Newcomb P M, Herman I M
Program in Cell, Molecular, and Developmental Biology, Tufts University Health Science Schools, Boston, Massachusetts 02111.
J Cell Physiol. 1993 May;155(2):385-93. doi: 10.1002/jcp.1041550220.
We compared the effects of endothelial-synthesized matrix and purified matrix molecules on pericyte (PC) and aortic smooth muscle cell (SMC) growth, heparin sensitivity, and contractile phenotype in vitro. When PC are plated on endothelial-synthesized (EC) matrix, cell number is, on average, 3.1-fold higher than identical populations grown on plastic. Under the same conditions, SMC proliferation is stimulated 1.6-fold. Purified matrix molecules, such as collagen type IV (COLL) or fibronectin (FN), both major components of the EC matrix, stimulate PC/SMC growth 1.2-1.7-fold. Heparin (100 micrograms/ml), which inhibits the growth of early passage SMC by 60%, inhibits PC growth approximately 50%, when cells were plated on plastic. However, PC plated on EC matrix in the presence of heparin (100 micrograms/ml) grow as well as parallel cultures grown on plastic (in the absence of heparin). Concomitant with matrix-stimulated proliferation, we observed a marked reduction in PC containing alpha vascular smooth muscle actin (alpha VSMA), as seen by immunofluorescence using affinity-purified antibodies (173/615 positive pericytes on DOC matrix (28%) vs. 221/285 (77%) positive on glass). SMC respond similarly. Whereas alpha VSMA protein is markedly altered when PC and SMC are cultured on EC matrix, similar reductions in mRNA are not observed. However, Northern blotting does reveal that PC contain 17-30 times the steady-state levels of alpha VSMA mRNA compared to SMC. When SMC and PC cultures on plastic are treated with heparin, the steady-state levels of vascular smooth muscle actin mRNA increase 5 and 1.5 fold, respectively. Similarly, heparin treatment of PC grown on plastic induces a 1.8 fold increase in nonmuscle actin mRNA. These heparin-induced alterations in isoactin mRNA levels are not seen when PC are cultured on EC matrix. We also observed reductions in alpha VSMA and beta actin mRNA levels when PC are plated on FN, where they maintain a ratio of 13:1 (alpha:beta). Similar ratios are found in SMC present in rat and bovine aortae in vivo. These steady-state isoactin mRNA ratios are slightly different from those seen in cultured PC (8-10:1; alpha:beta). These results suggest that selective synthesis and remodelling of the endothelial basal lamina may signal alterations in pericyte growth and contractile phenotype during normal vascular morphogenesis, angiogenesis, or during the microvascular remodelling that accompanies hypertensive onset.
我们比较了内皮细胞合成基质和纯化的基质分子对周细胞（PC）和主动脉平滑肌细胞（SMC）体外生长、肝素敏感性及收缩表型的影响。当将PC接种在内皮细胞合成（EC）基质上时，细胞数量平均比在塑料上生长的相同细胞群体高3.1倍。在相同条件下，SMC增殖受到1.6倍的刺激。纯化的基质分子，如IV型胶原（COLL）或纤连蛋白（FN），均为EC基质的主要成分，可刺激PC/SMC生长1.2 - 1.7倍。肝素（100微克/毫升）可使早期传代的SMC生长抑制60%，当细胞接种在塑料上时，可使PC生长抑制约50%。然而，当PC接种在存在肝素（100微克/毫升）的EC基质上时，其生长情况与在塑料上生长的平行培养物（无肝素）相同。伴随基质刺激的增殖，我们观察到含α血管平滑肌肌动蛋白（αVSMA）的PC显著减少，使用亲和纯化抗体进行免疫荧光检测可见（在DOC基质上173/615个周细胞呈阳性（28%），而在玻璃上221/285个（77%）呈阳性）。SMC有类似反应。当PC和SMC在EC基质上培养时，αVSMA蛋白明显改变，但未观察到mRNA有类似减少。然而，Northern印迹分析确实显示，与SMC相比，PC中αVSMA mRNA的稳态水平高17 - 30倍。当塑料上的SMC和PC培养物用肝素处理时，血管平滑肌肌动蛋白mRNA的稳态水平分别增加5倍和1.5倍。同样，用肝素处理塑料上生长的PC可使非肌肉肌动蛋白mRNA增加1.8倍。当PC在EC基质上培养时，未见到这些肝素诱导的肌动蛋白异构体mRNA水平的改变。当PC接种在FN上时，我们还观察到αVSMA和β肌动蛋白mRNA水平降低，此时它们维持13:1（α:β）的比例。在体内大鼠和牛主动脉中的SMC中也发现了类似比例。这些稳态肌动蛋白异构体mRNA比例与培养的PC中所见比例（8 - 10:1；α:β）略有不同。这些结果表明，在内皮细胞基底膜的选择性合成和重塑可能在正常血管形态发生、血管生成过程中，或在高血压发病时伴随的微血管重塑过程中，发出周细胞生长和收缩表型改变的信号。
