Myogenic Tone In Resistance Arteries Research Articles

Protection against angiotensin II-induced hypertension in mice lacking sphingosine kinase 1 in vascular smooth muscle cells

Abstract Background Sphingosine-1-phosphate (S1P), a lipid mediator produced by sphingosine kinases (Sphks), significantly contributes to the maintenance of cardiovascular homeostasis. Mice lacking Sphk1 globally are protected against angiotensin II-induced hypertension, yet, due to pleiotropic action of S1P, it has been challenging to identify a specific cell type responsible for this protection. Purpose The aim of this study was to investigate the role of Sphk1 expressed in vascular smooth muscle cells (VSMCs) in the development of angiotensin II (AngII)-induced hypertension in mice. Methods Male, 12-week-old C57BL/6J wild type (WT) or VSMC-specific, Cre-mediated, Sphk1 knockout (Sphk1VSMC−/−) mice underwent either Sham or AngII (490 ng/min/kg s.c.) treatment for 2 weeks, using a surgically implanted osmotic minipump. Blood pressure was measured by the tail-cuff method. Vascular structure and function were examined ex vivo using wire (vasoconstrictor/vasorelaxant responses) and pressure myography (myogenic tone, structural and elasticity parameters). Gene set enrichment analysis (GSEA) was performed using transcriptomic profiles of the isolated mesenteric arteries (MA, RNASeq). Expression of proteins was analyzed by Western blot. Results Deletion of Sphk1 in VSMCs resulted in 5x and 40x lower expression of Sphk1 mRNA in MA and aorta compared to WT mice respectively. A 2-week infusion of AngII resulted in a development of higher systolic blood pressure (SBP) in WT mice compared to Sphk1VSMC−/− mice (mean SBP ± SEM: 168±4.3 mmHg vs. 132±4.4 mmHg respectively, p<0.05). Studies of ex vivo mesenteric artery (MA) function revealed diminished both, depolarization-induced (in response to KCl), as well as G protein-coupled receptor-elicited (in response to phenylephrine and thromboxane A2 analogue) contraction of MA derived from hypertensive Sphk1VSMC−/− mice compared to WT mice. In the same time, significantly impaired endothelial vasorelaxation was observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice. Moreover, MA from Sphk1VSMC−/− mice were characterized by the significantly reduced myogenic response evoked by intravascular pressure elevation (maximal myogenic response ± SEM: 19.7±1.8% for WT normotensive, 26.2±1.8% for WT AngII vs. 19.0±2.5% for Sphk1VSMC−/− AngII mice), and an increased stiffness compared to WT mice. GSEA found that the lack of Sphk1 gene in VSMCs significantly downregulated molecular pathways related to smooth muscle contraction. In line with transcriptomic studies, significantly reduced level of the Rho-associated protein kinase (Rock) has been observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice using Western blot. Conclusions This study revealed that mice lacking Sphk1 gene in VSMCs are protected against the development of AngII-induced hypertension likely due to the lowered myogenic tone in resistance arteries, which may be mediated by autocrine effects of Sphk1 on Rock proteins. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The National Science Centre, Poland

Chronic 3D Vascular-Immune Interface Established by Coculturing Pressurized Resistance Arteries and Immune Cells.

[Figure: see text].

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

Increasing prevalence of obesity and its associated risk with cardiovascular diseases demands a better understanding of the contribution of different cell types within this complex disease for developing new treatment options. Previous studies could prove a fundamental role of FTO (fat mass and obesity-associated protein) within obesity; however, its functional role within different cell types is less understood. We identify endothelial FTO as a previously unknown central regulator of both obesity-induced metabolic and vascular alterations. We generated endothelial Fto-deficient mice and analyzed the impact of obesity on those mice. While the loss of endothelial FTO did not influence the development of obesity and dyslipidemia, it protected mice from high-fat diet-induced glucose intolerance and insulin resistance by increasing AKT (protein kinase B) phosphorylation in endothelial cells and skeletal muscle. Furthermore, loss of endothelial FTO prevented the development of obesity-induced hypertension by preserving myogenic tone in resistance arteries. In Fto-deficient arteries, microarray analysis identified upregulation of L-Pgds with significant increases in prostaglandin D2 levels. Blockade of prostaglandin D2 synthesis inhibited the myogenic tone protection in resistance arteries of endothelial Fto-deficient mice on high-fat diet; conversely, direct addition of prostaglandin D2 rescued myogenic tone in high-fat diet-fed control mice. Myogenic tone was increased in obese human arteries with FTO inhibitors or prostaglandin D2 application. These data identify endothelial FTO as a previously unknown regulator in the development of obesity-induced metabolic and vascular changes, which is independent of its known function in regulation of obesity.

Abstract P1120: Opioids Induce Proliferation in Vascular Smooth Muscle Cells Leading to an Increase in Myogenic Tone in Resistance Arteries

The United States of America is currently undergoing an opioid crisis. According to the 2018 CDC Annual Surveillance Report of Drug Related Risks and Outcomes there were 78,840 hospitalizations and an estimated 140,077 emergency room visits for opioid related poisonings in 2015 alone. It has been shown that opioid exposure increases vascular age relative to chronological age. Further, long-term abuse of opioids leads to changes in regional cerebral blood flow. However, there is a lack of understanding about acute and chronic opioid abuse and how it affects microvasculature structure and function; specifically on myogenic tone, a fundamental mechanism to maintain blood flow to important tissues including the brain, kidneys, and more. We hypothesize that opioids cause an increase in proliferation of vascular smooth muscle cells (VSMC) leading to an increase in myogenic tone in mesenteric resistance arteries. To test this hypothesis, aortic vascular smooth muscle cells were cultured and treated with the endogenous opioid (ENO) beta-endorphin or the exogenous opioid (EXO) morphine. ENO treatment increased cellular proliferation with chronic exposure (greater than 72 hours). We measured a maximum proliferation at 120 hours and performed 2 way ANOVA with Bonferroni posttests [confluence (%): DMSO vehicle 20.4 ± 4.8, 1 nM 34.9 ± 7.4*, and 1 μM 29.9 ± 7.0; * p<0.05]. Increased proliferation was also demonstrated in cells treated with EXO. We measured a maximum proliferation at 120 hours and performed 2 way ANOVA with Bonferroni posttests [confluence (%): DMSO vehicle 20.4 ± 4.8, 1 nM 32.6 ± 9.0, 1μM 33.3 ± 10.2*, and 10 μM 29.7 ± 4.4; * p<0.05]. We also observed that when acutely exposing mesenteric resistance arteries to EXO (1.0 μM) there was a left shift in myogenic tone to 80 mmHg as opposed to 100 mmHg in the in absence of EXO. In conclusion, our results suggest that chronic exposure to ENO and EXO increase VSMC proliferation. Further, acute exposure to EXO was able to increase myogenic tone in resistance arteries. This suggests that opioid dependence could contribute to the development of peripheral vascular disorders such as hypertension.

Abstract 058: Novel Mechanisms at Immune-Vascular Interfaces Regulate Myogenic Tone of Resistance Arteries and Induce Hypertension in Mice

Multiple reports recognized that immunity participates in hypertension (HTN), although direct mechanisms that orchestrate the immune-vascular interaction are still unknown. Mice infused with AngiotensinII (AngII) typically increase blood pressure (BP), concomitantly recruiting immune cells in the vasculature, with CD8 T cells playing a crucial role. To investigate the molecular regulators of vascular-immune interface, we established a system of vessel organ culture, enabling the study of physiological properties of resistance arteries via a modified pressure myograph which keeps vessels alive and functional for several days, while co-culturing immune cells of interest. We tested potential direct effects of CD8 T cells isolated from AngII-HTN or vehicle mice on resistance arteries from naïve mice. After co-incubation in the organ culture, CD8 T cells from HTN mice significantly increased myogenic tone (MT) of arteries, while no effect was induced by CD8 T of vehicle mice (%MT=CD8 AngII :27±2; CD8 veh :16±2, p<0.01). Our previous studies showed that an intracellular signaling involved in the acquisition of CD8 effectors functions, phosphatidylinositol-3-kinase-γ (PI3Kγ), was also involved in AngII-induced HTN, being its inhibition protective toward increase in BP and in vascular resistance. Here we took advantage of a mouse model expressing constitutively active PI3Kγ (PI3Kγ CX ), to test immune functions of PI3Kγ in HTN. PI3Kγ CX showed a spontaneous hypertensive phenotype (SBP mmHg :129±1 vs WT:104±2, p<0.001) accompanied by infiltration of activated CD8 T cells in renal vasculature. Then, CD8 T cells isolated from PI3Kγ CX mice and placed in co-culture with WT arteries increased their MT. Conversely, the CD8 T cells from WT mice had no effect when cultured with vessels (%MT=CD8 CX :31±1; CD8 WT :19±1, p<0.001). To test the in vivo relevance, we performed an adoptive transfer of CD8 CX in WT mice, finding that they developed spontaneous HTN, while no effect was induced by CD8 WT (SBP mmHg : CD8 CX 128±2 vs CD8 WT 103±2, p<0.001). Taken together these data show that CD8 T cells from AngII-HTN mice are able to directly increase MT of resistance arteries and that PI3Kγ signaling is a crucial modulator of this effector function, likely contributing to BP increase.

From pregnancy to cardiovascular disease: Lessons from relaxin-deficient animals to understand relaxin actions in the vascular system.

Early maternal vascular adaptations to pregnancy are predominantly driven by changes in vascular tone, reactivity, and remodeling. Failure of the maternal systemic vasculature to adapt sufficiently can lead to serious complications of pregnancy. The hormone relaxin is widely recognized for its contribution to the essential renal and systemic hemodynamic adaptations in early pregnancy through direct actions on the maternal vasculature. Studies in relaxin gene knockout mice revealed that endogenous relaxin is not only a "pregnancy hormone" but has pleiotropic actions in various tissues in males and non-pregnant females. There is strong interest in relaxin's actions in the vasculature and its utility in the treatment of vascular diseases. Relaxin treatment in rodents for 2-5days or acute intravenous injection enhances endothelium-dependent relaxation and decreases myogenic tone in resistance arteries. These vascular actions are prolonged, even in the absence of circulating relaxin, and are underpinned by the production of endothelium-derived relaxing factors including nitric oxide, endothelium-derived hyperpolarization, and prostacyclin. Relaxin is also capable of remodeling the vascular wall in a variety of blood vessels in disease conditions. Lessons learned in pregnancy research have aided studies investigating the potential therapeutic potential of relaxin in cardiovascular disease.

Abstract 485: Novel Mechanisms at Immune-vascular Interfaces Regulates Myogenic Tone of Resistance Arteries and Induce Hypertension in Mice

In the search for mechanisms regulating the pathophysiological relationship existing between hypertension and immune system, CD8 effector T cells emerge as possible mediators of target organ colonization. However, how the crosstalk at vascular interfaces is established still remains unidentified. Phosphatidylinositol-3-kinase gamma (PI3Kγ) is an intracellular signaling involved in the acquisition of CD8 effectors functions and was previously showed by our group as involved in hypertension. The hypothesis here tested rely on the possibility that PI3Kγ could regulate trafficking of immune cells at vascular interfaces relevant for hypertension. By taking advantage of a knock-in mouse model, expressing a constitutively active PI3Kγ isoform (PI3Kγ CX/CX ), we conceived an experimental setting allowing to assess the direct crosstalk between immune cells and vasculature, obtained by co-culturing CD8 T cells with isolated resistance arteries mounted in pressurized systems. Thus, CD8 isolated from PI3Kγ CX/CX mice were co-cultured for 3 days with WT resistance arteries and, after the incubation period, vascular function was tested by challenging arteries to increasing intraluminal pressure to detect myogenic tone (MT). CD8 T cells with constitutively active PI3Kγ spontaneously increased MT of mesenteric arteries, a behavior typically associated with hypertension. Conversely, the same T cell population from WT mice, had no effect when cultured with vessels. In addition, PI3Kγ CX/CX CD8 infiltrated naïve vessels upon co-culture suggesting an intrinsic ability to colonize resistance districts relevant for hypertension. To test the in vivo relevance of the observed phenotype, we performed an adoptive transfer of PI3Kγ CX/CX CD8 in WT mice, finding that they developed spontaneous hypertension, accompanied by infiltration of effector CD8 in perivascular spaces of resistance districts as kidneys. Taken together these data show that PI3Kγ signaling in CD8 T cells is crucial for their effector functions in target vasculature of hypertension, likely contributing to BP increase by increasing peripheral resistance. These results also suggest translational potential as PI3Kγ inhibitors could modulate the immune response involved in hypertension.

Dynamic regulation of TREK1 gating by Polycystin 2 via a Filamin A-mediated cytoskeletal Mechanism

Mechanosensing is essential for several physiological functions including touch and pain sensations, osmoregulation, and controlling the myogenic tone of resistance arteries. Understanding how mechanosensitive ion channels (MSCs) are gated can provide important information regarding these processes. We have previously demonstrated that during pathological conditions such as polycystic kidney disease, polycystin 2 (TRPP2) inhibits the activity of potassium-selective MSCs through a filamin A-mediated cytoskeletal effect, and renders tubular epithelial cells susceptible to apoptosis. However, the nature of this cytoskeletal inhibition remains poorly understood. In this study we use a combination of electrophysiology, structured illumination microscopy, and fluorescence recovery after photobleaching (FRAP) to examine the dynamic nature of the TRPP2-mediated cytoskeletal inhibition of the potassium-selective MSC TREK1. Our data indicate that this inhibition of MSC activity occurs through an accelerated cytoskeletal inhibition, and ultimately decreases the open probability of the TREK1 channel. These results shed light on a novel mode of regulation of MSCs gating, which may be at play in several physiological functions.

Abstract 064: Transactivation of Egfr by Tgfβ Induces Hypertension by Increasing Arteriolar Myogenic Response

Emilin1 (E1) is a protein of the extracellular matrix regulating TGFβ bioavailability through proTGFβ proteolysis. E1 KO mice are hypertensive with increased TGFβ activation. As E1 is expressed in vessels from embryonic life to adulthood, is still unknown whether the E1 KO phenotype results from a developmental defect or lack of homeostatic role in the adult. To dissect this issue, we used a conditional gene targeting inactivating E1 in smooth muscle cells (SMCs) of adult mice, by the use of E1flox/flox and tamoxifen (TAM) inducible Cre recombinase specific for SMCs. When E1fl/fl mice carrying the Smmhc-CreERT2 were given TAM blood pressure significantly increased (SBP: 123±2 vs basal condition 104±3;***p< 0.001) as well as myogenic tone (MT) of resistance arteries (16.3±0.7 vs basal condition 11.4±0.1 % at 125 mmHg). How increased TGFβ signaling in SMCs could determine an increased MT is still unknown. Relevant to this, we found that the higher TGFβ signaling in E1 KO SMCs stimulates heparin binding epidermal growth factor (HB-EGF) and subsequent transactivation of the EGF receptor, a mechanism typically implied in potentiating MT. When mesenteric resistance arteries from E1 were subjected to step-increases in intraluminal pressure, EGFR inhibition rescued the increased MT. At the molecular level, TGFβ-induced EGFR transactivation resulted into the activation of transient receptor potential classical type 6 (TRPC6) and melastatin type 4 (TRPM4) channels. To put our data into translational perspective, we measured MT of resistance arteries isolated from hypertensive patients and normotensive subjects, finding increased MT (HT 16.4±0.7; NT 11±0.4;***p< 0.001) and TGFβ signaling in the former group. By using a neutralizing anti-TGFβ or an anti-EGFR we found a normalization of the increased MT (HT+anti-TGFβ 11±1.3; NT+anti-TGFβ 11.2±1.2;***p< 0.001 and HT+anti-EGFR 9±0.6; NT+anti-EGFR 10.8±1.1;***p< 0.001), thus confirming the relevance of TGFβ-EGFR pathway in humans. Taken together these data suggest that primary increase of MT induced by TGFβ-EGFR transactivation can cause hypertension and that higher TGFβ signaling and MT are common alterations of resistance arteries of hypertensive patients.

Smooth Muscle Ion Channels and Regulation of Vascular Tone in Resistance Arteries and Arterioles.

Vascular tone of resistance arteries and arterioles determines peripheral vascular resistance, contributing to the regulation of blood pressure and blood flow to, and within the body's tissues and organs. Ion channels in the plasma membrane and endoplasmic reticulum of vascular smooth muscle cells (SMCs) in these blood vessels importantly contribute to the regulation of intracellular Ca2+ concentration, the primary determinant of SMC contractile activity and vascular tone. Ion channels provide the main source of activator Ca2+ that determines vascular tone, and strongly contribute to setting and regulating membrane potential, which, in turn, regulates the open-state-probability of voltage gated Ca2+ channels (VGCCs), the primary source of Ca2+ in resistance artery and arteriolar SMCs. Ion channel function is also modulated by vasoconstrictors and vasodilators, contributing to all aspects of the regulation of vascular tone. This review will focus on the physiology of VGCCs, voltage-gated K+ (KV) channels, large-conductance Ca2+-activated K+ (BKCa) channels, strong-inward-rectifier K+ (KIR) channels, ATP-sensitive K+ (KATP) channels, ryanodine receptors (RyRs), inositol 1,4,5-trisphosphate receptors (IP3Rs), and a variety of transient receptor potential (TRP) channels that contribute to pressure-induced myogenic tone in resistance arteries and arterioles, the modulation of the function of these ion channels by vasoconstrictors and vasodilators, their role in the functional regulation of tissue blood flow and their dysfunction in diseases such as hypertension, obesity, and diabetes. © 2017 American Physiological Society. Compr Physiol 7:485-581, 2017.

Abstract 169: Hypertension Down Regulates Emilin1 in the Extracellular Matrix of Resistance Arteries in Humans and Mice, in Order to Increase the Myogenic Tone Through Overactive TGFβ, Thus Contributing to Blood Pressure Regulation

Emilin1, a protein of the extracellular matrix of vessels, regulates TGFβ activity through proteolysis of the proTGFβ. Emilin1 KO mice, displaying increased TGFβ vascular activation, are hypertensive. As vascular Emilin1 is expressed from embryonic life to adulthood, we questioned whether the hypertensive phenotype results from a developmental defect or lack of a homeostatic role exerted in the adult. Thus, we inactivated Emilin1 in smooth muscle cells (VSMCs) of adult mice, with a tamoxifen inducible Cre -loxP, under the control of the SM myosin heavy chain promoter, finding that mice developed hypertension (HTN) as well as increased myogenic tone (MT) in resistance arteries. In order to evaluate the relevance of this mechanism in the human pathology, we enrolled 20 hypertensive (HT) and 20 normotensive (NT) patients, subjected to primary lumbar hernia neurosurgery, and dissected out resistance arteries from adipose tissue biopsies. We randomized vessels to receive vehicle, neutralizing antibody anti-TGFβ or non-relevant IgG and analyzed the MT. HT had a significantly increased MT, as compared to NT, rescued by the pretreatment with anti-TGFβ antibody. A pretreatment with the non-relevant IgG had no effect. Thus we hypothesized that Emilin1 could be down regulated by HTN to increase MT of resistances through overactive TGFβ, thus further sustaining high blood pressure (BP). In order to unravel this issue, we analyzed Emilin1 expression in the vessels walls of human arteries, finding a significant reduction in HT as compared to NT. We also evaluated Emilin1 expression in the resistance arteries of several murine models of HTN induced by: chronic AngII, DOCA-salt, L-NAME and obesity, finding that even in the experimental setting, HTN reduced Emilin1 in the vessel walls. Overall, we show that overactive TGFβ in VSMC increases the MT of resistance arteries, contributing to BP regulation. The data obtained in resistance arteries from HT and in the different murine models of HTN suggest that the down regulation of Emilin1 realized by HTN is a general mechanism to increase MT thus further sustaining high BP.

Abstract 18266: Overactive Tgfβ Signaling in Smooth Muscle Cells Induces Hypertension in Mice Through Increased Myogenic Tone and is Responsible for Increased Myogenic Tone in Resistance Vessels of Hypertensive Patients

Emilin1 (E1) is a protein of the extracellular matrix that regulates TGFβ activity through proteolysis of the proTGFβ. E1 KO mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1 KO phenotype results from a developmental defect or lack of a homeostatic role exerted in the adult. To dissect this issue, we inactivated E1 in smooth muscle cells (VSMCs) of adult mice, by the use of floxed E1 and CreERT2 [a tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. When Smmhc-CreERT2 E1fl/fl mice were given TAM, blood pressure significantly increased (124±1 vs basal condition 106±1 mmHg) as well as myogenic response in resistance arteries (16.3±0.7 vs basal condition 11.4±0.1 % at 125 mmHg). In order to evaluate the relevance of our findings in the human pathology, we enrolled 20 hypertensive and 20 normotensive patients that underwent neurosurgical intervention for primary lumbar hernia repair and dissected out resistance vessels from skeletal muscle biopsies. We analyzed the myogenic tone of human peripheral resistance vessels in a pressure myograph and randomized vessels to receive vehicle, a neutralizing antibody anti-TGFβ or a preimmune non relevant IgG. As first, we found that hypertensive patients had significantly increased myogenic tone as compared to normotensive (16.6±2.1% vs 10.5±0.4% at 125 mmHg). Moreover, the pretreatment of vessels with anti-TGFβ antibody normalized the myogenic tone of hypertensive patients (10.8±0.6%) to the levels of normotensives (11.8±1.3%). A pretreatment with the non relevant IgG did not modify the myogenic tone of hypertensives (17±0.6%), thus demonstrating that the effect was dependent of an increased TGFβ signaling in vessels of hypertensive patients. Overall, our results suggest that the TGFβ pathway in VSMC, is crucial for the myogenic tone of resistance arteries and is linked to the regulation of blood pressure. The data obtained in peripheral resistance vessels from hypertensive patients, that display an increased myogenic tone dependent on overactive TGFβ signaling, highlight the translational potential of our findings to the human pathology.

Inhibition of Myogenic Tone in Rat Cremaster and Cerebral Arteries by SKA-31, an Activator of Endothelial KCa2.3 and KCa3.1 Channels.

Endothelial KCa2.3 and KCa3.1 channels contribute to the regulation of myogenic tone in resistance arteries by Ca(2+)-mobilizing vasodilatory hormones. To define further the functional role of these channels in distinct vascular beds, we have examined the vasodilatory actions of the KCa channel activator SKA-31 in myogenically active rat cremaster and middle cerebral arteries. Vessels pressurized to 70 mm Hg constricted by 80-100 μm (ie, 25%-45% of maximal diameter). SKA-31 (10 μM) inhibited myogenic tone by 80% in cremaster and ∼65% in middle cerebral arteries, with IC50 values of ∼2 μM in both vessels. These vasodilatory effects were largely prevented by the KCa2.3 blocker UCL1684 and the KCa3.1 blocker TRAM-34 and abolished by endothelial denudation. Preincubation with N(G) nitro L-arginine methyl ester (L-NAME, 0.1 mM) did not affect the inhibitory response to SKA-31, but attenuated the ACh-evoked dilation by ∼45%. Penitrem-A, a blocker of BK(Ca) channels, did not alter SKA-31 evoked vasodilation but did reduce the inhibition of myogenic tone by ACh, the BKCa channel activator NS1619, and sodium nitroprusside. Collectively, these data demonstrate that SKA-31 produces robust inhibition of myogenic tone in resistance arteries isolated from distinct vascular beds in an endothelium-dependent manner.

Genetic interference with peroxisome proliferator-activated receptor γ in smooth muscle enhances myogenic tone in the cerebrovasculature via A Rho kinase-dependent mechanism.

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 γ has emerged as a regulator of vascular tone. We hypothesized that interference with peroxisome proliferator-activated receptor γ in smooth muscle would augment myogenic responses in cerebral arteries. We studied transgenic mice expressing a dominant-negative mutation in peroxisome proliferator-activated receptor γ 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 γ 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.

Abstract 051: The Myogenic Tone Of Resistance Arteries Is Controlled By A Molecular Pathway Driven By Tgfβ Through Emilin1 And Converging On The Regulation Of Trp Channels.

Emilin1 (E1) is a protein of the extracellular matrix that regulates TGFβ bioavailability through proteolysis of the proTGFβ. E1 KO mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1 KO phenotype results from a developmental defect or lack of a homeostatic role in the adult. To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (VSMCs) of adult mice, by the use of floxed E1 and CreERT2 [tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. When E1fl/fl mice carrying the Smmhc-CreERT2 were given TAM blood pressure significantly increased (124±1 vs basal condition 106±1 mmHg) as well as myogenic response in resistance arteries (16.3±0.7 vs basal condition 11.4±0.1 % at 125 mmHg). In order to understand how E1 ablation in VSMCs could functionally determine the increase in myogenic tone, we used an ex vivo approach in resistance arteries, to target the molecular pathway that could regulate intracellular calcium fluxes, the main regulator of myogenic contraction. Experiments performed in isolated VSMCs from E1 KO as compared to WT arteries, suggested the involvement of a ROCE (receptor operated calcium entry) mechanism, whose main final molecular determinant is represented by the TRP channels, known to be regulated by a canonical PLC-DAG pathway, but also by a parallel one controlled by the growth factors signaling cascade. By exploring this pathway, we found that E1 regulates the activity of TRPC3 channels and thus of the myogenic response. The molecular pathway delineated by our studies started from the transactivation of EGFR through metalloproteinases and converged on the Src kinases that increased TRPC3 function and thus calcium entry. In particular, with an in vivo targeting of TRPC3 in E1 KO with an adenovirus carrying a dominant negative mutation, we demonstrated a rescue of the increased myogenic response of E1 KO resistance arteries. Overall our results suggest that this pathway in VSMC, driven by TGFβ and regulated by E1, is crucial for the myogenic tone of resistance arteries and is linked to the regulation of blood pressure.

Diabetes and Microvascular Dysfunction

Microvascular reactivity is an important mechanism that regulates local blood flow and therefore tissue perfusion. The reactivity (vasorelaxation and vasoconstriction) of microvessels is mainly regulated by mechanical factors (shear stress for relaxation and blood pressure for myogenic tone, which is a vasoconstriction of microvessels in response to increase in intraluminal blood pressure) and neurohormones. We previously showed that epidermal growth factor receptor tyrosine kinase (EGFRtk) is an important factor in the regulation of myogenic tone in microvessels. In fact, pressure-induced myogenic tone in resistance arteries is regulated by the mechanism that involves metalloproteinases 2 and 9 activation with subsequent Heparin-BindingEGF release and EGFRtk transactivation [1]. This mechanism appears to be specific to myogenic tone, since the contractions to angiotensin and KCl are independent of EGFRtk transactivation. We previously reported that diabetes is associated with microvascular dysfunction [1,2]. We observed reduced microvascular endothelium-dependent relaxation and aberrant increase in pressure-induced myogenic tone in type 2 diabetes [3,4]. The impairment in microvascular function in type 2 diabetes is, in part, caused by the enhanced EGFRtk activity. In a murine model of type 2 diabetic mice, we found an increase of EGFRtk phosphorylation in microvessels. Interestingly, the inhibition of EGFRtk improves vascular function in type 2 diabetes independently of the diabetes and obesity [4]. We also demonstrated that advanced glycation end products cause microvascular dysfunction in type 2 diabetes through the enhance in oxidative stress [3]. In another study, we demonstrated an increase in poly (ADP-ribose) polymerase 1 (PARP1) and nuclear factor-κB activity in microvessels isolated from type 2 diabetic compared to control [2,5,6]. Interestingly, the inhibition of PARP-1 and NFκB pathways improves microvascular function in type 2 diabetic mice [2,6] indicating that PARP-1 and NFκB are important factors in microvascular dysfunction in type 2 diabetes. The aberrant increase in PARP-1 and NFκB activity is caused by the augmented EGFRtk activity [2,7]. Thus, we determined that the inhibition of EGFRtk activity improved microvascular function by PARP-1 and NFκB-dependent mechanism [2,7] suggesting that EGFRtk is up stream to PARP-1 and NFκB signaling. The aberrant increase in NFκB and PARP-1 activity impairs microvascular function in type 2 diabetes by Sp-1 and COX-2-dependent mechanism. In summary, our studies delineated the mechanism of type 2 diabetes induced microvascular dysfunction. Therefore, the EGFRtk, oxidative stress, NFκB and PARP1 pathways could be potential targets for novel therapeutic strategies to overcome diabetes-induced microvascular dysfunction.

Expression of microRNAs is essential for arterial myogenic tone and pressure-induced activation of the PI3-kinase/Akt pathway

The myogenic response is the intrinsic ability of small arteries to constrict in response to increased intraluminal pressure. Although microRNAs have been shown to play a role in vascular smooth muscle function, their importance in the regulation of the myogenic response is not known. In this study, we investigate the role of microRNAs in the regulation of myogenic tone by using smooth muscle-specific and tamoxifen-inducible deletion of the endonuclease Dicer in mice. In order to avoid effects of Dicer deletion on smooth muscle differentiation and growth, we used an early time point (5 weeks) after the tamoxifen-induction of Dicer knockout (KO). At this time point, we found that myogenic tone was completely absent in the mesenteric arteries of Dicer KO mice. This was associated with a reduced pressure-induced Akt-phosphorylation, possibly via increased phosphatase and tensin homologue (PTEN) expression, which was found to be a target of miR-26a. Furthermore, loss of myogenic tone was associated with a decreased depolarization-induced calcium influx, and was restored by the L-type channel agonist Bay K 8644 or by transient stimulation with angiotensin II (Ang II). The effect of Ang II was dependent on AT1-receptors and activation of the PI3-kinase/Akt pathway. In this study we have identified novel mechanisms that regulate myogenic tone in resistance arteries, which involves microRNA-dependent control of PI3-kinase/Akt signalling and L-type calcium influx. Furthermore, we have demonstrated that transient stimulation by Ang II can have long-lasting effects by potentiating myogenic tone.

Abstract 484: Augmented Myogenic Responses in Cerebral Arteries Following Interference with Peroxisome Proliferator-Activated Receptor γ (PPARγ) in Smooth Muscle via a Rho Kinase (ROCK)-Dependent Mechanism

Myogenic responses by resistance vessels in brain play a crucial role in regulating cerebral blood flow and protecting the brain parenchyma and blood-brain barrier against potentially detrimental elevations in blood pressure. PPARγ is a transcription factor that may protect the vasculature by suppressing increases in vascular tone. Using transgenic mice expressing a dominant negative mutation of human PPARγ only in smooth muscle cells (S-P467L; TG), we have shown that augmented myogenic responses in mesenteric arteries are mediated by a mechanism that involved protein kinase C, but not ROCK. As ROCK is thought to play a major role in cerebrovascular disease, we hypothesized that interference with PPARγ in smooth muscle would augment myogenic responses in cerebral arteries via a ROCK dependent mechanism. We studied S-P467L TG mice and non-transgenic littermates (NT). Middle cerebral arteries (MCA) from TG mice generated more myogenic tone compared with NT (75 mmHg: 32±2% vs. 13±2% of max diameter). Myogenic reactivity was similarly augmented in MCA from TG mice from 15-150 mmHg whereas vessel diameter in Ca2+ free conditions was similar between groups. We used two approaches to evaluate the impact of ROCK in this model. First, the ROCK inhibitor, Y27632 (3 μM), abolished the augmented myogenic tone in MCA from TG mice (34±5% vs. 9±4% of max diameter). Second, treatment with a cell permeable molecule that modifies RhoA to make it constitutively active (CN-03) elevated myogenic tone to ~60% in both groups, which was reduced to ~10% by Y27632. The large conductance potassium (BK) channel modulates myogenic tone. The BK channel inhibitor, iberiotoxin, caused greater constriction in MCA from NT compared with TG (100 nM: NT, 14±2% vs. TG, 8±1% Δ tone at 75 mmHg). Although angiotensin II can activate ROCK, the AT1 receptor antagonist (losartan) did not affect myogenic tone in MCA from TG mice. Gene expression analysis revealed no change in mRNA for RhoA or ROCK1/2 expression in cerebral arteries from TG mice. These findings support the concept that in cerebral arteries, activity of ROCK has substantial effects on myogenic tone. The data also suggest that under normal conditions, PPARγ in smooth muscle inhibits ROCK, influencing myogenic tone in resistance arteries in brain.

Hypotension in SM-Specific TRIC-A-Transgenic Mice

TRIC (trimeric intracellular cation) channel subtypes, namely TRIC-A and TRIC-B, likely mediate counter-ion movements coupled with rapid Ca2+ release from intracellular stores in various cell types. The Tric-a-knockout mice suffered from hypertension due to enhanced myogenic tone in resistance arteries. Conversely, smooth muscle (SM)-specific Tric-a-transgenic mice developed hypotension. To maintain vascular tonus, two Ca2+ release mechanisms are functioning in vascular smooth muscle cells (VSMCs); incidental opening of ryanodine receptors (RyRs) generates local Ca2+ sparks to induce hyperpolarization, while agonist-induced activation of inositol trisphosphate receptors (IP3Rs) evokes global Ca2+ transients causing contraction.To investigate the mechanism of hypotension in the Tric-a-transgenic mice, we performed Ca2+ imaging and Ca2+ spark measurements. Mutant VSMCs from the transgenic mice showed elevated Ca2+ spark generation, enhanced spontaneous transient outward currents (STOCs) and lowered resting Ca2+ levels.The observation may suggest that overexpression of TRIC-A channels activates RyRs and enhances hyperpolarization signaling generated by functional coupling between RyRs and BK channels in VSMCs. In this situation, deactivated voltage-dependent Ca2+ channels may reduce resting Ca2+ levels and spontaneous tonus in the mutant VSMCs.

Selective Involvement of Serum Response Factor in Pressure-Induced Myogenic Tone in Resistance Arteries

In resistance arteries, diameter adjustment in response to pressure changes depends on the vascular cytoskeleton integrity. Serum response factor (SRF) is a dispensable transcription factor for cellular growth, but its role remains unknown in resistance arteries. We hypothesized that SRF is required for appropriate microvascular contraction. We used mice in which SRF was specifically deleted in smooth muscle or endothelial cells, and their control. Myogenic tone and pharmacological contraction was determined in resistance arteries. mRNA and protein expression were assessed by quantitative real-time PCR (qRT-PCR) and Western blot. Actin polymerization was determined by confocal microscopy. Stress-activated channel activity was measured by patch clamp. Myogenic tone developing in response to pressure was dramatically decreased by SRF deletion (5.9±2.3%) compared with control (16.3±3.2%). This defect was accompanied by decreases in actin polymerization, filamin A, myosin light chain kinase and myosin light chain expression level, and stress-activated channel activity and sensitivity in response to pressure. Contractions induced by phenylephrine or U46619 were not modified, despite a higher sensitivity to p38 blockade; this highlights a compensatory pathway, allowing normal receptor-dependent contraction. This study shows for the first time that SRF has a major part to play in the control of local blood flow via its central role in pressure-induced myogenic tone in resistance arteries.