Date of Degree

9-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Engineering

Advisor(s)

John M. Tarbell

Committee Members

Bingmei Fu

Steven B. Nicoll

Sihong Wang

M. Lane Gilchrist

Subject Categories

Biomedical Engineering and Bioengineering

Keywords

shear stress; cyclic strain; glycoclayx; heparan sulfate proteoglycan; syndecan 4; integrins; mechanotransduction; embryonic stem cell; endothelial cell

Abstract

The isolation of human ES cells (hES) in 1998 remarkably elevated the interest in the cell therapy capability of ES cells and moved this concept one step closer to reality. Embryonic stem cells with their potential for both self-renewal and directed differentiation have received increasing attention in applications in tissue engineering and repair, cell therapy, and regenerative medicine.

The differentiation of embryonic stem cells into desired lineages depends on the chemical environment of the “niche”, and for certain lineages such as the vascular lineage that must function in a mechanical environment, the timely imposition of mechanical forces is critical. In order to develop functional endothelial cells from embryonic stem cells, we need to identify the imposed forces on the cells, demonstrate their effects and finally reveal the underlying mechanism for each force.

In the early stages of embryonic vasculogenesis, blood islands formed by the hemangioblasts give rise to the early vasculature needed to nourish the embryo. At this stage, fluid shear stress of blood flow is the principal mechanical force that drives further differentiation into endothelial cell (EC) phenotype.

It has been shown that shear stress plays a critical role in promoting endothelial cell (EC) differentiation from embryonic stem cell (ESC)-derived EC. However, the underlying mechanisms mediating shear stress effects in this process have yet to be investigated. It has been reported that the glycocalyx component heparan sulfate proteoglycans (HSPG) mediate shear stress mechanotransduction in mature EC.

In the first part of this study, we investigated whether cell surface HSPG play a role in shear stress modulation of ESC phenotype. Our result presents that shear stress enhanced expression of vascular endothelial cell-specific marker genes (vWF, VE-cadherin, PECAM-1), tight junction protein genes (ZO-1, OCLD, CLD5) and vasodilatory genes (eNOS, COX-2), while it attenuated the expression of the vasoconstrictive gene ET1. After reduction of HSPG with Hep III, the shear stress-induced expression of vWF, VE-cadherin, ZO-1, eNOS, and COX-2, were abolished, suggesting that shear stress-induced expression of these genes depends on HSPG. These findings indicate for the first time that HSPG is a mechanosensor mediating shear stress-induced EC differentiation from ESC-derived EC cells

As the fetal heart begins to beat, the vasculature starts to experience cyclic strain (CS) and associated stress in addition to shear stress. While cyclic strain has been shown to induce mESC and Flk-1+ cells differentiation into vascular smooth muscle cells, there have been no studies to elucidate the effect of CS on differentiation of ESC-derived endothelial cells to EC or the underlying mechanisms which mediate cyclic strain effects in this process.

The second part of our study shows that cyclic strain reduced expression of of vascular endothelial cell-specific marker genes (vWF, VE-cadherin, PECAM-1), tight junction protein genes (ZO-1, OCLD, CLD5) and vasoactive genes (eNOS, ET1), while it didn’t alter the expression of the vasodilatory gene COX2. Our study also reveals the first evidence that HSPG, integrin, and syndecan-4, as mechanosensors, can contribute to cyclic strain-induced regulation of ESC-derived EC.

Recent studies by different groups have acknowledged the effect of mechanical forces on ESC modulation. However, the underlying mechanisms mediating the effect of mechanical forces in this process are not yet elucidated. Our work presents the first evidence that HSPG, integrin, and syndecan-4, as mechanosensors, can contribute to shear stress and cyclic strain-induced regulation of ESC-derived EC.

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