Role of Gap Junctions in Blood Vessel Assembly

描述(申请人提供)：本研究的长期目标是了解缝隙连接在血管形成中的作用(S)。从这些研究中获得的见解将使成人血管形成的调节能够用于治疗流行的病理学，以及用于再生医学策略的体外组织生成。缝隙连接通道可以由~20个已知的连接蛋白(Cx)蛋白组成，但在维管系统中只有Cx37、Cx40、Cx43和Cx45表达。我们研究的主要假设是，在血管系统中表达的不同的缝隙连接通道蛋白在血管形成的不同阶段发挥着独特的作用。我们以前的研究提供了直接证据，表明由Cx43组成的缝隙连接通道在内皮细胞和它们招募的间充质细胞之间形成。此外，缝隙连接通道的形成和/或通讯是介导转化生长因子-2的激活所必需的，而转化生长因子-2是内皮细胞诱导壁细胞分化所必需的。在目前的授权期内，我们已经确定由Cx45组成的缝隙连接通道也参与了这一过程；而由Cx40组成的通道则不起作用。此外，Cx43的胞浆区域似乎需要特定的磷酸化位点，这表明在缝隙连接通道形成的下游，相关的细胞内信号可能是转化生长因子-2激活所必需的。我们进一步确定，在异质细胞缝隙连接形成时被激活的钙信号似乎在内皮诱导的壁细胞分化中发挥了作用。我们拟议的研究将进一步剖析Cx43和Cx45下游的细胞内信号通路，这些信号通路由缝隙连接通道组成，形成于内皮细胞和间充质细胞之间，并使内皮细胞诱导壁细胞分化。我们还将确定Cx37和Cx40在调节内皮细胞分化和生长控制中的作用(S)，这两个基因不介导内皮细胞诱导的壁细胞分化。两者在分化内皮细胞时都高度上调；而Cx43和Cx45则下调。因此，我们的研究将进一步剖析和确定在血管系统中表达的不同Cx蛋白的特定作用。与公共卫生相关：血管由两个细胞层组成；内皮细胞组成血管的内腔层，壁细胞(周细胞和血管平滑肌细胞)组成周围的血管壁，控制血流和血管的稳定性。我们正在研究内皮细胞和壁细胞之间的沟通如何有助于促进血管形成和稳定性。我们从发育研究中获得的见解可能有助于开发治疗常见疾病的临床策略，包括动脉粥样硬化、糖尿病和癌症。

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.