Mesenteric Vascular Smooth Muscle Cells Research Articles

Aberrant splicing of CaV1.2 calcium channel induced by decreased Rbfox1 enhances arterial constriction during diabetic hyperglycemia

Diabetic hyperglycemia induces dysfunctions of arterial smooth muscle, leading to diabetic vascular complications. The CaV1.2 calcium channel is one primary pathway for Ca2+ influx, which initiates vasoconstriction. However, the long-term regulation mechanism(s) for vascular CaV1.2 functions under hyperglycemic condition remains unknown. Here, Sprague–Dawley rats fed with high-fat diet in combination with low dose streptozotocin and Goto-Kakizaki (GK) rats were used as diabetic models. Isolated mesenteric arteries (MAs) and vascular smooth muscle cells (VSMCs) from rat models were used to assess K+-induced arterial constriction and CaV1.2 channel functions using vascular myograph and whole-cell patch clamp, respectively. K+-induced vasoconstriction is persistently enhanced in the MAs from diabetic rats, and CaV1.2 alternative spliced exon 9* is increased, while exon 33 is decreased in rat diabetic arteries. Furthermore, CaV1.2 channels exhibit hyperpolarized current–voltage and activation curve in VSMCs from diabetic rats, which facilitates the channel function. Unexpectedly, the application of glycated serum (GS), mimicking advanced glycation end-products (AGEs), but not glucose, downregulates the expression of the splicing factor Rbfox1 in VSMCs. Moreover, GS application or Rbfox1 knockdown dynamically regulates alternative exons 9* and 33, leading to facilitated functions of CaV1.2 channels in VSMCs and MAs. Notably, GS increases K+-induced intracellular calcium concentration of VSMCs and the vasoconstriction of MAs. These results reveal that AGEs, not glucose, long-termly regulates CaV1.2 alternative splicing events by decreasing Rbfox1 expression, thereby enhancing channel functions and increasing vasoconstriction under diabetic hyperglycemia. This study identifies the specific molecular mechanism for enhanced vasoconstriction under hyperglycemia, providing a potential target for managing diabetic vascular complications.

SMIT (Sodium-Myo-Inositol Transporter) 1 Regulates Arterial Contractility Through the Modulation of Vascular Kv7 Channels.

The SMIT1 (sodium:myo-inositol transporter 1) regulates myo-inositol movement into cells and responses to hypertonic stimuli. Alteration of myo-inositol levels has been associated with several diseases, including hypertension, but there is no evidence of a functional role of SMIT1 in the vasculature. Recent evidence showed that in the nervous system SMIT1 interacted and modulated the function of members of the Kv7 family of voltage-gated potassium channels, which are also expressed in the vasculature where they regulate arterial contractility. Therefore, in this study, we evaluated whether SMIT1 was functionally relevant in arterial smooth muscle. Approach and Results: Immunofluorescence and polymerase chain reaction experiments revealed that SMIT1 was expressed in rat renal and mesenteric vascular smooth muscle cells. Isometric tension recordings showed that incubation of renal arteries with raffinose and myo-inositol (which increases SMIT1 expression) reduced the contractile responses to methoxamine, an effect that was abolished by preincubation with the pan-Kv7 blocker linopirdine and by molecular knockdown of Kv7.4 and Kv7.5. Knockdown of SMIT1 increased the contraction of renal arteries induced by methoxamine, impaired the response to the Kv7.2-Kv7.5 activator ML213 but did not interfere with the relaxant responses induced by openers of other potassium channels. Proximity ligation assay showed that SMIT1 interacted with heteromeric channels formed by Kv7.4 and Kv7.5 proteins in both renal and mesenteric vascular smooth muscle cells. Patch-clamp experiments showed that incubation with raffinose plus myo-inositol increased Kv7 currents in vascular smooth muscle cells. SMIT1 protein is expressed in vascular smooth muscle cells where it modulates arterial contractility through an association with Kv7.4/Kv7.5 heteromers.

Prenatal hypoxia inhibited propionate-evoked BK channels of mesenteric artery smooth muscle cells in offspring.

As a common complication of pregnancy, gestational hypoxia has been shown to predispose offspring to vascular dysfunction. Propionate, one of short‐chain fatty acids, exerts cardioprotective effects via reducing blood pressure. This study examined whether prenatal hypoxia impaired propionate‐stimulated large‐conductance Ca2+‐activated K+ (BK) channel activities in vascular smooth muscle cells (VSMCs) of offspring. Pregnant rats were exposed to hypoxia (10.5% oxygen) and normoxia (21% oxygen) from gestational day 7‐21. At 6 weeks of age, VSMCs in mesenteric arteries of offspring were analysed for BK channel functions and gene expressions. It was shown firstly that propionate could open significantly BK single channel in VSMCs in a concentration‐dependent manner. Antagonists of G protein βγ subunits and inositol trisphosphate receptor could completely suppress the activation of BK by propionate, respectively. Gαi/o and ryanodine receptor were found to participate in the stimulation on BK. Compared to the control, vasodilation and increments of BK NPo (the open probability) evoked by propionate were weakened in the offspring by prenatal hypoxia with down‐regulated Gβγ and PLCβ. It was indicated that prenatal hypoxia inhibited propionate‐stimulated BK activities in mesenteric VSMCs of offspring via reducing expressions of Gβγ and PLCβ, in which endoplasmic reticulum calcium release might be involved.

Differential targeting and signalling of voltage-gated T-type Cav 3.2 and L-type Cav 1.2 channels to ryanodine receptors in mesenteric arteries.

In arterial smooth muscle, Ca2+ sparks are elementary Ca2+ -release events generated by ryanodine receptors (RyRs) to cause vasodilatation by opening maxi Ca2+ -sensitive K+ (BKCa ) channels. This study elucidated the contribution of T-type Cav 3.2 channels in caveolae and their functional interaction with L-type Cav 1.2 channels to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs). Our data demonstrate that L-type Cav 1.2 channels provide the predominant Ca2+ pathway for the generation of Ca2+ sparks in murine arterial VSMCs. T-type Cav 3.2 channels represent an additional source for generation of VSMC Ca2+ sparks. They are located in pit structures of caveolae to provide locally restricted, tight coupling between T-type Cav 3.2 channels and RyRs to ignite Ca2+ sparks. Recent data suggest that T-type Cav 3.2 channels in arterial vascular smooth muscle cells (VSMCs) and pits structure of caveolae could contribute to elementary Ca2+ signalling (Ca2+ sparks) via ryanodine receptors (RyRs) to cause vasodilatation. While plausible, their precise involvement in igniting Ca2+ sparks remains largely unexplored. The goal of this study was to elucidate the contribution of caveolar Cav 3.2 channels and their functional interaction with Cav 1.2 channels to trigger Ca2+ sparks in VSMCs from mesenteric, tibial and cerebral arteries. We used tamoxifen-inducible smooth muscle-specific Cav 1.2-/- (SMAKO) mice and laser scanning confocal microscopy to assess Ca2+ spark generation in VSMCs. Ni2+ , Cd2+ and methyl-β-cyclodextrin were used to inhibit Cav 3.2 channels, Cav 1.2 channels and caveolae, respectively. Ni2+ (50μmolL-1 ) and methyl-β-cyclodextrin (10mmolL-1 ) decreased Ca2+ spark frequency by ∼20-30% in mesenteric VSMCs in a non-additive manner, but failed to inhibit Ca2+ sparks in tibial and cerebral artery VSMCs. Cd2+ (200μmolL-1 ) suppressed Ca2+ sparks in mesenteric arteries by ∼70-80%. A similar suppression of Ca2+ sparks was seen in mesenteric artery VSMCs of SMAKO mice. The remaining Ca2+ sparks were fully abolished by Ni2+ or methyl-β-cyclodextrin. Our data demonstrate that Ca2+ influx through CaV 1.2 channels is the primary means of triggering Ca2+ sparks in murine arterial VSMCs. CaV 3.2 channels, localized to caveolae and tightly coupled to RyR, provide an additional Ca2+ source for Ca2+ spark generation in mesenteric, but not tibial and cerebral, arteries.

Angiotensin II Promotes KV7.4 Channels Degradation Through Reduced Interaction With HSP90 (Heat Shock Protein 90).

Voltage-gated Kv7.4 channels have been implicated in vascular smooth muscle cells' activity because they modulate basal arterial contractility, mediate responses to endogenous vasorelaxants, and are downregulated in several arterial beds in different models of hypertension. Angiotensin II (Ang II) is a key player in hypertension that affects the expression of several classes of ion channels. In this study, we evaluated the effects of Ang II on the expression and function of vascular Kv7.4. Western blot and quantitative polymerase chain reaction revealed that in whole rat mesenteric artery, Ang II incubation for 1 to 7 hours decreased Kv7.4 protein expression without reducing transcript levels. Moreover, Ang II decreased XE991 (Kv7)-sensitive currents and attenuated membrane potential hyperpolarization and relaxation induced by the Kv7 activator ML213. Ang II also reduced Kv7.4 staining at the plasma membrane of vascular smooth muscle cells. Proteasome inhibition with MG132 prevented Ang II-induced decrease of Kv7.4 levels and counteracted the functional impairment of ML213-induced relaxation in myography experiments. Proximity ligation assays showed that Ang II impaired the interaction of Kv7.4 with the molecular chaperone HSP90 (heat shock protein 90), enhanced the interaction of Kv7.4 with the E3 ubiquitin ligase CHIP (C terminus of Hsp70-interacting protein), and increased Kv7.4 ubiquitination. Similar alterations were found in mesenteric vascular smooth muscle cells isolated from Ang II-infused mice. The effect of Ang II was emulated by 17-AAG (17-demethoxy-17-(2-propenylamino) geldanamycin) that inhibits HSP90 interactions with client proteins. These results show that Ang II downregulates Kv7.4 by altering protein stability through a decrease of its interaction with HSP90. This leads to the recruitment of CHIP and Kv7.4 ubiquitination and degradation via the proteasome.

Vascular dysfunction and fibrosis in stroke-prone spontaneously hypertensive rats: The aldosterone-mineralocorticoid receptor-Nox1 axis

AimsWe questioned whether aldosterone and oxidative stress play a role in vascular damage in severe hypertension and investigated the role of Nox1 in this process. Materials and methodsWe studied mesenteric arteries, aortas and vascular smooth muscle cells (VSMC) from WKY and SHRSP rats. Vascular effects of eplerenone or canrenoic acid (CA) (mineralocorticoid receptor (MR) blockers), ML171 (Nox1 inhibitor) and EHT1864 (Rac1/2 inhibitor) were assessed. Nox1-knockout mice were also studied. Vessels and VSMCs were probed for Noxs, reactive oxygen species (ROS) and pro-fibrotic/inflammatory signaling. Key findingsBlood pressure and plasma levels of aldosterone and galectin-3 were increased in SHRSP versus WKY. Acetylcholine-induced vasorelaxation was decreased (61% vs 115%) and phenylephrine-induced contraction increased in SHRSP versus WKY (Emax 132.8% vs 96.9%, p<0.05). Eplerenone, ML171 and EHT1864 attenuated hypercontractility in SHRSP. Vascular expression of collagen, fibronectin, TGFβ, MCP-1, RANTES, MMP2, MMP9 and p66Shc was increased in SHRSP versus WKY. These changes were associated with increased ROS generation, 3-nitrotyrosine expression and Nox1 upregulation. Activation of vascular p66Shc and increased expression of Nox1 and collagen I were prevented by CA in SHRSP. Nox1 expression was increased in aldosterone-stimulated WKY VSMCs, an effect that was amplified in SHRSP VSMCs (5.2vs9.9 fold-increase). ML171 prevented aldosterone-induced VSMC Nox1-ROS production. Aldosterone increased vascular expression of fibronectin and PAI-1 in wild-type mice but not in Nox1-knockout mice. SignificanceOur findings suggest that aldosterone, which is increased in SHRSP, induces vascular damage through MR-Nox1-p66Shc-mediated processes that modulate pro-fibrotic and pro-inflammatory signaling pathways.

Purinergic signalling in mesenteric vascular smooth muscle in a mice model of essential hypertension

In vascular smooth muscle cells (VSMCs) non‐selective cation channels such as TRPC3 and TRPC6 have been proposed as the molecular constituents of receptor‐(ROCs) and stretch‐(SOCs) operated channels linking DAG signaling cascade pathway with increased vascular tone. The membrane depolarization caused by TRPCs can increase Ca2+ entry leading to augmented [Ca2+]I and VSMC contraction. Therefore, by influencing membrane potential, TRPC channels could play an important role controlling the increased vascular tone present in several pathologies, such as essential hypertension. We have previously described an increased functional expression of TRPC3 channels in mesenteric VSMCs from hypertensive mice that can contribute to increased vascular tone by favouring depolarization (Alvarez‐Miguel et al., J Physiol, in press). As several mechanosensitive G‐protein coupled receptors (GPCR) are linked to the DAG‐dependent activation of TRPC3/6 channels, we explore here the contribution of this signalling pathway to the increased vascular tone in hypertension.Using a mouse model of essential hypertension (BPH mice) and its normotensive control (BPN mice) we explored changes in the molecular components of the mechanotransduction pathway in hypertension. We analized the expression of mechanosensitive ion channels and some VSMCs sensor elements, including GPCRs, channels and transporters with qPCR. Their functional contribution was studied in isolated VSMCs (using electrophysiology) and in intact mesenteric arteries with pressure myography.Real‐time PCR showed an increased expression of some of the elements of the purinergic signalling pathway, including purinergic receptors P2Y2 and P2Y6, TRPC3 channels and the Ca2+‐activated Cl− channel Clca1. Pressure myography of mesenteric arteries after endothelium removal showed a vasoconstriction response to ATP, UTP and UDP. ATP‐induced vasoconstriction was not different between BPN and BPH arteries. The EC50 of the effect was around 7–10 μM in both preparations, and it could be blocked by α‐β‐methilene‐ATP. UTP‐induced vasoconstriction was not affected by α‐β‐methilene‐ATP and was significantly larger in BPH arteries: 50 μM UTP‐elicited vasoconstriction represented 40% of the maximal response to ATP in BPN arteries and 150% in BPH arteries. Our data indicate that mesenteric vascular reactivity to ATP is not altered in our hypertension model, but there is an increased contribution of the purinergic GPCR signalling pathway to the increased vascular tone. Together with the observed upregulation of P2YR receptors in BPH VSMCs, the augmented functional expression of TRPC3 channels could participate in the increased vasoconstriction observed in response to uridine nucleotides in BPH mesenteric arterieSupport or Funding InformationSupported by grants ISCIII‐FEDER RD12/0042/0006, (Heracles Program), BFU2013‐45867‐R (MINECO) and by a Junta de Castilla y Leon fellowship to IAM

Activation of TRPM3 in Perivascular Sensory Nerves Induces Dilation of Mouse Resistance Arteries

The vascular tone is determined by a complex interplay of vasodilator and vasoconstrictor stimuli that modulate the contractile state of vascular smooth muscle cells (VSMCs). Activation of the cation channel Transient Receptor Potential Melastatin 3 (TRPM3) has been shown to induce contraction of VSMCs in aorta. However, the contribution of this channel to the vascular tone in resistance arteries remains unknown. Real-time qPCR and immunochemistry experiments showed Trpm3 expression in mesenteric arteries isolated from wild type (WT) C57BL/6J mice. Myography experiments carried out in intact pressurized mesenteric arteries from WT mice showed that the TRPM3 agonist PS induces vasodilation at concentrations above ∼5 µM, with a concentration-dependency featuring two distinct increasing phases. In contrast, PS only induced vasodilation above 10 µM following a single Hill-type behavior in preparations from Trpm3 knockout (KO) mice. Recordings in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096 recapitulated the results of Trpm3 KO preparations, indicating that the TRPM3-mediated effect of PS entails CGRP release from perivascular nerve endings. The effect of 10 µM PS was inhibited to about 50% by the combination of potassium channel blockers (500 nM paxilline, 10 µM correolide and 50 nM stromatoxin). Electrophysiological recordings in freshly isolated mesenteric VSMCs, revealed that basal currents are not sensitive to PS (10 µM and 30 µM) and that PS has no effect on potassium currents. Our data indicates that activation of TRPM3 channels in perivascular sensory nerves induces CGRP release, which leads to activation of potassium channels in smooth muscle cells, resulting in dilation of mesenteric arteries. These findings reveal a potential role of TRPM3 in vascular tone regulation, and support the recent notion that this channel may play roles in neurogenic inflammation.

Differences in TRPC3 and TRPC6 channels assembly in mesenteric vascular smooth muscle cells in essential hypertension.

Canonical transient receptor potential (TRPC)3 and TRPC6 channels of vascular smooth muscle cells (VSMCs) mediate stretch- or agonist-induced cationic fluxes, contributing to membrane potential and vascular tone. Native TRPC3/C6 channels can form homo- or heterotetrameric complexes, which can hinder individual TRPC channel properties. The possibility that the differences in their association pattern may change their contribution to vascular tone in hypertension is unexplored. Functional characterization of heterologously expressed channels showed that TRPC6-containing complexes exhibited Pyr3/Pyr10-sensitive currents, whereas TRPC3-mediated currents were blocked by anti-TRPC3 antibodies. VSMCs from hypertensive (blood pressure high; BPH) mice have larger cationic basal currents insensitive to Pyr10 and sensitive to anti-TRPC3 antibodies. Consistently, myography studies showed a larger Pyr3/10-induced vasodilatation in BPN (blood pressure normal) mesenteric arteries. We conclude that the increased TRPC3 channel expression in BPH VSMCs leads to changes in TRPC3/C6 heteromultimeric assembly, with a higher TRPC3 channel contribution favouring depolarization of hypertensive VSMCs. Increased vascular tone in essential hypertension involves a sustained rise in total peripheral resistance. A model has been proposed in which the combination of membrane depolarization and higher L-type Ca2+ channel activity generates augmented Ca2+ influx into vascular smooth muscle cells (VSMCs), contraction and vasoconstriction. The search for culprit ion channels responsible for membrane depolarization has provided several candidates, including members of the canonical transient receptor potential (TRPC) family. TRPC3 and TRPC6 are diacylglycerol-activated, non-selective cationic channels contributing to stretch- or agonist-induced depolarization. Conflicting information exists regarding changes in TRPC3/TRPC6 functional expression in hypertension. However, although TRPC3-TRPC6 channels can heteromultimerize, the possibility that differences in their association pattern may change their functional contribution to vascular tone is largely unexplored. We probe this hypothesis using a model of essential hypertension (BPH mice; blood pressure high) and its normotensive control (BPN mice; blood pressure normal). First, non-selective cationic currents through homo- and heterotetramers recorded from transfected Chinese hamster ovary cells indicated that TRPC currents were sensitive to the selective antagonist Pyr10 only when TRPC6 was present, whereas intracellular anti-TRPC3 antibody selectively blocked TRPC3-mediated currents. In mesenteric VSMCs, basal and agonist-induced currents were more sensitive to Pyr3 and Pyr10 in BPN cells. Consistently, myography studies showed a larger Pyr3/10-induced vasodilatation in BPN mesenteric arteries. mRNA and protein expression data supported changes in TRPC3 and TRPC6 proportions and assembly, with a higher TRPC3 channel contribution in BPH VSMCs that could favour cell depolarization. These differences in functional and pharmacological properties of TRPC3 and TRPC6 channels, depending on their assembly, could represent novel therapeutical opportunities.

Homocysteine regulates endothelin type B receptors in vascular smooth muscle cells

Vascular smooth muscle endothelin type B (ETB) receptor is involved in the pathogenesis of cardiovascular diseases (CVDs). Hyperhomocysteinemia is an independent risk factor for CVDs. The present study was designed to examine the hypothesis that homocysteine (Hcy) up-regulates vascular smooth muscle ETB receptors. In vitro experiments were performed in rat superior mesenteric artery (SMA) and vascular smooth muscle cells (VSMCs). The rat SMA or VSMCs were cultured in serum-free medium for 24h in the presence and absence of Hcy with or without specific inhibitors for the ERK1/2 signaling pathway and NF-κB. In vivo, the rats received subcutaneous injections of Hcy in the presence or absence of specific inhibitors for the ERK1/2 signaling pathway (U0126) for 3weeks. Levels of protein expression were determined using Western blot analysis. The contractile responses to sarafotoxin 6c (an ETB receptor agonist) were studied using a sensitive myograph. The blood pressure of the rats was measured via a noninvasive tail-cuff plethysmography method. The results from in vitro experiments showed that Hcy concentration-dependently increased the ETB receptor-mediated contractile responses, and up-regulated ETB receptor expression, in rat SMA. Blockage of the ERK1/2 signaling pathway and NF-κB using the MEK1/2 inhibitor (PD98059 and U0126) or IκB kinase inhibitor (wedelolactone) significantly abolished Hcy-induced up-regulation of ETB receptor. Finally, we used VSMCs as a cellular model to further validate our finding. In vivo study found that hyperhomocysteinemia up-regulated ETB receptor expression, and elevated the blood pressure of rats via the ERK1/2 signaling pathway. In conclusion, Hcy up-regulated vascular smooth muscle ETB receptor via activation of the ERK1/2 signaling pathway and NF-κB.

Role of miR-124 and miR-141 in the regulation of vascular reactivity and the relationship to RhoA and Rac1 after hemorrhage and hypoxia

Recent studies show that hypoxia can alter expression levels of microRNAs (miRNAs). Whether hypoxia or hemorrhage-induced vascular hyporeactivity is related to miRNAs and the underlying mechanisms of this process is not clear. Using hypoxia-treated superior mesenteric arteries (SMAs) and vascular smooth muscle cells (VSMCs) of rats that underwent hemorrhage, we observed the regulatory effects of miR-124/miR-141 on vascular reactivity, the relationship of these miRNAs to RhoA and Rac1, and the mutual regulation of miR-124 and miR-141. The contractile responses of SMAs and VSMCs showed an increase in early stages and a decrease in late stages of hypoxia and hemorrhage. Forty-five miRNAs appeared to have been significantly changed in SMAs after hypoxia, and miR-124 and miR-141 underwent the most change. Overexpressed miR-124 or miR-141 and their antisenses appeared to alter both vascular reactivity and expression of the proteins RhoA and Rac1 after hypoxia. miR-124 inhibited Rac1 by acting at the Rac1 mRNA 3'-untranslated region (UTR), but it led to an increase in RhoA by inhibiting miR-141. miR-141 inhibited RhoA by acting at the RhoA mRNA 3'-UTR, but it led to an increase in Rac1 by inhibiting miR-124. Further study found that miR-124 inhibited miR-141 via transcription factor early growth response gene-1 (Egr-1), whereas miR-141 inhibited miR-124 via transcription of nuclear factor erythroid 2-related factor 2 (Nrf-2). These results suggest that miR-124 and miR-141 participate in the regulation of vascular reactivity after hypoxia and hemorrhage by regulating expression of the RhoA and Rac1 proteins, and in doing so, miR-124 and miR-141 are mutually regulated. These findings provide potential targets for restoring vascular function as part of the treatment protocol for hemorrhagic shock and some other critical illness.

Inhibition of TLR4 attenuates vascular dysfunction and oxidative stress in diabetic rats.

Hyperglycemia-induced reactive oxygen species (ROS) production plays a major role in the pathogenesis of diabetic vascular dysfunction. However, the underlying mechanisms remain unclear. Toll-like receptor 4 (TLR4), a key component of innate immunity, is known to be activated during diabetes. Therefore, we hypothesize that hyperglycemia activates TLR4 signaling in vascular smooth muscle cells (VSMCs) that triggers ROS production and causes vascular dysfunction. Rat mesenteric VSMCs exposed to high glucose (25 mmol/l) increased TLR4 expression and activated TLR4 signaling via upregulation of myeloid differentiation factor 88 (MyD88). TLR4 inhibitor CLI-095 significantly attenuated elevated levels of ROS and nuclear factor-kappa B (NF-κB) activity in VSMCs exposed to high glucose. Mesenteric arteries from streptozotocin-induced diabetic rats treated with CLI-095 (2 mg/kg/day) intraperitoneally for 2 weeks exhibited reduced ROS generation and attenuated noradrenaline-induced contraction. These results suggest that hyperglycemia-induced ROS generation and NF-κB activation in VSMCs are at least, in part, mediated by TLR4 signaling. Therefore, strategies to block TLR4 signaling pathways pose a promising avenue to alleviate diabetic-induced vascular complications. High glucose-induced TLR4 activation in vascular smooth muscle cells. Inhibition of TLR4 attenuated high glucose-induced ROS production and NF-κB activity in VSMC. Suppression of TLR4 signaling attenuated mesenteric contraction in diabetic rat.

Cysteinyl leukotriene 1 receptors as novel mechanosensors mediating myogenic tone together with angiotensin II type 1 receptors-brief report.

Myogenic vasoconstriction is mediated by vascular smooth muscle cells of resistance arteries sensing mechanical stretch. Angiotensin II AT1 receptors and in particular AT1BRs in murine vascular smooth muscle cells have been characterized as mechanosensors that cannot fully account for myogenic vasoconstriction observed. Therefore, we aimed at uncovering novel vascular mechanosensors by expression profiling and functional characterization of candidate proteins. Analyzing myogenic tone of isolated murine mesenteric arteries of AT1A and AT1B receptor double gene-deficient (AT1A/1B (-/-)) mice ex vivo, we observed a decreased myogenic tone at high intraluminal pressures and an unexpected hyper-reactivity at low intraluminal pressures because of upregulation of cysteinyl leukotriene 1 receptors (CysLT1Rs). Pharmacological blockade of CysLT1Rs with pranlukast significantly reduced myogenic tone not only in AT1A/1B (-/-) but also in wild-type arteries. In wild-type arteries, additional blockade of angiotensin II AT1 receptors with candesartan resulted in an additive reduction of myogenic tone. Furthermore, calcium imaging experiments were performed with fura-2-loaded human embryonic kidney 293 cells overexpressing CysLT1Rs and with isolated mesenteric vascular smooth muscle cells. Hypo-osmotically induced membrane stretch provoked calcium transients that were significantly reduced by pranlukast. Incubations of isolated mesenteric vascular smooth muscle cells with the 5-lipoxygenase inhibitor zileuton had no effect. Furthermore, the Gq/11-protein inhibitor YM 254890 profoundly reduced myogenic tone to the same extent as induced by the application of pranlukast plus candesartan. Here, we identify a novel, hitherto unappreciated role of CysLT1Rs in vascular regulation. We identified CysLT1Rs as novel mechanosensors in the vasculature involved in myogenic vasoconstriction. Moreover, our findings suggest that myogenic tone is determined by AT1 and CysLT1 receptors acting together as mechanosensors via Gq/11-protein activation.

Mitochondrial regulation of NADPH oxidase in hindlimb unweighting rat cerebral arteries.

Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU) rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001) but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001) in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001), decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) and the activity of NADPH oxidase (P<0.001) in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation.

Phenotypic Modulation of Mesenteric Vascular Smooth Muscle Cells from Type 2 Diabetic Rats is Associated with Decreased Caveolin-1 Expression

Aims: Diabetes-induced vascular complications are associated with vascular smooth muscle cell (VSMC) phenotypic modulation, switching from a contractile to a synthetic-proliferative phenotype. Loss of caveolin-1 is involved with proliferation of VSMCs. We tested the hypothesis that mesenteric VSMCs from type 2 diabetic Goto-Kakizaki (GK) rat undergo phenotypic modulation and it is linked to decreased caveolin-1 expression. Methods: VSMCs were isolated from mesenteric arteries from GK rats and age-matched control Wistar rats. Western blotting was used to determine expression of target proteins such as caveolin-1, calponin (marker of differentiation), and proliferating cell nuclear antigen (PCNA, marker of proliferation). In addition, we measured intracellular reactive oxygen species (ROS) production using H2DCF-DA and activation of extracellular signal-regulated kinase (ERK1/2) by western blotting in VSMCs from GK stimulated with lipopolysaccharide (LPS), an endotoxin upregulated in diabetes. Results: Mesenteric VSMCs from diabetic GK rats exhibited decreased caveolin-1 and calponin expression and increased PCNA expression compared to control. Increased levels of ROS and phospho-ERK1/2 expression were also found in GK VSMCs. LPS augmented ROS and phosphorylated ERK1/2 levels to a greater extent in GK VSMCs than in control. Likewise, high glucose decreased caveolin-1 and calponin expression, increased PCNA expression and augmented ROS production in control mesenteric VSMCs. Conclusion: These results suggest that mesenteric VSMCs from diabetic GK rats undergo phenotypic modulation and it is associated with decreased caveolin-1 expression. These alterations may be due to enhanced inflammatory stimuli and glucose levels present in diabetic milieu.

Differential Control of Calcium Homeostasis and Vascular Reactivity by Ca 2+ /Calmodulin-Dependent Kinase II

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 ( I Ca ), cytoplasmic and sarcoplasmic reticulum Ca 2+ , and vasoconstriction in mesenteric arteries. In mesenteric VSMC, CaMKII inhibition significantly reduced action potential duration and the residual I Ca 50 ms after peak amplitude, indicative of loss of L-type Ca 2+ channel–dependent I Ca 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.

Adiponectin vasodilates small arteries by increasing BK activity

We have previously shown that human perivascular adipose tissue (PVAT) vasodilates adjacent arteries, in part by release of adiponectin. This study was designed to further elucidate this adipose‐coupling at a cellular level. Adiponectin caused a small vasodilation of 3rd order mesenteric arteries constricted with pressure‐induced myogenic tone. The vasodilation was substantially reduced by pre‐incubation of arteries with ryanodine (Apn: 5.5%±0.7 vs Apn&amp;Ryr: 2.2%±1, p=0.03). Confocal microscopy of Fluo‐4 loaded mesenteric arteries showed that the presence of intact PVAT increased vascular smooth muscle cell (VSMC) calcium (Ca) spark frequency in mesenteric arteries. However, there was no effect on VSMC Ca spark frequency following incubation with adiponectin. Using patch clamp of mesenteric VSMC, we found that adiponectin significantly increased the BK current when cells were incubated with ryanodine (20μM). Consistent with this, adiponectin increased amplitude but not frequency of spontaneous transient outward currents in untreated cells. Adiponectin had no effect on the Kv component of the whole cell current. It is concluded that adiponectin vasodilates small arteries predominantly by modulating VSMC BK activity, independently of Ca sparks. Intact PVAT appears to have an additive effect by increasing Ca spark frequency, suggesting the contribution of alternative adipose derived vasodilators.

TNFα receptor 1 causes endocytosis‐dependent NF‐κB and ‐ independent JNK activation in vascular smooth muscle cells

Tumor necrosis factor‐α (TNFα), a pro‐inflammatory cytokine can cause either vascular smooth muscle cell (VSMC) apoptosis (JNK‐dependent) or proliferation (NF‐κB‐dependent). We hypothesized that TNFα receptor 1 (TNFR1) endocytosis balances these two pathways. We quantified NF‐κB activation and MAPK (JNK, p38, ERK1/2) phosphorylation by TNFα in the presence of siRNA targeting TNFR1 or 2 in cultured murine mesenteric VSMC. Both NF‐κB activation (Fig. 1, n=3–4) and JNK phosphorylation are TNFR1‐dependent, and NF‐κB activation is potentiated by JNK inhibition (SP600125, 3μM). Impairing endocytosis (Dynasore, 15μM) reduced NF‐κB activation by TNFα (1.5‐fold, n=5) and prevented this effect of SP600125 (2.2‐fold, n=4). TNFα also caused phosphorylation of p38 and ERK1/2. JNK and ERK1/2 phosphorylation were TNFR1‐dependent while p38 activation was altered by both TNFR1 and 2 siRNA. Thus, TNFR1 activates both NF‐κB and JNK activation and JNK modulates endocytosis‐dependent NF‐κB activation in VSMC.

Myogenic tone is impaired at low arterial pressure in mice deficient in the low‐voltage‐activated CaV3.1 T‐type Ca2+ channel

Using mice deficient in the CaV 3.1 T-type Ca(2+) channel, the aim of the present study was to elucidate the molecular identity of non-L-type channels involved in vascular tone regulation in mesenteric arteries and arterioles. We used immunofluorescence microscopy to localize CaV 3.1 channels, patch clamp electrophysiology to test the effects of a putative T-type channel blocker NNC55-0396 on whole-cell Ca(2+) currents, pressure myography and Ca(2+) imaging to test diameter and Ca(2+) responses of the applied vasoconstrictors, and Q-PCR to check mRNA expression levels of several Ca(2+) handling proteins in wild-type and CaV 3.1(-/-) mice. Our data indicated that CaV 3.1 channels are important for the maintenance of myogenic tone at low pressures (40-80mmHg), whereas they are not involved in high-voltage-activated Ca(2+) currents, Ca(2+) entry or vasoconstriction to high KCl in mesenteric arteries and arterioles. Furthermore, we show that NNC55-0396 is not a specific T-type channel inhibitor, as it potently blocks L-type and non-L-type high-voltage-activated Ca(2+) currents in mouse mesenteric vascular smooth muscle cell. Our data using mice deficient in the CaV 3.1 T-type channel represent new evidence for the involvement of non-L-type channels in arteriolar tone regulation. We showed that CaV 3.1 channels are important for the myogenic tone at low arterial pressure, which is potentially relevant under resting conditions in vivo. Moreover, CaV 3.1 channels are not involved in Ca(2+) entry and vasoconstriction to large depolarization with, for example, high KCl. Finally, we caution against using NNC 55-0396 as a specific T-type channel blocker in native cells expressing high-voltage-activated Ca(2+) channels.

High blood pressure associates with the remodelling of inward rectifier K+ channels in mice mesenteric vascular smooth muscle cells

The increased vascular tone that defines essential hypertension is associated with depolarization of vascular smooth muscle cells (VSMCs) and involves a change in the expression profile of ion channels promoting arterial contraction. As a major regulator of VSMC resting membrane potential (V(M)), K(+) channel activity is an important determinant of vascular tone and vessel diameter. However, hypertension-associated changes in the expression and/or modulation of K(+) channels are poorly defined, due to their large molecular diversity and their bed-specific pattern of expression. Moreover, the impact of these changes on the integrated vessel function and their contribution to the development of altered vascular tone under physiological conditions need to be confirmed. Hypertensive (BPH) and normotensive (BPN) mice strains obtained by phenotypic selection were used to explore whether changes in the functional expression of VSMC inward rectifier K(+) channels contribute to the more depolarized resting V(M) and the increased vascular reactivity of hypertensive arteries. We determined the expression levels of inward rectifier K(+) channel mRNA in several vascular beds from BPN and BPH animals, and their functional contribution to VSMC excitability and vascular tone in mesenteric arteries. We found a decrease in the expression of Kir2.1, Kir4.1, Kir6.x and SUR2 mRNA in BPH VSMCs, and a decreased functional contribution of both K(IR) and K(ATP) channels in isolated BPH VSMCs. However, only the effect of K(ATP) channel modulators was impaired when exploring vascular tone, suggesting that decreased functional expression of K(ATP) channels may be an important element in the remodelling of VSMCs in essential hypertension.