Effect of CTHRC1 on endothelial cell survival after acute ischemia

摘要 在以前的研究中，我们已经证明了CTHRC1(胶原蛋白三螺旋重复序列包含1)，一个因子 在我们实验室发现的，仅在脑和骨中结构性表达，而在其他组织中不表达 成人期。然而，CTHRC1在激活的成纤维细胞和组织的间质细胞中高表达 正在进行改建和修复。我们发现CTHRC1是一种循环因子，但仅约为 30%的健康受试者在血浆中检测到CTHRC1水平，浓度范围为 低pg/ml至近100 ng/ml。与大多数人类相似，在小鼠体内检测不到循环中的CTHRC1水平 而大鼠和PDGFRb启动子控制下的强制转基因CTHRC1过表达也没有结果 在可检测到的CTHRC1血浆水平中。因此，有两个CTHRC1池；一个是在 正在进行修复的组织需要48小时才能提供，以及第二个循环CTHRC1池 在任何时候都是可用的，并且只在少数人类受试者中发现。循环CTHRC1的意义 因为当我们从患者身上获取血浆样本时，心血管系统变得明显 心脏骤停，一种死亡率约为50%的情况。高CTHRc1水平(≥0.75 ng/ml)与 在经历心脏骤停的人类中存活率要高得多。而CTHRC1不表达 在成人心脏，它在心肌梗死后激活的成纤维细胞中高度诱导。要测试 CTHRc1在急性缺血性损伤中的作用，我们对Ctrc1基因缺失的小鼠和Ctrc1基因缺失的小鼠进行了冠状动脉结扎 Ctrc1缺失背景中具有生理相关CTHRC1血浆水平的转基因小鼠 在人类中发现的Ng/ml范围。令人惊讶的是，70%的Ctrc1基因缺失小鼠在缺血损伤后3-4天死亡 而所有转基因小鼠都存活了下来。为了确定这一戏剧性发现的机制，我们 在体外进行的研究发现，CTHRC1促进了多种细胞类型的细胞存活，包括 内皮细胞。完全代谢监测显示，Ctrc1基因缺失的小鼠能量增加 静息消耗和体外细胞代谢分析表明，在CTHRC1存在的情况下 线粒体呼吸显著增加，而糖酵解减少，这导致了我们的假设 CTHRC1是新陈代谢效率的调节因子。总体而言，这项提议将检验以下假设 CTHRC1通过增加新陈代谢在细胞应激条件下作为细胞存活的中介 效率，这反过来又保护免受急性缺血损伤的致命后果。我们将确定是否 增加循环或CTHRC1组织水平通过限制有害的 心肌缺血的后果。利用遗传小鼠模型和体外方法研究潜在的 作用机制将被确定，这将为新的治疗方法提供基础 改善急性缺血状况的转归。

Abstract In previous studies we have demonstrated that CTHRC1 (Collagen Triple Helix Repeat Containing 1), a factor discovered in our laboratory, is constitutively expressed only in brain and bone and not in other tissues during adulthood. However, CTHRC1 is highly expressed in activated fibroblasts and interstitial cells of tissues undergoing remodeling and repair. We discovered that CTHRC1 is a circulating factor but only approximately 30% of healthy human subjects have detectable levels of CTHRC1 in plasma, ranging in concentration from low pg/ml to almost 100ng/ml. Similar to most humans, circulating levels of CTHRC1 are not detectable in mice and rats and forced transgenic overexpression of CTHRC1 under Pdgfrb promoter control does also not result in detectable CTHRC1 plasma levels. Thus there are two pools of CTHRC1; one that is generated locally in tissues undergoing repair requiring >48 hours to be available, and a second pool of circulating CTHRC1 available at all times and found only in a minority of human subjects. The significance of circulating CTHRC1 for the cardiovascular system became apparent when we obtained plasma samples from patients experiencing cardiac arrest, a condition with approximately 50% mortality. High CTHRC1 levels (≥0.75ng/ml) are associated with substantially higher survival rates in humans experiencing cardiac arrest. While CTHRC1 is not expressed in the adult heart, it is highly induced in fibroblasts activated in response to myocardial infarction. To test the role of CTHRC1 in acute ischemic injury, we performed coronary artery ligation in Cthrc1 null mice and Cthrc1 transgenic mice on the Cthrc1 null background with physiologically relevant CTHRC1 plasma levels in the ng/ml range found in humans. Strikingly, 70% of Cthrc1 null mice died 3-4 days after the ischemic injury whereas all transgenic mice survived. With the goal of identifying the mechanism for this dramatic finding we performed in vitro studies and found that CTHRC1 promotes cell survival in variety of cell types including endothelial cells. Complete metabolic monitoring revealed that Cthrc1 null mice have increased energy expenditure at rest and analysis of cell metabolism in vitro revealed that in the presence of CTHRC1 mitochondrial respiration is significantly increased whereas glycolysis is reduced, leading us to hypothesize that CTHRC1 functions as a mediator of metabolic efficiency. Overall, this proposal will test the hypothesis that CTHRC1 functions as a mediator of cell survival under conditions of cell stress by increasing metabolic efficiency, which in turn protects from the deadly consequences of acute ischemic injury. We will determine if increasing circulating- or CTHRC1 tissue levels provides cardiovascular protection by limiting the deleterious consequences of myocardial ischemia. Using genetic mouse models and in vitro approaches the underlying mechanism of action will be identified, and this will provide the foundation for novel therapeutic approaches to improve outcomes of acute ischemic conditions.