Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

George E. Davis, M.D., Ph.D.

Committee Member

Matthias Majetschak, M.D., Ph.D.

Committee Member

Saulius Sumanas, Ph.D.

Committee Member

Lianchun Wang, M.D.

Committee Member

Ying Yang, Ph.D.

Keywords

Endothelial Cell, Capillary Formation, 3D, Serum-Free, EC sprouting, Lumen

Abstract

Endothelial cells (ECs) form the inner lining of blood capillary walls. Our laboratory has developed a serum-free, defined Factor system that allows for human ECs to sprout, form lumens and tube networks, recruit pericytes, and induce capillary basement membrane deposition in 3D extracellular matrices. We have shown that the Factor system drives morphogenesis through the activation of multiple signaling pathways; however, we do not know if all factors in the Factor system are needed to induce maximal EC morphogenesis. If so, we have yet to identify why the Factors are critical to the model other than that they induce activation of known EC formation signals, such as Src family kinase, Pak kinases, and Ras-MAPK. Therefore, we test the hypothesis that all factors within the Factor system are needed to achieve maximal EC morphogenesis and understand its mechanism. We report here that the factor components within the Factor system synergistically promote maximal tip cell sprouting and lumen formation through prolonged stimulation of pro-morphogenic signals,such as Src, Akt, B-Raf, C-Raf, Erk 1/2, Pak2, Pak4, Stat3, and Stat5, as well as repressed pro-regressive signals including phosphor-P38, JNK, and MLC2.

Functionally, ECs undergoing morphogenesis can be subgrouped into two cell types, the invasive tip cell up front, followed by the lumen-forming cell. A fundamental step for the development of the blood vascular system is proper interactions between ECs and mural cells, such as pericytes. In total, there are three major steps in capillary formation: EC sprouting, EC lumen formation, and pericyte recruitment. Thus, imbalanced tip sprouting, lumen formation, and pericyte recruitment, the three critical steps to the capillary formation, can lead to vascular diseases, including vascular malformations. A previous study has shown that activated k-RasV12- expressing ECs markedly drive lumen formation with strongly inhibited tip cell sprouting and pericyte recruitment, which are critical pathogenic features predisposing the vasculature to develop arteriovenous malformations (AVMs). Notably, inhibitors of lumen formation rescued the abnormality by strongly blocking this response with k-RasV12 ECs. All of these steps are required to form capillaries. What has yet to be unveiled is an understanding of the full spectrum of signaling differences between EC tips and lumens, as well as how these EC signals control pericyte recruitment. We hypothesize that EC sprouting and lumen formation are differentially regulated and that EC signaling affects pericyte recruitment. Three major signaling arms were targeted in this study: Jak-Stat, PI3K-Akt-mTOR, and Ras-MAPK. We report here that kRas and Akt1 mutation markedly accentuate EC lumen formation, reduce sprouting behavior, and demonstrate a substantial reduction in both pericyte recruitment and pericyte-induced EC basement membrane formation compared to controls. Active Mek1 mutation markedly accentuates both EC lumen formation and sprouting behavior. Active Jak2, Stat3, and Rheb mutations do not affect normal EC lumen formation but increase tip sprouting. Active PIK3CA mutation reduces overall lumen formation but promotes cystic lumen formation, as seen also with active Akt1 mutation, and tip sprouting. EC tubes composed of active Mek1, Stat3, Rheb, and PIK3CA mutations demonstrate reduced pericyte recruitment and basement membrane deposition. Active Jak2 mutation demonstrated reduced pericyte recruitment but with normal basement membrane deposition.

Of note, these pathways largely overlap with the known cancer pathways. We will be able to repurpose already clinically approved drugs for the treatment of vascular anomalies. With our knowledge of sprouting, lumen, and pericyte recruitment signals, we will be able to tailor drugs to target the individual steps of lumen formation. Overall, we will be able to target unwanted vascular formation in a more detailed manner.

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