Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries: role in vasomotor control

Endothelial cells (ECs) govern smooth muscle cell (SMC) tone via the release of paracrine factors (eg, NO and metabolites of arachidonic acid). We tested the hypothesis that ECs can promote SMC relaxation or contraction via direct electrical coupling. Vessels (resting diameter, 57+/-3 microm; length, 4 mm) were isolated, cannulated, and pressurized (75 mm Hg; 37 degrees C). Two microelectrodes were used to simultaneously impale 2 cells (ECs or SMCs) in the vessel wall separated by 500 microm. Impalements of one EC and one SMC (n=26) displayed equivalent membrane potentials at rest, during spontaneous oscillations, and during hyperpolarization and vasodilation to acetylcholine. Injection of -0.8 nA into an EC caused hyperpolarization ( approximately 5 mV) and relaxation of SMCs (dilation, approximately 5 microm) along the vessel segment. In a reciprocal manner, +0.8 nA caused depolarization ( approximately 2 mV) of SMCs with constriction ( approximately 2 microm). Current injection into SMCs while recording from ECs produced similar results. We conclude that ECs and SMCs are electrically coupled to each other in these vessels, such that electrical signals conducted along the endothelium can be directly transmitted to the surrounding smooth muscle to evoke vasomotor responses.     

Involvement of myoendothelial gap junctions in the actions of endothelium-derived hyperpolarizing factor

The nature of the vasodilator endothelium-derived hyperpolarizing factor (EDHF) is controversial, putatively involving diffusible factors and/or electrotonic spread of hyperpolarization generated in the endothelium via myoendothelial gap junctions (MEGJs). In this study, we investigated the relationship between the existence of MEGJs, endothelial cell (EC) hyperpolarization, and EDHF-attributed smooth muscle cell (SMC) hyperpolarization in two different arteries: the rat mesenteric artery, where EDHF-mediated vasodilation is prominent, and the femoral artery, where there is no EDHF-dependent relaxation. In the rat mesenteric artery, stimulation of the endothelium with acetylcholine (ACh) evoked hyperpolarization of both ECs and SMCs, and characteristic pentalaminar MEGJs were found connecting the two cell layers. In contrast, in the femoral artery, ACh evoked hyperpolarization in only ECs but not in SMCs, and no MEGJs were present. Selective hyperpolarization of ECs or SMCs evoked hyperpolarization in the other cell type in the mesenteric artery but not in the femoral artery. Disruption of gap junctional coupling using the peptide Gap 27 markedly reduced the ACh-induced hyperpolarization in SMCs, but not in ECs, of the mesenteric artery. These results show that transfer of EC hyperpolarization or of a small molecule to SMCs through MEGJs is essential and sufficient to explain EDHF.     

Stem cell origins of intimal cells in graft arterial disease

Long-term solid organ allografts develop diffuse arterial intimal lesions (graft arterial disease [GAD]), consisting of smooth muscle cells (SMCs), extracellular matrix, and admixed mononuclear leukocytes. Although the exact mechanisms are unknown, alloresponses likely induce inflammatory cells and/or dysfunctional vascular wall cells to secrete growth factors that promote SMC intimal recruitment, proliferation, and matrix synthesis. GAD eventually culminates in vascular stenosis and ischemic graft failure. Although prior work demonstrated that the endothelium and medial SMCs and the vast majority of endothelial cells (ECs) lining GAD lesions in cardiac allografts are derived from donors, the intimal SMC origin could not be determined. Recent reports suggest that intimal lesions in allograft vessels may also contain host-derived ECs and SMCs. In light of these findings, it is noteworthy that subpopulations of bone marrow and circulating cells have also been shown to differentiate into ECs and SMCs. Here we review recent developments in the understanding of vascular wall cell recruitment that are forcing a re-evaluation of the pathogenic mechanisms underlying GAD, as well as those occurring in more “conventional” atherosclerosis. The demonstration of the host origin of intimal SMCs in GAD lays the groundwork for future interventions where therapeutic genes or drugs may be targeted not to donor medial SMCs, but rather to recipient SM precursor cells.     

Endothelial cells lining transjugular intrahepatic portasystemic shunts originate in hepatic sinusoids: implications for pseudointimal hyperplasia

The phenotype of the endothelial cells (ECs) in the pseudointima of transjugular intrahepatic portasystemic shunts (TIPS) and the mechanisms of pseudointima formation after TIPS were unknown. We hypothesized that TIPS were lined by hepatic sinusoidal ECs, which stimulated the migration of smooth muscle cells (SMCs) into the pseudointima and their proliferation. Studies were done with the following specific aims: (1) isolation of ECs from TIPS pseudointima and comparison of their phenotype with human cirrhotic sinusoidal and vascular ECs derived from hepatic and portal veins as well as aorta, and (2) testing of the effects of TIPS ECs on TIPS-derived SMC migration and proliferation. ECs were isolated from eight TIPS retrieved from liver explants by immunomagnetic separation using monodispersed magnetizable polystyrene beads (Dynabeads M-450) coated with Ulex Europeus 1. EC phenotypes were examined by transmission electron microscopy, factor VIII-related antigen, CD31, CD14, and CD34 expression, uptake of acetylated LDL and secretion of type IV collagen. The effects of EC-conditioned media on SMC migration and proliferation were tested in multiwell chemotaxis chambers and by cell counting, respectively. ECs were obtained from TIPS pseudointima with >95% purity. The phenotype of TIPS-derived ECs matched that of cirrhotic sinusoidal endothelium (both expressed CD14) and differed from that of vascular endothelium (CD14 negative, Weibel-Palade positive). Conditioned media from both stenosed (n = 3) and nonstenosed (n = 3) TIPS-derived endothelial cells produced a marked (>100%) P <.001 increase in migration as well as (up to 88%) P <.01 proliferation of SMCs from both stenosed (n = 3) as well as nonstenosed TIPS (n = 3). These data indicate that TIPS pseudointima are lined by hepatic sinusoidal endothelial cells, which stimulate pseudointima formation by increasing SMC migration and proliferation.     

Cyclic tensile strain triggers a sequence of autocrine and paracrine signaling to regulate angiogenic sprouting in human vascular cells

Mechanical signals regulate blood vessel development in vivo, and have been demonstrated to regulate signal transduction of endothelial cell (EC) and smooth muscle cell (SMC) phenotype in vitro. However, it is unclear how the complex process of angiogenesis, which involves multiple cell types and growth factors that act in a spatiotemporally regulated manner, is triggered by a mechanical input. Here, we describe a mechanism for modulating vascular cells during sequential stages of an in vitro model of early angiogenesis by applying cyclic tensile strain. Cyclic strain of human umbilical vein (HUV)ECs up-regulated the secretion of angiopoietin (Ang)-2 and PDGF-ββ, and enhanced endothelial migration and sprout formation, whereas effects were eliminated with shRNA knockdown of endogenous Ang-2. Applying strain to colonies of HUVEC, cocultured on the same micropatterned substrate with nonstrained human aortic (HA)SMCs, led to a directed migration of the HASMC toward migrating HUVECs, with diminished recruitment when PDGF receptors were neutralized. These results demonstrate that a singular mechanical cue (cyclic tensile strain) can trigger a cascade of autocrine and paracrine signaling events between ECs and SMCs critical to the angiogenic process.     

JTT-705 blocks cell proliferation and angiogenesis through p38 kinase/p27(kip1) and Ras/p21(waf1) pathways

The excessive proliferation and migration of vascular smooth muscle cells (SMCs) participate in the growth and instability of atherosclerotic plaque. We examined the direct role of a newly developed chemical inhibitor of cholesteryl ester transfer protein, JTT-705, on SMC proliferation and angiogenesis in endothelial cells (ECs). JTT-705 inhibited human coronary artery SMC proliferation. JTT-705 induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular-signal-regulated kinases (ERK) in SMCs. In addition, the anti-proliferative effects of JTT-705 in SMCs were blocked by p38 MAPK inhibitor. JTT-705 induced the upregulation of p-p21(waf1), and this effect was blocked by dominant-negative Ras (N17), but not by inhibitors of p38 MAPK or ERK. In addition, JTT-705 also induced the upregulation of p27(kip1), and this effect was blocked by p38 MAPK inhibitor. Interestingly, culture medium from JTT-705-treated SMCs blocked human coronary artery EC tube formation in an in vitro model of angiogenesis indirectly via a decrease in vascular endothelial growth factor (VEGF) from SMCs and directly via an anti-proliferative effect in ECs. JTT-705 blocked the proliferation of SMCs through the activation of p38 kinase/p27(kip1) and Ras/p21(waf1) pathways, and simultaneously blocked EC tube formation associated with a decrease in VEGF production from SMCs and an anti-proliferative effect in ECs. Our results indicate that JTT-705 may induce a direct anti-atherogenic effect in addition to its inhibitory effect of CETP activity.     

Mesenchymal stem cells participating in ex vivo endothelium repair and its effect on vascular smooth muscle cells growth

BACKGROUND: Previous studies have shown that mesenchymal stem cells (MSCs) transplantation can promote neovascularization and regenerate damaged myocardium. However, it remains unknown whether MSCs seeding can be used to repair injured cellular components in vascular diseases. In this study we explored the feasibility of applying MSCs to endothelium repair in endothelial damage and vasoproliferative disorders. METHODS: Ex vivo model of endothelium repair was developed in which rabbit vascular smooth muscle cells (SMCs) were inoculated into the upper chamber and rabbit endothelial cells (ECs)/human MSCs into the lower chamber of a co-culture system. 3H-TdR incorporation and PCNA protein expression were assayed and migrated number of SMCs was calculated to evaluate the effect of MSCs seeding on SMCs growth. Flk-1 and vWF protein expressions were observed to analyze the plasticity of the seeded MSCs along endothelial lineage. RESULTS: In this co-culture system, no vWF protein but Flk-1 protein was observed in the 25.71% of MSCs after having been co-cultured with mature rabbit ECs for 5 days. Compared with the control group, the proliferation and migration of SMCs was significantly increased by proliferative ECs but decreased by confluent ECs (n=6, P<0.01). MSCs seeding decreased the proliferation and migration of SMCs compatible with the effect of proliferative ECs (n=6, P<0.001). However, no inhibition on SMCs growth was observed with MSCs seeding in comparison to the effect of confluent ECs. CONCLUSIONS: MSCs seeding can inhibit the proliferation and migration of SMCs. MSCs co-cultured with mature ECs have the ability to undergo milieu-dependent differentiation toward ECs.     

Hepatocyte growth factor mediates angiopoietin-induced smooth muscle cell recruitment

Communication between endothelial cells (ECs) and mural cells is critical in vascular maturation. Genetic studies suggest that angiopoietin/Tie2 signaling may play a role in the recruitment of pericytes or smooth muscle cells (SMCs) during vascular maturation. However, the molecular mechanism is unclear. We used microarray technology to analyze genes regulated by angiopoietin-1 (Ang1), an agonist ligand for Tie2, in endothelial cells (ECs). We observed that hepatocyte growth factor (HGF), a mediator of mural cell motility, was up-regulated by Ang1 stimulation. We confirmed this finding by Northern blot and Western blot analyses in cultured vascular endothelial cells. Furthermore, stimulation of ECs with Ang1 increased SMC migration toward endothelial cells in a coculture assay. Addition of a neutralizing anti-HGF antibody inhibited Ang1-induced SMC recruitment, indicating that the induction of SMC migration by Ang1 was caused by the increase of HGF. Interestingly, Ang2, an antagonist ligand of Tie2, inhibited Ang1-induced HGF production and Ang1-induced SMC migration. Finally, we showed that deletion of Tie2 in transgenic mouse reduced HGF production. Collectively, our data reveal a novel mechanism of Ang/Tie2 signaling in regulating vascular maturation and suggest that a delicate balance between Ang1 and Ang2 is critical in this process.     

Myoendothelial coupling is unidirectional in guinea pig spiral modiolar arteries

Gap junctions (GJs) facilitate communication and promote transfer of signaling molecules or current between adjacent cells in various organs to coordinate cellular activity. In arteries, homocellular GJs are present between adjacent smooth muscle cells (SMCs) and between adjacent endothelial cells (ECs), whilst many arteries also exhibit heterocellular GJs between SMCs and ECs. To test the hypothesis that there is differential cell coupling in guinea pig spiral modiolar arteries (SMA), we used intracellular recording technique to record cellular activities simultaneously in ECs or SMCs in acutely isolated guinea pig SMA preparations. Cell types were identified by injection of a fluorescent dye, propidium iodide (PI), through recording microelectrodes. Stable intracellular recordings were made in 120 cells among which 61 were identified as SMCs and 28 as ECs. Dual intracellular recordings were conducted to detect the coexistence of the two distinct levels of resting potential (RP) and to estimate the intensity of electrical coupling between two cells by a current pulse of up to 0.5-1.5 nA. The electrotonic potential was detected not only in the current-injected cell, but also in the majority of non-injected cells. The electrical coupling ratios (ECRs) of homocellular cells were not significant (P>0.05) (0.084±0.032 (n=6) and 0.069±0.031 (n=7) for EC-EC and SMC-SMC pairs, respectively). By contrast, the ECRs of heterocellular cells were significantly different when a current pulse (1.5 nA, 2s) was injected into EC and SMC respectively (0.072±0.025 for EC; 0.003±0.001 for SMC, n=5, P<0.01). The putative gap junction blocker 18β-glycyrrhetinic acid significantly attenuated electrical coupling in both homocellular and heterocellular forms. The results suggest that homocellular GJs within SMCs or ECs are well coordinated but myoendothelial couplings between ECs and SMCs are unidirectional.     

Coagulation factors X, Xa, and protein S as potent mitogens of cultured aortic smooth muscle cells.

Smooth muscle cells (SMCs) in the rat carotid artery leave the quiescent state and proliferate after balloon catheter injury. The precise signals responsible for this SMC mitogenesis need to be elucidated. Although platelet-derived growth factor (PDGF), a potent SMC mitogen, is released from activated platelets, damaged endothelium, and macrophages, it cannot be solely responsible for this proliferation. In search of other SMC growth factors, we have examined several proteins of the coagulation cascade. At nanomolar concentrations, factors X, Xa, and protein S promote cultured rat aortic SMC mitosis. In contrast, factor IX is only weakly mitogenic, whereas factor VII and protein C fail to stimulate SMC division. Protein S, the most mitogenic of these coagulation cascade factors, stimulates DNA synthesis in cultured SMCs with a time course similar to that of PDGF-AA and without the delay observed for transforming growth factor beta. Antistasin and tick anticoagulant peptide, two specific factor Xa inhibitors, inhibit SMC mitogenesis due to Xa and protein S. Coagulation factors that possess mitogenic activity may contribute to intimal SMC proliferation after vascular injury as a result of angioplasty or vascular compromise during atherogenesis.     

A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall

Reactive oxygen species (ROS) play an important role in regulating vascular tone and intracellular signaling; the enzymes producing ROS in the vascular wall are, however, poorly characterized. We investigated whether a functionally active NADPH oxidase similar to the leukocyte enzyme, ie, containing the subunits p22phox and gp91phox, is expressed in endothelial cells (ECs) and smooth muscle cells (SMCs). Phorbol 12-myristate 13-acetate (PMA), a stimulus for leukocyte NADPH oxidase, increased ROS generation in cultured ECs and endothelium-intact rat aortic segments, but not in SMCs or endothelium-denuded arteries. NADPH enhanced chemiluminescence in all preparations. p22phox mRNA and protein was detected in ECs and SMCs, whereas the expression of gp91phox was confined to ECs. Endothelial gp91phox was identical to the leukocyte form as determined by sequence analysis. In contrast, mitogenic oxidase-1 (mox1) was expressed in SMCs, but not in ECs. To determine the functional relevance of gp91phox expression, experiments were performed in aortic segments from wild-type, gp91phox(-/-), and endothelial NO synthase (eNOS)(-/-) mice. PMA-induced ROS generation was comparable in aortae from wild-type and eNOS(-/-) mice, but was attenuated in segments from gp91phox(-/-) mice. Endothelium-dependent relaxation was greater in aortae from gp91phox(-/-) than from wild-type mice. The ROS scavenger tiron increased endothelium-dependent relaxation in segments from wild-type, but not from gp91phox(-/-) mice. These data demonstrate that ECs, in contrast to SMCs, express a gp91phox-containing leukocyte-type NADPH oxidase. This enzyme is a major source for arterial ROS generation and affects the bioavailability of endothelium-derived NO.     

Galectin 1 modulates attachment, spreading and migration of cultured vascular smooth muscle cells via interactions with cellular receptors and components of extracellular matrix

Galectin 1 (Gal-1), a lactose-binding lectin, is a component of vascular extracellular matrix and secreted by human vascular smooth muscle cells (SMCs). The purpose of this study was to investigate a possible role of Gal-1 in controlling adhesion and migration of cultured human vascular SMCs. Gal-1 co-localised with laminin and cellular fibronectin in extracellular matrix (ECM) secreted by cultured human vascular SMCs. Recombinant glutathione S-transferase (GST)-Gal-1 fusion protein bound to laminin and cellular fibronectin in ELISA. GST-Gal-1 inhibited SMC attachment to laminin via interactions with both SMCs and laminin. GST-Gal-1 inhibited SMC spreading on plastic or on laminin, but not on cellular fibronectin. GST-Gal-1 modulated SMC migration on laminin and inhibited migration on cellular fibronectin. GST-Gal-1 bound to several 35S-labelled proteins in SMC extracts including laminin and alpha1beta1 integrin, identified by depletion of SMC protein extracts with respective antibodies. We conclude that Gal-1 is able to modulate SMC attachment, spreading and migration via interactions with ECM proteins and alpha1beta1 integrin.     

Collagens,integrins, and the discoidin domain receptors in arterial occlusive disease

The collagen matrix constitutes a major portion of the vascular extracellular matrix and imparts blood vessels with tensile strength and, even more important, modulates smooth muscle cell (SMC) responses via specific receptors and signaling pathways. This review is focused on the interactions of SMCs with the collagen matrix, how these interactions are involved in sensing the local environment, and the receptors that mediate these processes. Better understanding of the pathways involved in cell matrix interactions promises to provide novel therapeutic targets and treatment strategies for the prevention of arterial occlusive diseases such as atherosclerosis and restenosis.     

Smooth muscle cell and endothelial cell growth factors

Blood vessels are composed basically of two cell types, vascular endothelial cells (ECs) and vascular smooth muscle cells (SMCs), whose proliferation in vivo is tightly regulated. A number of growth regulatory polypeptides have been identified that stimulate the proliferation of vascular cells. This article reviews briefly the structural properties and biologic activities of the best-characterized vascular cell growth factors. A fuller understanding of the properties of vascular cell growth modulators is an important element in delineating the proliferative events that are associated with vascular injury; with SMC hyperplasia such as occurs in restenosis following angioplasty, in atherosclerosis, and in hypertension; and with angiogenesis, both physiologic and pathologic.     

Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cells and its inhibition by shear stress

E-selectin is a major adhesion molecule expressed by endothelial cells (ECs), which are exposed to shear stress and neighboring smooth muscle cells (SMCs). We investigated the mechanisms underlying the modulation of EC E-selectin expression by SMCs and shear stress. SMC coculture induced rapid and sustained increases in expression of E-selectin and phosphorylation of interleukin-1 (IL-1) receptor-associated kinase glycoprotein-130, as well as the downstream mitogen-activated protein kinases (MAPKs) and Akt. By using specific inhibitors, dominant-negative mutants, and small interfering RNA, we demonstrated that activations of c-Jun-NH(2)-terminal kinase (JNK) and p38 of the MAPK pathways are critical for the coculture-induced E-selectin expression. Gel shifting and chromatin immunoprecipitation assays showed that SMC coculture increased the nuclear factor-kappaB (NF-kappaB)-promoter binding activity in ECs; inhibition of NF-kappaB activation by p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced E-selectin promoter activity. Protein arrays and blocking assays using neutralizing antibodies demonstrated that IL-1beta and IL-6 produced by EC/SMC cocultures are major contributors to the coculture induction of EC signaling and E-selectin expression. Preshearing of ECs at 12 dynes/cm(2) inhibited the coculture-induced EC signaling and E-selectin expression. Our findings have elucidated the molecular mechanisms underlying the SMC induction of EC E-selectin expression and the shear stress protection against this SMC induction.     

Direct and indirect effects of pulsatile shear stress on the smooth muscle cell.

BACKGROUND: Anastomotic intimal hyperplasia is still an unsolved problem after small caliber prosthetic bypass grafting. Oscillatory turbulent flow occurs at the end to side anastomosis, and produces various effects on smooth muscle cells (SMCs) and endothelial cells (ECs), which compose intimal hyperplasia. We examined the influences of pulsatile oscillating shear stress on smooth muscle cells mitogenic activity induced by sheared endothelial cells. METHODS:1) Smooth muscle cells were cultured under three different pulsatile shear conditions (mean: 0, 6, and 60 dyne/cm2). 2) Endothelial cells were cultured under both static and sheared condition (mean: 60 dyne/cm2). Using the conditioned media from each well, SMCs were cultured under static and sheared conditions (60 dyne/cm2). Four groups of SMCs were devised by combining the two types of media and the two culture conditions. SMC colony spreading distances were measured as an index of combined migration and proliferation activity. An MTT assay and a cell counting assay were used to determine the proliferation activities of SMCs. RESULTS: 1) SMC spreading activity was suppressed by shear stress. SMC proliferative activity was stimulated by pulsatile turbulent shear stress. 2) SMC spreading activity was stimulated by mitogens derived from ECs under shear stress. However, this augmented SMC spreading activity was attenuated under sheared conditions. The mitogens derived from ECs under pulsatile shear stress had no effects on SMC proliferation activity. CONCLUSIONS: Pulsatile oscillating shear stress attenuates SMC migration activity induced by EC-denve mitogens and stimulates SMC proliferative activity.     

Shear stress inhibits adhesion molecule expression in vascular endothelial cells induced by coculture with smooth muscle cells

Vascular endothelial cells (ECs), which exist in close proximity to vascular smooth muscle cells (SMCs), are constantly subjected to blood flow-induced shear stress. Although the effect of shear stress on endothelial biology has been extensively studied, the influence of SMCs on endothelial response to shear stress remains largely unexplored. We examined the potential role of SMCs in regulating the shear stress-induced gene expression in ECs, using a parallel-plate coculture flow system in which these 2 types of cells were separated by a porous membrane. In this coculture system, SMCs tended to orient perpendicularly to the flow direction, whereas the ECs were elongated and aligned with the flow direction. Under static conditions, coculture with SMCs induced EC gene expression of intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin, while attenuating EC gene expression of endothelial nitric oxide synthase (eNOS). Shear stress significantly inhibited SMC-induced adhesion molecule gene expression. These EC responses under static and shear conditions were not observed in the absence of close communication between ECs and SMCs, and they were also not observed when ECs were cocultured with fibroblasts instead of SMCs. Our findings indicate that under static conditions, coculture with SMCs induces ICAM-1, VCAM-1, and E-selectin gene expression in ECs. These coculture effects are inhibited by shear stress and require specific interaction between ECs and SMCs in close contact.