Stimulation Of Smooth Muscle Cells Research Articles

Compartmentalized Connexin 43 S -Nitrosylation/Denitrosylation Regulates Heterocellular Communication in the Vessel Wall

To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle. Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall. The myoendothelial junction serves as a conduit to facilitate gap junction communication between endothelial cells and vascular smooth muscle cells within the resistance vasculature. By using isolated vessels and a vascular cell coculture, we found that Cx43 is constitutively S-nitrosylated on cysteine 271 because of active endothelial NO synthase compartmentalized at the myoendothelial junction. Conversely, we found that stimulation of smooth muscle cells with the constrictor phenylephrine caused Cx43 to become denitrosylated because of compartmentalized S-nitrosoglutathione reductase, which attenuated channel permeability. We measured S-nitrosoglutathione breakdown and NO(x) concentrations at the myoendothelial junction and found S-nitrosoglutathione reductase activity to precede NO release. This study provides evidence for compartmentalized S-nitrosylation/denitrosylation in the regulation of smooth muscle cell to endothelial cell communication.

Urotensin II-induced signaling involved in proliferation of vascular smooth muscle cells

The urotensin II receptor, bound by the ligand urotensin II, generates second messengers, ie, inositol triphosphate and diacylglycerol, which stimulate the subsequent release of calcium (Ca(2+)) in vascular smooth muscle cells. Ca(2+) influx leads to the activation of Ca(2+)-dependent kinases (CaMK) via calmodulin binding, resulting in cellular proliferation. We hypothesize that urotensin II signaling in pulmonary arterial vascular smooth muscle cells (Pac1) and primary aortic vascular smooth muscle cells (PAVSMC) results in phosphorylation of Ca(2+)/calmodulin-dependent kinases leading to cellular proliferation. Exposure of Pac1 cultures to urotensin II increased intracellular Ca(2+), subsequently activating Ca(2+)/calmodulin-dependent kinase kinase (CaMKK), and Ca(2+)/calmodulin-dependent kinase Type I (CaMKI), extracellular signal-regulated kinase (ERK 1/2), and protein kinase D. Treatment of Pac1 and PAVSMC with urotensin II increased proliferation as measured by (3)H-thymidine uptake. The urotensin II-induced increase in (3)H-thymidine incorporation was inhibited by a CaMKK inhibitor. Taken together, our results demonstrate that urotensin II stimulation of smooth muscle cells leads to a Ca(2+)/calmodulin-dependent kinase-mediated increase in cellular proliferation.

Leukotriene B 4 mediates vascular smooth muscle cell migration through αvβ3 integrin transactivation

Vascular injury leads to a local inflammatory response, characterized by endothelial damage, extracellular matrix exposition and aggregation/adhesion of platelets and circulating leukocytes. The release of inflammatory mediators amplifies the process, and can induce vascular smooth muscle cells (SMC) migration and proliferation. Released by leukocytes, leukotriene B 4 (LTB 4) induces reactive oxygen species production and SMC chemotaxis. This study was conducted to elucidate the molecular mechanisms involved in the effect of LTB 4 on SMC migration, and a rat linage of vascular SMC (A7r5) were used throughout. The chemotactic effect of LTB 4 was dependent on the concentration used, being comparable to AngII at 100 nM. Migration induced by LTB 4 was inhibited in the presence of pertussis toxin, CP-105696, a BLT1 receptor antagonist, and by LY294002 or PD98059, two inhibitors of PI3K and MEK1/2, respectively. Stimulation of SMC with LTB 4 triggered integrin-associated signaling pathways, inducing focal adhesion kinase (FAK) phosphorylation, mobilization of actin cytoskeleton, association of FAK to PI3K, ERK-2 phosphorylation and nuclear translocation, and also NFκB pathway activation. Pretreatment of SMC with a selective ligand of αvβ3 integrin, kistrin, inhibited LTB 4-induced chemotaxis, FAK phosphorylation, FAK-PI3K association, and also inhibited ERK-2 and NFκB pathways activation. Taken together, the data demonstrated, for the first time, that the effect of LTB 4 on SMC migration is modulated by integrin signaling activation, suggesting that these adhesion molecules might be important target for therapeutic intervention in cardiovascular diseases.

Inducible nitric oxide synthase gene deletion exaggerates MAPK-mediated cyclooxygenase-2 induction by inflammatory stimuli

Cyclooxygenase (COX)-2 and inducible nitric oxide (NO) synthase (iNOS) are responsive to a wide array of inflammatory stimuli, have been localized to vascular smooth muscle cells (SMCs), and are intimately linked to the progression of vascular disease, including atherosclerotic lesion formation. We and others have shown that the production and subsequent impact of COX products appear to be correlative with the status of NO synthesis. This study examined the impact of inflammation-driven NO production on COX-2 expression in SMCs. Concurrent stimulation of quiescent rat aortic SMCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma increased COX-2, iNOS, and nitrite production. Pharmacological inhibition of NO synthase (N(G)-monomethyl-l-arginine) concentration- and time-dependently magnified LPS + IFN-gamma-mediated COX-2 mRNA and protein induction in a cGMP-independent manner. COX-2 induction was associated with activation of the ERK, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. Interestingly, NO synthase inhibition enhanced ERK, p38, and to a lesser extent JNK phosphorylation but suppressed MAPK phosphatase (MKP)-1 induction in response to LPS + IFN-gamma. Similarly, the exposure of SMCs from iNOS(-/-) mice to LPS + IFN-gamma produced an enhancement of COX-2 induction, p38, and JNK phosphorylation and an attenuated upregulation of MKP-1 versus their wild-type counterparts. Taken together, our data indicate that NO, in part derived from iNOS, negatively regulates the immediate early induction of COX-2 in response to inflammatory stimuli.

PARACRINE STIMULATION OF VASCULAR SMOOTH MUSCLE PROLIFERATION BY DIADENOSINE: PP.21.326

Objectives: The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Methods and Results: Both diadenosine pentaphosphate and hexaphosphate were identified by matrixassisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomola concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. Conclusion: We conclude that diadenosine polyphosphates are endogenous purinergic agonis of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.

Compartmentalized nitric oxide signaling at the myoendothelial junction can regulate gap junction communication

Myoendothelial junctions (MEJ) couple endothelial cells (EC) and vascular smooth muscle cells (SMC) in resistance vessels via gap junctions (GJ). Throughout the MEJ, caveolin‐1, which regulates endothelial nitric oxide synthase (eNOS) derived nitric oxide (NO), is extensively localized. Therefore, we tested the hypothesis that eNOS is compartmentalized at the MEJ and that NO can regulate heterocellular communication by S‐nitrosylation of connexin (Cx) proteins. To test this, we immunolabeled for eNOS and identified its expression at the MEJ in vivo (by TEM) and in our vascular cell co‐culture (VCCC) model (by confocal). We found increased eNOS phosphorylation at serine 1177 at the MEJ compared to the EC monolayer. This eNOS phosphorylation was selectively increased at the MEJ upon stimulation of SMCs with phenylephrine (PE) but not at the EC monolayer. Using the biotin switch assay, we determined the degree of S‐nitrosylated Cx43 at the MEJ. One minute after PE stimulation of SMCs, de‐nitrosylation of Cx43 was observed, followed by progressive re‐nitrosylation up to 20 min post‐PE stimulation. Functionally, this correlated with an inhibition of UV‐uncaged IP3 movement from SMC to EC 1 min after PE stimulation, which was restored 20 mins post PE stimulation. These results suggest that S‐nitrosylation of Cx proteins likely maintain open GJ channels and can contribute to heterocellular communication at the MEJ.

Thrombin receptors in vascular smooth muscle cells – function and regulation by vasodilatory prostaglandins

The vast majority of thrombin (>95%) is generated after clotting is completed, suggesting that thrombin formation serves purposes beyond coagulation, such as tissue repair after vessel injury. Two types of vascular thrombin binding sites exist: protease-activated receptors (PARs) and thrombomodulin (TM). Their expression is low in contractile vascular smooth muscle cells (SMC), the dominating subendothelial cell population, but becomes markedly up-regulated upon injury. In human SMC, PAR-1, PAR-3, and PAR-4 mediate thrombin-induced proliferation, migration and matrix biosynthesis as well as generation of inflammatory and growth-promoting mediators. Thrombin-responsive PARs are transcriptionally down-regulated in human vascular SMC by vasodilatory prostaglandins (PGI2/PGE2). For PAR-1 and PAR-3 this mechanism involves cAMP-dependent inactivation of the transcription factor NFAT. The human PAR-4 promoter does not possess NFAT recognition motifs suggesting involvement of other cAMP-regulated effectors. Unlike PARs, TM is induced in SMC exposed to vasodilatory prostaglandins. Enhanced thrombin binding to TM might ameliorate PAR-mediated SMC stimulation. Also expressed in human SMC is the endothelial protein C receptor (EPCR), which serves as an anchor to facilitate generation of activated protein C (aPC) by TM-bound thrombin. Whether prostaglandins affect aPC-generation is not known. In SMC, thrombin and aPC act synergistically via PAR-1 to modify tissue remodelling, in contrast to their antagonistic interaction in the coagulation pathways. Overall, this will contribute to plaque stability and wound healing. The processes outlined here are likely to become clinically relevant after up-regulation of vascular cyclooxygenase2, the rate limiting step in vascular PGE2/PGI2 biosynthesis, such as in advanced atherosclerosis and acute coronary syndromes.

Oxytocin Immunoreactivity in the Human Urethral (Littre's) Glands

Oxytocin is a cyclic nonapeptide whose best known effects are stimulation of uterine smooth muscle cells during labor and of milk ejection during lactation. Circulating oxytocin originates from the hypothalamus, but its production has also been documented in peripheral tissues. Furthermore, seminal plasma also contains oxytocin, but its functional role is still unknown, although its secretion is generally ascribed to the prostate. In this study, we investigated the possibility that seminal oxytocin is also secreted by other exocrine glands of the human male genital tract. Intramural (Littrè's) glands isolated from bioptic specimens of normal urethrae were processed for immunogold localization of oxytocin. Immunostaining was detected in principal cells, with gold particles specifically found on secretory granules. Basal and endocrine cells were unstained. The present findings suggest that urethral glands not only produce the mucinous layer that protects and lubricates the urethral wall, but also are potential sources of other seminal components, such as oxytocin, which probably play still unclear roles in reproductive physiology.

Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid‐β variants in cells composing the cerebral vessel walls

Cerebral amyloid angiopathy (CAA) is an age-associated condition and a common finding in Alzheimer's disease in which amyloid-beta (Abeta) vascular deposits are featured in >80% of the cases. Familial Abeta variants bearing substitutions at positions 21-23 are primarily associated with CAA, although they manifest with strikingly different clinical phenotypes: cerebral hemorrhage or dementia. The recently reported Piedmont L34V Abeta mutant, located outside the hot spot 21-23, shows a similar hemorrhagic phenotype, albeit less aggressive than the widely studied Dutch E22Q variant. We monitored the apoptotic events occurring after stimulation of human brain microvascular endothelial and smooth muscle cells with nonfibrillar structures of both variants and wild-type Abeta40. Induction of analogous caspase-mediated mitochondrial pathways was elicited by all peptides, although within different time frames and intensity. Activated pathways were susceptible to pharmacological modulation either through direct inhibition of mitochondrial cytochrome c release or by the action of pan- and pathway-specific caspase inhibitors, giving a clear indication of the independent or synergistic engagement of both extrinsic and intrinsic mechanisms. Structural analyses of the Abeta peptides showed that apoptosis preceded fibril formation, correlating with the presence of oligomers and/or protofibrils. The data support the notion that rare genetic mutations constitute unique paradigms to understand the molecular pathogenesis of CAA.

Cdc42GAP, reactive oxygen species, and the vimentin network

Cdc42GAP (GTPase-activating protein) has been implicated in the regulation of cell motility, adhesion, proliferation, and apoptosis. In this study, Cdc42GAP was cloned from smooth muscle tissues. Cdc42GAP, but not inactive R282A Cdc42GAP (alanine substitution at arginine-282), enhanced the GTP hydrolysis of Cdc42 in an in vitro assay. Furthermore, we developed an assay to evaluate the activity of Cdc42GAP in vivo. Stimulation of smooth muscle cells with 5-hydroxytryptamine (5-HT) resulted in the decrease in Cdc42GAP activity. The agonist-induced GAP suppression was reversed by reactive oxygen species inhibitors. Treatment with hydrogen peroxide also inhibited GAP activity in smooth muscle cells. Because the vimentin cytoskeleton undergoes dynamic changes in response to contractile activation, we evaluated the role of Cdc42GAP in regulating vimentin filaments. Smooth muscle cells were infected with retroviruses encoding wild-type Cdc42GAP or its R282A mutant. Expression of wild-type Cdc42GAP, but not mutant R282A GAP, inhibited the increase in the activation of Cdc42 upon agonist stimulation. Phosphorylation of p21-activated kinase (PAK) at Thr-423 (an indication of PAK activation), vimentin phosphorylation (Ser-56), partial disassembly and spatial remodeling, and contraction were also attenuated in smooth muscle cells expressing Cdc42GAP. Our results suggest that the activity of Cdc42GAP is regulated upon contractile activation, which is mediated by intracellular ROS. Cdc42GAP regulates the vimentin network through the Cdc42-PAK pathway in smooth muscle cells during 5-HT stimulation.

Cinaciguat, a soluble guanylate cyclase activator, causes potent and sustained pulmonary vasodilation in the ovine fetus

Impaired nitric oxide-cGMP signaling contributes to severe pulmonary hypertension after birth, which may in part be due to decreased soluble guanylate cyclase (sGC) activity. Cinaciguat (BAY 58-2667) is a novel sGC activator that causes vasodilation, even in the presence of oxidized heme or heme-free sGC, but its hemodynamic effects have not been studied in the perinatal lung. We performed surgery on eight fetal (126 +/- 2 days gestation) lambs (full term = 147 days) and placed catheters in the main pulmonary artery, aorta, and left atrium to measure pressures. An ultrasonic flow transducer was placed on the left pulmonary artery to measure blood flow, and a catheter was placed in the left pulmonary artery for drug infusion. Cinaciguat (0.1-100 microg over 10 min) caused dose-related increases in pulmonary blood flow greater than fourfold above baseline and reduced pulmonary vascular resistance by 80%. Treatment with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an sGC-oxidizing inhibitor, enhanced cinaciguat-induced pulmonary vasodilation by >120%. The pulmonary vasodilator effect of cinaciguat was prolonged, decreasing pulmonary vascular resistance for >1.5 h after brief infusion. In vitro stimulation of ovine fetal pulmonary artery smooth muscle cells with cinaciguat after ODQ treatment resulted in a 14-fold increase in cGMP compared with non-ODQ-treated cells. We conclude that cinaciguat causes potent and sustained fetal pulmonary vasodilation that is augmented in the presence of oxidized sGC and speculate that cinaciguat may have therapeutic potential for severe neonatal pulmonary hypertension.

Cell‐free supernatants of Escherichia coli Nissle 1917 modulate human colonic motility: evidence from an in vitro organ bath study

Abstract Clinical studies have shown that probiotics influence gastrointestinal motility, e.g. Escherichia coli Nissle 1917 (EcN) (Mutaflor) proved to be at least as efficacious as lactulose and more potent than placebo in constipated patients. As the underlying mechanisms are not clarified, the effects of EcN culture supernatants on human colonic motility were assessed in vitro. Human colonic circular smooth muscle strips (n = 94, 17 patients) were isometrically examined in an organ bath and exposed to different concentrations of EcN supernatants. Contractility responses were recorded under (i) native conditions, (ii) electrical field stimulation (EFS), (iii) non-adrenergic non-cholinergic conditions, and (iv) enteric nerve blockade by tetrodotoxin (TTX). As concentrations of acetic acid were increased in EcN supernatants, contractility responses to acetic acid were additionally tested. EcN supernatants significantly increased the maximal tension forces both at low and high concentrations. Neither blockade of both adrenergic and cholinergic nerves nor application of TTX abolished these effects. EFS-induced contractility responses were not altered after exposure to EcN supernatants. Acetic acid elicited effects comparable to EcN supernatants only under TTX conditions. EcN supernatants modulate in vitro contractility of the human colon. As neither partial nor TTX blockade of enteric nerves abolished these effects, EcN supernatants appear to enhance colonic contractility by direct stimulation of smooth muscle cells. Active metabolites may include other substances than acetic acid, as acetic acid only partially resembled the effects elicited by EcN supernatants. The data provide a rationale for therapeutical application of probiotics in gastrointestinal motility disorders.

Urotensin II Induced Signal Involved in Proliferation of Vascular Smooth Muscle Cells

Urotensin II receptor (UIIR), bound by the ligand Urotensin II (UII), generates second messengers IP3 and DAG, which stimulates the subsequent release of calcium (Ca2+) in vascular smooth muscle cells (VSMC). Ca2+ influx leads to the activation of Ca2+ dependent kinases via calmodulin binding, resulting in cellular proliferation. We hypothesize that UII signaling in pulmonary arterial vascular smooth muscle cells (Pac1) and primary aortic vascular smooth muscle cells (PAVSMC) results in phosphorylation of Ca2+/ CaM dependent kinases leading to cellular proliferation. UII exposure to Pac1 cell cultures increased intracellular Ca2+, which subsequently activated Ca2+/CaM dependent kinases (CaMKK & CaMKI), ERK 1/ 2, and PKD (PKC‐µ). Pac1 and PAVSMC cells treated with UII resulted in increased proliferation as measured by 3H thymidine uptake. UII‐induced increase of 3H thymidine incorporation was inhibited by a CaMKK inhibitor. Taken together, our results demonstrate that UII stimulation of smooth muscle cells leads to cellular proliferation, utilizing signaling mechanisms involving Ca2+/CaM dependent kinases.

Inducible nitric oxide synthase contributes to MAPK‐phosphatase induction and abundance of cyclooxygenase‐2 expression in an inflammatory setting

Cyclooxygenase (COX)‐2 and inducible nitric oxide synthase (iNOS) are responsive to inflammatory stimuli and have been localized to vascular smooth muscle cells (SMCs) in pathophysiological settings, e.g. atherosclerosis. Furthermore, the production of, and subsequent impact of COX products appear to be correlative with the status of NO synthesis and bioavailability. This study examined the role of inflammation‐driven NO synthesis on COX‐2 expression in rat aortic SMCs. Concurrent stimulation of quiescent SMCs with lipopolysaccharide (LPS) and interferon (IFN)‐γ induced iNOS and COX‐2 expression maximally at 2‐4hr. Inhibition of NO synthesis (LNMMA) significantly reduced nitrite (140±8 vs. 19±7 nmol/mg protein) and enhanced LPS+IFN‐γ‐mediated COX‐2 mRNA (156‐ vs. 43‐fold increase) and protein expression (5‐fold) at 30 and 60min. COX‐2 induction was associated with activation of the ERK, p38 and JNK pathways. Importantly, LNMMA enhanced ERK and p38 phosphorylation, and suppressed MAPK‐phosphatase (MKP)‐1 induction. Consistent with these data, exposure of SMCs from iNOS‐/‐ mice to LPS+IFN‐γ produced a more robust induction (p<0.05) of COX‐2 and a dramatically attenuated upregulation of MKP‐1 vs. WT. These findings provide pharmacological and genetic evidence implicating an inhibitory influence of iNOS‐derived species on phosphorylation cascades associated with COX‐2‐induction in SMCs.

NAADP contributes to norepinephrine and endothelin‐1‐mediated Ca2+ stimulation in rat renal afferent arterioles

Nicotinic acid adenine dinucleotide phosphate (NAADP) may be produced by ADPR cyclase residing in acidic vesicles in response to G protein‐coupled receptor (GPCR) stimulation in vascular smooth muscle cells. We tested the hypothesis that NAADP receptors mediate increases in cytosolic Ca2+ ([Ca2+]i ) induced by norepinephrine (NE) and endothelin‐1 (ET‐1) in isolated renal afferent arterioles via a lysosome‐dependent pathway. To test this, we measured [Ca2+]i responses to NE and ET‐1 in individual arterioles of Sprague‐Dawley rats. ET‐1 increased peak [Ca2+]i by 100‐130 nM. This response was attenuated by lysosome disrupters concanamycin A1 (CCA) and bafilomycin A (Baf) to 58 ± 8 and 64 ± 10 nM, respectively (P < 0.05). The NAADP receptor inhibitor NAADP EON discovered molecule 14 (Ned‐14) reduced ET‐1 stimulation to 34 ± 11 nM (P < 0.005). Control NE increased immediate [Ca2+]i 70‐80 nM. CCA and Baf decreased the response to 37 ± 3 and 47 ± 7 nM, respectively (P < 0.05). Ned‐14 decreased NE responses to 25 ± 8 nM (P < 0.005). We conclude that NAADP receptors and lysosomal vesicles contribute to Ca2+ signaling pathways evoked acutely by ET‐1 and NE in renal afferent arterioles.

Beta]‐arrestin 1 mediates angiotensin II induced ubiquitination and down‐regulation of TRPV4

β‐arrestins were originally discovered to desensitize activated G protein‐coupled receptors (GPCRs), also known as seven transmembrane receptor (7TMRs). It was subsequently found that β‐arrestins also mediate 7TMR internalization by bringing the activated receptors to clathrin‐coated pits. More recently, it has been discovered that β‐arrestins can also interact with and regulate ion channels and transporters. Using a proteomic approach, we identified TRPV4 as an interacting partner of β‐arrestin 1 after stimulation of cells with angiotensin II. Here we demonstrate that β‐arrestin 1 is recruited to TRPV4 upon angiotensin II stimulation in HEK‐293 cells and vascular smooth muscle cells. Surprisingly, angiotensin II stimulation leads to ubiquitination of TRPV4 which is abolished by β‐arrestin 1 siRNA. β‐arrestin 1 shows constitutive interaction with AIP4, a known ubiquitin E3 ligase for TRPV4, which is further increased upon angiotensin stimulation. Moreover, the interaction of TRPV4 and AIP4 is also significantly reduced by β‐arrestin 1 siRNA. These findings suggest that β‐arrestin 1 serves as an adaptor for AIP4 and thereby facilitates TRPV4 ubiquitination and down‐regulation. Taken together with the previous reports of analogous function of β‐arrestins for 7TMRs, this study suggests a novel role of β‐arrestins as a general adaptor for ubiquitin E3 ligases.

CBP and p300 in renin homeostasis: can they drive the fate?

CBP and p300 in renin homeostasis: can they drive the fate?

The timing of intra-coronary infusion of G-CSF mobilized peripheral blood stem cells influences cardiac function and in-stent restenosis in patients with myocardial infarction

We hypothesized that delaying the timing of intra-coronary infusion of G-CSF mobilized stem cell until at least 4 weeks after coronary stenting should avoid the stimulation of vascular smooth muscle cells during the early active cellular proliferative phase, thus decreases in-stent restenosis while preserving the beneficial effect of stem cell therapy on cardiac function in patients with myocardial infarction (MI). 25 patients with ST-elevation myocardial infarction (STEMI) treated with stenting were enrolled in this pilot study. The ages of MI at the time of cell treatment were from 1 month to 59 months. At 6 months follow-up, the left ventricular ejection fraction (LVEF) increased from 32% to 37.7% and the stress thallium perfusion defect decreased from 31.4% to 28.1%. Cell treatment-related complications such as arrhythmias were not observed. 9 patients who underwent cell treatment less than 3 months after coronary stenting were evaluated for in-stent restenosis; it was found in only 1 patient. This pilot study shows that delayed more than 4 weeks after coronary stenting but less than 3 months after MI, intra-coronary infusion of G-CSF mobilized PBSCs may improve cardiac function without triggering in-stent restenosis.

Rho-kinase Contributes to Sustained RhoA Activation through Phosphorylation of p190A RhoGAP

RhoA is transiently activated by specific extracellular signals such as endothelin-1 (ET-1) in vascular smooth muscle cells. RhoGAP negatively regulates RhoA activity: thus, RhoA becomes the GDP-bound inactive form afterward. Sustained activation of RhoA is induced with high doses of the extracellular signals and is implicated in certain diseases such as vasospasms. However, it remains largely unknown how prolonged activation of RhoA is induced. Here we show that Rho-kinase, an effector of RhoA, phosphorylated p190A RhoGAP at Ser(1150) and attenuated p190A RhoGAP activity in COS7 cells. Binding of Rnd to p190A RhoGAP is thought to enhance its activation. Phosphorylation of p190A RhoGAP by Rho-kinase impaired Rnd binding. Stimulation of vascular smooth muscle cells with a high dose of ET-1 provoked sustained RhoA activation and p190A RhoGAP phosphorylation, both of which were prohibited by a Rho-kinase inhibitor. The phosphomimic mutation of p190A RhoGAP weakened Rnd binding and RhoGAP activities. Taken together, these results suggest that ET-1 induces Rho-kinase activation and subsequent phosphorylation of p190A RhoGAP, leading to prolonged RhoA activation.

Force Transduction In Smooth Muscle Cells

Mechanical forces directly affect the form and function of tissues. Transmission of force from outside the cell through focal and junctional adhesions controls the maturation or disassembly of these adhesion sites and initiates intracellular signaling cascades that alter cellular behavior. To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a critical first step is to develop a new technology able to investigate cellular behavior at sub-cellular level that integrates an atomic force microscope (AFM) with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy, providing high spatial and temporal resolution. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time.Thus, we are able to: (i) image with high spatial resolution or stimulate the apical cell surface using AFM, and (ii) quantitatively time-lapse image the cell-coverslip interface using TIRF, or FSD confocal image to study molecular dynamics and protein translocation between different sub-cellular structures. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical smooth muscle cell stimulation at the apical cell membrane that induced changes into the cellular structure throughout the cell body.By exploring innovative approaches like those used in these investigations, new information for understanding live cell restructuring and dynamics in response to mechanical force can be provided. Understanding how live cells adapt to mechanical force and how they are able to recognize and respond to mechanical stimuli represents an important biophysical problem.