Atherothrombosis and Vascular Biology Laboratory, Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia (J.Z., J.L.-S., S.B., A.L., A.R., A. Watson, N.D., P.S., A.B.-W., Y.C.C., M.M., M.L.P.V., A.H., N.M.H., X.W., G.P., J.D.M., K.P.).
Department of Plastic and Hand Surgery, Medical Center, Faculty of Medicine, University of Freiburg, Breisgau, Germany (J.Z., J.T., D.B., S.U.E.).
Circ Res. 2024 Nov 8;135(11):1033-1047. doi: 10.1161/CIRCRESAHA.124.324248. Epub 2024 Oct 18.
CRP (C-reactive protein) is a prototypical acute phase reactant. Upon dissociation of the pentameric isoform (pCRP [pentameric CRP]) into its monomeric subunits (mCRP [monomeric CRP]), it exhibits prothrombotic and proinflammatory activity. Pathophysiological shear rates as observed in aortic valve stenosis (AS) can influence protein conformation and function as observed with vWF (von Willebrand factor). Given the proinflammatory function of dissociated CRP and the important role of inflammation in the pathogenesis of AS, we investigated whether shear stress can modify CRP conformation and induce inflammatory effects relevant to AS.
To determine the effects of pathological shear rates on the function of human CRP, pCRP was subjected to pathophysiologically relevant shear rates and analyzed using biophysical and biochemical methods. To investigate the effect of shear on CRP conformation in vivo, we used a mouse model of arterial stenosis. Levels of mCRP and pCRP were measured in patients with severe AS pre- and post-transcatheter aortic valve implantation, and the presence of CRP was investigated on excised valves from patients undergoing aortic valve replacement surgery for severe AS. Microfluidic models of AS were then used to recapitulate the shear rates of patients with AS and to investigate this shear-dependent dissociation of pCRP and its inflammatory function.
Exposed to high shear rates, pCRP dissociates into its proinflammatory monomers (mCRP) and aggregates into large particles. Our in vitro findings were further confirmed in a mouse carotid artery stenosis model, where the administration of human pCRP led to the deposition of mCRP poststenosis. Patients undergoing transcatheter aortic valve implantation demonstrated significantly higher mCRP bound to circulating microvesicles pre-transcatheter aortic valve implantation compared with post-transcatheter aortic valve implantation. Excised human stenotic aortic valves display mCRP deposition. pCRP dissociated in a microfluidic model of AS and induces endothelial cell activation as measured by increased ICAM-1 (intercellular adhesion molecule 1) and P-selectin expression. mCRP also induces platelet activation and TGF-β (transforming growth factor beta) expression on platelets.
We identify a novel mechanism of shear-induced pCRP dissociation, which results in the activation of cells central to the development of AS. This novel mechanosensing mechanism of pCRP dissociation to mCRP is likely also relevant to other pathologies involving increased shear rates, such as in atherosclerotic and injured arteries.
CRP（C-反应蛋白）是典型的急性期反应物。当五聚体形式（pCRP [五聚体 CRP]）解离为其单体亚基（mCRP [单体 CRP]）时，它表现出促血栓形成和促炎活性。在主动脉瓣狭窄（AS）中观察到的病理剪切速率可以影响蛋白构象和功能，如 vWF（血管性血友病因子）。鉴于解离的 CRP 的促炎功能以及炎症在 AS 发病机制中的重要作用，我们研究了剪切应力是否可以改变 CRP 的构象并诱导与 AS 相关的炎症作用。
为了确定病理剪切速率对人 CRP 功能的影响，将 pCRP 置于病理相关的剪切速率下，并使用生物物理和生化方法进行分析。为了研究剪切对 CRP 体内构象的影响，我们使用了动脉狭窄的小鼠模型。在经导管主动脉瓣植入术（TAVI）前和 TAVI 后，严重 AS 患者的 mCRP 和 pCRP 水平进行了测量，并在接受严重 AS 主动脉瓣置换术的患者的切除瓣膜上研究了 CRP 的存在。然后使用 AS 的微流控模型来再现 AS 患者的剪切速率，并研究这种依赖于剪切的 pCRP 解离及其炎症功能。
暴露于高剪切速率下，pCRP 会解离为促炎单体（mCRP）并聚集成大颗粒。我们的体外发现进一步在小鼠颈动脉狭窄模型中得到了证实，其中人 pCRP 的给药导致狭窄后 mCRP 的沉积。与 TAVI 后相比，接受 TAVI 的患者在 TAVI 前的循环微泡中 mCRP 与循环微泡结合的水平显著升高。切除的人狭窄主动脉瓣显示 mCRP 沉积。在 AS 的微流控模型中，pCRP 解离并诱导内皮细胞活化，表现为 ICAM-1（细胞间黏附分子 1）和 P-选择素表达增加。mCRP 还诱导血小板活化和血小板上 TGF-β（转化生长因子β）的表达。
我们确定了一种新的剪切诱导 pCRP 解离的机制，这导致了 AS 发展中核心细胞的激活。这种 pCRP 向 mCRP 解离的新型机械感应机制可能也与其他涉及剪切率增加的病理有关，如动脉粥样硬化和受损的动脉。
