Moore Michayla, Ryzhov Sergey, Sawyer Douglas B, Gartner Carlos, Vary Calvin P H
Center for Molecular Medicine, MaineHealth Institute for Research, MaineHealth 81 Research Drive, Scarborough, Maine, USA.
Graduate School of Biomedical Science and Engineering, University of Maine Orono, Maine, USA.
J Cell Signal. 2024;5(3):122-142. doi: 10.33696/signaling.5.119.
Pro-angiogenic paracrine/autocrine signaling impacts myocardial repair in cell-based therapies. Activin A receptor-like type 1 (, ALK1) signaling plays a pivotal role in cardiovascular development and maintenance, but its importance in human-derived therapeutic cardiac cells is not well understood. Here, we isolated a subpopulation of human highly proliferative cells (hHiPCs) from adult epicardial tissue and found that they express ALK1, a high affinity receptor for bone morphogenetic protein-9 (BMP9), which signals via SMAD1/5 to regulate paracrine/autocrine signaling and angiogenesis. We show that in humans, circulating BMP9 level is negatively associated with the number of epicardial hHiPC and positively associated with endothelial cell (EC) number in the adult heart, implicating the potential importance of this signaling pathway in cardiac cell fate and vascular maintenance. To investigate BMP9/ALK1 signaling in hHiPCs, we selected a primary cell population of hHiPC from each of 3 individuals and studied their responses to BMP9 and BMP10 treatment . Proteins were collected in conditioned media (CM) for mass spectrometry and cell-based assays on human ECs and hHiPCs. Proteomic analysis of the hHiPC secretome following BMP9 or BMP10 treatment demonstrates that the secreted proteins, sclerostin (SOST), meflin/immunoglobulin superfamily containing leucine rich repeat (ISLR), and insulin-like growth factor binding protein-3 (IGFBP3), are novel regulated targets of BMP9/ALK1 signaling. Lentiviral shRNA and pharmacological inhibition of ALK1 in hHiPCs suppressed transcription and secretion of SOST, ISLR, and IGFBP3 following BMP9 treatment. Moreover, the BMP9-treated secretome of hHiPC increased capillary-like tube formation of ECs and hHiPCs. Treatment of hHiPCs with recombinant SOST increased expression, increased tube formation and enhanced expression of EC receptor marker annexin A2 (ANXA2). These data provide the first proteomic characterization of hHiPC, identifying BMP9/ALK1-mediated target protein secretion in hHiPCs, and underscore the complex role of BMP9/ALK1 signaling in paracrine/autocrine mediated angiogenesis. Data are available via ProteomeXchange with identifier PXD055302.
促血管生成的旁分泌/自分泌信号影响基于细胞疗法中的心肌修复。激活素A受体样1型（ALK1）信号在心血管发育和维持中起关键作用，但其在人源治疗性心脏细胞中的重要性尚未得到充分了解。在这里，我们从成人的心外膜组织中分离出一群人高增殖细胞（hHiPCs），发现它们表达ALK1，这是骨形态发生蛋白-9（BMP9）的高亲和力受体，其通过SMAD1/5信号传导来调节旁分泌/自分泌信号和血管生成。我们表明，在人类中，循环BMP9水平与心外膜hHiPC的数量呈负相关，与成人心脏中的内皮细胞（EC）数量呈正相关，这表明该信号通路在心脏细胞命运和血管维持中的潜在重要性。为了研究hHiPCs中的BMP9/ALK1信号传导，我们从3名个体中分别选择了hHiPC的原代细胞群体，并研究了它们对BMP9和BMP10处理的反应。收集条件培养基（CM）中的蛋白质用于质谱分析以及对人ECs和hHiPCs进行基于细胞的检测。对BMP9或BMP10处理后的hHiPC分泌组进行蛋白质组学分析表明，分泌蛋白硬化蛋白（SOST）、含富亮氨酸重复序列的膜连蛋白/免疫球蛋白超家族（ISLR）和胰岛素样生长因子结合蛋白-3（IGFBP3）是BMP9/ALK1信号传导的新调控靶点。hHiPCs中ALK1的慢病毒shRNA和药物抑制作用抑制了BMP9处理后SOST、ISLR和IGFBP3的转录和分泌。此外，hHiPC经BMP9处理后的分泌组增加了ECs和hHiPCs的毛细血管样管形成。用重组SOST处理hHiPCs增加了表达，增加了管形成并增强了EC受体标志物膜联蛋白A2（ANXA2）的表达。这些数据首次提供了hHiPC的蛋白质组学特征，确定了hHiPCs中BMP9/ALK1介导的靶蛋白分泌，并强调了BMP9/ALK1信号传导在旁分泌/自分泌介导的血管生成中的复杂作用。数据可通过ProteomeXchange获得，标识符为PXD055302。
