Cysteine-rich with EGF-like domains 2 – Exodosis and angiogenesis after myocardial infarction

急性心肌梗死对冠脉微循环造成大量损伤，导致血管崩解和毛细血管稀疏。在几天内，一个新的血管系统在梗死区发展，并有助于限制疤痕形成和左心室重构的程度。内质网(ER)在许多自分泌和旁分泌作用的血管生成因子的折叠、翻译后修饰和分泌中起着关键作用。然而，心肌梗死后的缺血再灌注损伤和分泌活动的增加可能会压倒内质网稳态(内质网应激)。与KDEL受体的结合决定了蛋白质是保留在内质网/高尔基体系统中还是被分泌出来。因此，许多内质网驻留的伴侣蛋白具有经典的C端Lys-Asp-Glu-Leu(KDEL)内质网滞留信号。缺少这个基序的生长因子是分泌的。相反，具有弱ER保留信号的蛋白质在基础条件下保留在内质网中，并在内质网应激(外源)下优先分泌。我们认为，外引流提供了一种刺激血管生成的机制，特别是在梗死区。我们在小鼠的梗死区进行了基于单个内皮细胞RNA测序的生物信息分泌组分析，以确定内质网应激内皮细胞产生的以前未确定的生长因子。因此，我们鉴定了富含半胱氨酸的EGF样结构域2。CRELD2具有微弱的ER保留信号(RET1)，并且在ER胁迫下被强烈诱导。根据我们的初步数据，我们假设内皮细胞在梗死区通过外分泌释放CRELD2，以自分泌的方式刺激血管生成和伤口愈合。这两个假设都将在我们的项目中进行调查。我们打算检测CRELD2在急性心肌梗死小鼠和人类中的表达和分泌。我们想研究整体或条件性Creld2基因缺陷小鼠的心肌梗死后血管生成和伤口愈合。我们的目的是确定CRELD2是否在梗死区作为内质网驻留和/或分泌蛋白，并探索重组CRELD2在心肌梗死后的治疗潜力。最后，我们计划鉴定内皮细胞中假定的CRELD2细胞表面受体，并表征磷蛋白质组下游的变化。以CRELD2为例，我们的项目将确立外引流作为心肌梗死后血管生成和伤口愈合的重要刺激因素。

Acute myocardial infarction inflicts massive injury to the coronary microcirculation leading to vascular disintegration and capillary rarefication. Within days, a new vascular system develops in the infarct area and helps limiting the extent of scar formation and left ventricular remodelling. The endoplasmic reticulum (ER) plays a pivotal role in the folding, posttranslational modification, and secretion of many autocrine- and paracrine-acting angiogenic growth factors. However, ischemia-reperfusion injury and the increased secretory activity after myocardial infarction may overwhelm ER homeostasis (ER stress). Binding to KDEL receptors determines whether proteins are retained in the ER/Golgi system or are secreted. Accordingly, many ER-resident chaperones possess a classical, C-terminal Lys-Asp-Glu-Leu (KDEL) ER retention signal. Growth factors lacking this motif are secreted. Conversely, proteins with a weak ER retention signal are retained in the ER under basal conditions and are preferentially secreted under ER stress (exodosis). We propose that exodosis provides a mechanism to stimulate angiogenesis specifically in the infarct area. We have performed a single endothelial cell RNA-sequencing-based bioinformatic secretome analysis in the infarct area of mice to identify previously uncharacterised growth factors produced by ER-stressed endothelial cells. We thus identified cysteine-rich with EGF-like domains 2. CRELD2 has a weak ER retention signal (REDL) and is strongly induced during ER stress. Based on our preliminary data, we postulate that endothelial cells release CRELD2 in the infarct area via exodosis to stimulate angiogenesis and wound healing in an autocrine manner. Both hypotheses will be investigated in our project. We intend to measure the expression and secretion of CRELD2 in mice and humans with acute myocardial infarction. We want to study angiogenesis and wound healing after myocardial infarction in globally or conditionally Creld2-deficient mice. We aim to define if CRELD2 acts as an ER resident and/or secreted protein in the infarct area and to explore the therapeutic potential of recombinant CRELD2 after myocardial infarction. Finally, we plan to identify the putative CRELD2 cell surface receptor in endothelial cells and to characterise downstream alterations in the phosphoproteome. Using CRELD2 as an example, our project shall establish exodosis as an important stimulus of angiogenesis and wound healing after myocardial infarction.