Indiana University-Purdue University Indianapolis (IUPUI) / Obesity increases cardiovascular disease risk and is associated with factors of the “metabolic syndrome” (MetS), a disorder including hypertension, hypercholesterolemia and/or impaired glucose tolerance. Expanding adipose and subsequent inflammation is implicated in vascular dysfunction in MetS. Perivascular adipose tissue (PVAT) surrounds virtually every artery and is capable of releasing factors that influence vascular reactivity, but the effects of PVAT in the coronary circulation are unknown. Accordingly, the goal of this investigation was to delineate mechanisms by which lean vs. MetS coronary PVAT influences vasomotor tone and the coronary PVAT proteome. We tested the hypothesis that MetS alters the functional expression and vascular contractile effects of coronary PVAT in an Ossabaw swine model of the MetS. Utilizing isometric tension measurements of coronary arteries in the absence and presence of PVAT, we revealed the vascular effects of PVAT vary according to anatomical location as coronary and mesenteric, but not subcutaneous adipose tissue augmented coronary artery contractions to KCl. Factors released from coronary PVAT increase baseline tension and potentiate constriction of isolated coronary arteries relative to the amount of adipose tissue present. The effects of coronary PVAT are elevated in the setting of MetS and occur independent of endothelial function. MetS is also associated with substantial alterations in the coronary PVAT proteome and underlying increases in vascular smooth muscle Ca2+ handling via CaV1.2 channels, H2O2-sensitive K+ channels and/or upstream mediators of these ion channels. Rho-kinase signaling participates in the increase in coronary artery contractions to PVAT in lean, but not MetS swine. These data provide novel evidence that the vascular effects of PVAT vary according to anatomic location and are influenced by the MetS phenotype.

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.

Indiana University-Purdue University Indianapolis (IUPUI). / Tumor necrosis factor-induced protein 6 (TSG-6) has been shown to mitigate inflammation. Its presence in the secretome of adipose stem / stromal cells (ASC) and its role in activities of ASC have been overlooked. This thesis described for the first time the release of TSG-6 from ASC, and its modulation by endothelial cells. It also revealed that protection of endothelial barrier function was a novel mechanism underlying the anti-inflammatory activity of both ASC and TSG-6. Moreover, TSG-6 was found to inhibit mitogen-activated lymphocyte proliferation, extending the understanding of its pleiotropic effects on major cell populations involved in inflammation. Next, enzyme-linked immunosorbent assays (ELISA) were established to quantify secretion of TSG-6 from human and murine ASC. To study the importance of TSG-6 to specific activities of ASC, TSG-6 was knocked down in human ASC by siRNA. Murine ASC from TSG-6-/- mice were isolated and the down-regulation of TSG-6 was verified by ELISA. The subsequent attempt to determine the efficacy of ASC in ameliorating ischemic limb necrosis and the role of TSG-6, however, was hampered by the highly variable ischemic tissue necrosis in the BALB/c mouse strain. Afterwards in a mouse model of cigarette smoking (CS), in which inflammation also plays an important role, it was observed, for the first time, that 3-day CS exposure caused an acute functional exhaustion and cell cycle arrest of hematopoietic progenitor cells; and that 7-week CS exposure led to marked depletion of phenotypic bone marrow stem and progenitor cells (HSPC). Moreover, a dynamic crosstalk between human ASC and murine host inflammatory signals was described, and specifically TSG-6 was identified as a necessary and sufficient mediator accounting for the activity of the ASC secretome to ameliorate CS-induced myelotoxicity. These results implicate TSG-6 as a key mediator for activities of ASC in mitigation of inflammation and protection of HSPC from the myelotoxicity of cigarette smoke. They also prompt the notion that ASC and TSG-6 might potentially play therapeutic roles in other scenarios involving myelotoxicity.