Molecular mechanisms of heparanase-2 (hpa-2) in endothelial cell activation, inflammation and albuminuria

血管内皮细胞被一层高度糖基化的糖蛋白所覆盖，这层糖蛋白被称为糖萼。内皮糖基化在许多生理性肾小球机制中起着重要作用，糖基化损伤会导致肾脏疾病和蛋白尿。蛋白多糖可由乙酰肝素酶调节，乙酰肝素酶是唯一已知的能够降解硫酸乙酰肝素(HS)糖胺聚糖的哺乳动物内切糖苷酶。乙酰肝素酶对HS的酶降解作用深刻地影响着许多生理和病理过程，包括形态发生、新生血管、肿瘤发生以及免疫反应和炎症。最近，一种新的肝素酶HPA-2被克隆出来。最重要的是，Hpa2c抑制乙酰肝素酶的活性，可能是因为它对肝素和HS有很高的亲和力，并且能够物理上与肝素酶结合。然而，HPA-2在肾脏疾病中的功能作用及其在内皮细胞功能和蛋白尿发病机制中的可能作用尚未被研究。因此，我们想要研究肝素酶-2(HPA-2)的分子功能，并检验HPA-2的假设，即：(1)防止肝素酶-1脱落，(2)拮抗和减少肝素酶-1的细胞内效应，(3)减少细胞因子和生长因子(血管内皮生长因子、成纤维细胞生长因子)的细胞内信号和效应机制，以及(4)从而防止糖尿病和脓毒症等病理条件下的肾脏和血管损伤。为此，我们将使用(1)新开发的斑马鱼蛋白尿动物模型，(2)新的内皮细胞微循环系统，以及(3)新的糖萼染色技术和(4)HPA-2在小鼠肾脏疾病模型中的病毒转染法。我们的研究不仅有助于理解血管内皮细胞糖基化及其调控，还可能导致糖尿病肾病和败血症的新的治疗策略。

The endothelium is covered with a layer of heavily glycosylated glycoproteins called the glycocalyx. The endothelial glycocalyx plays an important role in many physiological glomerular mechanisms and damage to the glycocalyx results in renal disease and proteinuria. Proteoglycans can be regulated by heparanase, the only known mammalian endoglycosidase capable of degrading heparan sulfate (HS) glycosaminoglycan. Enzymatic degradation of HS by heparanase profoundly affects numerous physiological and pathological processes, including morphogenesis, neovascularization, tumorigenesis as well as immune reactivity and inflammation. Recently, a novel heparanase, hpa-2 has been cloned. Most importantly, Hpa2c inhibits heparanase enzymatic activity, likely due to its high affinity to heparin and HS and its ability to associate physically with heparanase. However, the functional role of hpa-2 in renal disease and its possible role in the pathogenesis of endothelial cell function and albuminuria have not been investigated so far. We therefore want to study the molecular function of heparanase-2 (hpa-2) and test the hypothesis that hpa-2 (1) prevents the shedding of the endothelial glycocalyx by heparanase-1, (2) antagonizes and reduces the intracellular effects of heparanase-1, (3) reduces the intracelluar signaling and effector mechanisms of cytokines and growth factors (VEGF, FGF), and (4) thereby prevents renal and vascular damage under pathological conditions such as diabetes and sepsis. For this purpose, we will use (1) a newly developed animal model for proteinuria in the zebrafish, (2) a novel microcirculation system for endothelial cells, and we have established (3) novel staining techiques for glycocalyx and (4) viral transfection of hpa-2 in mouse models of renal disease. Our research is not only improtnat for the understanding of endothelial glycocalyx and ist regulation but may also lead to novel treatment strategies in diabetic nephropathy and sepsis.