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Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006. / Vito, Raymond, Committee Member ; Nerem, Robert, Committee Chair ; Eskin, Suzanne, Committee Member ; Hanson, Stephen, Committee Member ; Gibbons, Gary, Committee Member.

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2006. / Hanjoong Jo, Ph.D., Committee Chair ; Don P. Giddens, Ph.D., Committee Member ; W. Robert Taylor, M.D., Ph.D., Committee Member ; Ajit Yoganathan, Ph.D., Committee Member.

The importance of shear stress in vascular biology and pathophysiology has been highlighted by the focal development patterns of atherosclerosis, abdominal aortic aneurysms, and heart valve disease in regions exposed to disturbed flow leading to low or oscillatory shear stress at the wall of the blood vessel or the surface of the valve leaflet. The novel and significant finding of this study is that mouse aortic endothelial cell exposure to pro-atherogenic oscillatory shear stress (OS) (+/- 5 dynes/cm2) increased their production of cathepsins, the family of lysosomal cysteine proteases that are potent elastases and collagenases leading to protease degradation and remodeling of the extracellular matrix structural components. Conversely, atheroprotective unidirectional laminar shear stress (LS) (15 dynes/cm2) decreased elastase and gelatinase activities of endothelial cells through a shear stress mediated reduction in cathepsins K, L, and S activity. Their endogenous inhibitor, cystatin C, was found to be inversely regulated by shear stress; LS increased its secretion by endothelial cells while OS decreased it. Binding of free cystatin C in the conditioned media to carboxymethylated papain coated agarose beads led to an increase in cathepsin activity since the available cathepsin was not inhibited. To verify these findings in human samples, immunohistochemical analysis of cystatin C and cathepsin K was performed on human coronary arteries. Cathepsin K stained strongly in the endothelial layer of vessels with degraded internal elastic lamina while cystatin C staining intensity was strongest overlying minimally diseased vessels. Additional roles for cathepsins K, L, and S were found in endothelial cell alignment in response to unidirectional laminar shear stress, endothelial cell migration, and programmed cell death. We conclude that there is an inverse regulation of cathepsins and cystatin C in endothelial cells by LS and OS and identify the cathepsin family of proteases as potential targets for therapeutic intervention of cardiovascular disease development at sites of disturbed flow.

Indiana University-Purdue University Indianapolis (IUPUI) / In the US, more than 1 million burn injuries are reported annually. About 45,000 injuries due to fires and burns result in hospitalization and ten percent of these result in death every year. Advances in burn treatment have led to a reduction in mortality rate over the last decades. Since more patients are surviving the initial resuscitation phase even with very large areas of skin being burned away, wound care has become increasingly important to ensure continued patient survival and improvement. While currently a common treatment for third degree burn wounds, skin grafts have several drawbacks. The availability of donor sites for autografts may be limited, especially in incidences of extensive skin loss. The rejection associated with the use of allografts and xenografts may render them inadequate or undesirable. Even if a suitable graft is found, poor retention due to infection, hematoma, and low vascularity at the recipient site are other drawbacks associated with the use of skin grafts as a primary treatment for severe burn wounds. As such, research has been done into alternative treatments, which include but are not limited to artificial skin, cell therapy, and growth factor application. We propose the delivery of adipose derived stem cells (ASC) in combination with endothelial progenitor cells (EC) via Integra Dermal Regenerative Template (DRT) to promote faster graft vascularization and thus faster healing of wounds. Integra DRT is an acellular skin substitute that consists of a dermal layer composed of bovine collagen and chondroitin-6-sulfate glycosaminoglycan, and an "epidermal" layer, which consists of silicone polymer. This silicone layer is removed after the collagen matrix is adequately vascularized (usually takes 2-3 weeks), and then a thin layer autograft is applied to the top of the neo-dermis. ASC are derived from the stromal-vascular fraction (SVF) of adipose tissue and are a readily available, pluripotent, mesenchymal cell known to promote angiogenesis. They are being explored as a treatment for a myriad of diseases and conditions, including wound healing. In combination with ECs, they form stable microvessel networks in vitro and in vivo. In our work, we found that ASC+EC form stable microvessel networks when cultured on Integra DRT. Also, ASC and ASC+EC conditioned media promoted both survival and migration of human epidermal keratinocytes compared to control medium. In a full thickness wound healing model, using healthy NSG mice, the ASC+EC case showed a significantly higher rate of wound closure compared to control. Based on best linear unbiased estimates (BLUE), the difference between the healing rates of ASC alone treatment and the Control treatment group is -0.45 +/- 0.22 mm²/day (p=0.041), which is not less than 0.025 and thus not statistically significant (Bonferroni Adjusted). However, the BLUE for the difference between the ASC+EC group and the Control group healing rates is -0.55 +/- 0.28 mm²/day (p = 0.017<0.025, Bonferroni Adjusted), which is statistically significant. Histology revealed a significantly higher number of vessels compared to control in both ASC alone and ASC+EC case. CD31 staining revealed the presence of human vessels in ASC+EC treatment scaffolds. We conclude that the combination of ASC and EC can be used to accelerate healing of full-thickness wounds when delivered to site of the wound via Integra. This result is especially compelling due to the fact that the mice used were all healthy. Thus our treatment shows an improvement in healing rate even compared to normal wound healing.