**4. Regulation of differentiation of vascular wall-resident multipotent stem cells into smooth muscle cells**

Epigenetic regulation was shown to play a crucial role in SMC differentiation [107]. High levels of histone modifications were found in promoters of SMC-specific genes as compared to undifferentiated embryonic stem cells [108, 109]. Of the epigenetic regulation mechanisms, histone acetylation, which is adjusted through acetyltransferases (HATs) and histone deace‐ tylases (HDACs), primarily promotes the expression of target genes [110]. However, whether the differentiation of MSCs to SMCs was affected by such histone modifications remains unresolved. HDACs, however, can arrest stem cell proliferation and induce cell differentiation and apoptosis [111]. A histone deacetylase inhibitor (sodium butyrate) was further found to effectively promote rat BM-MSC differentiation into SMCs; a strategy that could potentially be applied in clinical tissue engineering and cell transplantation, for example for the treatment of bladder function disorders such as stress urinary incontinence [112, 113].

In order to identify molecular mechanisms governing the differentiation of the vascular wall-resident MPSCs into SMCs, cDNA microarray analyses on MPSCs isolated from human internal thoracic artery fragments in comparison to mature SMC of human aorta were performed (unpublished data). Among several genes being differentially expressed in VW-

MPSCs, the HOX genes HOXB7, HOXC6 and HOXC8 were found to be expressed in VW-MPSCs at a clearly higher level than in mature hAoSMC [60]. The HOX genes are a family of regulatory transcription factors that control the activity of other functionally related genes in the course of individual development, and are expressed variously in the adult organ‐ ism [114]. Because of their central role in the development of body parts, limbs and organs, mutation of these genes can cause serious changes in body parts at points in the body that they do not physiologically occur, such as the conversion of complete limbs. In humans, so far, HOX-39 transcription factors have been identified in the four separate clusters (HOXA-D) that are located on four different chromosomes. Together with accessory factors, HOX proteins bind to specific DNA sequences in order to activate or repress genes [115]. HOX genes are thought to act as micromanagers orchestrating cell differentiation after embryonic development in many different cell types and developmental pathways [116]. In the adult, it is already known that colony-forming unit-fibroblasts (CFU-F) derived from different organs have characteristic HOX expression signatures that are heterogeneous but highly specific for their anatomical origin [117]. The topographic specificity of HOX code is maintained during differentiation, which indeed suggests that the pattern of expression is an intrinsic property of MSCs. Furthermore, stem and progenitor cells from mesoder‐ mal tissues have HOX-specific gene expression profiles. This so-called biological finger‐ print can be used to differentiate functionally different MSC populations from bone marrow and umbilical cord blood [118]. Thus, HOX proteins have a role in specifying the cellular identity of MSC. A differential analysis of 39 HOX genes in vascular wall-resident MPSCs compared to terminally differentiated endothelial cells, SMC and less differentiated (pluripotent) embryonic stem cells showed that HOX family members HOXB7, HOXC6 and HOXC8 are overexpressed in the vessel-resident MPSCs. This suggests that these HOX genes are involved in the development and differentiation of the VW-MPSCs [60]. To gain further insights into the molecular role of these HOX genes for VW-MPSC differentiation as well as to identify potential downstream regulated genes of HOXB7, HOXC6 and HOXC8 activity, VW-MPSCs were transfected with HOXB7, HOXC6 and HOXC8-specific siRNAs both individually and in defined combinations using non-specific siRNAs as controls. Interestingly, silencing these HOX genes in VW-MPSCs significantly reduced their sprout‐ ing capacity and increased expression of the SMC differentiation and maturation markers transgelin (TAGLN) and calponin (CNN1), and the histone gene histone H1. Further‐ more, the methylation pattern of the TAGLN promoter was altered, which clearly indi‐ cates a differentiation of VW-MPSCs to a more mature SMC phenotype. A restricted expression of HOX genes, in particular HOXB7, had already been reported in the 1990s to distinguish foetal from adult human SMC, whereby HOXB7 was expressed at markedly higher levels in embryonic vascular SMC as compared to mature SMC of adult vessels [119]. These data suggest that HOXB7 initiates a differentiation from multipotent cell type towards SMC, but stops the further differentiation of these cells into mature SMC. Further strik‐ ing evidence is that H1 is also involved in the regulation of VW-MPSC differentiation into SMC [60]. H1 expression in VW-MPSCs is significantly enhanced upon differentiation towards SMC, as shown after gene silencing for HOXB7, HOXC6 and HOXC8, respective‐ ly. In general, H1 function can alter the chromatin structure and serves as both a positive

and negative regulator of transcription, depending on the gene. H1 can further influence DNA methylation and regulate specific gene expression [120-122]. We may conclude that the interaction of H1 and HOXB7 might be a more specific mechanism regulating gene expression and differentiation of VW-MPSCs to SMCs and then to mature SMCs in physiological remodelling processes of the vessel wall and vascular diseases. Indeed, in human atherosclerotic lesions, where mature SMCs revert to a more immature and less contractile phenotype, HOXB7 mRNA was detected at a higher level than in normal artery wall [123]. An even closer relationship seems to exist between VW-MPSCs and mature SMCs. SMC differentiation is accompanied by enhanced ACTA2, TAGLN and CNN1 expression. TAGLN is expressed exclusively in smooth muscle-containing tissues of adult mammals, and is one of the earliest markers of differentiating SMCs [124]. While the expression of these markers is a common feature of SMC regardless of their anatomical position, it has been shown that even SMCs of different parts of adult arteries, e.g., aortic arch, abdominal aorta and femoral artery, exhibit different codes of HOX gene expres‐ sion, indicating the close relation between HOX code and the anatomical positional identity of SMC in each part of the blood vessels [125].

MPSCs, the HOX genes HOXB7, HOXC6 and HOXC8 were found to be expressed in VW-MPSCs at a clearly higher level than in mature hAoSMC [60]. The HOX genes are a family of regulatory transcription factors that control the activity of other functionally related genes in the course of individual development, and are expressed variously in the adult organ‐ ism [114]. Because of their central role in the development of body parts, limbs and organs, mutation of these genes can cause serious changes in body parts at points in the body that they do not physiologically occur, such as the conversion of complete limbs. In humans, so far, HOX-39 transcription factors have been identified in the four separate clusters (HOXA-D) that are located on four different chromosomes. Together with accessory factors, HOX proteins bind to specific DNA sequences in order to activate or repress genes [115]. HOX genes are thought to act as micromanagers orchestrating cell differentiation after embryonic development in many different cell types and developmental pathways [116]. In the adult, it is already known that colony-forming unit-fibroblasts (CFU-F) derived from different organs have characteristic HOX expression signatures that are heterogeneous but highly specific for their anatomical origin [117]. The topographic specificity of HOX code is maintained during differentiation, which indeed suggests that the pattern of expression is an intrinsic property of MSCs. Furthermore, stem and progenitor cells from mesoder‐ mal tissues have HOX-specific gene expression profiles. This so-called biological finger‐ print can be used to differentiate functionally different MSC populations from bone marrow and umbilical cord blood [118]. Thus, HOX proteins have a role in specifying the cellular identity of MSC. A differential analysis of 39 HOX genes in vascular wall-resident MPSCs compared to terminally differentiated endothelial cells, SMC and less differentiated (pluripotent) embryonic stem cells showed that HOX family members HOXB7, HOXC6 and HOXC8 are overexpressed in the vessel-resident MPSCs. This suggests that these HOX genes are involved in the development and differentiation of the VW-MPSCs [60]. To gain further insights into the molecular role of these HOX genes for VW-MPSC differentiation as well as to identify potential downstream regulated genes of HOXB7, HOXC6 and HOXC8 activity, VW-MPSCs were transfected with HOXB7, HOXC6 and HOXC8-specific siRNAs both individually and in defined combinations using non-specific siRNAs as controls. Interestingly, silencing these HOX genes in VW-MPSCs significantly reduced their sprout‐ ing capacity and increased expression of the SMC differentiation and maturation markers transgelin (TAGLN) and calponin (CNN1), and the histone gene histone H1. Further‐ more, the methylation pattern of the TAGLN promoter was altered, which clearly indi‐ cates a differentiation of VW-MPSCs to a more mature SMC phenotype. A restricted expression of HOX genes, in particular HOXB7, had already been reported in the 1990s to distinguish foetal from adult human SMC, whereby HOXB7 was expressed at markedly higher levels in embryonic vascular SMC as compared to mature SMC of adult vessels [119]. These data suggest that HOXB7 initiates a differentiation from multipotent cell type towards SMC, but stops the further differentiation of these cells into mature SMC. Further strik‐ ing evidence is that H1 is also involved in the regulation of VW-MPSC differentiation into SMC [60]. H1 expression in VW-MPSCs is significantly enhanced upon differentiation towards SMC, as shown after gene silencing for HOXB7, HOXC6 and HOXC8, respective‐ ly. In general, H1 function can alter the chromatin structure and serves as both a positive

38 Muscle Cell and Tissue

Further candidate factors were reported to be important for MSC differentiation to SMC. The most prominent one is the morphogenetic transforming growth factor-beta (TGFb) [126]. TGFb stimulation alone is sufficient for the induction of a rapid SMC differentiation of MPSC and MSC-like cells [46, 127, 128]. Isolated VW-MPSCs exposed to exogenous TGFβ1 during culturing exhibited alterations in the gene expression profile in the form of significantly increased expression of the SMC markers TAGLN, hyaluronan and proteoglycan binding link protein (HAPLN), and thrombospondin 1 (THSP1) [46]. In embryonic stem cell-derived MSCs (hES-MCs), TGF-β-treatment resulted in SMC differentiation in a dose- and time-dependent manner as demonstrated by the expression of SMC-specific genes ACTA2, CNN1, and smooth muscle myosin heavy chain (SM-MHC) [127]. Mechanistically, TGFb-induced differentiation was Smad- and serum response factor/myocardin-dependent. Furthermore, the treatment of adipose tissue-derived MSCs (hASCs) with TGFb dramatically increased the contraction of a collagen-gel lattice and the expression levels of SMC-specific genes including ACTA2, CNN1, SM-MHC, smoothelin-B, myocardin and h-caldesmon, as well as causing an increased expression of vascular SMC-like ion channels, indicating differentiation of hASCs into contractile vascular SMCs [128]. Beside the direct action of growth and differentiation factors, either by direct, exogenous application to cultured MPSC and MSC-like cells, or by stimulation of vascular MPSC in their native niche (e.g., by tumour secretion), other factors were described as decisive for the SMC differentiation of vascular multipotent stem cells. The differential expression of these cell-type-specific factors seems to act more indirectly, and to prime the cell somehow to differentiate along the SMC lineage. The basic molecular mechanism behind these cell-type-specific factors remains elusive at present. A prominent EphA3 expression in endometrial spiral arterioles and surrounding stroma, but not in other human tissues, suggests EphA3 as a unique marker of perivascular MSCs that are implicated in rapid neovasculariza‐ tion and vascular remodelling [129]. This selective EphA3 expression was further observed in actively growing rather than established blood vessels in the vascular microenvironment of solid tumours. In addition, a strong expression of CD146 within a BM-MSC subpopulation was associated with a commitment to a vascular smooth muscle cell lineage characterized by a strong up-regulation of calponin-1 and SM22α expression and an ability to contract the collagen matrix [130].
