Kumar V B Sameer, Viji R I, Kiran M S, Sudhakaran P R
Department of Biochemistry, University of Kerala, Thiruvananthapuram, Kerala 695581, India.
J Cell Biochem. 2008 Apr 15;103(6):1808-25. doi: 10.1002/jcb.21567.
Endothelial cell (EC) matrix interaction is critical in angiogenesis. Although matrix components can regulate the process of angiogenesis by acting as a reservoir of various cytokines, it is not clear if extracellular matrix (ECM) can modulate the production and activity of angiogenic cytokines. Investigations were therefore carried out to study the influence of the basement membrane (BM) protein, laminin (Ln) on the activity of vascular endothelial growth factor (VEGF), the major angiogenic cytokine, using isolated human umbilical vein ECs (HUVECs) in culture. Analysis of the biochemical markers of angiogenesis confirmed proangiogenic effect of Ln. The levels of VEGF protein and mRNA were not different in cells maintained on Ln, collagen I or polylysine substrata. Chorioallantoic membrane assay using VEGF isolated from cell extracts however revealed that Ln increased its angiogenic potency. Immunoblotting and HPLC analysis showed considerable reduction in poly adenosyl ribosylation of VEGF associated with a significant decrease in the levels of NAD+, in cells maintained on Ln substrata. Further, a shift in the isoenzymic pattern of LDH towards the B rich forms and an upregulation of LDH B gene were observed in cells maintained on Ln. Ln modulates expression of LDH gene through alpha(6)beta(4) integrin mediated downstream signaling involving p38 mitogen activated protein kinases (MAPK) pathway. It thus appears that Ln can affect aerobic metabolism of ECs by modulating the expression of LDH isoenzymes resulting in a decrease in the level of NAD+ that can cause a reduction in the poly adenosyl ribosylation of VEGF altering its angiogenic potency.
内皮细胞（EC）与基质的相互作用在血管生成中至关重要。尽管基质成分可作为多种细胞因子的储存库来调节血管生成过程，但细胞外基质（ECM）是否能调节血管生成细胞因子的产生和活性尚不清楚。因此，利用培养的分离人脐静脉内皮细胞（HUVECs）开展研究，以探讨基底膜（BM）蛋白层粘连蛋白（Ln）对主要血管生成细胞因子血管内皮生长因子（VEGF）活性的影响。血管生成生化标志物分析证实了Ln的促血管生成作用。在Ln、I型胶原或聚赖氨酸基质上培养的细胞中，VEGF蛋白和mRNA水平并无差异。然而，使用从细胞提取物中分离出的VEGF进行的鸡胚绒毛尿囊膜试验表明，Ln提高了其血管生成能力。免疫印迹和高效液相色谱分析显示，在Ln基质上培养的细胞中，VEGF的多聚腺苷酸化显著减少，同时NAD+水平显著降低。此外，在Ln上培养的细胞中观察到乳酸脱氢酶（LDH）同工酶模式向富含B的形式转变，且LDH B基因上调。Ln通过α(6)β(4)整合素介导的下游信号传导（涉及p38丝裂原活化蛋白激酶（MAPK）途径）调节LDH基因的表达。因此，Ln似乎可以通过调节LDH同工酶的表达来影响内皮细胞的有氧代谢，导致NAD+水平降低，进而使VEGF的多聚腺苷酸化减少，改变其血管生成能力。
