Angiopoietin 1, PDGF-B, and TGF-beta gene regulation in endothelial cell and smooth muscle cell interaction

The vascular wall is mainly composed of endothelial cells (ECs) and smooth muscle cells (SMCs). The crosstalking between these two cell types is critical in the vascular maturation process. Genetic studies suggest that the Tie2/angiopoietin 1 (Ang1) pathway regulates vascular remodeling. However, the molecular mechanism is unclear. PDGF is a potent chemoattractant for SMCs, and TGF-beta regulates SMC differentiation. Here, we examined gene regulation. PDGF-B stimulation upregulated Ang1 expression in SMCs through the PI3K and PKC pathways. PDGF-B stimulation also produced an acute induction of TGF-beta expression in SMCs through the MAPK/ERK pathway. Interestingly, TGF-beta negatively regulated Ang1 expression induced by the PDGF-B stimulation in SMCs. Reciprocally, we observed that stimulation of ECs with either Ang1 or TGF-beta slightly downregulated PDGF expression. A combination of both TGF-beta with Ang1 produced much stronger downregulation of PDGF. Our data showed complex gene regulations that include both positive and negative regulations between ECs and SMCs to maintain vascular homeostasis.     

RhoA modulates Smad signaling during transforming growth factor-beta-induced smooth muscle differentiation

We recently reported that transforming growth factor (TGF)-beta induced the neural crest stem cell line Monc-1 to differentiate into a spindle-like contractile smooth muscle cell (SMC) phenotype and that Smad signaling played an important role in this phenomenon. In addition to Smad signaling, other pathways such as mitogen-activated protein kinase (MAPK), phosphoinositol-3 kinase, and RhoA have also been shown to mediate TGF-beta actions. The objectives of this study were to examine whether these signaling pathways contribute to TGF-beta-induced SMC development and to test whether Smad signaling cross-talks with other pathway(s) during SMC differentiation induced by TGF-beta. We demonstrate here that RhoA signaling is critical to TGF-beta-induced SMC differentiation. RhoA kinase (ROCK) inhibitor Y27632 significantly blocks the expression of multiple SMC markers such as smooth muscle alpha-actin, SM22alpha, and calponin in TGF-beta-treated Monc-1 cells. In addition, Y27632 reversed the cell morphology and abolished the contractility of TGF-beta-treated cells. RhoA signaling was activated as early as 5 min following TGF-beta addition. Dominant negative RhoA blocked nuclear translocation of Smad2 and Smad3 because of the inhibition of phosphorylation of both Smads and inhibited Smad-dependent SBE promoter activity, whereas constitutively active RhoA significantly enhanced SBE promoter activity. Consistent with these results, C3 exotoxin, an inhibitor of RhoA activation, significantly attenuated SBE promoter activity and inhibited Smad nuclear translocation. Taken together, these data point to a new role for RhoA as a modulator of Smad activation while regulating TGF-beta-induced SMC differentiation.     

Effects of transforming growth factor-beta 1 and ascorbic acid on differentiation of human bone-marrow-derived mesenchymal stem cells into smooth muscle cell lineage

Bone-marrow-derived mesenchymal stem cells (MSCs) can differentiate into a variety of cell types including smooth muscle cells (SMCs). We have attempted to demonstrate that, following treatment with transforming growth factor-beta 1 (TGF-beta1) and ascorbic acid (AA), human bone-marrow-derived MSCs differentiate into the SMC lineage for use in tissue engineering. Quantitative polymerase chain reaction for SMC-specific gene (alpha smooth muscle actin, h1-calponin, and SM22alpha) expression was performed on MSCs, which were cultured with various concentrations of TGF-beta1 or AA. TGF-beta1 had a tendency to up-regulate the expression of SMC-specific genes in a dose-dependent manner. The expression of SM22alpha was significantly up-regulated by 30 muM AA. We also investigated the additive effect of TGF-beta1 and AA for differentiation into SMCs and compared this effect with that of other factors including platelet-derived growth factor BB (PDGF-BB). In addition to SMC-specific gene expression, SMC-specific proteins increased by two to four times when TGF-beta1 and AA were used together compared with their administration alone. PDGF did not increase the expression of SMC-specific markers. MSCs cultured with TGF-beta1 and AA did not differentiate into osteoblasts and adipocytes. These results suggest that a combination of TGF-beta1 and AA is useful for the differentiation of MSCs into SMCs for use in tissue engineering.     

Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

Background

During vascular injury, vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) are exposed to altered luminal blood flow or transmural interstitial flow. We investigate the effects of these two types of fluid flows on the phenotypes of SMCs and MFBs and the underlying mechanotransduction mechanisms.

Methodology/Principal Findings

Exposure to 8 dyn/cm² laminar flow shear stress (2-dimensional, 2-D) for 15 h significantly reduced expression of α-smooth muscle actin (α-SMA), smooth muscle protein 22 (SM22), SM myosin heavy chain (SM-MHC), smoothelin, and calponin. Cells suspended in collagen gels were exposed to interstitial flow (1 cmH₂O, ∼0.05 dyn/cm², 3-D), and after 6 h of exposure, expression of SM-MHC, smoothelin, and calponin were significantly reduced, while expression of α-SMA and SM22 were markedly enhanced. PD98059 (an ERK1/2 inhibitor) and heparinase III (an enzyme to cleave heparan sulfate) significantly blocked the effects of laminar flow on gene expression, and also reversed the effects of interstitial flow on SM-MHC, smoothelin, and calponin, but enhanced interstitial flow-induced expression of α-SMA and SM22. SMCs and MFBs have similar responses to fluid flow. Silencing ERK1/2 completely blocked the effects of both laminar flow and interstitial flow on SMC marker gene expression. Western blotting showed that both types of flows induced ERK1/2 activation that was inhibited by disruption of heparan sulfate proteoglycans (HSPGs).

Conclusions/Significance

The results suggest that HSPG-mediated ERK1/2 activation is an important mechanotransduction pathway modulating SMC marker gene expression when SMCs and MFBs are exposed to flow. Fluid flow may be involved in vascular remodeling and lesion formation by affecting phenotypes of vascular wall cells. This study has implications in understanding the flow-related mechanobiology in vascular lesion formation, tumor cell invasion, and stem cell differentiation.     

Platelet-derived growth factor-BB represses smooth muscle cell marker genes via changes in binding of MKL factors and histone deacetylases to their promoters

A hallmark of smooth muscle cell (SMC) phenotypic switching is suppression of SMC marker gene expression. Although myocardin has been shown to be a key regulator of this process, the role of its related factors, MKL1 and MKL2, in SMC phenotypic switching remains unknown. The present studies were aimed at determining if: 1) MKL factors contribute to the expression of SMC marker genes in cultured SMCs; and 2) platelet-derived growth factor-BB (PDGF-BB)-induced repression of SMC marker genes is mediated by suppression of MKL factors. Results of gain- and loss-of-function experiments showed that MKL factors regulated the expression of single and multiple CArG [CC(AT-rich)(6)GG]-containing SMC marker genes, such as smooth muscle (SM) alpha-actin and telokin, but not CArG-independent SMC marker genes such as smoothelin-B. Treatment with PDGF-BB reduced the expression of CArG-containing SMC marker genes, as well as myocardin expression in cultured SMCs, while it had no effect on expression of MKL1 and MKL2. However, of interest, PDGF-BB induced the dissociation of MKL factors from the CArG-containing region of SMC marker genes, as determined by chromatin immunoprecipitation assays. This dissociation was caused by the competition between MKL factors and phosphorylated Elk-1 at early time points, but subsequently by the reduction in acetylated histone H4 levels at these promoter regions mediated by histone deacetylases, HDAC2, HDAC4, and HDAC5. Results provide novel evidence that PDGF-BB-induced repression of SMC marker genes is mediated through combinatorial mechanisms, including downregulation of myocardin expression and inhibition of the association of myocardin/MKL factors with CArG-containing SMC marker gene promoters.     

Construction of vascular tissues with macro-porous nano-fibrous scaffolds and smooth muscle cells enriched from differentiated embryonic stem cells

Vascular smooth muscle cells (SMCs) have been broadly used for constructing tissue-engineered blood vessels. However, the availability of mature SMCs from donors or patients is very limited. Derivation of SMCs by differentiating embryonic stem cells (ESCs) has been reported, but not widely utilized in vascular tissue engineering due to low induction efficiency and, hence, low SMC purity. To address these problems, SMCs were enriched from retinoic acid induced mouse ESCs with LacZ genetic labeling under the control of SM22α promoter as the positive sorting marker in the present study. The sorted SMCs were characterized and then cultured on three-dimensional macro-porous nano-fibrous scaffolds in vitro or implanted subcutaneously into nude mice after being seeded on the scaffolds. Our data showed that the LacZ staining, which reflected the corresponding SMC marker SM22α expression level, was efficient as a positive selection marker to dramatically enrich SMCs and eliminate other cell types. After the sorted cells were seeded into the three-dimensional nano-fibrous scaffolds, continuous retinoic acid treatment further enhanced the SMC marker gene expression level while inhibited pluripotent maker gene expression level during the in vitro culture. Meanwhile, after being implanted subcutaneously into nude mice, the implanted cells maintained the positive LacZ staining within the constructs and no teratoma formation was observed. In conclusion, our results demonstrated the potential of SMCs derived from ESCs as a promising cell source for therapeutic vascular tissue engineering and disease model applications.