Endothelial fate switches and function via matrix control of adherens junctions - PROJECT SUMMARY Fibrogenesis is essential for wound healing but, when dysregulated, contributes to fatal diseases like solid cancers, pulmonary fibrosis, and cirrhosis. All tissues or organs that are susceptible to fibrosis contain not only resident fibroblasts but also microvasculature critical to fluidic transport of nutrients and waste exchange. Although the cellular precursors and signaling that engender myofibroblasts (MF), mediators of wound healing that excessively secrete extracellular matrix (ECM), have been a major target to date, the current lack of effective anti-fibrotic therapies suggests the critical involvement of other cellular players. Endothelial-mesenchymal transition (EndMT) is believed to portend the expansion of functional neovessels to support wound healing, but how this process can be dysregulated by fibrosis-driven matrix remodeling has not been established. In fact, while EndMT has previously been implicated as a precursor to endothelial cell (EC) differentiation into MFs, alternative mechanisms through which EndMT contributes to fibrosis have not been considered. Here, motivated by strong preliminary data acquired by the investigative team, we demonstrate, using mouse models and in an in vitro human microphysiological system (MPS) of arteriole/venule-scale microvessels embedded within a tunable stromal mimetic matrix, that fibrotic physical cues activate ECs, drive EndMT, and promote aberrant single EC (sEC) invasion into the matrix. sECs remain adherent to fibrotic matrix and may reinforce fibrogenesis through signaling interactions with other stromal cells. Critically, we identify that the formation of sECs is gated by the destabilization of EC adherens junction (AJs) upon EC adhesion to fibrous matrix. We find that quiescent EC microvessels in healthy matrix do not respond to TGFβ, but adhesion to fibrotic matrix destabilizes AJs to promote TGFβ-mediated signaling. Further, using unbiased proteomics, we have uncovered a novel interaction between the AJ adhesion receptor CDH5 (VE-cadherin) and TGFβR2 that is controlled by EC-ECM interactions. This project leverages two highly complementary labs, next generation MPS culture models, genetically engineered mouse models, and mechanistic studies to dissect EC decision-making and function in response to fibrotic cues, testing the central hypothesis that fibrotic ECM drives EC fate switching to reinforce fibrogenic signals underlying tissue fibrosis. In Aim 1, we will identify what fibrogenic physical and soluble signals are associated with sECs and test whether these signals allow sECs to modulate the states of other stromal cells to promote fibrogenesis. In Aim 2, we deeply investigate the mechanism by which EC adhesion to fibrous matrix promotes sEC formation and test whether a critical CDH5-TGFβR2 association is modulated by fibrotic ECM to potentiate EC TGFβ signaling. Completion of these studies will define: new mechanisms by which ECM control EC fate, new functional contributions of ECs to fibrogenesis, and a previously-unappreciated mechanism governing EC fate and function operating at EC AJs. These insights offer the potential for new anti-fibrotic therapeutics and associated mechanism of action for future investigation in pre-clinical studies.
