The in vitro production and expression of ECM proteins

The in vitro production and expression of ECM proteins collagens I and IV and laminin differed between the perivascular derivatives. In the body, pericytes produce ECM in the subendothelial basement membrane of capillaries, while both vSMCs and pericytes produce ECM in the tunica media layer of larger blood vessels (Niland, 2009). Collagen I, a fibrillar collagen, is a substantial component of the interstitial connective tissue in contrast to collagen IV, which is present in all basal lamina, forming the basic irregular fibrous 2D network of vasculature (Eble and Niland, 2009). Similarly, laminin is an indispensible component of the vascular basement membrane, the primary site where collagen IV and laminin form an interdependent network (Eble and Niland, 2009). In human arteries, collagen I, deposited between vSMCs, was reported to exhibit sparse thin fiber morphology in the media of small hepititis b while being organized in fibrillar structures in larger arteries. We report that in vitro, hiPSC pericytes, associated with small vasculature, have a greatly diminished collagen I expression compared to both hiPSC syn-vSMCs and hiPSC con-vSMCs, found in larger vessels (Shekhonin et al., 1987). Both our in vitro findings and in vivo studies illustrate that perivascular cells associated with larger vessels express more collagen I. From our in vitro study, we also observed that a morphologically distinct high density globular collagen IV expression is deposited by both hiPSC syn-vSMCs and pericytes, while a more fibrous collagen IV deposition as well as increased collagen IV expression is exhibited by hiPSC con-vSMCs. Similarly, in vivo, collagen IV deposition varies between the two phenotypes of vSMCs. For instance, fibrous plaques of atherosclerotic human arteries, known to mainly contain syn-vSMCs, have been reported to have greatly decreased collagen IV deposition and increased collagen I deposition around vSMCs compared to healthy arteries containing contractile vSMCs (Rekhter, 1999; Shekhonin et al., 1985, 1987). However, the loci of the plaques have been shown to have large quantities of collagen IV, correlating with the vSMCs being surrounded by layers of basement membrane material in this region (Rekhter, 1999; Shekhonin et al., 1987). Additionally, the in vitro laminin expression was different in hiPSC pericytes compared to both phenotypes of hiPSC vSMCs. hiPSC con-vSMCs had diffuse cytoplasmic expression of laminin, while hiPSC pericytes had punctate expression around the cell membrane. hiPSC syn-vSMCs had mostly diffuse cytoplasmic expression of laminin with few instances of punctate expression. In vivo, rat synthetic vSMCs lost the ability to produce laminin unlike contractile vSMCs (Thyberg et al., 1997). Fibronectin was expressed and deposited by all tested perivascular cell types, with hiPSC pericytes expressing the highest fibronectin mRNA levels. Human iPSC con-vSMCs expressed higher levels of fibronectin mRNA compared to hiPSC syn-vSMCs. Similarly, the hiPSC con-vSMCs also expressed higher levels of ED-A fibronectin compared to the hiPSC syn-vSMCs, while in vivo, ED-A fibronectin was suggested to be associated with the synthetic phenotype (Glukhova et al., 1989). Examination of the expression pattern of ED-A fibronectin in differentiating stem cells highlights the need for further investigation of stem cell derivatives and the conceivable differences between in vitro and reported in vivo phenotypes, thus warranting additional studies correlating fibronectin slice variants to perivascular phenotypes both in vitro and in vivo. Elastin was also highly expressed by con-vSMCs compared to all other perivascular cell types. Not only is elastin production characteristic of the contractile phenotype, but also, interestingly, in vivo studies demonstrated that increasing elastin production itself promoted a contractile vSMC phenotype by inhibiting vSMC proliferation (Karnik et al., 2003; Urbán et al., 2002).