +/+ mice, consistent with impeding tone development. Conclusion: RSK2 signaling contributes to myogenic tone and vessel stiffness by regulating sensitivity to NO, increasing Ca 2+ events, and RLC 20 activity. RSK2 signaling pathway is a potential therapeutic target for hypertension.

Functionally linked potassium channel activity in cerebral endothelial and smooth muscle cells is compromised in Alzheimer’s disease

The brain microcirculation is increasingly viewed as a potential target for disease-modifying drugs in the treatment of Alzheimer's disease patients, reflecting a growing appreciation of evidence that cerebral blood flow is compromised in such patients. However, the pathogenic mechanisms in brain resistance arteries underlying blood flow defects have not yet been elucidated. Here we probed the roles of principal vasodilatory pathways in cerebral arteries using the APP23 mouse model of Alzheimer's disease, in which amyloid precursor protein is increased approximately sevenfold, leading to neuritic plaques and cerebrovascular accumulation of amyloid-β similar to those in patients with Alzheimer's disease. Pial arteries from APP23 mice (18 mo old) exhibited enhanced pressure-induced (myogenic) constriction because of a profound reduction in ryanodine receptor-mediated, local calcium-release events ("Ca2+ sparks") in arterial smooth muscle cells and a consequent decrease in the activity of large-conductance Ca2+-activated K+ (BK) channels. The ability of the endothelial cell inward rectifier K+ (Kir2.1) channel to cause dilation was also compromised. Acute application of amyloid-β 1-40 peptide to cerebral arteries from wild-type mice partially recapitulated the BK dysfunction seen in APP23 mice but had no effect on Kir2.1 function. If mirrored in human Alzheimer's disease, these tandem defects in K+ channel-mediated vasodilation could account for the clinical cerebrovascular presentation seen in patients: reduced blood flow and crippled functional hyperemia. These data direct future research toward approaches that reverse this dual vascular channel dysfunction, with the ultimate aim of restoring healthy cerebral blood flow and improving clinical outcomes.

Smooth Muscle TRPV4 Channels Signaling Nanodomains in Blood Pressure Regulation

Transient receptor potential vanilloid 4 (TRPV4) channel is a crucial Ca2+influx pathway in arterial smooth muscle cells (SMCs). However, the effect of SMC TRPV4 (TRPV4SMC) channel activity on resting blood pressure has not been investigated. We hypothesized that SMC TRPV4 (TRPV4SMC) channels regulate blood pressure under homeostatic and hypertensive conditions. Radiotelemetric recordings showed lower resting blood pressure in SMC‐specific TRPV4‐/‐ (TRPV4SMC‐/‐) mice than control mice, suggesting that TRPV4SMC channels elevate the resting blood pressure. α1 adrenergic receptor (α1AR) stimulation–induced constriction was blunted in small mesenteric arteries (MAs) from TRPV4SMC‐/‐ mice, whereas intraluminal pressure–induced (myogenic) constriction was increased. Moreover, both α1AR stimulation and increased intraluminal pressure activated elementary Ca2+ influx signals through TRPV4SMC channels (TRPV4SMC sparklets) in MAs from control mice. These data supported the concept that TRPV4SMC channels contribute to α1AR–induced vasoconstriction but oppose myogenic vasoconstriction. Inhibition of Ca2+‐sensitive K+ (BK) channels abolished the effect of TRPV4SMC channels on myogenic vasoconstriction, indicating a crucial role for TRPV4SMC–BK channel signaling in limiting myogenic vasoconstriction. Accordingly, a direct activation of TRPV4SMC channels with GSK1016790A (30 nM) increased BK channel currents in SMC patch‐clamp experiments. α1AR stimulation, however, was unable to increase BK channel currents, implying two functionally opposite pools of TRPV4SMC channels. Direct STORM superresolution imaging revealed a1AR:TRPV4SMC and TRPV4SMC:BK channel co‐localization, and almost no co‐localization of a1ARs and BK channels. Together, these data supported the idea of spatially separated and functionally opposite nanodomains of TRPV4SMC channels. We further hypothesized that an imbalance between these two nanodomains contributes to elevated blood pressure in hypertension. In skeletal muscle arteries from hypertensive patients, a1AR activation of TRPV4SMC channel currents was accentuated whereas TRPV4SMC activation of BK channel currents was impaired. Similarly, MAs from angiotensin II‐infused hypertensive mice showed increased activity of constrictor a1AR–TRPV4SMC signaling and impairment of dilatory TRPV4SMC–BK channel signaling. Collectively, our data identify novel smooth muscle Ca2+ signaling nanodomains that regulate blood pressure and demonstrate their impairment in hypertension.

βENaC and ASIC2 associate in VSMCs to mediate pressure-induced constriction in the renal afferent arteriole.

In independent studies, our laboratory has shown the importance of the degenerin proteins β-epithelial Na+ channel (βENaC) and acid-sensing ion channel 2 (ASIC2) in pressure-induced constriction (PIC) in renal interlobar arteries. Most, but not all, of the PIC response is abolished in mice lacking normal levels of βENaC or in ASIC2-null mice, indicating that the functions of βENaC and ASIC2 cannot fully compensate for the loss of the other. Degenerin proteins are known to associate and form heteromeric channels in expression systems, but whether they interact biochemically and functionally in vascular smooth muscle cells is unknown. We hypothesized that βENaC and ASIC2 interact to mediate PIC responses in renal vessels. To address this possibility, we 1) used biochemical approaches to show that βENaC associates into high-molecular-weight complexes and immunoprecipitants with ASIC2 in vascular smooth muscle cells and then 2) examined PIC in renal afferent arterioles in mice lacking normal levels of βENaC (βENaCm/m) or/and ASIC2 (ASIC2-/-) using the isolated afferent arteriole-attached glomerulus preparation. We found that the sensitivity of the PIC response (slope of the relationship between intraluminal pressure and percent myogenic tone) decreased to 26%, 27%, and -8% of wild-type controls in ASIC2-/-, βENaCm/m, and ASIC2-/-/βENaCm/m groups, respectively, suggesting that the PIC response was totally abolished in mice deficient in both ASIC2 and βENaC. Surprisingly, we found that resting internal diameters were 20-30% lower (60 mmHg, Ca2+ free) in ASIC2-/-/βENaCm/m (11.3 ± 0.5 µm) mice compared with control (14.4 ± 0.6 µm, P = 0.0007, independent two-tailed t test) or singly modified (15.7 ± 1.0 to 16.3 ± 1.1 µm) mice, suggesting compensatory vasoconstriction or remodeling. We then examined mean arterial blood pressure (MAP) using radiotelemetry and glomerular injury using histological examination of renal sections. We found that 24-h MAP was mildly elevated (+8 mmHg) in ASIC2-/-/βENaCm/m mice versus wild-type controls and the glomerular injury score was modestly increased by 38%. These findings demonstrate that myogenic constriction in afferent arterioles is dependent on normal expression of βENaC and ASIC2 and that mice lacking normal levels of ASIC2 and βENaC have mild renal injury and increased MAP.NEW & NOTEWORTHY Transmission of systemic blood pressure to delicate renal microvessels is a primary determinant of vascular injury in chronic kidney disease progression to end-stage renal disease. Here, we identified two degenerin family members, with an evolutionary link to mechanosensing, that interact biochemically and functionally to regulate systemic blood pressure and renal injury. Thus, degenerin proteins may serve as a target for the development of therapies to prevent or delay renal disease progression.

Abstract 12772: Regulation of Blood Pressure by Smooth Muscle TRPV4 Channels Under Normal and Hypertensive Conditions

Introduction: Recent studies suggest that transient receptor potential vanilloid 4 (TRPV4) channel is a functionally important Ca 2+ influx pathway in arterial smooth muscle cells (SMCs). Hypothesis: We hypothesized that SMC TRPV4 (TRPV4 SMC ) channels regulate blood pressure under normal conditions and in hypertension. Results: Radiotelemetric recordings showed lower blood pressures in the SMC-specific TRPV4 -/- (TRPV4 SMC -/- ) mice than the control mice. Phenylephrine (PE, α1 adrenergic receptor or α1AR agonist)-induced constriction was blunted in small mesenteric arteries (MAs) from TRPV4 SMC -/- mice, whereas intraluminal pressure-induced constriction was increased. These data demonstrated that TRPV4 SMC channels contribute to α1AR-induced vasoconstriction but oppose pressure-induced vasoconstriction. Both PE and intraluminal pressure increased the activity of elementary Ca 2+ influx signals through TRPV4 SMC channels (TRPV4 SMC sparklets). TRPV4 agonist GSK1016790A (30 nM) activated the currents through large-conductance, Ca 2+ -sensitive K + (BK) channels in SMCs, whereas phenylephrine had no effect on BK channel currents. These data implied the presence of two functionally distinct pools of TRPV4 SMC channels: 1) α1AR-activated, constrictor TRPV4 SMC channels; and 2) pressure-activated, dilator TRPV4 SMC channels. Next, direct STORM imaging demonstrated that ~30% TRPV4 SMC channels co-localize with α1ARs and ~10% TRPV4 SMC channels co-localize with BK channels. Importantly, only ~3% TRPV4 SMC channels co-localized with both α1ARs and BK channels, supporting the concept of spatially distinct constrictor and dilator TRPV4 SMC channels. Angiotensin II (Ang II, 7-14 days) infusion resulted in a lower increase in blood pressure in TRPV4 SMC -/- mice than the control mice, demonstrating a crucial role of TRPV4 SMC channels in blood pressure elevation in hypertension. Moreover, Ang II infusion in C57BL6 mice increased α1AR-TRPV4 SMC signaling; reduced pressure-activation of TRPV4 SMC channels; and reduced TRPV4 SMC -BK channel signaling. Conclusions: Our results show that increased constrictor TRPV4 SMC channel activity and reduced dilator TRPV4 SMC channel activity combine to elevate blood pressure in hypertension.

Abstract 14369: Smooth Muscle Specific Deletion of RSK2 Suppresses Vasoconstriction of Resistance Arteries

Introduction: Intracellular Ca 2+ is a vital second messenger that plays a fundamental role in regulating intraluminal pressure- and agonist-induced vasoconstriction. Our laboratory previously demonstrated that intraluminal pressure and G-protein coupled receptor agonist stimulation activate p90 ribosomal S6 kinase 2 (RSK2). RSK2, in turn, phosphorylates the regulatory light chain of myosin (RLC 20 ) to induce constriction of small, resistance-sized arteries. More recently, we discovered that resistance arteries from global KO RSK2 mice exhibited a more dilated luminal diameter, reduced myogenic tone and RLC 20 phosphorylation, and decreased blood pressure. However, it is unclear whether the observed effects are a result of RSK2 global deletion in the mouse which may cause developmental abnormalities affecting the cardiovascular system. Hypothesis: We hypothesize that smooth muscle-specific RSK2 signaling in mouse resistance arteries is a significant regulator of myogenic tone and arterial stiffness. Methods: Inducible smooth muscle-specific KO mice for RSK2 (RSK2 SMC-/- ) were generated to study the role of RSK2 signaling in the regulation of vascular tone. Pressure myography studies assessed vascular reactivity in 4 th - and 5 th -order mesenteric arteries (MAs) isolated from RSK2 SMC-/- and RSK2 fl/fl mice. Results: The downregulation of normal RSK2 expression in resistance arteries was confirmed by Western blot. Our data shows that the intraluminal pressure-induced constriction of MAs is significantly attenuated in RSK2 SMC-/- mice (n=7 each group). Additionally, RSK2 SMC-/- resistance arteries experienced a delay in development of myogenic tone at 40 mmHg indicating that RSK2 signaling is sensitive to small changes in intraluminal pressure. Furthermore, MAs from RSK2 SMC-/- mice exhibited decreased vascular stiffness (indicated by enhanced strain, and rightward shift of stress-strain relationship) when compared to MAs from RSK2 fl/fl mice. Conclusions: This study provides novel evidence that smooth muscle RSK2 signaling contributes to pressure-induced vasoconstriction, and vascular stiffness. Inhibiting smooth muscle RSK2 activity may represent a novel strategy for rescuing vascular function in hypertensive patients.

Expression of Exogenous Epithelial Sodium Channel Beta Subunit in the Mouse Middle Cerebral Artery Increases Pressure-Induced Constriction.

Pressure-induced constriction (PIC) is inherent to small arteries and arterioles, in which intraluminal pressure-induced vascular smooth muscle cell stretch elicits vasoconstriction. Degenerin (Deg) proteins, such as beta-epithelial Na+ channel (βENaC), have been studied in the PIC response because they are evolutionarily linked to known mechanosensors. While loss of Deg function phenotypes are plentiful, a gain-of-function phenotype has not been studied. The aim of this study was to determine if expression of exogenous βENaC in the isolated middle cerebral artery (MCA) enhances the PIC response. Isolated MCA segments from female mice (24 weeks, n = 5) were transfected with enhanced green fluorescent protein-βENaC (EGFP-βENaC) or with EGFP alone, incubated overnight at 37 °C, then studied in a pressure myograph. Mechanical/morphological properties and vasoconstrictor responses to KCl and phenylephrine were identical in EGFP-βENaC and EGFP MCAs. In contrast, PIC responses were greater in EGFP-βENaC segments with ~2-fold greater peak myogenic tone. These data confirm previous findings that βENaC is critical in the PIC response. These data provide proof-of-concept that upregulating βENaC can enhance PIC responses and lay the foundation to test the hypothesis that inflammation-mediated downregulation of βENaC contributes to cerebrovascular dysfunction.

Upregulation of Epithelial Na + Channel Beta Subunit in Isolated Mouse Middle Cerebral Artery Enhances Pressure‐Induced Constriction

Pressure-induced constriction (PIC), also referred to as myogenic constriction, is an intrinsic response to the vascular smooth muscle cell (VSMC) of small arteries and arterioles. The response is mechano-dependent and initiated by intraluminal pressure-induced VSMC stretch. While numerous molecules are required for the overall signaling response, our laboratories have focused on the role of Degenerin (Deg) proteins, such as Epithelial Na+Channel (ENaC), because of their close evolutionary link to known mechanosensors in the nematode. Loss of function phenotypes, using broad spectrum pharmacological inhibition and specific gene silencing, have been demonstrated in cerebral arteries. However, a gain of function phenotype has not been shown. Demonstration of a gain of function phenotype is critical to establishing a role for loss of vascular degenerin function in certain diseases, such as pre-eclampsia. Thus, the aim of this study was to determine if upregulation of full-length βENaC in the isolated middle cerebral artery (MCA) enhances the PIC response. To address this aim, MCA segments were dissected from mature, female mice (24 weeks old, mixed genetic background) and transiently transfected with βENaC enhanced green fluorescent protein (EGFP) fusion vector (βENaC-EGFP, n=5) or with EGFP alone (EGFP, n=5) using Lipofectamine 3000 (Thermo Fisher Scientific) using a 1:1 ratio. Vessel segments were incubated for 18 hours at 37 °C and 5% CO2, then pressurized in a pressure-flow myograph system with physiological salt solution. Responses to KCl (20-80 mM), phenylephrine (PE, 10-7-10-4 M), and active (+Ca2+) and passive (-Ca2+) diameters measured under graded pressure steps (15-90 mm Hg, 15 mm Hg increments, 5 min) were determined to assess vasoconstriction to depolarizing and α-adrenergic agonists and intraluminal pressure, respectively. Data were analyzed using repeated measures ANOVA. MCA vasoconstrictor responses to KCl (80 mM, 30.3±2.2% vs 34.3±4.2%, main effect of treatment p=0.55) and PE (10-4 M, 40.3±4.0% vs 35.6.0±3.1%, main effect of treatment p=0.73) were identical in βENaC-EGFP and EGFP transfected segments, respectively. Circumferential stress/strain, wall thickness and wall-to-lumen ratio were also similar. In contrast, PIC responses were greater in βENaC-EGFP group at >30 mm Hg (main effect of treatment p=0.0015). Peak myogenic tone was nearly 2 fold greater in βENaC-EGFP treated segments (11.6±0.4% vs 6.3±0.4% at 75 mmHg, p=0.0006). These data confirm previous findings that βENaC is an important mediator of the PIC response. Moreover, they provide proof-of-concept that upregulating βENaC can potentially improve PIC responses in cerebral vessels and will allow us to test the hypothesis that downregulation of vascular βENaC in inflammatory conditions, such as preeclampsia, contributes to cerebrovascular dysfunction.

Dichotomous effects of smooth muscle TRPV4 channels on vascular contractility

Ca2+ signaling mechanisms in smooth muscle cells (SMCs) determine vascular contractility. Transient receptor potential vanilloid 4 (TRPV4) ion channels have been described as a crucial Ca2+ influx pathway in SMCs from small, resistance-sized arteries. However, the impact of SMC TRPV4 (TRPV4SMC) channel activity on blood pressure remains unknown, mostly due to the lack of studies in SMC-specific knockout mice. We hypothesized that TRPV4SMC channels control the resting blood pressure. Radiotelemetric blood pressure recordings showed reduced daytime and nighttime blood pressures in the inducible, SMC-specific TRPV4 knockout (TRPV4SMC-/-) mice compared to the control mice. The effect of TRPV4SMC-/- on vascular contractility was assessed using two endogenous vasoconstrictor mechanisms- 1) SMC α1 adrenergic receptor (α1AR) stimulation and 2) increased intraluminal pressure. Phenylephrine (α1AR agonist)-induced constriction of pressurized mesenteric arteries (MAs, 80 mm Hg, 1 nM-10 mM phenylephrine) and blood pressure elevation (10 mg/kg phenylephrine, i.p.) were blunted in TRPV4SMC-/- mice, identifying a central role of TRPV4SMC channels in α1AR-induced vasoconstriction and blood pressure elevation. Individual Ca2+ influx signals through TRPV4SMC channels (TRPV4SMC sparklets) were recorded using spinning disk confocal imaging in fluo-4-loaded MAs pressurized to 80 mm Hg. Phenylephrine (1 mM) activated TRPV4SMC sparklets, an effect abolished by protein kinase C (PKC) inhibitor (Go6976, 1 mM). These results indicated that PKC-TRPV4SMC channel signaling is a major contributor to α1AR-induced vasoconstriction. In direct contrast with PE-induced constriction, intraluminal pressure-induced constriction was higher in MAs from TRPV4SMC-/- mice, suggesting that SMC TRPV4 channels limit pressure-induced vasoconstriction. Previous studies have shown that TRPV4SMC channels can activate large-conductance, Ca2+-sensitive K+ (BK) channels, thereby decreasing pressure-induced vasoconstriction. Consistent with these findings, vasoconstriction in response to BK channel inhibitor (paxilline, 1 mM) was reduced in MAs from TRPV4SMC-/- mice, supporting a dilatory TRPV4SMC channel-BK channel signaling under resting conditions. In situ proximity ligation assay revealed co-localization of TRPV4SMC channels with both α1ARs and BK channels. Overall, TRPV4SMC channels increase the resting blood pressure but have divergent effects on vascular contractility. TRPV4SMC channels contribute to α1AR-induced vasoconstriction but limit pressure-induced vasoconstriction. Therefore, selective vasoactive stimulus-TRPV4SMC channel coupling may determine the impact of TRPV4SMC channels on vascular resistance.

Abstract 16955: Contrasting Effects of Smooth Muscle TRPV4 Channels on Vascular Function

Introduction: Transient receptor potential vanilloid 4 (TRPV4) channels are a major Ca 2+ entry pathway in vascular endothelial cells. TRPV4 channels are also expressed in vascular smooth muscle cells (SMC). However, the effect of SMC TRPV4 (TRPV4 SMC ) channels on vascular resistance and blood pressure remains controversial. Hypothesis: We hypothesized that TRPV4 SMC channels are essential regulators of vascular function and blood pressure. Methods: Inducible, SMC-specific TRPV4 knockout (TRPV4 SMC -/- ) mice were used. Blood pressure was monitored using radiotelemetry catheters. SMC Ca 2+ signals were recorded with a spinning disk confocal imaging system. Pressure myography studies assessed vascular reactivity in MAs. Results: Resting diastolic blood pressure and mean arterial pressure were lower in TRPV4 SMC -/- mice when compared to control mice. Selective TRPV4 channel agonist, GSK1016790A (GSK101), induced Ca 2+ influx signals in SMCs that were inhibited by TRPV4 channel inhibitor GSK2193874 and were absent in MAs from TRPV4 SMC -/- mice. In pressure myography studies, GSK101 constricted the MAs in control mice, but not in TRPV4 SMC -/- mice. Moreover, MAs from TRPV4 SMC -/- showed lower vasoconstriction in response to phenylephrine, a α1 adrenergic receptor agonist. These results indicated that Ca 2+ influx through TRPV4 SMC channels contributes to vasoconstriction and increased vascular resistance. However, studies of pressure-induced constriction or myogenic tone indicated that MAs from TRPV4 SMC -/- mice exhibit a higher myogenic constriction at pressures ≥ 60 mm Hg. Moreover, membrane depolarization-induced vasoconstriction (KCl, 33 and 60 mM) was also increased in TRPV4 SMC -/- mice when compared to the control mice, indicating that TRPV4 SMC channels oppose pressure- or membrane depolarization-induced vasoconstriction. Conclusions: TRPV4 SMC channels are essential regulators of resting blood pressure. While TRPV4 SMC channels contribute to α1 adrenergic receptor-induced vasoconstriction, they negatively regulate pressure-induced vasoconstriction. The divergent effects of TRPV4 SMC channels on vascular function may be determined by the selective stimulus-TRPV4 SMC channel coupling and downstream signaling mechanisms.

Pressure-induced constriction of the middle cerebral artery is abolished in TrpC6 knockout mice.

Pressure-induced constriction (PIC) is an inherent response of small arteries and arterioles in which increases in intraluminal pressure evoke vasoconstriction. It is a critical mechanism of blood flow autoregulation in the kidney and brain. Degenerin (Deg) and transient receptor potential (Trp) protein families have been implicated in transduction of PIC because of evolutionary links to mechanosensing in the nematode and fly. While TrpC6 has been suggested to contribute to PIC signaling, direct supporting evidence is contradictory. Therefore, the aim of this study was to determine the importance of TrpC6 in PIC signaling using a mouse model lacking TrpC6. To address this aim, we evaluated graded pressure (20-90 mmHg), depolarization (4-80 mM KCl)-, and adrenergic receptor (phenylephrine; PE 10-7-10-4 M)-mediated constriction of isolated middle cerebral artery (MCA) segments from 9-wk-old male wild-type (TrpC6+/+, n = 7) and homozygous null (TrpC6-/-, n = 9) TrpC6 mice (Jackson Laboratories). Isolated MCA segments were cannulated and pressurized with physiological salt solution using pressure myography (Living Systems). Vasoconstrictor responses to KCl and PE were identical in TrpC6-/- and TrpC6+/+ mice. In contrast, PIC responses were totally abolished in TrpC6-/- mice. At 90 mmHg, the calculated myogenic tone was -0.8 ± 0.5 vs. 10.7 ± 1.7%, P = 0.0002 in TrpC6-/- and TrpC6+/+ mice, respectively. Additionally, there were no changes in mechanical properties of circumferential wall strain and stress or morphological properties of wall thickness and wall-to-lumen ratio at 50 mmHg between TrpPC6-/- and TrpC6+/+ mice. Although these results demonstrate that TrpC6 is critical for the integrated PIC response, they do not identify whether TrpC6 acts as a mechanosensor or a downstream signaling component.NEW & NOTEWORTHY Pressure-induced, but not agonist-induced, vasoconstriction is abolished in the middle cerebral artery (MCA) of TrpC6 null mice. TrpC6 localization in dissociated cerebral vascular smooth muscle cells is primarily cytoplasmic and not associated with the surface membrane where a mechanoelectrical coupler might be expected. These findings suggest that TrpC6 is required for transduction of pressure-induced constriction in the MCA; however, its role as a mechanoelectrical coupler or downstream signal amplifier remains unresolved.

Interleukin-17 Reduces βENaC via MAPK Signaling in Vascular Smooth Muscle Cells.

Degenerin proteins, such as the beta epithelial Na+ channel (βENaC), are essential in the intracellular signaling of pressure-induced constriction, an important vascular smooth muscle cell (VSMC) function. While certain cytokines reduce ENaC protein in epithelial tissue, it is unknown if interleukin-17 (IL-17), a potent pro-inflammatory cytokine, directly mediates changes in membrane-associated βENaC in VSMCs. Therefore, we tested the hypothesis that exposure to IL-17 reduces βENaC in VSMCs through canonical mitogen-activated protein kinase (MAPK) signaling pathways. We treated cultured rat VSMCs (A10 cell line) with IL-17 (1–100 ng/mL) for 15 min to 16 h and measured expression of βENaC, p38MAPK, c-jun kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). IL-17 reduced βENaC protein expression in a concentration-dependent fashion and increased phosphorylation of p38MAPK by 15 min and JNK by 8 h. NFκB was unaffected by IL-17 in VSMCs. IL-17 treatment reduced VSMC viability but had no effect on cell death. To determine the underlying signaling pathway involved in this response, VSMCs were treated before and during IL-17 exposure with p38MAPK or JNK inhibitors. We found that JNK blockade prevented IL-17-mediated βENaC protein suppression. These data demonstrate that the pro-inflammatory cytokine IL-17 regulates VSMC βENaC via canonical MAPK signaling pathways, raising the possibility that βENaC-mediated loss of VSMC function may occur in inflammatory disorders.

Pressure‐Induced Constriction of the Middle Cerebral Artery is Abolished in TrpC6 Knockout Mice

Pressure‐induced constriction (PIC) is an inherent response of small arteries and arterioles in which increases in intraluminal pressure evoke vasoconstriction. It is a critical mechanism of blood flow autoregulation in the kidney and brain. Degenerin (Deg) and transient receptor potential (Trp) protein families have been implicated in transduction of PIC because of evolutionary links to mechanosensing in the nematode and fly. We hypothesize that Deg and Trp proteins associate to form a multi‐meric mechanosensing complex in vascular smooth muscle cells (VSMCs), in which Deg and Trp proteins play different, yet equally critical, roles as sensors and amplifiers, in PIC molecular signaling. Our laboratory has provided direct evidence that the Deg protein bENaC acts as a sensor by mediating mechanoreceptor currents in isolated VSMCs. While TrpC6 has been suggested to mediate PIC, direct supporting evidence is lacking. Therefore, the aim of this study was to determine the importance of TrpC6 in PIC signaling using a mouse model lacking TrpC6. To address this aim, we evaluated graded pressure (20–90 mm Hg), depolarization‐ (20–80 mM KCl) and adrenergic receptor‐(phenylephrine, PE 10−7–10−4 M) mediated constriction of isolated middle cerebral artery (MCA) segments from 9 weeks old male wild‐type (+/+, n=7) and homozygous null (−/−, n=9) TrpC6 mice (Jackson Laboratories). Isolated MCA segments were cannulated and pressurized with physiological salt solution using pressure myography (Living Systems). The data were analyzed preliminarily using independent 2‐tailed t‐test. Normalized MCA vasoconstrictor responses to KCl (80 mM, 45.5±1.1% vs 40.9±1.2%, p=0.2) and PE (10−4 M, 37.4±1.7% vs 38.7.0±1.2%, p=0.8) were similar in TrpC6−/− and TrpC6+/+ mice, respectively. In contrast, PIC responses were totally abolished in TrpC6 −/− mice; at 90 mmHg calculated myogenic tone was 0.8±0.5% vs 10.7±1.7%, p=0.0002, in TrpC6−/− and TrpC6+/+ mice, respectively. Additionally, there were no changes in mechanical properties of circumferential wall strain (0.2±0.02 vs. 0.2±0.02, p=0.8) and stress (2.7±0.1 vs. 3.1±0.4, p=0.4), at 90 mmHg; or morphological properties wall thickness (13.4±0.8μm vs. 14.3±1.5μm, p=0.6) and wall‐to‐lumen ratio (0.3±0.02 vs. 0.3±0.04, p=0.7), at 50 mmHg between TrpC6−/− and TrpC6+/+ mice. Although these results suggest that TrpC6 is required for the PIC response, they do not identify the role of TrpC6. Studies in the nematode suggest that Deg and Trp channels play different, yet equally critical roles in mechano‐signaling; Deg channels mediate mechanoreceptor currents, while Trp channels mediate post‐transduction signal amplification. Future studies will determine the role of TrpC6 in isolated VSMCs and cerebral blood flow autoregulation. This research was supported by P20GM104357, P20121334, P01HL051971, R01HL1136684.Support or Funding InformationP20GM104357, P20121334, P01HL051971, R01HL1136684.

Tumor necrosis factor-α impairs cerebral blood flow in pregnant rats: role of vascular β-epithelial Na+ channel.

Preeclampsia is a pregnancy-related disorder characterized by hypertension, vascular dysfunction and an increase in circulating inflammatory factors including the cytokine, tumor necrosis factor-α (TNF-α). Studies have shown that placental ischemia is associated with 1) increased circulating TNF-α, 2) attenuated pressure-induced cerebral vascular tone, and 3) suppression of β-epithelial Na+ channel (βENaC) protein in cerebral vessels. In addition to its role in epithelial Na+ and water transport, βENaC is an essential signaling element in transduction of pressure-induced (aka "myogenic") constriction, a critical mechanism of blood flow autoregulation. While cytokines inhibit expression of certain ENaC proteins in epithelial tissue, it is unknown if the increased circulating TNF-α associated with placental ischemia mediates the loss of cerebrovascular βENaC and cerebral blood flow regulation. Therefore, the purpose of this study was to test the hypothesis that increasing plasma TNF-α in normal pregnant rats reduces cerebrovascular βENaC expression and impairs cerebral blood flow (CBF) regulation. In vivo TNF-α infusion (200 ng/day, 5 days) inhibited cerebrovascular expression of βENaC and impaired CBF regulation in pregnant rats. To determine the direct effects of TNF-α and underlying pathways mediating vascular smooth muscle cell βENaC reduction, we exposed cultured VSMCs (A10 cell line) to TNF-α (1-100 ng/mL) for 16-24 h. TNF-α reduced βENaC protein expression in a concentration-dependent fashion from 0.1 to 100 ng/mL, without affecting cell death. To assess the role of canonical MAPK signaling in this response, VSMCs were treated with p38MAPK or c-Jun kinase (JNK) inhibitors in the presence of TNF-α. We found that both p38MAPK and JNK blockade prevented TNF-α-mediated βENaC protein suppression. These data provide evidence that disorders associated with increased circulating TNF-α could lead to impaired cerebrovascular regulation, possibly due to reduced βENaC-mediated vascular function.NEW & NOTEWORTHY This manuscript identifies TNF-α as a possible placental-derived cytokine that could be involved in declining cerebrovascular health observed in preeclampsia. We found that infusion of TNF-α during pregnancy impaired cerebral blood flow control in rats at high arterial pressures. We further discovered that cerebrovascular β-epithelial sodium channel (βENaC) protein, a degenerin protein involved in mechanotransduction, was reduced by TNF-α in pregnant rats, indicating a potential link between impaired blood flow and this myogenic player. We next examined this effect in vitro using a rat vascular smooth muscle cell line. TNF-α reduced βENaC through canonical MAPK-signaling pathways and was not dependent on cell death. This study demonstrates the pejorative effects of TNF-α on cerebrovascular function during pregnancy and warrants future investigations to study the role of cytokines on vascular function during pregnancy.