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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
by 1, 7 and 14 days reperfusion (DR). At each time point, mice were evaluated for functional limb recovery, histologic evidence of acute injury, regenerating fibers, tissue levels of ATP, and molecular markers of regeneration (MyoD, Myogenin). Results: At 1DR, SIR resulted in a 5 fold greater number of injured fibers than those receiving MIR (p < 0.001). At 1 and 7DR, mice subjected to SIR had less ATP than mice receiving MIR (p ¼ 0.0006). By 14DR, ATP levels in both groups of mice were equivalent, but less than the contralateral limb (p < 0.05). Despite differences in injured fibers and ATP content, there was no difference in functional recovery between the groups at 1DR. In contrast, by 7 and 14DR mice subjected to MIR recovered to normal function, but the SIR mice sustained ongoing deficits (p ¼ 0.004). SIR mice had greater numbers of immature (centrally nucleated) myofibers at 7 and 14DR as compared to MIR mice (p < 0.05). MyoD and Myogenin expression were greater in the MIR group at 7DR, but were equivalent by 14DR. Conclusions: The chronic phase of reperfusion injury is characterized by depletion of muscle ATP in the setting of both moderate and severe ischemia. Functional recovery after moderate IR was associated with greater expression of MyoD and fewer immature fibers at 7DR. In the time interval leading up to 7DR, there may be a therapeutic window where pharmacologic interventions might facilitate functional and metabolic recovery. 50.8. A Novel Role of Estrogen in Diabetic Wound Healing: Improved Function of Bone Marrow-Derived Endothelial Progenitor Cells. Y. Zhuge, Z. Liu, D. J. Pincus, S. R. Thaller, S. Elliot, O. C. Velazquez; University of Miami Miller School of Medicine, Miami, FL Introduction: Endothelial progenitor cells (EPC) are mobilized into circulation from the bone marrow (BM) in response to wound repair signals and contribute to neovascularization. Diabetes delays wound healing and decreases the number/function of EPC while estrogen has beneficial healing effects. However, estrogen’s roles in countering delayed healing and modulating EPC biology in diabetes are unknown. We investigated the effect of estrogen on BM-EPC number and function using a murine diabetic wound healing model. Methods: Twenty-one diabetic (dbþ/dbþ) mice were given cutaneous wounds and treated with topical estrogen, tamoxifen, or placebo cream. On day five post-wounding, BM cells were harvested to quantify EPC number and assess survival and proliferative ability via colony-forming units (CFU) in vitro. CFUs were counted on day ten after plating. Wound healing and neovascularization were concurrently studied via digital photography and immunohistochemistry staining, respectively. Data were analyzed using ANOVA. Results: FACS analysis of fresh BM showed that diabetes did not significantly affect the number of BM-EPC (CD31þ/CD34þ). However, viability of these cells was significantly decreased, as demonstrated by reduced CFUs in vitro (p ¼ 0.0009). Although treatment with estrogen did not change the number of BM-EPC, it significantly increased CFUs of EPC compared to tamoxifen and placebo cream (p ¼ 0.030), indicating increased EPC viability and proliferative capacity. Consistently, estrogen significantly enhanced wound angiogenesis and healing in the diabetic mice, as demonstrated by faster healing rate and increased vessel density in wound tissue. Conclusions: Estrogen treatment increases wound healing and wound neovascularization in diabetic mice. Our data indicates that these beneficial effects may be mediated through increasing the viability and proliferative capacity of BM-EPC. Other progenitor cells may also be involved and require additional study.
against invading pathogens through pattern recognition receptors such as TLR4 and RAGE. It also mediates inflammation following tissue injury through endogenous ligands called danger signals that bind the same receptors. We have previously shown that HMGB-1, a highly conserved nuclear protein and danger signal, is released by ischemic skeletal muscle and hypoxic cells and promotes angiogenic activity in vitro. HMGB-1 blocking antibody dramatically inhibits endothelial tube formation and increases tissue necrosis in murine hindlimb ischemia, suggesting that innate immunity mediates angiogenesis. Receptors for HMGB-1 include TLR4 and RAGE. Our hypothesis is that signaling through these receptors may be necessary for ischemia-induced angiogenesis. Methods: C3H/HeJ (TLR4 mutant; N ¼ 6) and C3H/HeOuJ (wildtype; N ¼ 6) mice underwent femoral artery ligation on the right hind limb and sham dissection on the left. Laser doppler perfusion imaging (LDPI) was used to measure tissue perfusion and results are expressed as a ratio of right limb:left limb perfusion. Hind-limb ischemia was also created in RAGE knockout mice (N ¼ 4) and control C57/B6 mice (N ¼ 4). Muscle samples were collected at 2 wks, sectioned, and stained for von Willebrand’s factor. Results: Femoral artery ligation resulted in significant ischemia in both HeJ and HeOuJ mice, resulting in limping and toe necrosis. By 2 wks, the ratio of perfusion in ischemic compared to nonischemic limbs was w2-fold greater in HeJ mice than in HeOuJ mice (0.89 6 0.30 vs. 0.48 6 0.23, p ¼ 0.023; Figure). In RAGE KO and control mice, femoral artery ligation resulted in initial limb dysfunction. By 2 wks, the function of ischemic legs returned essentially to normal in both RAGE KO and control mice. Vascular density in the ischemic muscle was similar in RAGE KO and control mice (13.1 6 0.5 vs 14.9 6 0.8 vessels/HPF). Interestingly, the severity of ischemic injury and degree of recovery varied significantly between the mouse strains with C3H demonstrating greater tissue injury with less recovery vs C57/ B6 mice. Conclusions: Our results reveal greater recovery of perfusion following hind-limb ischemia in mice lacking functional TLR4. Our previous studies revealed HMGB-1 to be pro-angiogenic and contrary to our hypothesis, our current experiments suggest that angiogenesis following ischemia does not require intact TLR4 signaling. Similarly, the presence of functional RAGE did not increase vascular density after ischemic injury. Thus, other HMGB-1 receptors may be involved in mediating effects on angiogenesis. Alternatively, the absence of TLR4 or RAGE may upregulate other innate immune pathways that compensate for the absence of the receptor. Finally, differences in the response to hind-limb ischemia between C3H and C57/B6 mice may compromise the ability to compare the angiogenic responses between TLR4 and RAGE KO mice.
50.9. TLR4 and RAGE Function Are Not Essential for Angiogenesis in a Model of Hind-Limb Ischemia. U. Sachdev, X. Cui, D. Chen, T. Wang, E. Tzeng; University of Pittsburgh Medical Center, Pittsburgh, PA
50.10. Improved Number and Function of Bone Marrow Mesenchymal Stem Cells by Topical Estrogen in Diabetic Murine Wound Healing. Y. Zhuge, Z. Liu, D. J. Pincus, S. R. Thaller, S. Elliot, O. C. Velazquez; University of Miami Miller School of Medicine, Miami, FL
Introduction: Inflammation is an important feature of the angiogenic response. Innate immunity is the first line of defense
Objectives: Mesenchymal stem cells (MSCs) are a heterogeneous population of stem/progenitor cells with pluripotent differentiation