Skip to main content
eScholarship
Open Access Publications from the University of California

Regulation Of Vascular Stem Cells By Transcription Factor And Microenvironment

  • Author(s): Chang, Julia Haewon
  • Advisor(s): Li, Song
  • Ngai, John
  • et al.
Abstract

Cardiovascular diseases (CVDs) cause about 31% of all global deaths. As people age, CVD progress due to the accumulation of fatty materials, immune cells, and smooth muscle cells form stabilized fatty plaques in blood vessels through a process known as atherosclerosis. Atherosclerosis plaques affect vessel wall integrity, elasticity, and occlude blood flow, which can result in death. Top risk factors of CVDs include tobacco use, alcohol, blood pressure, and blood cholesterol levels. In order to develop better drug treatment strategies, it is important to elucidate the cellular mechanisms that drive CVD progression within the vessel wall. In healthy blood vessels, smooth muscle cells have a low proliferating rate, low synthesis activity, and high expression of contractility proteins and ion channels important in responding to vessel contraction, tone, and blood pressure.

In chapter one, I discuss current theories on what cells directly contribute to vessel wall repair and/or disease progression. The de-differentiation hypothesis proposes that vascular smooth muscle cells directly contribute to CVDs. This phenotypic switch, or de-differentiation, results in smooth muscle cells that down-regulate expression of contractility proteins, rapidly proliferate and migrate into the damaged area. The heterogeneous population of cells observed in diseased vessels is the result of smooth muscle cell de-differentiation. The isolation and characterization of side populations of vascular stem cells (VSCs) in the vessel wall presents an additional cell source that may also contribute to healthy and diseased vessel wall repair. Previous work from our group characterized a population of multipotent VSCs. In healthy vessel repair, VSCs are activated and differentiate to smooth muscle cell end fates. However, in diseased vessels, aberrant differentiation of VSCs could contribute to the heterogeneous population of fat, bone, and chondrogenic cells.

In chapter two, we characterized how matrix elasticity regulates VSC proliferation, expression of SMC markers, and cellular localization of mechanical signaling factors. We began by confirming trends of previous DNA microarray results when rat VSCs were seeded on polyacrylamide hydrogels (pAAm). While average nuclear area also decreases as matrix elasticity decreases, the rate of cell proliferation was approximately equal compared to glass control. Based on previous work, we tested whether global decreases in nuclear area also led to global chromatin remodeling, primarily detected through histone tail modifications. We did not find global changes in response to matrix elasticity. Instead, we were able to show that smooth muscle marker protein expression decreases as a function of matrix elasticity. As matrix elasticity decreases, F-actin stress fibers decrease, which increases relative pools of G-actin. G-actin can bind to Myocardin-related transcription factor A, which activates gene transcription of contractile and cytoskeletal proteins, including smooth muscle a-actin.

In chapter three, we describe multiple attempts to characterize the role of Sox family of transcription factor by either targeted knockdown or overexpression. We hypothesized that high expression of two Sox family members, Sox10 and Sox17, in early passage rat VSCs affects vascular stem cell maintenance and/or prevents differentiation. We attempted to use short- and long-term targeted knockdown of Sox protein in rat VSCs. Specifically, we characterized the effects on VSCs proliferation, smooth muscle cell gene expression, cell migration, and directed differentiation. Due to the recovery of Sox gene and protein expression, our results were inconclusive. When we tried to overexpress a GFP-tagged Sox10, we saw dramatic decreases in GFP+ cells after 7 days in culture.

In this work, we were able to determine: (1) Rat VSCs respond to matrix elasticity, a mechanical signal, and decrease expression of smooth muscle cell markers. (2) Matrix elasticity decreases MRTF-A nuclear localization in rat VSCs. (3) As a result, direct downstream targets of MRTF-A, like SMA, also decrease.

Our data for targeted knockdown of Sox10 and Sox17 in rat VSCs were inconclusive. Rat VSCs showed recovery of Sox protein expression after siRNA transfection. Instead, we tried to generate stable shRNA rat VSC lines, but did not see reduced Sox10 staining compared to scrambled control.

Main Content
Current View