Medial Vascular Smooth Muscle Cells Research Articles

BMP type I receptor inhibition attenuates endothelial dysfunction in mice with chronic kidney disease

The molecular mechanisms of endothelial dysfunction and vascular calcification have been considered independently and potential links are currently unknown in chronic kidney disease (CKD). Bone morphogenetic protein (BMP) receptor signaling mediates calcification of atherosclerotic plaques. Here we tested whether BMP receptor signaling contributes to endothelial dysfunction, as well as to osteogenic differentiation of vascular smooth muscle cells (VSMCs), in a model of short-term CKD. In C57BL/6 mice, subtotal nephrectomy activated BMP receptor and increased phosphatase-and-tensin homolog (PTEN) protein in the endothelial cells and medial VSMCs without vascular remodeling in the aorta. In the endothelial cells, PTEN induction led to inhibition of the Akt-endothelial nitric oxide synthase (eNOS) pathway and endothelial dysfunction. In VSMCs, the PTEN increase induced early osteogenic differentiation. CKD-induced inhibition of eNOS phosphorylation and the resultant endothelial dysfunction were inhibited in mice with endothelial cell-specific PTEN ablation. Knockout of the BMP type I receptor abolished endothelial dysfunction, the inhibition of eNOS phosphorylation, and VSMC osteogenic differentiation in mice with CKD. A small molecule inhibitor of BMP type I receptor, LDN-193189, prevented endothelial dysfunction and osteogenic differentiation in CKD mice. Thus, BMP receptor activation is a mechanism for endothelial dysfunction in addition to vascular osteogenic differentiation in a short-term CKD model. PTEN may be key in linking BMP receptor activation and endothelial dysfunction in CKD.

Connexins and intercellular communication in arterial growth and remodeling

Gap junction functionality and intercellular exchange of chemical messengers and other biologically important cellular signals makes communication between adjacent cells possible and is essential for multicellular living systems. This intercellular communication is carried out through gap junction channels that are comprised of multimeric connexin (Cx) proteins and that provide physical linkages between neighboring cells. This Cx-specific gap junction intercellular communication (GJIC) helps perform numerous essential physiological functions in different organ systems. In vascular tissues, reciprocal GJIC between intimal vascular endothelial cells (VECs) and medial vascular smooth muscle cells (VSMCs) helps to control blood flow and arterial pressures by signaling contraction or relaxation events. In the vascular system appropriate GJIC also regulates responses to circulating hormones and other vasoactive factors as well as growth during episodes of stimulated angiogenesis and/or vasculogenesis. Deleterious GJIC, however, serves as a contributing element in vascular pathologies and plays key roles in abnormal vascular cell proliferation, migration, and inflammation, foundational mechanisms of arterial remodeling and neointima development. In this review we discuss the critical roles of vascular gap junction Cx proteins and GJIC in regulating pathologic arterial remodeling.

Adventitial inflammation and its interaction with intimal atherosclerotic lesions.

The presence of adventitial inflammation in correlation with atherosclerotic lesions has been recognized for decades. In the last years, several studies have investigated the relevance and impact of adventitial inflammation on atherogenesis. In the abdominal aorta of elderly Apoe−/− mice, adventitial inflammatory structures were characterized as organized ectopic lymphoid tissue, and therefore termed adventitial tertiary lymphoid organs (ATLOs). These ATLOs possess similarities in development, structure and function to secondary lymphoid organs. A crosstalk between intimal atherosclerotic lesions and ATLOs has been suggested, and several studies could demonstrate a potential role for medial vascular smooth muscle cells in this process. We here review the development, phenotypic characteristics, and function of ATLOs in atherosclerosis. Furthermore, we discuss the possible role of medial vascular smooth muscle cells and their interaction between plaque and ATLOs.

Tumor necrosis factor-like weak inducer of apoptosis or Fn14 deficiency reduce elastase perfusion-induced aortic abdominal aneurysm in mice.

BackgroundAbdominal aortic aneurysm (AAA) involves leukocyte recruitment, inflammatory cytokine production, vascular cell apoptosis, neovascularization, and vascular remodeling, all of which contribute to aortic dilatation. Tumor necrosis factor‐like weak inducer of apoptosis (TWEAK) is a cytokine implicated in proinflammatory responses, angiogenesis, and matrix degradation but its role in AAA formation is currently unknown.Methods and ResultsExperimental AAA with aortic elastase perfusion in mice was induced in wild‐type (WT), TWEAK deficient (TWEAK KO), or Fn14‐deficient (Fn14 KO) mice. TWEAK or Fn14 KO deficiency reduced aortic expansion, lesion macrophages, CD3+ T cells, neutrophils, CD31+ microvessels, CCL2 and CCL5 chemokines expression, and MMP activity after 14 days postperfusion. TWEAK and Fn14 KO mice also showed a reduced loss of medial vascular smooth muscle cells (VSMC) that was related to a reduced number of apoptotic cells in these animals compared with WT mice. Aortas from WT animals present a higher disruption of the elastic layer and MMP activity than those from TWEAK or Fn14 KO mice, indicating a diminished vascular remodeling in KO animals. In vitro experiments unveiled that TWEAK induces CCL5 secretion and MMP‐9 activation in both VSMC and bone marrow‐derived macrophages, and decrease VSMC viability, effects dependent on Fn14.ConclusionsTWEAK/Fn14 axis participates in AAA formation by promoting lesion inflammatory cell accumulation, angiogenesis, matrix‐degrading protease expression, and vascular remodeling. Blocking TWEAK/Fn14 interaction could be a new target for the treatment of AAA.

Roles of the kinase TAK1 in CD40-mediated effects on vascular oxidative stress and neointima formation after vascular injury.

Although TAK1 has been implicated in inflammation and oxidative stress, its roles in vascular smooth muscle cells (VSMCs) and in response to vascular injury have not been investigated. The present study aimed to investigate the role of TAK1 in modulating oxidative stress in VSMCs and its involvement in neointima formation after vascular injury. Double immunostaining reveals that vascular injury induces a robust phosphorylation of TAK1 (Thr187) in the medial VSMCs of injured arteries in wildtype mice, but this effect is blocked in CD40-deficient mice. Upregulation of TAK1 in VSMCs is functionally important, as it is critically involved in pro-oxidative and pro-inflammatory effects on VSMCs and eventual neointima formation. In vivo, pharmacological inhibition of TAK1 with 5Z-7-oxozeaenol blocked the injury-induced phosphorylation of both TAK1 (Thr187) and NF-kB/p65 (Ser536), associated with marked inhibition of superoxide production, 3-nitrotyrosine, and MCP-1 in the injured arteries. Cell culture experiments demonstrated that either siRNA knockdown or 5Z-7-oxozeaenol inhibition of TAK1 significantly attenuated NADPH oxidase activation and superoxide production induced by CD40L/CD40 stimulation. Co-immunoprecipitation experiments indicate that blockade of TAK1 disrupted the CD40L-induced complex formation of p22phox with p47phox, p67phox, or Nox4. Blockade of TAK1 also inhibited CD40L-induced NF-kB activation by modulating IKKα/β and NF-kB p65 phosphorylation and this was related to reduced expression of proinflammatory genes (IL-6, MCP-1 and ICAM-1) in VSMCs. Lastly, treatment with 5Z-7-oxozeaenol attenuated neointimal formation in wire-injured femoral arteries. Our findings demonstrate previously uncharacterized roles of TAK1 in vascular oxidative stress and the contribution to neointima formation after vascular injury.

ZIPK is critical for the motility and contractility of VSMCs through the regulation of nonmuscle myosin II isoforms

Migration of medial vascular smooth muscle cells (VSMCs) into the intimal layer contributes to pathological remodeling of the blood vessel in arterial hypertension and atherosclerosis. It is well established that reorganization of cytoskeletal networks is an essential component of cellular motile events. However, there is currently a lack of insight into the cellular characteristics of VSMC migration under three-dimensional environments. Here, we investigated the mechanisms of VSMC migration and remodeling using two different collagen matrix assays as in vitro models: migration of VSMCs within a collagen matrix for VSMC invasion and contraction of a collagen gel by VSMCs for VSMC remodeling and contraction. We found that nonmuscle myosin IIA (NMIIA) and nonmuscle myosin IIB (NMIIB) differentially contribute to the migratory capacity of VSMCs via NMII isoform-dependent cytoskeletal reorganization. Depletion of NMIIA by short hairpin RNA revealed a unique interplay between actomyosin and microtubules during VSMC migration. On the other hand, NMIIB was required for the structural maintenance of migrating VSMC. Interestingly, there was a significant difference between NMIIA and NMIIB knockdown in the VSMC migration but not in the VSMC-mediated collagen gel contraction. Furthermore, depletion of zipper-interacting protein kinase by short hairpin RNA resulted in an impairment of VSMC migration and a substantial decrease of VSMC-mediated collagen gel contraction. These results suggest that NMIIA and NMIIB uniquely control VSMC migration and may contribute to vascular remodeling, which are both regulated by zipper-interacting protein kinase.

Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury.

BackgroundThe origins of neointimal smooth muscle cells that arise following vascular injury remains controversial. Studies have suggested that these cells may arise from previously differentiated medial vascular smooth muscle cells, resident stem cells or blood born progenitors. In the current study we examined the contribution of the previously differentiated vascular smooth muscle cells to the neointima that forms following carotid artery ligation.MethodsWe utilized transgenic mice harboring a cre recombinase-dependent reporter gene (mTmG). These mice express membrane targeted tandem dimer Tomato (mTomato) prior to cre-mediated excision and membrane targeted EGFP (mEGFP) following excision. The mTmG mice were crossed with transgenic mice expressing either smooth muscle myosin heavy chain (Myh11) or smooth muscle α-actin (Acta2) driven tamoxifen regulated cre recombinase. Following treatment of adult mice with tamoxifen these mice express mEGFP exclusively in differentiated smooth muscle cells. Subsequently vascular injury was induced in the mice by carotid artery ligation and the contribution of mEGFP positive cells to the neointima determined.ResultsAnalysis of the cellular composition of the neointima that forms following injury revealed that mEGFP positive cells derived from either Mhy11 or Acta2 tagged medial vascular smooth muscle cells contribute to the majority of neointima formation (79 ± 17% and 81 ± 12%, respectively).ConclusionThese data demonstrate that the majority of the neointima that forms following carotid ligation is derived from previously differentiated medial vascular smooth muscle cells.

Forgetting to Switch Off SMAD2 in Aneurysmal Disease

Vascular smooth muscle cells (VSMCs) are key component cells of the vascular wall that are critical for contractility of the aorta.1 They respond to biochemical and mechanical signals and play an important role in the regulation of blood pressure. Dysregulation of VSMCs is a hallmark of cardiovascular diseases such as restenosis, hypertension, aneurysms, and atherosclerosis. Investigating the signaling pathways in both normal and dysregulated VSMCs is important for understanding how these cardiovascular diseases develop and for the development of more effective therapies for these potentially life-threatening disorders. Article, see p 881 Aneurysms are dangerous vascular diseases characterized by aortic dilatation with thinning of the medial VSMC layer. Several mechanisms have been implicated in the development of human aortic aneurysms, including the role of transforming growth factor-β1 (TGF-β1), although there are some controversies in the field.2–6 Evidence shows that VSMCs from thoracic aortic aneurysms (TAA) exhibit enhanced activity of SMAD2, a transcription factor that is part of the canonical TGF-β1 pathway and a key effector of the actions of TGF-β1.7 TGF-β1 signaling results in the phosphorylation and translocation of cytoplasmic SMAD2 into the nucleus and consequent transcriptional activation of its target genes.8,9 However, the activation of SMAD2 in TAA has previously been shown to be dissociated from the TGF-β1 pathway. Instead, the SMAD2 hyperactivity is suggested to be attributed to the constitutive overexpression of SMAD2 at the transcriptional level.10 Furthermore, SMAD2 overexpression was associated with key chromatin histone modifications at the SMAD2 promoter, suggesting an element of epigenetic control.10 In the nucleus of mammalian cells, chromosomal DNA is tightly packaged into chromatin, a higher-order structure comprised of subunits called nucleosomes. Each nucleosome consists of an …

Bone morphogenetic protein receptor activation plays a crucial role in endothelial dysfunction and osteogenic differentiation in mice with chronic kidney disease

Background: Endothelial dysfunction and vascular calcification are the major cardiovascular complications in patients with chronic kidney disease (CKD). Recently, we showed that endothelial dysfunction is caused by reduced endothelial nitric oxide synthase (eNOS) phosphorylation at Ser1177 in CKD mice. However, little is known regarding the mechanisms of reduction of eNOS phosphorylation in CKD. Bone morphogenetic protein (BMP) receptor signaling is a key pathway of bone metabolism through Smad1/5/8 phosphorylation. The aim of this study was to examine the role of BMP receptor signaling in endothelial dysfunction and vascular osteogenic differentiation in CKD. Methods and results: We created a CKD model by performing 5/6 nephrectomy in wild-type (WT) mice, which increased serum creatinine levels 2-fold without changing blood pressure. WT mice with CKD (WT+CKD) showed impaired endothelium-dependent relaxation and reduced eNOS and Akt phosphorylations in the aorta. Immunohistostaining revealed that phosphorylated Smad1/5/8 was increased in the endothelial cells and medial vascular smooth muscle cells (VSMCs) of the aorta in WT+CKD mice. To investigate the causal relationship between BMP receptor activation and endothelial dysfunction, CKD was created in mice with heterozygous ablation of Alk3 (Alk3+/-), a BMP type 1 receptor. Basal and CKD-induced increases in serum creatinine levels were similar between WT and Alk3+/- mice. CKD-induced deterioration of endothelium-dependent relaxation and reduction of eNOS were abolished in Alk3+/- mice. In addition, alkaline phosphatase activity was increased in medial VSMCs of WT+CKD mice, but not in those of Alk3+/- +CKD mice. Conclusions: BMP receptor activation plays a crucial role in CKD-induced endothelial dysfunction and VSMC osteogenic differentiation. Thus, inhibition of BMP receptor signaling may be a therapeutic target for the prevention and treatment of endothelial dysfunction and vascular calcification in CKD patients.

Abstract 421: Anti-Atherosclerotic Effect of Ellagic Acid

Introduction Abnormal proliferation and migration of medial vascular smooth muscle cells (VSMC) of vessel wall result in atherosclerotic plaque growth. Targeting proliferation of VSMC could reduce atherosclerotic complications. Therefore, the objective was to study the anti-proliferative effect of Ellagic acid (EA) on primary rat aortic smooth muscle cells (RASMCs) against an atherogenic agonist PDGF in vitro and also anti atherosclerotic effect in streptozotocin (STZ) induced diabetic rats. Methods Proliferation of cells was measured via Alamar blue assay and through propidium iodide based cell cycle analysis in flow cytometer. Reactive oxygen species (ROS) were measured via 2’, 7’-dichlorofluorescin diacetate (DCF-2DA) and Amplex red methods. Expression of proliferation markers and activation of kinases were assessed by immunoblot analysis. Immunohistochemistry was done on aortic tissue sections obtained from in vivo experiments. Results Co-treatment of primary cultures of RASMCs with 25μmol/L of EA significantly reduced PDGF-BB (20ng/ml) induced proliferation by blocking S-phase entry. EA effectively blocked PDGF receptor-β (PDGFR-β) tyrosine phosphorylation, intracellular ROS and downstream activation of extra cellular signal-regulated kinase 1/2. It also blocked PDGF-BB induced expression of cyclin D1. Docking analysis with AutoDock4 revealed two binding pockets for EA in the PDGF-BB-PDGFR-β complex with predicted free energy of bindings similar to known inhibitor, AG1295. In diabetic rats, supplementation of diet with 2% EA significantly blocked diabetes-induced medial thickness, and lipid and collagen deposition in the arch of aorta as well as proliferation marker cyclin D1 expression. Conclusion Therefore, EA is effective as anti atherogenic nutrient therapy.

Engineering of an ω-3 polyunsaturated fatty acid-containing nanoemulsion system for combination C6-ceramide and 17β-estradiol delivery and bioactivity in human vascular endothelial and smooth muscle cells

Delayed endothelial cell (EC) regeneration and the medial vascular smooth muscle cells (VSMCs) proliferation contribute to arterial restenosis. Although ω-3-polyunsaturated fatty acids (PUFAs), 17β-estradiol (17-βE) and C6-ceramide (CER) have shown therapeutic promise in addressing restenosis, extensive protein binding and lipophilicity complicate their (co-)delivery to cellular targets. We report engineering of an ω-3-PUFA-rich oil-in-water nanoemulsion formulation that effectively delivers 17-βE and CER cargo to cultured vascular cells. The cargo-free, ω-3-PUFA-rich nanoemulsion itself typically reduced growth factor-stimulated cellular proliferation, as did nanoemulsion-delivered CER alone, through enhanced pro-apoptotic caspase 3/7 activity. 17-βE loaded nanoemulsion inhibited VSMC proliferation and supported EC proliferation, responses associated with the mitogen-activated-protein-kinase (MAPK) signaling. Co-administration of 17-βE and CER loaded nanoemulsions exerted an anti-proliferative effect more pronounced on VSMCs than ECs. These therapeutically beneficial responses to ω-3-PUFA, CER, and/or 17-βE in our nanoemulsion formulation invite evaluation of this novel approach in animal models of restenosis and other occlusive vasculopathies. From the Clinical EditorThis team of investigators report the engineering of an ω-3-PUFA-rich oil-in-water nanoemulsion formulation that effectively delivers 17-βE and C6-ceramide cargo to cultured vascular cells in an effort to address vascular restenosis. Further preclinical studies will be needed in animal models before this approach could be considered for clinical trials.

Deregulation of smooth muscle cell cytoskeleton within the human atherosclerotic coronary media layer

Fatal events derived from coronary atherosclerosis are the major cause of mortality in the developed countries. Proteomic analysis of the atherosclerotic coronary artery has been mainly carried out with whole tissue extracts, making it difficult to distinguish the alterations present in every region of the plaque. For this reason, we have recently described proteins altered in the human coronary intima layer as a consequence of the atherosclerotic disease. In order to complement this work, we aimed here to analyze proteomic alterations occurring within the human coronary media layer. Media layers from human atherosclerotic and preatherosclerotic coronary arteries were isolated by laser microdissection and compared by means of two-dimensional differential in-gel electrophoresis (2D-DIGE). Twelve proteins were found altered, 5 of which were cytoskeleton proteins decreased in the atherosclerotic coronary media. Among these, 4 proteins (filamin A, gelsolin, vinculin and vimentin) were further analyzed by immunohistochemistry and its alteration validated. Such cytoskeleton deregulation evidence, at the molecular level, explains how medial vascular smooth muscle cells (VSMCs) switch from a contractile to a synthetic phenotype. Moreover, an oxidative stress response within the media, leaded by superoxide dismutase 3 and glycolysis activation, may have been triggered by atherosclerosis development. Although atherosclerosis is mainly a disease of the intima layer, the media plays an important role in the initiation of the pathology, as a source of vascular smooth muscle cells (VSMCs), which migrate into the intima and may additionally be affected by intima layer degeneration through pathogenesis. In fact, intimal thickening has been related to a mechanical compression of the media layer, resulting on a significant thinning of the latter in the atherosclerotic carotid and coronary arteries, which may provoke alterations at a molecular level. Here we provide the first differential proteomic analysis of atherosclerotic coronary media layer, reporting important alterations of this sub-proteome with pathogenesis. It is important to remark a cytoskeleton deregulation observed at the molecular level within VSMCs, which may be explained by a contractile to synthetic phenotype switch. Moreover, atherosclerosis seems to trigger an oxidative stress response within the coronary media layer.

Notch Signaling in Descending Thoracic Aortic Aneurysm and Dissection

BackgroundDescending thoracic aortic aneurysm and dissection (DTAAD) is characterized by progressive medial degeneration, which may result from excessive tissue destruction and insufficient repair. Resistance to tissue destruction and aortic self-repair are critical in preventing medial degeneration. The signaling pathways that control these processes in DTAAD are poorly understood. Because Notch signaling is a critical pathway for cell survival, proliferation, and tissue repair, we examined its activation in DTAAD.MethodsWe studied descending thoracic aortic tissue from patients with sporadic thoracic aortic aneurysm (TAA; n = 14) or chronic thoracic aortic dissection (TAD; n = 16) and from age-matched organ donors (n = 12). Using western blot, real-time RT-PCR, and immunofluorescence staining, we examined aortic tissue samples for the Notch ligands Delta-like 1, Delta-like 4 (DLL1/4), and Jagged1; the Notch receptor 1 (Notch1); the Notch1 intracellular domain (NICD); and Hes1, a downstream target of Notch signaling.ResultsWestern blots and RT-PCR showed higher levels of the Notch1 protein and mRNA and the NICD and Hes1 proteins in both TAA and TAD tissues than in control tissue. However, immunofluorescence staining showed a complex pattern of Notch signaling in the diseased tissue. The ligand DLL1/4 and Notch1 were significantly decreased and NICD and Hes1 were rarely detected in medial vascular smooth muscle cells (VSMCs) in both TAA and TAD tissues, indicating downregulation of Notch signaling in aortic VSMCs. Interestingly Jagged1, NICD, and Hes1 were highly present in CD34+ stem cells and Stro-1+ stem cells in aortas from TAA and TAD patients. NICD and Hes1 were also detected in most fibroblasts and macrophages that accumulated in the aortic wall of DTAAD patients.ConclusionsNotch signaling exhibits a complex pattern in DTAAD. The Notch pathway is impaired in medial VSMCs but activated in stem cells, fibroblasts, and macrophages.

Enhanced expression of glucose transporter-1 in vascular smooth muscle cells via the Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) pathway in experimental renal failure

Chronic renal failure (CRF) is associated with increased cardiovascular mortality, and medial vascular smooth muscle cell (VSMC) hypertrophy, proliferation, and calcification play a pivotal role in uremic vasculopathy. Glucose transporter-1 (GLUT1) facilitates the transport of glucose into VSMCs, and GLUT1 overexpression associated with high glucose influx leads to a stimulation of VSMC proliferation. However, the role of GLUT1 in uremic vasculopathy remains unclear. This study aimed to identify changes in the expression of GLUT1 in VSMCs in the setting of experimental uremia and investigate whether Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) signaling, which plays a crucial role in VSMC proliferation and glucose metabolism, is involved in the regulation of GLUT1 expression. In vivo experimental CRF was induced in Wistar rats by 5/6 nephrectomy, and the GLUT1 expression in aortic tissue was determined by the reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemical staining. Indoxyl sulfate (IS) is a uremic retention solute proven with pro-proliferative effect on rat VSMCs, and we further studied the expression of GLUT1 in rat A7r5 rat embryonic aortic cells stimulated by IS in the presence or absence of phloretin, a GLUT1 inhibitor, to explore the pathogenic role of GLUT1 in uremic vasculopathy. The contribution of Akt/TSC2/mTOR/S6K signaling in modifying the GLUT1 expression was also assessed. Eight weeks after 5/6 nephrectomy, aortic tissue obtained from CRF rats exhibited increased wall thickness and VSMC hypertrophy, hyperplasia, and degeneration. Compared with the sham-operated control group, the messenger (m)RNA and protein abundance of GLUT1 were both markedly increased in CRF rats. In vitro, IS induced a significant increase in expression of GLUT1 protein as well as pro-proliferative cyclin D1 and p21 mRNA and a modest increase in expression of antiapoptotic p53 mRNA in A7r5 cells, whereas inhibition of GLUT1 mediated glucose influx reduced the pro-proliferative and antiapoptotic effects of IS. In addition to increased GLUT1 expression, IS significantly suppressed Akt and TSC2 phosphorylation after 6-hour and 12-hour treatment, but increased S6K phosphorylation after 3-hour treatment. Inactivation of mTOR downstream signaling by rapamycin treatment inhibited S6K phosphorylation and abolished the stimulatory effect of IS on GLUT1 expression. In vivo and in vitro experimental CRF displayed prominent GLUT1 upregulation in VSMCs. The uremic toxin IS stimulated proliferation of VSMCs possibly through induction of GLUT1 expression. The Akt/TSC/mTOR/S6K signaling pathway may be one of the mechanisms underlying the upregulation of GLUT1 expression in uremic VSMCs.

Plasticity-related gene-1 inhibits lysophosphatidic acid-induced vascular smooth muscle cell migration and proliferation and prevents neointima formation

Plasticity-related gene-1 (PRG-1) protects neuronal cells from lysophosphatidic acid (LPA) effects. In vascular smooth muscle cells (VSMCs), LPA was shown to induce phenotypic modulation in vitro and vascular remodeling in vivo. Thus we explored the role of PRG-1 in modulating VSMC response to LPA. PCR, Western blot, and immunofluorescence experiments showed that PRG-1 is expressed in rat and human vascular media. PRG-1 expression was strongly inhibited in proliferating compared with quiescent VSMCs both in vitro and in vivo (medial vs. neointimal VSMCs), suggesting that PRG-1 expression is dependent on the cell phenotype. In vitro, adenovirus-mediated overexpression of PRG-1 specifically inhibited LPA-induced rat VSMC proliferation and migration but not platelet-derived growth factor-induced proliferation. This effect was abolished by mutation of a conserved histidine in the lipid phosphate phosphatase family that is essential for interaction with lipid phosphates. In vivo, balloon-induced neointimal formation in rat carotid was significantly decreased in vessels infected with PRG-1 adenovirus compared with β-galactosidase adenovirus (-71%; P < 0.05). PRG-1 overexpression abolished the activation of the p42/p44 signaling pathway in LPA-stimulated rat VSMCs in culture and in balloon-injured rat carotids. Taken together, these findings provide the first evidence of a protective role of PRG-1 in the vascular media under pathophysiological conditions.

The Cell Motility Modulator Slit2 Is a Potent Inhibitor of Platelet Function

Vascular injury and atherothrombosis involve vessel infiltration by inflammatory leukocytes, migration of medial vascular smooth muscle cells to the intimal layer, and ultimately acute thrombosis. A strategy to simultaneously target these pathological processes has yet to be identified. The secreted protein, Slit2, and its transmembrane receptor, Robo-1, repel neuronal migration in the developing central nervous system. More recently, it has been appreciated that Slit2 impairs chemotaxis of leukocytes and vascular smooth muscle cells toward diverse inflammatory attractants. The effects of Slit2 on platelet function and thrombus formation have never been explored. We detected Robo-1 expression in human and murine platelets and megakaryocytes and confirmed its presence via immunofluorescence microscopy and flow cytometry. In both static and shear microfluidic assays, Slit2 impaired platelet adhesion and spreading on diverse extracellular matrix substrates by suppressing activation of Akt. Slit2 also prevented platelet activation on exposure to ADP. In in vivo studies, Slit2 prolonged bleeding times in murine tail bleeding assays. Using intravital microscopy, we found that after mesenteric arteriolar and carotid artery injury, Slit2 delayed vessel occlusion time and prevented the stable formation of occlusive arteriolar thrombi. These data demonstrate that Slit2 is a powerful negative regulator of platelet function and thrombus formation. The ability to simultaneously block multiple events in vascular injury may allow Slit2 to effectively prevent and treat thrombotic disorders such as myocardial infarction and stroke.

Relaxin mediates uterine artery compliance during pregnancy and increases uterine blood flow

Normal pregnancy involves dramatic remodeling of the uterine vasculature, with abnormal vascular adaptations contributing to pregnancy diseases such as preeclampsia. The peptide hormone relaxin is important for the renal and systemic hemodynamic adaptations to pregnancy, and has been shown to increase arterial compliance and outward hypertrophic remodeling. Therefore, we investigated the possibility that relaxin acts on its receptor, RXFP1, to mediate uterine artery compliance in late pregnancy and increase uterine blood flow velocity in rats. RXFP1 was predominantly localized to the tunica media vascular smooth muscle cells in the uterine artery, although receptors were also detected in endothelial cells. Highest expression of Rxfp1 in the uterine artery occurred in estrus and early pregnancy. Isolated uterine arteries from late pregnant rats treated with a monoclonal antibody against circulating relaxin (MCA1) had significantly increased vessel wall stiffness compared with controls, with no reduction in wall thickness. Chronic infusion of relaxin (4 μg/h, osmotic minipump) for 5 d in nonpregnant rats significantly increased uterine artery blood flow velocity. Overall, these data demonstrate a functional role for relaxin in mediating uterine artery compliance in pregnant rats, which may be necessary to maintain adequate uterine blood flow to the uterus and placenta.

Abstract 427: Transforming Growth Factor β/Smad3 Stimulates Smooth Muscle Cell Migration and Proliferation Through a Signaling Pathway that Involves C-X-C Chemokine Receptor Type 4 Expression and Activation of Mitogen-Activated Protein Kinase

Introduction: Intimal hyperplasia is a complex process that requires vascular smooth muscle cell (SMC) migration and proliferation. We have previously shown that levels of transforming growth factor beta (TGF-β) and its signaling protein Smad3 are elevated following arterial injury and contribute to intimal hyperplasia. In this study we exam whether these effects might be mediated through a mechanism that involves the chemokine stromal cell derived factor (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4). Methods and Results: A microarray was used to better understand the signaling pathways through which TGF-β and Smad3 stimulate SMC proliferation and migration. Overexpression of Smad3 in SMCs followed by stimulation with TGF-β (5 ng/ml) for 6 hours resulted in significant alteration of ∼300 genes, the most dramatic effect being a &gt;500 fold upregulation of CXCR4. Quantitative RT-PCR and Western blotting confirmed that Smad3/TGF- β increased CXCR4 transcription and protein expression by 155±9 and 24±5 -fold, respectively. Immunocytochemistry and flowcytometry analysis revealed a 6-fold increase in CXCR4 positive cells in TGF-β stimulated Smad3 overexpressing SMCs compared to control (P&lt;0.05). TGF-β/Smad3 treated SMCs exhibited enhanced migration (4-fold VS control, P&lt;0.01) in response to SDF-1α (100 ng/ml) which was eliminated after preincubation of SMCs with CXCR4 inhibitor AMD3100 (10 μM). Moreover, there was a 45±9% increase in proliferation (BrdU) in response to SDF-1α in TGF-β/Smad3 treated SMCs compared to GFP control (P&lt;0.05). To explore the mechanism by which SDF-1α/CXCR4 produces proliferation and migration in SMCs, we studied the intracelluar signaling protein mitogen-activated protein kinase (MAPK), which is a known intermediate for both processes. Incubation of TGF-β/Smad3 SMCs with SDF-1α led to activation of MAPK (11-fold) as well as nuclear translocation of CXCR4 in a dose and time dependent manner. Conclusions: Our data demonstrate that TGF-β through Smad3 stimulates expression of CXCR4 in medial vascular SMCs. These SMCs in response to SDF-1α then proliferate and migrate to the subintimal layer. These findings suggest a novel pathway through which TGF-β enhances intimal hyperplasia at sites of arterial injury.

The vascular smooth muscle cell in arterial pathology: a cell that can take on multiple roles

Vascular smooth muscle cells (VSMCs) are the stromal cells of the vascular wall, continually exposed to mechanical signals and biochemical components generated in the blood compartment. They are involved in all the physiological functions and the pathological changes taking place in the vascular wall. Owing to their contractile tonus, VSMCs of resistance vessels participate in the regulation of blood pressure and also in hypertension. VSMCs of conduit arteries respond to hypertension-induced increases in wall stress by an increase in cell protein synthesis (hypertrophy) and extracellular matrix secretion. These responses are mediated by complex signalling pathways, mainly involving RhoA and extracellular signal-regulated kinase1/2. Serum response factor and miRNA expression represent main mechanisms controlling the pattern of gene expression. Ageing also induces VSMC phenotypic modulation that could have influence on cell senescence and loss of plasticity and reprogramming. In the early stages of human atheroma, VSMCs support the lipid overload. Endocytosis/phagocytosis of modified low-density lipoproteins, free cholesterol, microvesicles, and apoptotic cells by VSMCs plays a major role in the progression of atheroma. Migration and proliferation of VSMCs in the intima also participate in plaque progression. The medial VSMC is the organizer of the inwardly directed angiogenic response arising from the adventitia by overexpressing vascular endothelial growth factor in response to lipid-stimulated peroxisome proliferator-activated receptor-γ, and probably also the organizer of the adventitial immune response by secreting chemokines. VSMCs are also involved in the response to proteolytic injury via their ability to activate blood-borne proteases, to secrete antiproteases, and to clear protease/antiprotease complexes.

Vascular Potency of Sus Scrofa Bone Marrow–Derived Mesenchymal Stem Cells: A Progenitor Source of Medial but Not Endothelial Cells

Short-term thrombotic occlusion and compliance mismatch hamper clinical use of synthetic small-diameter tissue engineered vascular grafts. It is felt that preconditioning of the graft with intimal (endothelial) and medial (vascular smooth muscle) cells contributes to patency of the graft. Autologous, non-vessel-derived cells are preferred because of systemic vascular pathology and immunologic concerns. We tested in a porcine model whether cultured bone marrow-derived mononuclear cells, also referred to as mesenchymal stem cells (MSC), are a potential source of intimal or medial cells in vascular tissue engineering. We show that MSC cultured in endothelial medium do not gain an endothelial phenotype or functional characteristics, even after enrichment for CD31, culturing under flow, treatment with additional growth factors (vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-2), or co-culture with microvascular endothelial cells (EC). On the other hand, we show that MSC cultured in MSC medium, but not in smooth muscle cell medium, show phenotypical and functional characteristics of vascular smooth muscle cells. We conclude that bone marrow-derived MSCs can be used as a bona fide source of medial, but not EC in small-diameter vascular tissue engineering.