Smooth Muscle Cell Proangiogenic Phenotype Induced by Cyclopentenyl Cytosine Promotes Endothelial Cell Proliferation and Migration.
ABSTRACT: Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) are in close contact with blood vessels. SMC phenotypes can be altered during pathological vascular remodeling. However, how SMC phenotypes affect EC properties remains largely unknown. In this study, we found that PDGF-BB-induced synthetic SMCs suppressed EC proliferation and migration while exhibiting increased expression of anti-angiogenic factors, such as endostatin, and decreased pro-angiogenic factors, including CXC motif ligand 1 (CXCL1). Cyclopentenyl cytosine (CPEC), a CTP synthase inhibitor that has been reported previously to inhibit SMC proliferation and injury-induced neointima formation, induced SMC redifferentiation. Interestingly, CPEC-conditioned SMC culture medium promoted EC proliferation and migration because of an increase in CXCL1 along with decreased endostatin production in SMCs. Addition of recombinant endostatin protein or blockade of CXCL1 with a neutralizing antibody suppressed the EC proliferation and migration induced by CPEC-conditioned SMC medium. Mechanistically, CPEC functions as a cytosine derivate to stimulate adenosine receptors A1 and A2a, which further activate downstream cAMP and Akt signaling, leading to the phosphorylation of cAMP response element binding protein and, consequently, SMC redifferentiation. These data provided proof of a novel concept that synthetic SMC exhibits an anti-angiogenic SMC phenotype, whereas contractile SMC shows a pro-angiogenic phenotype. CPEC appears to be a potent stimulator for switching the anti-angiogenic SMC phenotype to the pro-angiogenic phenotype, which may be essential for CPEC to accelerate re-endothelialization for vascular repair during injury-induced vascular wall remodeling.
Project description:Background Sepsis, a leading cause of death in intensive care units, is generally associated with vascular dysfunction. However, its pathophysiological process has not been fully clarified, lacking in-depth knowledge of its pathophysiological process may hinder the improvement of diagnosis and therapy for sepsis. Hence, as the key parts of the vascular wall, the interaction between endothelial cells (ECs) and smooth muscle cells (SMCs) under septic situation need to be further studied. Methods ECs and SMCs were co-cultured using Transwell plates. Lipopolysaccharide (LPS) was used to induce sepsis. A scratch-wound assay was used to assess cell migration, and western blotting was used to assess the level of redifferentiation of SMCs as well as the expression of PDGFR-β and IQGAP1. Results Co-culture with ECs reduced the redifferentiation of SMCs induced by LPS (10 μg/ml), which was characterized by increased migration ability and decreased expression of contractile proteins (e.g., SM22 and α-SMA). The production of TNF-α could decrease the level of PDGFR-β in SMCs. Treatment of SMCs with the PDGFR-β inhibitor imatinib (5 μM) was able to counteract LPS-induced SMC redifferentiation and reduce IQGAP1 protein expression, especially when SMCs were co-cultured with ECs. Conclusion The phenotype of vascular SMCs co-cultured with ECs was modulated by IQGAP1 through the PDGFR-β pathway, which may lead to vascular remodeling and homeostasis in LPS-induced intravascular injury. This pathway could be a novel target for the treatment of vascular damage.
Project description:Phenotypic modulation of smooth muscle cells (SMCs), which are located in close proximity to endothelial cells (ECs), is critical in regulating vascular function. The role of flow-induced shear stress in the modulation of SMC phenotype has not been well defined.The objective was to elucidate the role of shear stress on ECs in modulating SMC phenotype and its underlying mechanism.Application of shear stress (12 dyn/cm2) to ECs cocultured with SMCs modulated SMC phenotype from synthetic to contractile state, with upregulation of contractile markers, downregulation of proinflammatory genes, and decreased percentage of cells in the synthetic phase. Treating SMCs with media from sheared ECs induced peroxisome proliferator-activated receptor (PPAR)-alpha, -delta, and -gamma ligand binding activities; transfecting SMCs with specific small interfering (si)RNAs of PPAR-alpha and -delta, but not -gamma, inhibited shear induction of contractile markers. ECs exposed to shear stress released prostacyclin (PGI2). Transfecting ECs with PGI2 synthase-specific siRNA inhibited shear-induced activation of PPAR-alpha/delta, upregulation of contractile markers, downregulation of proinflammatory genes, and decrease in percentage of SMCs in synthetic phase. Mice with PPAR-alpha deficiency (compared with control littermates) showed altered SMC phenotype toward a synthetic state, with increased arterial contractility in response to angiotensin II.These results indicate that laminar shear stress induces synthetic-to-contractile phenotypic modulation in SMCs through the activation of PPAR-alpha/delta by the EC-released PGI2. Our findings provide insights into the mechanisms underlying the EC-SMC interplays and the protective homeostatic function of laminar shear stress in modulating SMC phenotype.
Project description:Local modulation of vascular mammalian target of rapamycin (mTOR) signaling reduces smooth muscle cell (SMC) proliferation after endovascular interventions but may be associated with endothelial cell (EC) toxicity. The trilaminate vascular architecture juxtaposes ECs and SMCs to enable complex paracrine coregulation but shields SMCs from flow. We hypothesized that flow differentially affects mTOR signaling in ECs and SMCs and that SMCs regulate mTOR in ECs.SMCs and/or ECs were exposed to coronary artery flow in a perfusion bioreactor. We demonstrated by flow cytometry, immunofluorescence, and immunoblotting that EC expression of phospho-S6 ribosomal protein (p-S6RP), a downstream target of mTOR, was doubled by flow. Conversely, S6RP in SMCs was growth factor but not flow responsive, and SMCs eliminated the flow sensitivity of ECs. Temsirolimus, a sirolimus analog, eliminated the effect of growth factor on SMCs and of flow on ECs, reducing p-S6RP below basal levels and inhibiting endothelial recovery. EC p-S6RP expression in stented porcine arteries confirmed our in vitro findings: Phosphorylation was greatest in ECs farthest from intact SMCs in metal stented arteries and altogether absent after sirolimus stent elution.The mTOR pathway is activated in ECs in response to luminal flow. SMCs inhibit this flow-induced stimulation of endothelial mTOR pathway. Thus, we now define a novel external stimulus regulating phosphorylation of S6RP and another level of EC-SMC crosstalk. These interactions may explain the impact of local antiproliferative delivery that targets SMC proliferation and suggest that future stents integrate design influences on flow and drug effects on their molecular targets.
Project description:RATIONALE:Endothelial microRNA-126 (miR-126) modulates vascular development and angiogenesis. However, its role in the regulation of smooth muscle cell (SMC) function is unknown. OBJECTIVE:To elucidate the role of miR-126 secreted by endothelial cells (ECs) in regulating SMC turnover in vitro and in vivo, as well as the effects of shear stress on the regulation. METHODS AND RESULTS:Coculture of SMCs with ECs or treatment of SMCs with conditioned media from static EC monoculture (EC-CM) increased SMC miR-126 level and SMC turnover; these effects were abolished by inhibition of endothelial miR-126 and by the application of laminar shear stress to ECs. SMC miR-126 did not increase when treated with EC-CM from ECs subjected to inhibition of miR biogenesis, or with CM from sheared ECs. Depletion of extracellular/secreted vesicles in EC-CM did not affect the increase of SMC miR-126 by EC-CM. Biotinylated miR-126 or FLAG (DYKDDDDK epitope)-tagged Argonaute2 transfected into ECs was detected in the cocultured or EC-CM-treated SMCs, indicating a direct EC-to-SMC transmission of miR-126 and Argonaute2. Endothelial miR-126 represses forkhead box O3, B-cell lymphoma 2, and insulin receptor substrate 1 mRNAs in the cocultured SMCs, suggesting the functional roles of the transmitted miR-126. Systemic depletion of miR-126 in mice inhibited neointimal lesion formation of carotid arteries induced by cessation of blood flow. Administration of EC-CM or miR-126 mitigated the inhibitory effect. CONCLUSIONS:Endothelial miR-126 acts as a key intercellular mediator to increase SMC turnover, and its release is reduced by atheroprotective laminar shear stress.
Project description:Diffusion is a limiting factor in regenerating large tissues (100-200 ?m) due to reduced nutrient supply and waste removal leading to low viability of the regenerating cells as neovascularization of the implant by the host is a slow process. Thus, generating prevascularized tissue engineered constructs, in which endothelial (ECs) and mural (MCs) cells, such as smooth muscle cells (SMCs), and pericytes (PCs), are preassembled into functional in vitro vessels capable of rapidly connecting to the host vasculature could overcome this obstacle. Toward this purpose, using feeder-free and low serum conditions, we developed a simple, efficient and rapid in vitro approach to induce the differentiation of human pluripotent stem cells-hPSCs (human embryonic stem cells and human induced pluripotent stem cells) to defined SMC populations (contractile and synthetic hPSC-SMCs) by extensively characterizing the cellular phenotype (expression of CD44, CD73, CD105, NG2, PDGFR?, and contractile proteins) and function of hPSC-SMCs. The latter were phenotypically and functionally stable for at least 8 passages, and could stabilize vessel formation and inhibit vessel network regression, when co-cultured with ECs in vitro. Subsequently, using a methylcellulose-based hydrogel system, we generated spheroids consisting of EC/hPSC-SMC (vascular organoids), which were extensively phenotypically characterized. Moreover, the vascular organoids served as focal starting points for the sprouting of capillary-like structures in vitro, whereas their delivery in vivo led to rapid generation of a complex functional vascular network. Finally, we investigated the vascularization potential of these vascular organoids, when embedded in hydrogels composed of defined extracellular components (collagen/fibrinogen/fibronectin) that can be used as scaffolds in tissue engineering applications. In summary, we developed a robust method for the generation of defined SMC phenotypes from hPSCs. Fabrication of vascularized tissue constructs using hPSC-SMC/EC vascular organoids embedded in chemically defined matrices is a significant step forward in tissue engineering and regenerative medicine.
Project description:Endothelial dysfunction is widely implicated in cardiovascular pathological changes and development of vascular disease. In view of the fact that the spontaneous endothelial cell (EC) regeneration is a slow and insufficient process, it is of great interest to explore alternative cell sources capable of generating functional ECs. Vascular smooth muscle cell (SMC) composes the majority of the vascular wall and retains phenotypic plasticity in response to various stimuli. The aim of this study is to test the feasibility of the conversion of SMC into functional EC through the use of reprogramming factors. Human SMCs are first dedifferentiated for 4 days to achieve a vascular progenitor state expressing CD34, by introducing transcription factors OCT4, SOX2, KLF4 and c-MYC. These SMC-derived progenitors are then differentiated along the endothelial lineage. The SMC-converted ECs exhibit typical endothelial markers expression and endothelial functions in vitro, in vivo and in disease model. Further comprehensive analysis indicates that mesenchymal-to-epithelial transition is requisite to initiate SMCs reprogramming into vascular progenitors and that members of the Notch signalling pathway regulate further differentiation of the progenitors into endothelial lineage. Together, we provide the first evidence of the feasibility of the conversion of human SMCs towards endothelial lineage through an intermediate vascular progenitor state induced by reprogramming.
Project description:Vascular diseases are characterized by the over-proliferation and migration of aortic smooth muscle cells (SMCs), and degradation of extracellular matrix (ECM) within the vessel wall, leading to compromise in cell-cell and cell-matrix signaling pathways. Tissue engineering approaches to regulate SMC over-proliferation and enhance healthy ECM synthesis showed promise, but resulted in low crosslinking efficiency. Here, we report the benefits of exogenous nitric oxide (NO) cues, delivered from S-Nitrosoglutathione (GSNO), to cell proliferation and matrix deposition by adult human aortic SMCs (HA-SMCs) within three-dimensional (3D) biomimetic cocultures. A coculture platform with two adjacent, permeable 3D culture chambers was developed to enable paracrine signaling between vascular cells. HA-SMCs were cultured in these chambers within collagen hydrogels, either alone or in the presence of human aortic endothelial cells (HA-ECs) cocultures, and exogenously supplemented with varying GSNO dosages (0-100?nM) for 21 days. Results showed that EC cocultures stimulated SMC proliferation within GSNO-free cultures. With increasing GSNO concentration, HA-SMC proliferation decreased in the presence or absence of EC cocultures, while HA-EC proliferation increased. GSNO (100?nM) significantly enhanced the protein amounts synthesized by HA-SMCs, in the presence or absence of EC cocultures, while lower dosages (1-10?nM) offered marginal benefits. Multi-fold increases in the synthesis and deposition of elastin, glycosaminoglycans, hyaluronic acid, and lysyl oxidase crosslinking enzyme (LOX) were noted at higher GSNO dosages, and coculturing with ECs significantly furthered these trends. Similar increases in TIMP-1 and MMP-9 levels were noted within cocultures with increasing GSNO dosages. Such increases in matrix synthesis correlated with NO-stimulated increases in endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expression within EC and SMC cultures, respectively. Results attest to the benefits of delivering NO cues to suppress SMC proliferation and promote robust ECM synthesis and deposition by adult human SMCs, with significant applications in tissue engineering, biomaterial scaffold development, and drug delivery.
Project description:Vascular remodeling as a result of smooth muscle cell (SMC) proliferation and neointima formation is a major medical challenge in cardiovascular intervention. However, antineointima drugs often indistinguishably block re-endothelialization, an essential step toward successful vascular repair, because of their nonspecific effect on endothelial cells (ECs). The objective of this study is to identify a therapeutic target that differentially regulates SMC and EC proliferation.Using both rat balloon injury and mouse wire injury models, we identified CTP synthase 1 (CTPS1) as one of the potential targets that may be used for developing therapeutics for treating neointima-related disorders. CTPS1 was induced in proliferative SMCs in vitro and neointima SMCs in vivo. Blockade of CTPS1 expression by small hairpin RNA or activity by cyclopentenyl cytosine suppressed SMC proliferation and neointima formation. Surprisingly, cyclopentenyl cytosine had much less effect on EC proliferation. Of importance, blockade of CTPS1 in vivo sustained the re-endothelialization as a result of induction of CTP synthesis salvage pathway enzymes nucleoside-diphosphate kinase A and B in ECs. Diphosphate kinase B seemed to preserve EC proliferation via use of extracellular cytidine to synthesize CTP. Indeed, blockade of both CTPS1 and diphosphate kinase B suppressed EC proliferation in vitro and the re-endothelialization in vivo.Our study uncovered a fundamental difference in CTP biosynthesis between SMCs and ECs during vascular remodeling, which provided a novel strategy by using cyclopentenyl cytosine or other CTPS1 inhibitors to selectively block SMC proliferation without disturbing or even promoting re-endothelialization for effective vascular repair after injury.
Project description:RATIONALE:Regeneration of denuded or injured endothelium is an important component of vascular injury response. Cell-cell communication between endothelial cells and smooth muscle cells (SMCs) plays a critical role not only in vascular homeostasis but also in disease. We have previously demonstrated that PKC? (protein kinase C-delta) regulates multiple components of vascular injury response including apoptosis of SMCs and production of chemokines, thus is an attractive candidate for a role in SMC-endothelial cells communication. OBJECTIVE:To test whether PKC?-mediated paracrine functions of SMCs influence reendothelialization in rodent models of arterial injury. METHODS AND RESULTS:Femoral artery wire injury was performed in SMC-conditional Prkcd knockout mice, and carotid angioplasty was conducted in rats receiving transient Prkcd knockdown or overexpression. SMC-specific knockout of Prkcd impaired reendothelialization, reflected by a smaller Evans blue-excluding area in the knockout compared with the wild-type controls. A similar impediment to reendothelialization was observed in rats with SMC-specific knockdown of Prkcd. In contrast, SMC-specific gene transfer of Prkcd accelerated reendothelialization. In vitro, medium conditioned by AdPKC?-infected SMCs increased endothelial wound closure without affecting their proliferation. A polymerase chain reaction-based array analysis identified Cxcl1 and Cxcl7 among others as PKC?-mediated chemokines produced by SMCs. Mechanistically, we postulated that PKC? regulates Cxcl7 expression through STAT3 (signal transducer and activator of transcription 3) as knockdown of STAT3 abolished Cxcl7 expression. The role of CXCL7 in SMC-endothelial cells communication was demonstrated by blocking CXCL7 or its receptor CXCR2, both significantly inhibited endothelial wound closure. Furthermore, insertion of a Cxcl7 cDNA in the lentiviral vector that carries a Prkcd shRNA overcame the adverse effects of Prkcd knockdown on reendothelialization. CONCLUSIONS:SMCs promote reendothelialization in a PKC?-dependent paracrine mechanism, likely through CXCL7-mediated recruitment of endothelial cells from uninjured endothelium.
Project description: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-betabeta, 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.