Role of Gap Junctions in Blood Vessel Assembly

描述（由申请人提供）：本研究的长期目标是了解间隙连接在血管形成中的作用。从这些研究中获得的见解将能够调节成体血管形成，以治疗常见的病理，以及为再生医学策略产生离体组织。间隙连接通道可由约 20 种已知的连接蛋白 (Cx) 蛋白中的任何一种组成；然而，只有 Cx37、Cx40、Cx43 和 Cx45 在脉管系统中表达。我们研究的总体假设是，脉管系统中表达的不同间隙连接通道蛋白在血管形成的不同阶段发挥独特的作用。我们之前的研究提供了直接证据，表明内皮细胞和它们招募的间充质细胞之间形成了由 Cx43 组成的间隙连接通道。此外，间隙连接通道的形成和/或通讯对于介导TGF-2的激活是必要的，而TGF-2是内皮诱导的壁细胞分化所必需的。在当前的资助期内，我们确定由Cx45组成的间隙连接通道也介导了这一过程；而由 Cx40 组成的通道则不然。此外，Cx43 细胞质区域内的特定磷酸化位点似乎是必需的，这表明间隙连接通道形成下游的相关细胞内信号传导可能是 TGF-2 激活所必需的。我们进一步确定，在异细胞间隙连接形成时被激活的钙信号传导似乎在内皮诱导的壁细胞分化中发挥作用。我们提出的研究将进一步剖析 Cx43 和 Cx45 组成的间隙连接通道下游的细胞内信号通路，这些间隙连接通道在内皮细胞和间充质细胞之间形成，并实现内皮诱导的壁细胞分化。我们还将确定 Cx37 和 Cx40 在内皮细胞分化和生长控制的调节中的作用，它们不介导内皮诱导的壁细胞分化。两者在内皮细胞分化过程中均高度上调；而 Cx43 和 Cx45 则下调。因此，我们的研究将进一步剖析和定义脉管系统中表达的不同 Cx 蛋白的具体作用。公共卫生相关性：血管由两层细胞组成；内皮细胞构成血管内腔层，壁细胞（周细胞和血管平滑肌细胞）构成周围血管壁并控制血流和血管稳定性。我们正在研究内皮细胞和壁细胞之间的通讯如何有助于促进血管形成和稳定性。从我们的发育研究中获得的见解可能有助于制定治疗动脉粥样硬化、糖尿病和癌症等常见疾病的临床策略。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the role(s) of gap junctions in blood vessel formation. Insights gained from these studies will enable modulation of adult vessel formation for the treatment of prevalent pathologies, as well as for the generation of tissues ex vivo for regenerative medicine strategies. Gap junction channels can be composed of any of ~20 known connexin (Cx) proteins; however, only Cx37, Cx40, Cx43 and Cx45 are expressed in the vasculature. The overarching hypothesis of our studies is that distinct gap junction channel proteins expressed in the vasculature play unique roles at different stages of blood vessel formation. Our previous studies provide direct evidence that gap junction channels composed of Cx43 form between endothelial cells and mesenchymal cells that they recruit. Furthermore, gap junction channel formation and/or communication is necessary to mediate the activation of TGF- 2, which is required for endothelial-induced mural cell differentiation. In the current grant period, we have determined that gap junction channels composed of Cx45 also mediate this process; whereas, channels composed of Cx40 do not. Furthermore, it appears that specific phosphorylation sites within the cytoplasmic region of Cx43 are required, suggesting that associated intracellular signaling downstream of gap junction channel formation may be necessary for TGF-2 activation. We have further determined that calcium signaling, which is activated upon heterocellular gap junction formation, appears to play a role in endothelial- induced mural cell differentiation. Our proposed studies will further dissect the intracellular signaling pathways downstream of Cx43- and Cx45-composed gap junction channels that form between endothelial and mesenchymal cells and enable endothelial-induced mural cell differentiation. We will also determine the role(s) of Cx37 and Cx40, which do not mediate endothelial-induced mural cell differentiation, in the regulation of endothelial cell differentiation and growth control. Both are highly upregulated in differentiating endothelial cells; whereas, Cx43 and Cx45 are downregulated. Thus, our studies will further dissect and define the specific roles of distinct Cx proteins expressed in the vasculature. PUBLIC HEALTH RELEVANCE: Blood vessels are made up of two cell layers; endothelial cells comprise the inner luminal layer of vessels and mural cells (pericytes and vascular smooth muscle cells) make up the surrounding vessel wall and control blood flow and vessel stability. We are studying how communication between endothelial cells and mural cells helps to promote vessel formation and stability. Insights gained from our developmental studies may help to develop clinical strategies for treating common diseases including atherosclerosis, diabetes, and cancer.