Montesano R, Kumar S, Orci L, Pepper M S
Department of Morphology, University Medical Center, Geneva, Switzerland.
Lab Invest. 1996 Aug;75(2):249-62.
The aim of the present study was to determine whether hyaluronan (HA) degradation products, which have been shown to be angiogenic in vivo, influence endothelial cell invasion of a 3-dimensional matrix, an essential component of the neovascularization process. Using a previously described in vitro assay, we demonstrate that like the angiogenic cytokines basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), HA oligosaccharides (OHA) induce bovine microvascular endothelial cells to invade a 3-dimensional collagen gel within which they form capillary-like tubes, with an optimal effect at approximately 0.5 to 2 micrograms/ml. Strikingly, co-addition of OHA (0.5 - 2 micrograms/ml) and VEGF (30 ng/ml), but not co-addition of OHA and bFGF (10 ng/ml), induced an in vitro angiogenic response that was greater than the sum of the effects elicited by either agent separately. In contrast to OHA, native high molecular weight HA was consistently inactive, whether added alone or in combination with VEGF or bFGF. Because endothelial cell invasion is believed to require extracellular proteolytic activity, we also investigated the effect of OHA on the plasminogen activator (PA)-plasmin system. OHA (0.01 to 1 microgram/ml) but not native high molecular weight HA induced a dose-dependent increase in mRNA levels of urokinase type PA (uPA), urokinase type PA receptor and PA inhibitor type 1, and a parallel increase in the functional activity of urokinase type PA and PA inhibitor type 1, as determined by zymography and reverse zymography, respectively. The effects of OHA on proteolytic activity were additive with those of VEGF, but not with those of bFGF. Taken together, these results demonstrate that OHA modulate the invasive and proteolytic properties of bovine microvascular endothelial cells and synergize specifically with VEGF in the induction of angiogenesis in vitro. We suggest that the synergism between OHA and VEGF plays a role in the regulation of angiogenesis and that it may be exploited therapeutically in situations that would benefit from stimulation of new blood vessel growth.
本研究的目的是确定已证实在体内具有血管生成作用的透明质酸（HA）降解产物是否会影响三维基质中的内皮细胞侵袭，而内皮细胞侵袭是新血管形成过程的一个重要组成部分。使用先前描述的体外试验，我们证明，与血管生成细胞因子碱性成纤维细胞生长因子（bFGF）和血管内皮生长因子（VEGF）一样，HA寡糖（OHA）可诱导牛微血管内皮细胞侵袭三维胶原凝胶，并在其中形成毛细血管样管，在约0.5至2微克/毫升时效果最佳。引人注目的是，共同添加OHA（0.5 - 2微克/毫升）和VEGF（30纳克/毫升），而非共同添加OHA和bFGF（10纳克/毫升），可诱导出一种体外血管生成反应，该反应大于单独使用任一试剂所产生的效应之和。与OHA相反，天然高分子量HA无论是单独添加还是与VEGF或bFGF联合添加，始终无活性。由于内皮细胞侵袭被认为需要细胞外蛋白水解活性，我们还研究了OHA对纤溶酶原激活剂（PA）-纤溶酶系统的影响。OHA（0.01至1微克/毫升）而非天然高分子量HA可诱导尿激酶型PA（uPA）、尿激酶型PA受体和PA抑制剂1型的mRNA水平呈剂量依赖性增加，并且分别通过酶谱分析和反向酶谱分析确定，尿激酶型PA和PA抑制剂1型的功能活性也相应增加。OHA对蛋白水解活性的影响与VEGF的影响具有相加性，但与bFGF的影响不具有相加性。综上所述，这些结果表明，OHA可调节牛微血管内皮细胞的侵袭和蛋白水解特性，并在体外血管生成诱导中与VEGF特异性协同作用。我们认为，OHA与VEGF之间的协同作用在血管生成调节中发挥作用，并且在受益于新血管生长刺激的情况下可能具有治疗应用价值。
