TRPC proteins as determinants of endothelial proliferation

Development, maintainance and function of the vascular system requires the ability of endothelial cells to switch between a quiescent, differentiated and a proliferating phenotype, which is able to migrate and form novel capillary tube networks as a key process of angiogenesis. This "angiogenic switch" plays a pivotal role in variety of diseases and is controlled by stimuli that are associated with phopsholipase C-mediated Ca2+ entry. Canonical transient receptor potential (TRPC) proteins are typical downstream targets of phospholipase C signalling providing a route for Ca2+ entry due to formation of homo- and/or heteromeric cation channel complexes. Such TRPC channels may be dynamically recruited into specific signalplexes in response to phopsholipase C activation via receptor tyrosine kinases or G protein-coupled receptors. Moreover, specific targeting and assembly mechanisms are considered to enable TRPC-mediated local cellular Ca2+ signals and phopsholipase C-dependent control of a variety of endothelial functions. So far, the role of TRPC channels as determinants of endothelial proliferation, migration and cell-cell adhesion is elusive. The aims in this project are: i) To determine which TRPC channel complexes are involved in Ca2+ signalling of proliferating and quiescent endothelial cells, ii) to characterize the cellular localization and the protein interaction partners of TRPC proteins in these endothelial phenotypes, and to analyze mechanisms of cellular trafficking, assembly and/or disassembly of endothelial TRPC complexes in response to angiogenic stimuli, iii) to test the concept that specific TRPC signalplexes and processes of cellular TRPC recruitment govern endothelial proliferation, migration and/or integrity of cell-cell juntions. These topics will be investigated using endothelial progenitor cells and microvascular endothelial cells, isolated and cultured from human adipose stroma as well as immortalized human microvascular endothelial cells. The employed methods comprise classical techniques for subcellular localization of signalling molecules and for the analysis of protein- protein interactions such as immunocytochemistry, immunoprecipitation and GST-pulldown experiments as well as functional analysis by expression of dominant negative protein fragments. These classical methods will be complemented by analysis of protein-protein interactions by FRET- and TIRF/FRET-microscopy and by proteomic approaches. The physiological/pathophysiological significance of particular endothelial TRPC signalplexes will be evaluated by dominant negative as well as si/shRNA knock-down strategies and by use of blocking antibodies. The expected gain in knowledge on the cellular response of endothelial cells to angiogenic stimuli is considered as an important step towards exploiting endothelial TRPC proteins therapeutic targets.

The work of this project identified novel molecular mechanisms involved in the control of proliferation and differentiation/maturation of endothelial cells. These mechanisms are important for repair of blood vessels as well as for expansion of organ vascularization. The endothelium constitutes the innermost layer of blood vessels and is indispenisble for function, integrity and dynamic remodeling of the vascular system. We explored specifically the cellular role of ion transport proteins of the TRPC (canonical transient receptor potential) family in human endothelium and identified the TRPC4 molecule as a pivotal signaling element that enables the endothelial cells to respond to growth factors at a certain stage of proliferation. TRPC4 was found to play a crucial role in the process of endothelial phenotype switching, which is the transition between the quiescent, barrier-forming state and a proliferating and migrating cellular state. This phenotype switching of endothelium is important for angiogenesis. Thus, our results suggest TRPC4 as a potential target for pharmacological modulation of angiogenesis, a process involved in prevention of ischemic tissue damage but also tumor growth. A key finding of this project was the discovery of a novel signaling mechanism by which TRPC proteins control endothelial gene transcription. Besides the well-recognized Ca2+ channel function of TRPC proteins, we demonstrated that TRPC4 is a binding partner of the junctional protein and transcriptional regulator ß-catenin. We found that TRPC4 governs nuclear translocation of beta catenin, which is a key mechanism involved in phenotype switching and in the process of vascular regeneration and extension of vascular beds. Moreover, this project focused not only on mature vascular endothelium but also on adult stem cells, which serve as endothelial precursors in processes of vascular repair and generation of new blood vessels. De-novo formation of blood vessels as well as vascular repair requires the proliferation and subsequent differentiation of endothelial progenitor cells, which are localized in the perivascular connective tissue. We identified TRPC3 as an important player in proliferation/expansion of adipose tissue-resident endothelial progenitor cells. Another important finding of this project was the observation that TRPC and ß-catenin signaling in endothelial progenitors and mature endothelium was strikingly affected by specific nanostructured extracellular contact surfaces (extracellular matrix). We identified a certain nanostructured surface pattern that profoundly promotes endothelial proliferation by induction of nuclear translocation of ß-catenin. These findings may be exploited in terms of controlled ex-vivo expansion of endothelial cells for autologous cell therapy or tissue engineering application.