Enhancer of polycomb1 lessens neointima formation by potentiation of myocardin-induced smooth muscle differentiation

ObjectivePreviously, we reported that enhancer of polycomb1 (Epc1) induces skeletal muscle differentiation through the serum response factor (SRF). Considering that SRF plays a critical role in vascular smooth muscle cell (VSMC) differentiation, we expected that Epc1 also works in VSMCs. Here we examined the effect of Epc1 on neointima formation after arterial balloon injury and the underlying mechanism.MethodsEpc1 expression was examined in carotid artery injury or VSMC models. Interaction with myocardin (Myocd), a master regulator of smooth muscle differentiation, was examined by immunoprecipitation or promoter analysis with smooth muscle (SM) 22α promoter. Finally, we investigated whether local delivery of Epc1 regulated neointimal formation after injury.ResultsEpc1 expression was down-regulated during proliferation induced by platelet-derived growth factor BB, whereas it was upregulated during differentiation in VSMCs. Forced expression of Epc1 induced VSMC differentiation. Epc1 physically interacted with Myocd to synergistically activate SM22α promoter activity. Transient transfection of Epc1 enhanced the physical interaction between Myocd and SRF, whereas that interaction was reduced when A10 cells were treated with siRNA for Epc1. Local delivery of Epc1 significantly reduced neointima formation induced by balloon injury.ConclusionsOur results indicate that Epc1 induces VSMC differentiation by interacting with Myocd to induce SRF-dependent smooth muscle genes. We propose that Epc1 acts as a novel negative regulator of neointima formation after carotid injury.     

CREG promotes a mature smooth muscle cell phenotype and reduces neointimal formation in balloon-injured rat carotid artery

AIM: We previously showed that cellular repressor of E1A-stimulated genes (CREG) is up-regulated during serum starvation-induced vascular smooth muscle cell (SMC) differentiation. The aim of this study was to determine the role of CREG in maintaining the quiescent, differentiated phenotype of SMCs both in culture and in balloon-injured rat carotid artery. METHODS AND RESULTS: In cultured SMCs recombinant virus-mediated CREG expression enhanced cellular differentiation, inhibited proliferation, and reduced synthesis of extracellular matrix component fibronectin. In contrast, CREG knockdown via retroviral transfer of short hairpin RNAs abrogated serum starvation-induced SMC differentiation and growth arrest. Both immunostaining and Western analysis demonstrated marked down-regulation of CREG in the vascular media after balloon injury to the rat carotid artery. Retrovirus-mediated CREG transfer to the injured artery inhibited SMC dedifferentiation and proliferation, and reduced neointimal hyperplasia. CONCLUSION: These results suggest that CREG participates in the maintenance of quiescent mature SMC phenotype in the arterial media by promoting SMC differentiation and growth arrest and that CREG gene transfer has therapeutic potential for vascular diseases associated with neointimal hyperplasia.     

Stage-specific modulation of skeletal myogenesis by inhibitors of nuclear deacetylases

Nuclear acetyltransferases promote and deacetylases inhibit skeletal muscle-gene expression, suggesting the potential effectiveness of deacetylase inhibitors (DIs) in modulating skeletal myogenesis. Surprisingly, previous studies have indicated that DIs suppress myogenesis. The recent observations that histone deacetylases associate with the muscle-regulatory proteins MyoD and MEF2C only in undifferentiated myoblasts prompted us to evaluate the effect of DIs at distinct stages of the myogenic program. We found that exposure of established rodent and human muscle cells to distinct DIs has stage-specific effects. Exposure of undifferentiated skeletal myoblasts to DIs, followed by incubation in differentiation medium, enhanced the expression of muscle-specific reporters and increased the levels of endogenous muscle proteins, leading to a dramatic increase in the formation of multinucleated myotubes. By contrast, simultaneous exposure of muscle cells to differentiation medium and DIs inhibited the myogenic program. Likewise, embryos exposed in utero to nonteratogenic doses of DI at the early stages of somitic myogenesis (embryonic day 8.5) exhibited an increased number of somites and augmented expression of a muscle-specific transgene as well as endogenous muscle genes. The functional effects induced by DIs were mirrored by changes in the state of acetylation of histones present at a muscle-gene enhancer and of MyoD itself. These results represent the first evidence that DIs can enhance muscle differentiation and suggest the rationale for their use in manipulating adult and embryonic skeletal myogenesis.     

MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes.

Shortly after their birth, postmitotic mononucleated myoblasts in myotomes, limb buds, and conventional muscle cultures elongate and assemble a cohort of myofibrillar proteins into definitively striated myofibrils. MyoD induces a number of immortalized and/or transformed nonmuscle cells to express desmin and several myofibrillar proteins and to fuse into myosacs. We now report that MyoD converts normal dermal fibroblasts, chondroblasts, gizzard smooth muscle, and pigmented retinal epithelial cells into elongated postmitotic mononucleated striated myoblasts. The sarcomeric localization of antibodies to desmin, alpha-actinin, titin, troponin-I, alpha-actin, myosin heavy chain, and myomesin in these converted myoblasts are indistinguishable from in vivo and in vitro normal myoblasts. Converted myoblasts fuse into typical anisodiametric multinucleated myotubes that often contract spontaneously. Conversion and subsequent expression of the skeletal myogenic program are autonomous events, occurring in four nonmuscle microenvironments consisting of different combinations of foreign extracellular matrix molecules. Early events associated with conversion by MyoD involve (i) withdrawal from the cell cycle, (ii) down-regulation of the subverted cell's ongoing differentiation program, and (iii) initiation of desmin synthesis in presumptive myoblasts and dramatic redistribution of microtubules and desmin intermediate filaments in postmitotic myoblasts.