Michael G. Galvez

The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues

Diabetes is associated with poor outcomes following acute vascular occlusive events. This results in part from a failure to form adequate compensatory microvasculature in response to ischemia. Since vascular endothelial growth factor (VEGF) is an essential mediator of neovascularization, we examined whether hypoxic up-regulation of VEGF was impaired in diabetes. Both fibroblasts isolated from type 2 diabetic patients, and normal fibroblasts exposed chronically to high glucose, were defective in their capacity to up-regulate VEGF in response to hypoxia. In vivo, diabetic animals demonstrated an impaired ability to increase VEGF production in response to soft tissue ischemia. This resulted from a high glucose-induced decrease in transactivation by the transcription factor hypoxia-inducible factor-1α (HIF-1α), which mediates hypoxia-stimulated VEGF expression. Decreased HIF-1α functional activity was specifically caused by impaired HIF-1α binding to the coactivator p300. We identify covalent modification of p300 by the dicarbonyl metabolite methylglyoxal as being responsible for this decreased association. Administration of deferoxamine abrogated methylglyoxal conjugation, normalizing both HIF-1α/p300 interaction and transactivation by HIF-1α. In diabetic mice, deferoxamine promoted neovascularization and enhanced wound healing. These findings define molecular defects that underlie impaired VEGF production in diabetic tissues and offer a promising direction for therapeutic intervention.

HIF-1α dysfunction in diabetes

Diabetic wounds are a significant public health burden, with slow or non-healing diabetic foot ulcers representing the leading cause of non-traumatic lower limb amputation in developed countries. These wounds heal poorly as a result of compromised blood vessel formation in response to ischemia. We have recently shown that this impairment in neovascularization results from a high glucose-induced defect in transactivation of hypoxia-inducible factor-1α (HIF-1α), the transcription factor regulating vascular endothelial growth factor (VEGF) expression. HIF-1 dysfunction is the end result of reactive oxygen species-induced modification of its coactivator p300 by the glycolytic metabolite methylglyoxal. Use of the iron chelator-antioxidant deferoxamine (DFO) reversed these effects and normalized healing of humanized diabetic wounds in mice. Here, we present additional data demonstrating that HIF-1α activity, not stability, is impaired in the high glucose environment. We demonstrate that high glucose-induced impairments in HIF-1α transactivation persist even in the setting of constitutive HIF-1α protein overexpression. Further, we show that high glucose-induced hydroxylation of the C-terminal transactivation domain of HIF-1α (the primary pathway regulating HIF-1α/p300 binding) does not alter HIF-1α activity. We extend our study of DFO’s therapeutic efficacy in the treatment of impaired wound healing by demonstrating improvements in tissue viability in diabetic mice with DFO-induced increases in VEGF expression and vascular proliferation. Since DFO has been in clinical use for decades, the potential of this drug to treat a variety of ischemic conditions in humans can be evaluated relatively quickly.

Imaging the Unfolded Protein Response in Primary Tumors Reveals Microenvironments with Metabolic Variations that Predict Tumor Growth

Cancer cells exist in harsh microenvironments that are governed by various factors, including hypoxia and nutrient deprivation. These microenvironmental stressors activate signaling pathways that affect cancer cell survival. While others have previously measured microenvironmental stressors in tumors, it remains difficult to detect the real-time activation of these downstream signaling pathways in primary tumors. In this study, we developed transgenic mice expressing an X-box binding protein 1 (XBP1)-luciferase construct that served as a reporter for endoplasmic reticulum (ER) stress and as a downstream response for the tumor microenvironment. Primary mammary tumors arising in these mice exhibited luciferase activity in vivo. Multiple tumors arising in the same mouse had distinct XBP1-luciferase signatures, reflecting either higher or lower levels of ER stress. Furthermore, variations in ER stress reflected metabolic and hypoxic differences between tumors. Finally, XBP1-luciferase activity correlated with tumor growth rates. Visualizing distinct signaling pathways in primary tumors reveals unique tumor microenvironments with distinct metabolic signatures that can predict for tumor growth.

SDF-1α expression during wound healing in the aged is HIF dependent

Background: Age-related impairments in wound healing are associated with decreased neovascularization, a process that is regulated by hypoxia-responsive cytokines, including stromal cell–derived factor (SDF)-1&agr;. Interleukin-1&bgr; is an important inflammatory cytokine involved in wound healing and is believed to regulate SDF-1&agr; expression independent of hypoxia signaling. Thus, the authors examined the relative importance of interleukin (IL)-1&bgr; and hypoxia-inducible factor (HIF)-1&agr; on SDF-1&agr; expression in aged wound healing. Methods: Young and aged mice (n = 4 per group) were examined for wound healing using a murine excisional wound model. Wounds were harvested at days 0, 1, 3, 5, and 7 for histologic analysis, immunohistochemistry, enzyme-linked immunosorbent assay, and Western blot. An engineered wild-type and mutated SDF luciferase reporter construct were used to determine HIF transactivation. Results: Aged mice demonstrated significantly impaired wound healing, reduced granulation tissue, and increased epithelial gap compared with young controls. Real-time polymerase chain reaction demonstrated reduced SDF-1&agr; levels in aged wounds that correlated with reduced CD31+ neovessels. Western blots revealed decreased HIF-1&agr; protein in aged wounds. However, both IL-1&bgr; and macrophage infiltrate were unchanged between young and aged animals. Using the wild-type and mutated SDF luciferase reporter construct in which the hypoxia response element was deleted, only young fibroblasts were able to respond to IL-1&bgr; stimulation, and this response was abrogated by mutating the HIF-binding sites. This suggests that HIF binding is essential for SDF-1 transactivation in response to both inflammatory and hypoxic stimuli. Conclusions: SDF-1&agr; deficiency observed during aged wound healing is attributable predominantly to decreased HIF-1&agr; levels rather than impaired IL-1&bgr; expression.

Vascular anastomosis using controlled phase transitions in poloxamer gels

Vascular anastomosis is the cornerstone of vascular, cardiovascular and transplant surgery. Most anastomoses are performed with sutures, which are technically challenging and can lead to failure from intimal hyperplasia and foreign body reaction. Numerous alternatives to sutures have been proposed, but none has proven superior, particularly in small or atherosclerotic vessels. We have developed a new method of sutureless and atraumatic vascular anastomosis that uses US Food and Drug Administration (FDA)-approved thermoreversible tri-block polymers to temporarily maintain an open lumen for precise approximation with commercially available glues. We performed end-to-end anastomoses five times more rapidly than we performed hand-sewn controls, and vessels that were too small (<1.0 mm) to sew were successfully reconstructed with this sutureless approach. Imaging of reconstructed rat aorta confirmed equivalent patency, flow and burst strength, and histological analysis demonstrated decreased inflammation and fibrosis at up to 2 years after the procedure. This new technology has potential for improving efficiency and outcomes in the surgical treatment of cardiovascular disease.

Transdermal deferoxamine prevents pressure-induced diabetic ulcers

Significance Diabetes is the leading cause of nontraumatic amputations. There are no effective therapies to prevent diabetic ulcer formation and only modestly effective technologies to help with their healing. To enhance diabetic wound healing we designed a transdermal delivery system containing the FDA-approved small molecule deferoxamine, an iron chelator that increases defective hypoxia inducible factor-1 alpha transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. This system overcomes the challenge of delivering hydrophilic molecules through the normally impermeable stratum corneum and both prevents diabetic ulcer formation and improves the healing of existing diabetic wounds. This represents a prophylactic pharmacological agent to prevent ulcer formation that is rapidly translatable into the clinic and has the potential to ultimately transform the care and prevention of diabetic complications. There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.

Tissue engineering in flexor tendon surgery: current state and future advances.

Tissue engineering of flexor tendons addresses a challenge often faced by hand surgeons: the restoration of function and improvement of healing with a limited supply of donor tendons. Creating an engineered tendon construct is dependent upon understanding the normal healing mechanisms of the tendon and tendon sheath. The production of a tendon construct includes: creating a three-dimensional scaffold; seeding cells within the scaffold; encouraging cellular growth within the scaffold while maintaining a gliding surface; and finally ensuring mechanical strength. An effective construct incorporates these factors in its design, with the ultimate goal of creating tendon substitutes that are readily available to the reconstructive hand surgeon.

A Novel Mouse Model for Frostbite Injury

BACKGROUND Frostbite injury occurs when exposure to cold results in frozen tissue. To screen drugs and other field therapies that might improve the outcome for a frostbite victim, it would be helpful to have a reliable and cost-effective preclinical in vivo model. OBJECTIVE We sought to create a novel mouse skin model of induced frostbite injury. This model would allow quantification of the surface area of involved skin, histology of the wound, rate of wound healing, and skin loss in a standardized fashion after the frostbite injury. METHODS Thirty-six mice were studied. Standardized 2.9-cm diameter circles were tattooed on the mouse dorsum. Magnets frozen in dry ice (-78.5°C) were used to create a frostbite injury on skin within the circle, either as a continuous 5-minute freeze or as 3 repeated freeze (1-minute) and thaw (3-minute) cycles. Appearance, healing rate, skin surface area loss, and histology were recorded until the wounds were healed. RESULTS The amount of skin surface area loss was approximately 50% for both freeze methods. Although the time to surface skin healing was similar for both freeze methods, the initial healing rate was significantly (P = .001) slower in mice exposed to the freeze-thaw cycles compared with the continuous freeze model. Histopathology reflected inflammatory changes, cell death, and necrosis. CONCLUSIONS This novel in vivo mouse model for frostbite allows quantification of affected skin surface area, histology, healing rate, and skin loss and has the potential of being utilized to screen future treatment modalities.

Reconstruction of First Web Space Contractures.

ASSH Disclaimer: The material presented in this CME activity is mad ASSH for educational purposes only. This material is not intended to methods or the best procedures appropriate for the medical situation rather it is intended to present an approach, view, statement, or opin that may be helpful, or of interest, to other practitioners. Examinees agre this medical education activity, sponsored by the ASSH, with full knowled that they waive any claim they may have against the ASSH for reliance o presented. The approval of the US Food and Drug Administration is requ and drugs that are considered experimental. Instrumentation syst reviewed during this educational activity may not yet have received F

Flow-through omental flap to free anterolateral thigh flap for complex chest wall reconstruction: Case report and review of the literature.

Despite the options currently available for chest wall reconstruction, patients with complex composite defects may still pose a significant challenge for the reconstructive surgeon when only using conventional methods. In particular, prior radiotherapy and/or large en bloc resection may leave inadequate regional flaps and recipient vessels for free tissue transfer. Here, we describe a case in which we reconstruct a 14 cm × 18 cm complex chest wall defect, secondary to tumor resection and infected sternum debridement, with a pedicled flow‐through omental flap to a 14 cm × 22 cm free anterolateral thigh flap using the omental gastroepiploic vessels as recipient vessels. Reconstruction was successful with excellent flap viability, and no complications at recipient or donor sites. We review the literature on complex chest wall reconstruction and introduce this valuable option of utilizing a pedicled omental flap as a flow‐through flap to a free flap for patients without viable recipient vessels or local flaps.