Stimulation Of Smooth Muscle Cells Research Articles

Analysis of Vascular Cell Response to Hypertension Induced by Pressure Loading and Its Application as a Tool for Exploring Pharmacological Modes of Action.

Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression is induced by interleukin-1β (IL-1β) stimulation in vascular smooth muscle cells (VSMCs), resulting in the production of nitric oxide and prostaglandins such as PGI2. The expression of iNOS and COX-2 in cultured VSMCs isolated from 6-7-week-old stroke-prone spontaneously hypertensive rats (SHRSP) is significantly lower than in cells of normotensive Wistar Kyoto rats (WKY). These reductions are also found in cells exposed to pulsatile atmospheric pressure between 80-160 mmHg at a rate of 4 cycles/min, which simulates systolic hypertension. Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, potentiates IL-1β-induced iNOS and COX-2 expression in VSMCs isolated from WKY, but not those from SHRSP. In response to endothelial injury at a local site, iNOS and COX-2 induction in VSMCs may function primarily as a defensive and compensatory mechanism for endothelial dysfunction by preventing the development of pathological conditions. Thus, in certain pathological conditions associated with hypertension, vascular walls with reduced iNOS and COX-2 expression may aggravate or initiate further vascular injury. In this situation, DHA may contribute to maintaining homeostasis in VSMCs by potentiating iNOS and COX-2 expression. Using cells isolated from a genetic pathological animal model alongside cells exposed to experimental pathological conditions can be an effective tool for the analysis of cell response to hypertension and exploring pharmacological modes of action in vitro.

Increased Intimal Hyperplasia After Vascular Injury in Male Androgen Receptor-Deficient Mice.

Intimal hyperplasia is a vascular pathological process involved in the pathogenesis of atherosclerosis. Data suggest that T, the most important sex steroid hormone in males, protects men from atherosclerotic cardiovascular disease. T mainly acts via the androgen receptor (AR), and in this study we evaluated formation of intimal hyperplasia in male AR knockout (ARKO) mice using a vascular injury model. Two weeks after ligation of the carotid artery, male ARKO mice showed increased intimal area and intimal thickness compared with controls. After endothelial denudation by an in vivo scraping injury, there was no difference in the reendothelialization in ARKO compared with control mice. Ex vivo, we observed increased outgrowth of vascular smooth muscle cells from ARKO compared with control aortic tissue explants; the number of outgrown cells was almost doubled in ARKO. In vitro, stimulation of human aortic vascular smooth muscle cells with a physiological T concentration inhibited both migration and proliferation of the cells. Analyzing the expression of central regulators of cell proliferation and migration, we found that mRNA and protein levels of p27 were lower in uninjured arteries from ARKO mice and that T replacement to castrated male mice increased p27 mRNA in an AR-dependent manner. In conclusion, AR deficiency in male mice increases intimal hyperplasia in response to vascular injury, potentially related to the effects of androgens/AR to inhibit proliferation and migration of smooth muscle cells.

Protective effects of MCR-1329, a dual α1 and angII receptor antagonist, in mineralocorticoid-induced hypertension.

With the prototypical structures of losartan and prazosin as the axis of our research, MCR-1329 emerged as a potential designed multiple ligand from a series of compounds designed to possess dual antagonistic activity on the α1 and AT1 receptor. After confirming the activity of MCR-1329 in in vitro and acute in vivo models, the present study was undertaken to determine the efficacy of MCR-1329 in a mammalian test system. A rat model of deoxycorticosterone acetate (DOCA)-salt induced renal hypertension following unilateral nephrectomy was utilized to determine the effect of MCR-1329. For mechanistic evaluations, MCR-1329 was evaluated on rat aortic strips in vitro and on rat aortic smooth muscle cells to determine the role of MCR-1329 on phosphoinositide 3 kinase (PI3K) signaling. Results of the study showed that MCR-1329 prevents development of arterial hypertension. It was also observed that MCR-1329 upheld the intimal structures of major arteries like the thoracic aorta. Acetylcholine (Ach)-mediated relaxation remained intact in arteries from MCR-1329 treated animals. It was observed that MCR-1329 partially prevents Thr-308 phosphorylation of Akt following ligand-mediated receptor stimulation in vascular smooth muscle cells. Addition of LY294002 to the reaction medium caused a near-complete inhibition of Akt-phosphorylation. This suggested that MCR-1329 elicits its antihypertensive role by blocking activation of receptor-mediated PI3K-Akt downstream signaling as well as through preservation of arterial integrity. MCR-1329 has the potential to be evaluated further for clinical development as a potential antihypertensive agent with multiple mechanisms of action.

Tracheal Smooth Muscle Cells Stimulated by Stem Cell Factor-c-Kit Coordinate the Production of Transforming Growth Factor-β1 and Fibroblast Growth Factor-2 Mediated by Chemokine (C-C Motif) Ligand 3.

The aim of this study was to evaluate the mechanism involved in the stem cell factor (SCF)-induced production of fibroblast growth factor-2 (FGF-2), transforming growth factor-β1 (TGF-β1), and chemokine (C-C motif) ligand 3 (CCL3) in tracheal smooth muscle cells (tSMCs) and the signaling pathway involved in the process. tSMC primary cultures were stimulated with SCF and evaluated at 24 h. Cells treated with specific antibodies did not show any immunolabeling for cytokeratin or fibroblast activation protein, but were positive for α-smooth muscle actin, indicating the purity of the primary cell line. Western blot analysis showed constitutive phosphorylation of c-Kit, as well as increased total protein and phosphorylated c-Kit levels in tSMCs after SCF stimulation. Flow cytometry analysis also showed an increase in cell-surface c-Kit expression in the presence of SCF. SCF induced TGF-β mRNA expression in tSMCs, as well as the production of TGF-β1, CCL3, and FGF-2. Pretreatment with anti-CCL3 antibody blocked TGF-β1 expression and partially inhibited FGF-2 production. On the other hand, anti-c-Kit antibody blocked TGF-β1 expression and FGF-2 production. Thus, TGF-β1 and FGF-2 production were mediated by CCL3 production through c-Kit. Pretreatment with mitogen-activated protein kinase kinase 1, p38, and Jun N-terminal kinase inhibitors showed that the effects mediated by SCF were involved with the modulation of mitogen-activated protein kinase (MAPK) pathways. Development of inhibitors targeting CCL3 through MAPK activation could thus be an attractive strategy to inhibit tSMC activation during asthma.

Anti-Proliferative Effects of Rutin on OLETF Rat Vascular Smooth Muscle Cells Stimulated by Glucose Variability

PurposeProliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. Rutin is a major representative of the flavonol subclass of flavonoids and has various pharmacological activities. Currently, data are lacking regarding its effects on VSMC proliferation induced by intermittent hyperglycemia. Here, we demonstrate the effects of rutin on VSMC proliferation and migration according to fluctuating glucose levels.Materials and MethodsPrimary cultures of male Otsuka Long-Evans Tokushima Fatty (OLETF) rat VSMCs were obtained from enzymatically dissociated rat thoracic aortas. VSMCs were incubated for 72 h with alternating normal (5.5 mmol/L) and high (25.0 mmol/L) glucose media every 12 h. Proliferation and migration of VSMCs, the proliferative molecular pathway [including p44/42 mitogen-activated protein kinases (MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2), p38 MAPK, phosphoinositide 3-kinase (PI3K), c-Jun N-terminal protein kinase (JNK), nuclear factor kappa B (NF-κB), and Akt], the migratory pathway (big MAPK 1, BMK1), reactive oxygen species (ROS), and apoptotic pathway were analyzed.ResultsWe found enhanced proliferation and migration of VSMCs when cells were incubated in intermittent high glucose conditions, compared to normal glucose. These effects were lowered upon rutin treatment. Intermittent treatment with high glucose for 72 h increased the expression of phospho-p44/42 MAPK (extracellular signal regulated kinase 1/2, ERK1/2), phospho-MEK1/2, phospho-PI3K, phospho-NF-κB, phospho-BMK1, and ROS, compared to treatment with normal glucose. These effects were suppressed by rutin. Phospho-p38 MAPK, phospho-Akt, JNK, and apoptotic pathways [B-cell lymphoma (Bcl)-xL, Bcl-2, phospho-Bad, and caspase-3] were not affected by fluctuations in glucose levels.ConclusionFluctuating glucose levels increased proliferation and migration of OLETF rat VSMCs via MAPK (ERK1/2), BMK1, PI3K, and NF-κB pathways. These effects were inhibited by the antioxidant rutin.

Vascular amyloidosis impairs the gliovascular unit in a mouse model of Alzheimer's disease.

Reduced cerebral blood flow impairs cognitive function and ultimately causes irreparable damage to brain tissue. The gliovascular unit, composed of neural and vascular cells, assures sufficient blood supply to active brain regions. Astrocytes, vascular smooth muscle cells, and pericytes are important players within the gliovascular unit modulating vessel diameters. While the importance of the gliovascular unit and the signals involved in regulating local blood flow to match neuronal activity is now well recognized, surprisingly little is known about this interface in disease. Alzheimer's disease is associated with reduced cerebral blood flow. Here, we studied how the gliovascular unit is affected in a mouse model of Alzheimer's disease, using a combination of ex vivo and in vivo imaging approaches. We specifically labelled vascular amyloid in living mice using the dye methoxy-XO4. We elicited vessel responses ex vivo using either pharmacological stimuli or cell-specific calcium uncaging in vascular smooth muscle cells or astrocytes. Multi-photon in vivo imaging through a cranial window allowed us to complement our ex vivo data in the presence of blood flow after label-free optical activation of vascular smooth muscle cells in the intact brain. We found that vascular amyloid deposits separated astrocyte end-feet from the endothelial vessel wall. High-resolution 3D images demonstrated that vascular amyloid developed in ring-like structures around the vessel circumference, essentially forming a rigid cast. Where vascular amyloid was present, stimulation of astrocytes or vascular smooth muscle cells via ex vivo Ca(2+) uncaging or in vivo optical activation produced only poor vascular responses. Strikingly, vessel segments that were unaffected by vascular amyloid responded to the same extent as vessels from age-matched control animals. We conclude that while astrocytes can still release vasoactive substances, vascular amyloid deposits render blood vessels rigid and reduce the dynamic range of affected vessel segments. These results demonstrate a mechanism that could account in part for the reduction in cerebral blood flow in patients with Alzheimer's disease.media-1vid110.1093/brain/awv327_video_abstractawv327_video_abstract.

Abstract 17947: Regulator of G-Protein Signaling 5 Prevents Smooth Muscle Cell Proliferation and Attenuates Neointima Formation

Introduction: Regulator of G-protein signaling 5 (RGS5) is abundantly and specifically expressed in vascular smooth muscle cells (SMCs) and inhibits G-protein signaling by enhancing the GTP-hydrolyzing activity of Gα-subunits. Hypothesis: In the present study, we aimed to investigate the effects of RGS5 on vascular SMC function in vitro and neointima formation in vivo and to determine the underlying molecular mechanisms. Methods: Expression of RGS5 was assessed in vitro and in vivo by qPCR, immunoblotting and immunohistochemistry. Proliferation and migration of human coronary artery SMC were assessed in vitro following the adenoviral-mediated over expression of wild type (WT) RGS5, of a non-degradable mutant (C2A-RGS5) or siRNA-mediated knock down. In vivo, the proliferation and neointima formation were assessed in a wire-mediated injury model of the femoral artery in C57BL/6J mice. Results: We found a robust expression of RGS5 in native arteries of C57BL/6 mice and a significant down-regulation within neointimal lesions 10 and 21 days after vascular injury as assessed by qPCR, immunohistochemistry and immunoblotting. In vitro, RGS5 was found significantly down-regulated after mitogenic stimulation of SMCs. Overexpression of RGS5 or, even more prominently, the C2A-RGS5 mutant prevented SMC proliferation and migration. In contrast, the siRNA-mediated knockdown of RGS5 significantly augmented SMC proliferation. Following overexpression of RGS5, FACS analysis of propidium iodide-stained cells indicated cell cycle arrest in G0/G1 phase. Mechanistically, inhibition of ERK1/2 phosphorylation and MAPK downstream signaling was found to be responsible for the anti-proliferative effect since over expression of a constitutive active form of MEK reversed the growth-inhibitory effect of of RGS5. Following wire-induced injury, adenoviral-mediated overexpression of RGS5 or C2A-RGS5 significantly decreased ERK phosphorylation, SMC proliferation and neointima formation in vivo (n=8; p<0.001). Conclusions: Down-regulation of RGS5 is an important prerequisite for SMC proliferation and neointima Formation. Therefore, reconstitution of RGS5 levels might represent a promising therapeutic option to prevent vascular remodeling processes.

Vascular amyloidosis impairs the gliovascular unit in a mouse model of Alzheimer’s disease

Reduced cerebral blood flow impairs cognitive function and ultimately causes irreparable damage to brain tissue. The gliovascular unit, composed of neural and vascular cells, assures sufficient blood supply to active brain regions. Astrocytes, vascular smooth muscle cells, and pericytes are important players within the gliovascular unit modulating vessel diameters. While the importance of the gliovascular unit and the signals involved in regulating local blood flow to match neuronal activity is now well recognized, surprisingly little is known about this interface in disease. Alzheimer's disease is associated with reduced cerebral blood flow. Here, we studied how the gliovascular unit is affected in a mouse model of Alzheimer's disease, using a combination of ex vivo and in vivo imaging approaches. We specifically labelled vascular amyloid in living mice using the dye methoxy-XO4. We elicited vessel responses ex vivo using either pharmacological stimuli or cell-specific calcium uncaging in vascular smooth muscle cells or astrocytes. Multi-photon in vivo imaging through a cranial window allowed us to complement our ex vivo data in the presence of blood flow after label-free optical activation of vascular smooth muscle cells in the intact brain. We found that vascular amyloid deposits separated astrocyte end-feet from the endothelial vessel wall. High-resolution 3D images demonstrated that vascular amyloid developed in ring-like structures around the vessel circumference, essentially forming a rigid cast. Where vascular amyloid was present, stimulation of astrocytes or vascular smooth muscle cells via ex vivo Ca(2+) uncaging or in vivo optical activation produced only poor vascular responses. Strikingly, vessel segments that were unaffected by vascular amyloid responded to the same extent as vessels from age-matched control animals. We conclude that while astrocytes can still release vasoactive substances, vascular amyloid deposits render blood vessels rigid and reduce the dynamic range of affected vessel segments. These results demonstrate a mechanism that could account in part for the reduction in cerebral blood flow in patients with Alzheimer's disease.media-1vid110.1093/brain/awv327_video_abstractawv327_video_abstract.

Biophysical Processes in a Urinary Bladder Detrusor Smooth Muscle Cell during Rehabilitation Electrostimulation: a Simulation Study

The work was aimed at the search for approaches to solving the problem of biophysically reasonable selection of the parameters of electrical stimulation of smooth muscle cells (SMCs) of the urinary bladder detrusor (UBD). Such stimulation is widely used in the rehabilitation of patients with surgical correction of congenital malformations accompanied by total or partial deficiency of the M2/M3 cholinergic receptors in the UBD. A computer model built on the basis of experimental data on ion channels and pumps of the sarcolemma and mechanisms of regulation of the intracellular calcium concentration ([Ca22+]i), providing both electrogenesis and the contractile function of the cell inherent to the biological prototype, was used. We studied changes in the membrane potential, partial transmembrane currents, and [Ca2+]i, caused by depolarizing current pulses applied with constant frequencies and combined in “packs” or “envelopes” typical of the protocols of rehabilitation stimulation; the stimuli had constant or trapezoid-modulated amplitudes. The examined UBD SMC responded to a single pulse by generation of the action potential (AP) close in its properties to the prototype. Stimulation by both packs and envelopes of identical pulses eventually led to the establishing of equal forced electrical and concentration oscillations with the parameters depending on the duration of interpulse intervals (IPIs). Such oscillations caused by stimulation with 5- and 50-msec-long IPIs, typical of the rehabilitation protocols and comparable with the durations of the absolute and relative refractoriness of the model SMC, significantly differed in the pattern of the regenerative responses (APs) and in the range and mean levels of depolarization shifts of the membrane potential and those of [Ca2+]i, which were greater at high-frequency stimulation. In the case of short IPIs, [Ca2+]i, having no time to return to the basal level, oscillated within a range of values which in other excitable cells are considered to exceed significantly the physiological norm. These data emphasize the necessity to estimate the exact kinetic characteristics of the mechanisms underlying the inflow and extrusion of Ca2+ in the UBD SMC necessary for a biophysically justified choice of the parameters of rehabilitation stimulation that would prevent possible cytotoxic side effects associated with excessively long-lasting high levels of [Ca2+]i. Essential for the observed processes and, therefore, requiring targeted studies, was such a parameter of UBD SMCs as the reversal potential for Ca2+-dependent chloride current (E Cl); this current is activated, in particular, by parasympathetic action on the M2/M3 receptors. When high-frequency oscillations of the membrane potential periodically exceeded the E Cl level, the mentioned current changed its main (depolarizing) direction to the opposite (hyperpolarizing) one.

Abstract 439: Biomechanical Stimulation Promotes Translocation of Nuclear Factor of Activated T-cells 5 Into The Nucleus of Vascular Smooth Muscle Cells

Chronic alterations in the biomechanical stimulation of vascular smooth muscle cells (VSMC) as experienced during hypertension lead to changes in VSMC phenotype and function and further enable the structural remodeling of the vessel wall. In this context, we recently reported that an increase in wall stress or biomechanical stretch is sufficient to activate nuclear factor of activated T-cells 5 (NFAT5). This transcription factor promotes the expression of gene products such as tenascin-C and κ-actin, both involved in VSMC migration. Based on these findings, we hypothesized that biomechanical stretch elicits NFAT5 mRNA expression and induces biochemical modifications of NFAT5 on the post-translational level, a prerequisite for its entry into the nucleus and transcriptional activity. To scrutinize this hypothesis, human arterial VSMC were exposed to biomechanical stretch (13%, 0.5 Hz) and subjected to detailed mRNA expression analyses. While a ~3-fold reduction in NFAT5 splice variant 1 (isoform A) mRNA expression was observed in stretch-stimulated VSMC as compared to the static controls (n=3, p<0.05), splice variant 3 (isoform C) mRNA levels were induced ~1.8-fold (n=3, p<0.05). Overexpression of corresponding Flag-tagged NFAT5 proteins in VSMC and subsequent immunofluorescence as well as biochemical analyses revealed that isoform A was primarily located in the cytoplasm of static and stretch-stimulated VSMC while isoform C was preferentially localized in the nucleus under baseline conditions and further accumulated in the nucleus upon biomechanical stimulation (n=3, p<0.05). Nuclear translocation of isoform C was amplified for phosphorylation-deficient mutants generated by exchanging serine to alanine at position 1197 even under static culture conditions while a phosphomimetic mutation at this residue (serine to glutamate) inhibited NFAT5c nuclear translocation (n=3, p<0.05). Collectively, our findings indicate that exposure of VSMC to biomechanical stretch triggers the expression of NFAT5 isoform C and controls its entry into the nucleus via phosphorylation at S-1197. Current investigations are focusing on the impact of NFAT5 on hypertensive remodeling utilizing inducible smooth muscle cell-specific NFAT5-deficient mice.

Involvement of interleukin-1 receptor-associated kinase-1 in vascular smooth muscle cell proliferation and neointimal formation after rat carotid injury.

Reduced frequency of atherosclerotic plaques is observed in interleukin-1 receptor-associated kinase-1 (IRAK1)-deficient mice; however, the underlying mechanism is not clear. Therefore, this study investigate the role of IRAK1 in vascular smooth muscle cell proliferation and neointimal hyperplasia. Stimulation of rat primary vascular smooth muscle cells with fetal bovine serum (10%) or platelet-derived growth factor-BB (20 ng/mL) for 15 minutes to 24 hours induced a time-dependent increase in IRAK1 and extracellular signal-regulated kinase (ERK) activation, proliferating cell nuclear antigen upregulation and p27Kip1 downregulation as assessed by Western blotting. Inhibitors of ERK pathway (U0126, 10 μmol/L), IRAK (IRAK1/4, 3 μmol/L), protein kinase C (PKC; Ro-31-8220, 1 μmol/L), siRNA of toll-like receptor-4 (200 nmol/L), and PKC-ε (200 nmol/L) significantly attenuated these changes. Platelet-derived growth factor induced endogenous IRAK-ERK-PKC-ε association in a toll-like receptor-4 and PKC-ε-dependent manner. A time-dependent increase in IRAK1 and ERK activation was observed after 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, and 24 hours of carotid balloon injury in rats. Balloon injury induced endogenous IRAK-ERK-PKC-ε interaction. Perivascular application of IRAK1/4 inhibitor (100 μmol/L), U0126 (100 μmol/L), and IRAK1 siRNA (220 and 360 nmol/L) in pluronic gel abrogated balloon injury-induced ERK phosphorylation, activation, and p27Kip1 downregulation. Hematoxylin and eosin staining and immunohistochemistry of proliferating cell nuclear antigen and smooth muscle actin demonstrated that balloon injury-induced intimal thickening and neointimal vascular smooth muscle cell proliferation were significantly abrogated in the presence of IRAK1/4 inhibitor, IRAK1 siRNA, and U0126. IRAK1 mediates vascular smooth muscle cell proliferation and neointimal hyperplasia by regulating PKC-ε-IRAK1-ERK axis.

Angiotensin II Activates the RhoA Exchange Factor Arhgef1 in Humans

Although a causative role for RhoA-Rho kinase has been recognized in the development of human hypertension, the molecular mechanism(s) and the RhoA guanine exchange factor(s) responsible for the overactivation of RhoA remain unknown. Arhgef1 was identified as a RhoA guanine exchange factor involved in angiotensin II (Ang II)-mediated regulation of vascular tone and hypertension in mice. The aim of this study was to determine whether Arhgef1 is activated and involved in the activation of RhoA-Rho kinase signaling by Ang II in humans. In vitro stimulation of human coronary artery smooth muscle cells and human peripheral blood mononuclear cells by Ang II (0.1 μmol/L) induced activation of Arhgef1 attested by its increased tyrosine phosphorylation. Silencing of Arhgef1 expression by siRNA inhibited Ang II-induced activation of RhoA-Rho kinase signaling. In normotensive subjects, activation of the renin-angiotensin system by a low-salt diet for 7 days increased RhoA-Rho kinase signaling and stimulated Arhgef1 activity in peripheral blood mononuclear cells. In conclusion, our results strongly suggest that Arhgef1 mediates Ang II-induced RhoA activation in humans. Moreover, they show that measurement of RhoA guanine exchange factor activity in peripheral blood mononuclear cells might be a useful method to evaluate RhoA guanine exchange factor activity in humans.

Angiotensin II에 의해 유도되는 활성산소발생 기전에 대한 연구

Angiotensin II (AngII) is an essential hormone that affects vascular physiology. For example, stimulation of vascular smooth muscle cells (VSMCs) rapidly induces vasoconstriction and results in the up-regulation of blood pressure. Chronic stimulation of VSMCs with AngII also results in hypertrophy. In this study, we confirmed an involvement of phosphatidylinositol 3-kinase (PI3K)-dependent calcium mobilization in AngII-induced generation of reactive oxygen species (ROS). Stimulation of rat aortic smooth muscle cells (RASMCs) with AngII significantly induced the generation of ROS in a dose- and time-dependent manner. AngII-induced generation of ROS was completely abolished by pharmacological inhibition of PI3K (with LY294002), but inhibition of the ERK signaling pathway had no effect. AngII-induced calcium mobilization was completely blocked by inhibition of PI3K, whereas inhibition of the ERK signaling pathway by PD98059 was ineffective. Depletion of extracellular calcium or inhibition of the L-type calcium channel by nifedipine completely blocked AngII-induced calcium mobilization. Depletion of extracellular calcium by EGTA and incubation of RASMCs with calcium- free medium both significantly blocked AngII-induced ROS generation. Inhibition of the L-type calcium channel also significantly blocked AngII-induced ROS generation. These results suggest that AngII-induced ROS generation is regulated by calcium mobilization, which, in turn, is modulated by a PI3K/L-type calcium channel signaling pathway.

Origins of variation in conducted vasomotor responses.

Regulation of blood flow in the microcirculation depends on synchronized vasomotor responses. The vascular conducted response is a synchronous dilatation or constriction, elicited by a local electrical event that spreads along the vessel wall. Despite the underlying electrical nature, however, the efficacy of conducted responses varies significantly between different initiating stimuli within the same vascular bed as well as between different vascular beds following the same stimulus. The differences have stimulated proposals of different mechanisms to account for the experimentally observed variation. Using a computational approach that allows for introduction of structural and electrophysiological heterogeneity, we systematically tested variations in both arteriolar electrophysiology and modes of stimuli. Within the same vessel, our simulations show that conduction efficacy is influenced by the type of cell being stimulated and, in case of depolarization, by the stimulation strength. Particularly, simultaneous stimulation of both endothelial and vascular smooth muscle cells augments conduction. Between vessels, the specific electrophysiology determines membrane resistance and conduction efficiency-notably depolarization or radial currents reduce electrical spread. Random cell-cell variation, ubiquitous in biological systems, only cause small or no reduction in conduction efficiency. Collectively, our simulations can explain why CVRs from hyperpolarizing stimuli tend to conduct longer than CVRs from depolarizing stimuli and why agonists like acetylcholine induce CVRs that tend to conduct longer than electrical injections. The findings demonstrate that although substantial heterogeneity is observed in conducted responses, it can be largely ascribed to the origin of electrical stimulus combined with the specific electrophysiological properties of the arteriole. We conclude by outlining a set of "principles of electrical conduction" in the microcirculation.

Abstract 18390: Ang II activates the RhoA Exchange Factor Arhgef1 in Humans

Introduction: Although a causative role for RhoA-Rho kinase signaling has been recognized in the development of human hypertension, the molecular mechanism(s) as well as the RhoA exchange factors (GEFs) responsible for the over activation of RhoA remain unknown. Arhgef1 has been recently identified as a RhoA GEF involved in Ang II-mediated regulation of vascular tone and hypertension in mice. Hypothesis: Here we assessed the hypothesis that Arhgef1 is activated and involved in the activation of RhoA-Rho kinase signaling by Ang II in humans. Methods: We used in vitro stimulation of human coronary artery smooth muscle cells and human peripheral blood cells (PBMC) by Ang II (0.1 μmol/L), and PBMC isolated from normotensive subjects before and after activation of the renin-angiotensin system by a low-salt diet for 7 days (checked by the increase in plasma aldosterone and active renine). Activation of Arhgef1 was monitored by measuring its tyrosine phosphorylation by western blot and phosphorylation of the Rho kinase target MYPT was used to measure the activity of RhoA/Rho kinase signaling. Results: In vitro, Ang II induced activation of Arhgef1 in human vascular smooth muscle cells and PBMC. Silencing of Arhgef1 expression by siRNA in human vascular smooth muscle cells inhibited Ang II-induced activation of RhoA-Rho kinase signaling (0.67±0.12 vs 3.21±0.91relative to unstimulated condition, n=4, P<0.05). In normotensive subjects, activation of the renin-angiotensin system by a low-salt diet stimulated Arhgef1 activity in PBMC (1.26±0.12-fold over basal level, n=39, P<0.05). This activation was associated with an increase activity of RhoA-Rho kinase signaling (1.33±0.14-fold over basal level, n=39, P<0.05). Conclusions: Our results show that Ang II stimulates Arhgef1 activity and strongly suggest that Arhgef1 mediates Ang II-induced RhoA activation in humans. Moreover, we show for the first time that measurement of RhoA GEF activity in PBMC might be a useful method to evaluate RhoA GEF activity in humans.

Cross-talk between p(38)MAPK and G iα in regulating cPLA 2 activity by ET-1 in pulmonary smooth muscle cells.

Endothelin-1 (ET-1) is known as the most potent vasoconstrictor yet described. Infusion of ET-1 into isolated rabbit lung has been shown to cause pulmonary vasoconstriction with the involvement of arachidonic acid metabolites. Given the potency of arachidonic acid metabolites, the activity of phospholipase A2 must be tightly regulated. Herein, we determined the mechanisms by which ET-1 stimulates cPLA2 activity during ET-1 stimulation of bovine pulmonary artery smooth muscle cells. We demonstrated that (i) treatment of bovine pulmonary artery smooth muscle cells with ET-1 stimulates cPLA2 activity in the cell membrane; (ii) ET-1 caused increase in O 2 (·-) production occurs via NADPH oxidase-dependent mechanism; (iii) ET-1-stimulated NADPH oxidase activity is markedly prevented upon pretreatment with PKC-ζ inhibitor, indicating that PKC-ζ plays a prominent role in this scenario; (iv) ET-1-induced NADPH oxidase-derived O 2 (·-) stimulates an aprotinin sensitive protease activity due to prominent increase in [Ca(2+)]i; (v) the aprotinin sensitive protease plays a pivotal role in activating PKC-α, which in turn phosphorylates p(38)MAPK and subsequently Giα leading to the activation of cPLA2. Taken together, we suggest that cross-talk between p(38)MAPK and Giα with the involvement of PKC-ζ, NADPH oxidase-derived O 2 (·-) , [Ca(2+)]i, aprotinin-sensitive protease and PKC-α play a pivotal role for full activation of cPLA2 during ET-1 stimulation of pulmonary artery smooth muscle cells.

TGF-β/Smad3 inhibit vascular smooth muscle cell apoptosis through an autocrine signaling mechanism involving VEGF-A.

We have previously shown that in the presence of elevated Smad3, transforming growth factor-β (TGF-β) transforms from an inhibitor to a stimulant of vascular smooth muscle cell (SMC) proliferation and intimal hyperplasia (IH). Here we identify a novel mechanism through which TGF-β/Smad3 also exacerbates IH by inhibiting SMC apoptosis. We found that TGF-β treatment led to inhibition of apoptosis in rat SMCs following viral expression of Smad3. Conditioned media from these cells when applied to naive SMCs recapitulated this effect, suggesting an autocrine pathway through a secreted factor. Gene array of TGF-β/Smad3-treated cells revealed enhanced expression of vascular endothelial growth factor (VEGF), a known inhibitor of endothelial cell apoptosis. We then evaluated whether VEGF is the secreted mediator responsible for TGF-β/Smad3 inhibition of SMC apoptosis. In TGF-β/Smad3-treated cells, VEGF mRNA and protein as well as VEGF secretion were increased. Moreover, recombinant VEGF-A inhibited SMC apoptosis and a VEGF-A-neutralizing antibody reversed the inhibitory effect of conditioned media on SMC apoptosis. Stimulation of SMCs with TGF-β led to the formation of a complex of Smad3 and hypoxia-inducible factor-1α (HIF-1α) that in turn activated the VEGF-A promoter and transcription. In rat carotid arteries following arterial injury, Smad3 and VEGF-A expression were upregulated. Moreover, Smad3 gene transfer further enhanced VEGF expression as well as inhibited SMC apoptosis. Finally, blocking either the VEGF receptor or Smad3 signaling in injured carotid arteries abrogated the inhibitory effect of Smad3 on vascular SMC apoptosis. Taken together, our study reveals that following angioplasty, elevation of both TGF-β and Smad3 leads to SMC secretion of VEGF-A that functions as an autocrine inhibitor of SMC apoptosis. This novel pathway provides further insights into the role of TGF-β in the development of IH.

The Effect of Phosphodiesterase Inhibitor On Vascular Smooth Muscle Cells Stimulated By Cyclosporine and High Glucose.

The Effect of Phosphodiesterase Inhibitor On Vascular Smooth Muscle Cells Stimulated By Cyclosporine and High Glucose.

Decorin mimic regulates platelet-derived growth factor and interferon-γ stimulation of vascular smooth muscle cells.

Following balloon injury, smooth muscle cells (SMCs) serve as targets for many of the pro-inflammatory and pro-fibrotic factors, including platelet-derived growth factor (PDGF) and interferon-γ (IFN-γ) released from activated inflammatory cells and platelets. Previously, our lab designed a mimic of the proteoglycan decorin, termed DS-SILY20, that suppressed vascular SMC proliferation, migration, and protein synthesis in vitro, and injured vessels treated with DS-SILY20 demonstrated reduced hyperplasia in vivo. Here we characterize the effects of DS-SILY20 on modulating PDGF and IFN-γ stimulation in both proliferative and quiescent human SMCs to further evaluate the potential impact of DS-SILY20-SMC interaction on restenosis. Nanomolar dissociation constants were observed between DS-SILY20 and both PDGF and IFN-γ. PDGF significantly increased migration, proliferation, and protein and cytokine expression, as well as increased ERK-1/2 and p38 MAPK phosphorylation in both quiescent and proliferative cultures. However, DS-SILY20 inhibited these increases, presumably through sequestration of the PDGF. Consistent with the complex responses seen with IFN-γ in SMC physiology in the literature, the response of SMC cultures to IFN-γ was variable and complex. However, where increased activity was seen with IFN-γ, DS-SILY20 attenuated this activity. Overall, the results suggest that DS-SILY20 would be an ideal alternative to traditional therapeutics used and may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.

Abstract 169: Smad3 Drives Cross-Talk Between TGFß and Wnt/ß-Catenin Signaling Pathways in Smooth Muscle Cells

Restenosis (neo-intimal hyperplasia) occurs in approximately 25-50% of patients undergoing arterial interventions, primarily due to the proliferation and migration of arterial smooth muscle cells (SMCs) into the peri-luminal area. Recently, Wnt/β-catenin signaling has been shown to promote SMC proliferation and enhance neo-intimal hyperplasia but its mechanism of activation is unclear. Interestingly, Wnt/β-catenin has been shown to be activated by TGFβ in mesenchymal stem cells and fibroblasts. We have shown that TGFβ and its downstream signaling protein, Smad3, are upregulated following vascular injury and that Smad3 overexpressing SMCs display enhanced proliferation, migration, and neo-intimal hyperplasia. These results led us to hypothesize that TGFβ, through Smad3, activates Wnt/β-catenin to regulate SMC behavior following arterial injury . In primary rat SMCs, TGFβ (5ng/mL) led to β-catenin activation and relocalization from the plasma membrane to the cytoplasm / nucleus within 24 hours. Furthermore, qRT-PCR results demonstrated that expression of Wnt11 (22 fold) and Wnt9a (3.9 fold) were significantly upregulated after 24 hours of TGFβ stimulation (p<0.05, n=3). In addition, 24 hours of TGFβ stimulation in SMCs overexpressing Smad3 (TGFβ/Smad3) further enhanced the gene expression of Wnt11 (>300 fold) and Wnt9a (14 fold) and also stimulated significant increases in Wnt2b (41 fold), Wnt5a (2.9 fold), and Wnt4 (3.2 fold) (p<0.05, n=3) as measured by qRT-PCR. Western blot results demonstrated that the combined TGFβ/Smad3 stimulation increased β-catenin protein levels, suggesting that TGFβ activates canonical Wnt signaling leading to stabilization of β-catenin protein. In normal rat carotid arteries, β-catenin protein was undetectable via immunohistochemistry but could be seen in SMCs of the vessel media at 3 days post-balloon angioplasty and in neo-intimal cells at 7 and 14 days. Smad3 was also expressed in neo-intimal cells at 7 and 14 days post-angioplasty suggesting that TGFβ, through Smad3, is responsible for Wnt/β-Catenin activation during vascular injury. In conclusion, this work describes a novel cross-talk in SMCs between TGFβ and Wnt signaling which may provide a viable target for future anti-restenotic treatments.