The Role of Heparan Sulfate in Vascular Remodeling

说明(申请人提供)：硫酸乙酰肝素蛋白多糖(HSPG)由蛋白多糖核心蛋白共价连接到硫酸乙酰肝素(HS)链上。这些HS链含有许多趋化因子和生长因子的结合位点。新出现的大量证据表明，趋化因子和生长因子结合和活性的特异性取决于沿着HS链长度的硫酸盐化表位的独特模式。我们已经证明，血管损伤会引起HS链上硫酸盐化表位模式的显着变化，这是一种基于高效液相的灵敏方法。伴随着这些变化的是调节HS硫酸盐化的酶的表达强劲增加。最初的硫化反应是由一系列N-脱乙酰酶-N-磺酸转移酶(NDSTs)催化的。我们发现，在小鼠血管损伤后，最常见的异构体NDST1增加了20倍。这项修订提案的总体目标是确定HS硫酸盐化在血管损伤中的作用。为了做到这一点，我们将使用新的小鼠基因模型，在平滑肌中定向删除NDST1，如果必要，还将额外定向条件删除调节HS硫酸盐化和合成的酶。遗传和生化方法的结合将在体内和体外使用，以及新建立的基于FACS的方法来仔细定义HS硫酸盐化如何影响损伤反应的重塑过程。具体地说，我们将测试以下假设：减少HS硫酸盐化会减少损伤反应中的病变形成。II.检验假设，减少HS的硫化作用会损害趋化因子的结合、表达和活性。了解体内HS结构和功能的操纵如何影响血管重塑，可能会通过设计专门修饰HS的新型合成化合物来治疗血管疾病，具有重要的治疗价值。这些研究将为血管平滑肌细胞内源性HS结构对损伤程度和趋化因子调节的影响提供第一个关键的观察。

DESCRIPTION (provided by applicant): Heparan sulfate proteoglycans (HSPG) consist of a proteoglycan core protein covalently attached to heparan sulfate (HS) chains. These HS chains contain binding sites for numerous chemokines and growth factors. An emerging body of evidence suggests that specificity of chemokine and growth factor binding and activity is dependent upon the unique patterning of sulfated epitopes along the length of the HS chains. We have demonstrated that vascular injury invokes significant changes in the pattern of sulfated epitopes along the HS chains utilizing a sensitive state-of-the-art HPLC-based approach. These changes were accompanied by robust increases in the expression of enzymes that regulate HS sulfation. The initial sulfation reaction is catalyzed by a family of N-deacetylase-N-sulfotransferases (NDSTs). We identified a 20- fold increase in the most prevalent isoform, NDST1, following vascular injury in mice. The overall goal of this revised proposal is to establish the role of HS sulfation in vascular injury. To do this we will employ novel genetic murine models with targeted deletion of NDST1 in smooth muscle, and if necessary, additional targeted conditional deletions of enzymes that regulate HS sulfation and synthesis. A combination of genetic and biochemical approaches will be used in vivo and in vitro along with a newly established FACS-based approach to carefully define how HS sulfation influences the process of remodeling in response to injury. Specifically, we will: Test the hypothesis that decreasing HS sulfation leads to a reduction in lesion formation in response to injury. II. Test the hypothesis that decreasing HS sulfation impairs chemokine binding, expression, and activity. Understanding how the manipulation of HS structure and function in vivo affects vascular remodeling could have significant therapeutic value in treating vascular disease through the design of novel synthetic compounds that specifically modify HS. These studies will provide the first critical look at the impact of endogenous HS structure in vascular smooth muscle cells on the extent of injury and the regulation of chemokines.