Theses and Dissertations

Date of Award

5-2023

Document Type

Dissertation

Degree Name

Ph.D.

Department

Basic Medical Sciences

Committee Chair

Dhananjay T. Tambe, Ph.D. and Troy Stevens, Ph.D.

Advisor(s)

Dr. Ji Young Lee, Dr. Mark Taylor, Dr. Natalie Bauer, Dr. Richard Honkanon, Dr. Thomas Rich

Abstract

Endothelial cells (ECs) adhere to their neighboring cells via adherens and tight junctions, and to the basement membrane via focal adhesions. These structural elements are constantly remodeling i.e., even in a quiescent state, cells are in constant motion. ECs sense the stiffness of the basement membrane, and this stiffness is a physiological signal that control the cellular phenotype. Whereas the normotensive pulmonary circulation has a substrate stiffness that is ~1.25-4 kPa, vascular remodeling in pulmonary arterial hypertension (PAH) increases substrate stiffness to ~30 kPa. The impact that this increase in substrate stiffness has no cell size, shape, and speed was unknown and was the focus of this dissertation. For this study, we cultured rat PAECs (RPAECs) isolated from the normotensive circulation, and human PAECs (HPAECs) isolated from human subjects with PAH, on collagen-coated polyacrylamide hydrogels with stiffnesses ranging from 0.22 kPa to 30 kPa. To examine cellular morphology and motion, phase-contrast time-lapse images were acquired at 5-minute intervals for 7 hours. These images were analyzed using a new inhouse image analysis toolkit: Integrative Toolkit to Analyze Cellular Signals. To examine paracellular membrane fluctuations, phase-contrast time-lapse images were acquired at xxiii 10-second intervals for 15 minutes on two different substrate stiffnesses, 1.25 kPa and 30 kPa. These images were analyzed using a novel technique called the Paracellular Fluctuation Analyzer. In addition, HPAECs: were examined for their biological characteristics, including proliferation, replication competence, anoikis resistance, and lectin binding specificity. Cellular motion and membrane fluctuation were maximal in normal RPAECs at a physiological substrate stiffness of 1.25 kPa and were impaired at both low (0.22 kPa) and high (30 kPa) substrate stiffnesses. The HPAECs exhibited varying degrees of anoikis resistance. Among all cell types, cells from the heritable PAH subjects had the highest degree of anoikis resistance. These anoikis-resistant cells had a high proliferation capacity and exhibited characteristics of macrovascular ECs. A subpopulation of these cells exhibited highly proliferative single-cell growth and interacted with Griffonia simplicifolia, a lectin that interacts with rapidly growing cells that exhibit a progenitor phenotype. Moreover, HPAECs from subjects with higher pulmonary vascular resistance (PVR) had a faster motion on a stiffer substrate compared to HPAECs from subjects with lower PVR. HPAECs from the pulmonary veno-occlusive disease (PVOD) subject had the highest PVR, and these cells had the fastest cell motion on the stiffest substrate. Overall, normal PAECs exhibit maximum membrane motion and speed when they are grown on substrates with stiffnesses that resemble the normotensive blood vessels. In contrast, PAECs from hypertensive patients showed the following traits: (1) they were anoikis-resistant, and the anoikis-resistant cells were hyperproliferative and exhibited characteristics of progenitor cells; and (2) their ability to move was adapted to stiffer substrates i.e., cell motion and PVR were associated.

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