Elevated plasma catecholamines functionally compensate for the reduced myogenic tone in smooth muscle STIM1 knockout mice but with deleterious cardiac effects.
ABSTRACT: Aims:Stromal interaction molecule 1 (STIM1) has emerged as an important player in the regulation of growth and proliferation of smooth muscle cells. Therefore, we hypothesized that STIM1 plays a crucial role in the maintenance of vascular integrity. The objective of this study was to evaluate whether reduced expression of STIM1 could modify the structure and function of the vasculature, leading to changes in blood pressure (BP). Methods and results:Smooth muscle-specific STIM1 knockout (sm-STIM1 KO) in mice resulted in arteries with ∼80% reduced STIM1 protein expression as compared with control mice. Mesenteric vessels exposed to increasing transmural pressure revealed attenuated myogenic reactivity and reduced vasoconstrictor response to phenylephrine in sm-STIM1 KO arteries. BP monitored via telemetry in sm-STIM1 KO and matched controls did not reveal differences. However, heart rate was significantly increased in sm-STIM1 KO mice. Consistent with these findings, plasma catecholamine levels were higher in sm-STIM1 KO than in control mice. Increased sympathetic activity in sm-STIM1 KO mice was unmasked by apha1-adrenergic receptor inhibitor (prazosin) and by treatment with the ganglion-blocking agent, hexamethonium. Both treatments resulted in a greater reduction of BP in sm-STIM1 KO mice. Cytoskeleton of cultured smooth muscle cells was studied by immunocytochemistry using specific antibodies. Staining for actin and vinculin revealed significant alterations in the cytoskeletal architecture of cells isolated from sm-STIM1 KO arteries. Finally, although sm-STIM1 KO mice were protected from Ang II-induced hypertension, such treatment resulted in significant fibrosis and a rapid deterioration of cardiac function. Conclusions:STIM1 deletion in smooth muscle results in attenuated myogenic tone and cytoskeletal defects with detrimental effects on the mechanical properties of arterial tissue. Although BP is maintained by elevated circulating catecholamine, this compensatory stimulation has a deleterious long-term effect on the myocardium.
Project description:The Ca(2+)-sensing stromal interaction molecule (STIM) proteins are crucial Ca(2+) signal coordinators. Cre-lox technology was used to generate smooth muscle (sm)-targeted STIM1-, STIM2-, and double STIM1/STIM2-knockout (KO) mouse models, which reveal the essential role of STIM proteins in Ca(2+) homeostasis and their crucial role in controlling function, growth, and development of smooth muscle cells (SMCs). Compared to Cre(+/-) littermates, sm-STIM1-KO mice showed high mortality (50% by 30 d) and reduced bodyweight. While sm-STIM2-KO was without detectable phenotype, the STIM1/STIM double-KO was perinatally lethal, revealing an essential role of STIM1 partially rescued by STIM2. Vascular and intestinal smooth muscle tissues from sm-STIM1-KO mice developed abnormally with distended, thinned morphology. While depolarization-induced aortic contraction was unchanged in sm-STIM1-KO mice, ?1-adrenergic-mediated contraction was 26% reduced, and store-dependent contraction almost eliminated. Neointimal formation induced by carotid artery ligation was suppressed by 54%, and in vitro PDGF-induced proliferation was greatly reduced (79%) in sm-STIM1-KO. Notably, the Ca(2+) store-refilling rate in STIM1-KO SMCs was substantially reduced, and sustained PDGF-induced Ca(2+) entry was abolished. This defective Ca(2+) homeostasis prevents PDGF-induced NFAT activation in both contractile and proliferating SMCs. We conclude that STIM1-regulated Ca(2+) homeostasis is crucial for NFAT-mediated transcriptional control required for induction of SMC proliferation, development, and growth responses to injury.-Mancarella, S., Potireddy, S., Wang, Y., Gao, H., Gandhirajan, K., Autieri, M., Scalia, R., Cheng, Z., Wang, H., Madesh, M., Houser, S. R., Gill, D. L. Targeted STIM deletion impairs calcium homeostasis, NFAT activation, and growth of smooth muscle.
Project description:Mice with smooth muscle (SM)-specific knockout of Na(+)/Ca(2+) exchanger type-1 (NCX1(SM-/-)) and the NCX inhibitor, SEA0400, were used to study the physiological role of NCX1 in mouse mesenteric arteries. NCX1 protein expression was greatly reduced in arteries from NCX1(SM-/-) mice generated with Cre recombinase. Mean blood pressure (BP) was 6-10 mmHg lower in NCX1(SM-/-) mice than in wild-type (WT) controls. Vasoconstriction was studied in isolated, pressurized mesenteric small arteries from WT and NCX1(SM-/-) mice and in heterozygotes with a global null mutation (NCX1(Fx/-)). Reduced NCX1 activity was manifested by a marked attenuation of responses to low extracellular Na(+) concentration, nanomolar ouabain, and SEA0400. Myogenic tone (MT, 70 mmHg) was reduced by approximately 15% in NCX1(SM-/-) arteries and, to a similar extent, by SEA0400 in WT arteries. MT was normal in arteries from NCX1(Fx/-) mice, which had normal BP. Vasoconstrictions to phenylephrine and elevated extracellular K(+) concentration were significantly reduced in NCX1(SM-/-) arteries. Because a high extracellular K(+) concentration-induced vasoconstriction involves the activation of L-type voltage-gated Ca(2+) channels (LVGCs), we measured LVGC-mediated currents and Ca(2+) sparklets in isolated mesenteric artery myocytes. Both the currents and the sparklets were significantly reduced in NCX1(SM-/-) (vs. WT or NCX1(Fx/-)) myocytes, but the voltage-dependent inactivation of LVGCs was not augmented. An acute application of SEA0400 in WT myocytes had no effect on LVGC current. The LVGC agonist, Bay K 8644, eliminated the differences in LVGC currents and Ca(2+) sparklets between NCX1(SM-/-) and control myocytes, suggesting that LVGC expression was normal in NCX1(SM-/-) myocytes. Bay K 8644 did not, however, eliminate the difference in myogenic constriction between WT and NCX1(SM-/-) arteries. We conclude that, under physiological conditions, NCX1-mediated Ca(2+) entry contributes significantly to the maintenance of MT. In NCX1(SM-/-) mouse artery myocytes, the reduced Ca(2+) entry via NCX1 may lower cytosolic Ca(2+) concentration and thereby reduce MT and BP. The reduced LVGC activity may be the consequence of a low cytosolic Ca(2+) concentration.
Project description:Nitric oxide regulates BP by binding the reduced heme iron (Fe2+) in soluble guanylyl cyclase (sGC) and relaxing vascular smooth muscle cells (SMCs). We previously showed that sGC heme iron reduction (Fe3+ ? Fe2+) is modulated by cytochrome b5 reductase 3 (CYB5R3). However, the in vivo role of SMC CYB5R3 in BP regulation remains elusive. Here, we generated conditional smooth muscle cell-specific Cyb5r3 KO mice (SMC CYB5R3-KO) to test if SMC CYB5R3 loss affects systemic BP in normotension and hypertension via regulation of the sGC redox state. SMC CYB5R3-KO mice exhibited a 5.84-mmHg increase in BP and impaired acetylcholine-induced vasodilation in mesenteric arteries compared with controls. To drive sGC oxidation and elevate BP, we infused mice with angiotensin II. We found that SMC CYB5R3-KO mice exhibited a 14.75-mmHg BP increase, and mesenteric arteries had diminished nitric oxide-dependent vasodilation but increased responsiveness to sGC heme-independent activator BAY 58-2667 over controls. Furthermore, acute injection of BAY 58-2667 in angiotensin II-treated SMC CYB5R3-KO mice showed greater BP reduction compared with controls. Together, these data provide the first in vivo evidence to our knowledge that SMC CYB5R3 is an sGC heme reductase in resistance arteries and provides resilience against systemic hypertension development.
Project description:Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Our study is the first to assess the role of Ephb6 in blood pressure (BP) regulation. We observed that EphB6 and all three of its Efnb ligands were expressed on vascular smooth muscle cells (VSMC) in mice. We discovered that small arteries from castrated Ephb6 gene KO males showed increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo compared with their WT counterparts. Consistent with this finding, castrated Ephb6 KO mice presented heightened BP compared with castrated WT controls. In vitro experiments in VSMC revealed that cross-linking Efnbs but not Ephb6 resulted in reduced VSMC contractions, suggesting that reverse signaling through Efnbs was responsible for the observed BP phenotype. The reverse signaling was mediated by an adaptor protein Grip1. Additional experiments demonstrated decreased 24-h urine catecholamines in male Ephb6 KO mice, probably as a compensatory feedback mechanism to keep their BP in the normal range. After castration, however, such compensation was abolished in Ephb6 KO mice and was likely the reason why BP increased overtly in these animals. It suggests that Ephb6 has a target in the nervous/endocrine system in addition to VSMC, regulating a testosterone-dependent catecholamine compensatory mechanism. Our study discloses that Ephs and Efns, in concert with testosterone, play a critical role in regulating small artery contractility and BP.
Project description:BACKGROUND: Increasing evidence implicates overactivation of RhoA as a critical component of the pathogenesis of hypertension. Although a substantial body of work has established that Rac1 functions antagonize RhoA in a broad range of physiological processes, the role of Rac1 in the regulation of vascular tone and blood pressure is not fully elucidated. METHODS AND RESULTS: To define the role of Rac1 in vivo in vascular smooth muscle cells (vSMC), we generated smooth muscle (SM)-specific Rac1 knockout mice (SM-Rac1-KO) and performed radiotelemetric blood pressure recordings, contraction measurements in arterial rings, vSMC cultures and biochemical analyses. SM-Rac1-KO mice develop high systolic blood pressure sensitive to Rho kinase inhibition by fasudil. Arteries from SM-Rac1-KO mice are characterized by a defective NO-dependent vasodilation and an overactivation of RhoA/Rho kinase signaling. We provide evidence that Rac1 deletion-induced hypertension is due to an alteration of cGMP signaling resulting from the loss of Rac1-mediated control of type 5 PDE activity. Consequently, cGMP-dependent phosphorylation and binding of RhoA with its inhibitory partner, the phosphatase-RhoA interacting protein (p116(RIP3)), are decreased. CONCLUSIONS: Our data reveal that the depletion of Rac1 in SMC decreases cGMP-dependent p116(RIP3)/RhoA interaction and the subsequent inhibition of RhoA signaling. Thus, we unveil an in vivo role of Rac1 in arterial blood pressure regulation and a new pathway involving p116(RIP3) that contributes to the antagonistic relationship between Rac1 and RhoA in vascular smooth muscle cells and their opposite roles in arterial tone and blood pressure.
Project description:The role of the smoothelin-like 1 (SMTNL1) protein in mediating vascular smooth muscle contractile responses to intraluminal pressure was examined in resistance vessels. Mesenteric arterioles from wild type (WT) and SMTNL1 global knock-out (KO) mice were examined with pressure myography. SMTNL1 deletion was associated with enhanced myogenic tone in vessels isolated from male, but not female, mice. Intraluminal pressures greater than 40?mmHg generated statistically significant differences in myogenic reactivity between WT and KO vessels. No overt morphological differences were recorded for vessels dissected from KO animals, but SMTNL1 deletion was associated with loss of myosin phosphatase-targeting protein MYPT1 and increase in the myosin phosphatase inhibitor protein CPI-17. Additionally, we observed altered contractile responses of isolated arteries from SMTNL1 KO mice to phenylephrine, KCl-dependent membrane depolarization and phorbol 12,13-dibutyrate (PDBu). Using pharmacological approaches, myogenic responses of both WT and KO vessels were equally affected by Rho-associated kinase (ROCK) inhibition; however, augmented protein kinase C (PKC) signaling was found to contribute to the increased myogenic reactivity of SMTNL1 KO vessels across the 60-120 mmHg pressure range. Based on these findings, we conclude that deletion of SMTNL1 contributes to enhancement of pressure-induced contractility of mesenteric resistance vessels by influencing the activity of myosin phosphatase.
Project description:The multifunctional Ca(2+)/calmodulin-dependent kinase II (CaMKII) is activated by vasoconstrictors in vascular smooth muscle cells (VSMC), but its impact on vasoconstriction remains unknown. We hypothesized that CaMKII inhibition in VSMC decreases vasoconstriction. Using novel transgenic mice that express the inhibitor peptide CaMKIIN in smooth muscle (TG SM-CaMKIIN), we investigated the effect of CaMKII inhibition on L-type Ca(2+) channel current (ICa), cytoplasmic and sarcoplasmic reticulum Ca(2+), and vasoconstriction in mesenteric arteries. In mesenteric VSMC, CaMKII inhibition significantly reduced action potential duration and the residual ICa 50 ms after peak amplitude, indicative of loss of L-type Ca(2+) channel-dependent ICa facilitation. Treatment with angiotensin II or phenylephrine increased the intracellular Ca(2+) concentration in wild-type but not TG SM-CaMKIIN VSMC. The difference in intracellular Ca(2+) concentration was abolished by pretreatment with nifedipine, an L-type Ca(2+) channel antagonist. In TG SM-CaMKIIN VSMC, the total sarcoplasmic reticulum Ca(2+) content was reduced as a result of diminished sarcoplasmic reticulum Ca(2+) ATPase activity via impaired derepression of the sarcoplasmic reticulum Ca(2+) ATPase inhibitor phospholamban. Despite the differences in intracellular Ca(2+) concentration, CaMKII inhibition did not alter myogenic tone or vasoconstriction of mesenteric arteries in response to KCl, angiotensin II, and phenylephrine. However, it increased myosin light chain kinase activity. These data suggest that CaMKII activity maintains intracellular calcium homeostasis but is not required for vasoconstriction of mesenteric arteries.
Project description:Myogenic responses by resistance vessels are a key component of autoregulation in brain, thus playing a crucial role in regulating cerebral blood flow and protecting the blood-brain barrier against potentially detrimental elevations in blood pressure. Although cerebrovascular disease is often accompanied by alterations in myogenic responses, mechanisms that control these changes are poorly understood. Peroxisome proliferator-activated receptor ? has emerged as a regulator of vascular tone. We hypothesized that interference with peroxisome proliferator-activated receptor ? in smooth muscle would augment myogenic responses in cerebral arteries. We studied transgenic mice expressing a dominant-negative mutation in peroxisome proliferator-activated receptor ? selectively in smooth muscle (S-P467L) and nontransgenic littermates. Myogenic tone in middle cerebral arteries from S-P467L was elevated 3-fold when compared with nontransgenic littermates. Rho kinase is thought to play a major role in cerebrovascular disease. The Rho kinase inhibitor, Y-27632, abolished augmented myogenic tone in middle cerebral arteries from S-P467L mice. CN-03, which modifies RhoA making it constitutively active, elevated myogenic tone to ?60% in both strains, via a Y-27632-dependent mechanism. Large conductance Ca(2+)-activated K(+) channels (BKCa) modulate myogenic tone. Inhibitors of BKCa caused greater constriction in middle cerebral arteries from nontransgenic littermates when compared with S-P467L. Expression of RhoA or Rho kinase-I/II protein was similar in cerebral arteries from S-P467L mice. Overall, the data suggest that peroxisome proliferator-activated receptor ? in smooth muscle normally inhibits Rho kinase and promotes BKCa function, thus influencing myogenic tone in resistance arteries in brain. These findings have implications for mechanisms that underlie large- and small-vessel disease in brain, as well as regulation of cerebral blood flow.
Project description:Notch3 is critically important for the structure and myogenic response of distal arteries, particularly of cerebral arteries. However, signaling pathways acting downstream of Notch3 remain largely unknown.Transcriptome analysis using tail arteries of Notch3-null mice identified a core set of 17 novel Notch3-regulated genes confirmed in tail or brain arteries. Postnatal deletion of RBP-J? in smooth muscle cells recapitulated the structural, functional, and molecular defects of brain arteries induced by Notch3 deficiency. Transient in vivo blockade of the Notch pathway with ?-secretase inhibitors uncovered, in addition to Notch3, 6 immediate responders, including the voltage-gated potassium channel Kv1.5, which opposes to myogenic constriction of brain arteries, and the glutamate receptor-interacting protein 2 (Grip2) with no previously established role in the cerebrovasculature. We identified a vascular smooth muscle cell isoform of Grip2. We showed that Notch3-RBP-J? specifically regulates this isoform. Finally, we found that cerebral arteries of Grip2 mutant mice, which express an N-terminally truncated Grip2 protein, exhibited selective attenuation of pressure-induced contraction.Our data provide insight into how Notch3 signals in the brain arteries, establishing the postnatal requirement of smooth muscle RBP-J? in this context. Notch3-regulated transcriptome provides potential for modulating myogenic response in the cerebrovasculature.
Project description:In resistance arteries, there is an emerging view that smooth muscle CaV3.2 channels restrain arterial constriction through a feedback response involving the large-conductance Ca(2+)-activated K(+) channel (BKCa). Here, we used wild-type and CaV3.2 knockout (CaV3.2(-/-)) mice to definitively test whether CaV3.2 moderates myogenic tone in mesenteric arteries via the CaV3.2-ryanodine receptor-BKCa axis and whether this regulatory mechanism influences blood pressure regulation.Using pressurized vessel myography, CaV3.2(-/-) mesenteric arteries displayed enhanced myogenic constriction to pressure but similar K(+)-induced vasoconstriction compared with wild-type C57BL/6 arteries. Electrophysiological and myography experiments subsequently confirmed the inability of micromolar Ni(2+), a CaV3.2 blocker, to either constrict arteries or suppress T-type currents in CaV3.2(-/-) smooth muscle cells. The frequency of BKCa-induced spontaneous transient outward K(+) currents dropped in wild-type but not in knockout arterial smooth muscle cells upon the pharmacological suppression of CaV3.2 channel. Line scan analysis performed on en face arteries loaded with Fluo-4 revealed the presence of Ca(2+) sparks in all arteries, with the subsequent application of Ni(2+) only affecting wild-type arteries. Although CaV3.2 channel moderated myogenic constriction of resistance arteries, the blood pressure measurements of CaV3.2(-/-) and wild-type animals were similar.Overall, our findings establish a negative feedback mechanism of the myogenic response in which CaV3.2 channel modulates downstream ryanodine receptor-BKCa to hyperpolarize and relax arteries.