Role of Tbc1d2b in vessel formation and neovascularization after ischemic injury - Project title: Role of Tbc1d2b in vessel formation and neovascularization after ischemic injury Project Summary: Neovascularization, the formation of new blood vessels, is a fundamental process in development, wound healing, and cardiovascular diseases. While vascular sprouting and outgrowth, i.e., angiogenesis, has been extensively studied, the intricate process of how vessel tube-like structures are formed, crucial for determining the functional and structural integrity of vessels, remains largely unexplored. Uncover the underlying mechanism for vessel tube formation may lead to development of new strategies for regenerative medicine, complementary to current angiogenic therapies that primarily focus on promoting vessel sprouting and outgrowth. Epithelial tube morphogenesis, in contrast, has been well characterized, in which phosphatidylinositols PIP2 and PIP3 play a central role. However, the role of PIP2 and PIP3 in endothelial tube formation remains unclear. Our preliminary studies showed that PIP2, but not PIP3, is required for vessel formation of endothelial cells (ECs). To investigate the underlying regulatory mechanisms, we assessed PIP2-interacting proteins by mass spectrometry. We identified that Tbc1d2b specifically interacts with PIP2 during EC tube formation. Importantly, consistent with its preferential expression and localization in tubular structures in various human and mouse tissues, knockdown of Tbc1d2b by siRNA or CRISPR/sgRNA inhibits EC tube formation. Moreover, injection of Tbc1d2b morpholinos (MOs) into the zebrafish embryos blocks blood vessel formation and zebrafish development. In addition, analysis of the sequence of Tbc1d2b identified the presence of PH (pleckstrin homology) and TBC (Tre2/Bub2/Cdc16) domains. Consistent with these findings, our mass spectrometry analysis revealed that Tbc1d2b interacts with small GTPases proteins including Rac1 and Rab2/7 confirmed by our immunoprecipitation assay, suggesting Tbc1d2b may regulate EC tube formation through phosphatidylinositol-binding and GTPase-activating protein (GAP) activation. Notably, using a newly generated EC-specific Tbc1d2b knockout mouse line, namely, Cdh5- CreERT2;Tbc1d2bflox/flox, we showed that Tbc1d2b deletion in ECs abrogates neovascularization and post- ischemic hindlimb perfusion. Furthermore, our single-cell RNAseq analysis of cardiac tissue in Cdh5- CreERT2;LSL-tdTomato endothelial lineage-tracing mice showed a temporally decrease in Tbc1d2b after myocardial infarction (MI). Based on these results, we hypothesize that Tbc1d2b is a critical regulator of vessel formation and neovascularization in tissue repair. We will test this hypothesis by pursuing the following Aims: (1) To define the mechanisms by which Tbc1d2b regulates vessel formation, with a focus on Tbc1d2b interaction with Rac1 and Rab as well as regulation by PIP2, and (2) To determine the in vivo role of Tbc1d2b in neovascularization and tissue repair after myocardial infarction in mice. 3) To test experiment therapy that targets Tbc1d2b for MI treatment with EC-specific Tbc1d2b mRNA nanoparticles. In sum, vessel lumen formation is an important but under-investigated area. In this study, we will define a previously unidentified molecular mechanism of vessel formation, which may lead to identification of new targets for regenerative therapy.
