ELUCIDATION OF THE UNIQUE TRANSLATION OF ANGIOGENIC SIGNALING BY AORTIC VALVE CELLS
Grande-Allen, Kathryn J
Doctor of Philosophy
Angiogenesis is a fundamental biological process but is a critical step in the progression of calcific aortic valve disease (CAVD). However, the process through which native valve cells, valve endothelial cells (VECs) and valve interstitial cells (VICs), form the neovascularization exhibited during CAVD is unclear due to their atypical translation of several angiogenesis related signals. Therefore, the in vitro angiogenic capacity of valve endothelial cells was characterized and compared to vascular derived endothelial cells. Their vasculogenic networks were demonstrated to be quantitatively and morphologically different from the networks generated by a vascular endothelial cell line, but the geometry of the VECs’ networks could be manipulated with small molecule Rho GTPase inhibitors, similar to previous studies of vascular endothelial cells, thus demonstrating typical and atypical ways in which VECs translate angiogenic signals. Next, the pericyte-like capacity of VICs was demonstrated by tracking fluorescently marked VECs and VICs in a long term in vitro angiogenesis co-culture assay. VICs regulated early VEC network organization in a ROCK-dependent manner, wrapping themselves around VEC network edges in a manner similar to a pericyte cell line. Using a novel method to quantify the Lagrangian-corrected chemoattraction of one cell type towards another in a mixed population, we identified and quantified a subpopulation of VICs that demonstrated a pericyte-like chemoattraction towards VECs. Directly comparing valve cell co-cultures to vascular cell co-cultures revealed that unlike vascular control cells, the valve cell cultures ultimately formed invasive spheroids with 3D sprouts. These 3D sprouts were found to have several markers typical of in vivo angiogenic root sprouts such as delta-like ligand 4 and β-catenin polarity. VECs co-cultured with VICs displayed significantly more invasion than VECs alone; interestingly, VICs were found leading and wrapping around VEC invasive sprouts demonstrating both tip cell and pericyte behaviors. Angiopoietin1-Tie2 signaling was found to regulate valve cell organization during VEC/VIC spheroid formation and invasion. Long term co-cultures demonstrated pronounced deviation of several angiogenesis and pericyte markers when measured with qRT-PCR. In the next study, mechanical stimulation is known to be a strong regulator of vascular endothelial cell angiogenic capacity, but its role in regulating VEC angiogenic capacity physiologically or pathologically was unknown. Therefore, experiments were performed to examine the effect of cyclic uni-axial strain on regulating the response of valve endothelial cells to an in vitro angiogenesis model. Network analysis revealed a strong pattern that strain decreases the propensity of VECs to form networks. Finally, the factors that govern VEC angiogenesis were investigated from the network scale down using tissue engineering strategies. Vascular networks of varying complexity can be designed within engineered tissues, but the amount of biological complexity necessary for proper biological functionality is unclear, as more complex is not necessarily better or necessary. Since VEC network complexity had been demonstrated to be sensitive to changes in actin regulators, this study used VECs as a framework to examine the fundamental relationship between network structure and endothelial cell biology more specifically it was tested whether the internal signaling biology of endothelial cells could be tuned based upon spatially-defined synthetic networks. Notable differences in several angiogenesis related markers were found as a function of the pattern the cells were seeded on, including several markers for actin activity regulators as well as changes in actin alignment, mimicking changes in signaling previously observed in in vitro angiogenesis models with VECs. Overall, this work has contributed to the understanding of the translation of angiogenic signals by valve cells and its potential role in the pathogenesis of valvular disease. Understanding from these studies can be applied to future studies of valve diseases with a similar framework to clarify the role of angiogenic signaling in the pathology of CAVD. This insight will allow development of targeted therapeutic strategies for the treatment of valvular diseases, as well as strategies to assess what level of complexity is sufficient to induce functional angiogenesis in tissue engineered constructs, such as those needed in pediatric aortic valve replacements.
For more information on supplemental movies included with this dissertation, see Supplemental Movie 1 page 76; Supplemental Movies 3-9 page 97; Supplemental Movie 10 page 101; Supplemental Movie 11 page 103; Supplemental Movies 12-14 page 105.
aortic valve; angiogenesis; valve endothelial cell; valve interstitial cell