Edward I. Chang

Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis

Hypertrophic scars occur following cutaneous wounding and result in severe functional and esthetic defects. The pathophysiology of this process remains unknown. Here, we demonstrate for the first time that mechanical stress applied to a healing wound is sufficient to produce hypertrophic scars in mice. The resulting scars are histopathologically identical to human hypertrophic scars and persist for more than six months following a brief (one‐week) period of augmented mechanical stress during the proliferative phase of wound healing. Resulting scars are structurally identical to human hypertrophic scars and showed dramatic increases in volume (20‐fold) and cellular density (20‐fold). The increased cellularity is accompanied by a four‐fold decrease in cellular apoptosis and increased activation of the prosurvival marker Akt. To clarify the importance of apoptosis in hypertrophic scar formation, we examine the effects of mechanical loading on cutaneous wounds of animals with altered pathways of cellular apoptosis. In p53‐null mice, with down‐regulated cellular apoptosis, we observe significantly greater scar hypertrophy and cellular density. Conversely, scar hypertrophy and cellular density are significantly reduced in proapoptotic BclII‐null mice. We conclude that mechanical loading early in the prolifer‐ative phase of wound healing produces hypertrophic scars by inhibiting cellular apoptosis through an Akt‐dependent mechanism.—Aarabi S., Bhatt, K. A., Shi, Y., Paterno, J., Chang, E. I., Loh, S. A., Holmes, J. W., Longaker, M. T., Yee, H., Gurtner G. C. Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis. FASEB J. 21, 3250–3261 (2007)

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.

Age Decreases Endothelial Progenitor Cell Recruitment Through Decreases in Hypoxia-Inducible Factor 1α Stabilization During Ischemia

Background— Advanced age is known to impair neovascularization. Because endothelial progenitor cells (EPCs) participate in this process, we examined the effects of aging on EPC recruitment and vascular incorporation. Methods and Results— Murine neovascularization was examined by use of an ischemic flap model, which demonstrated aged mice (19 to 24 months) had decreased EPC mobilization (percent mobilized 1.4±0.2% versus 0.4±0.1%, P<0.005) that resulted in impaired gross tissue survival compared with young mice (2 to 6 months). This decrease correlated with diminished tissue perfusion (P<0.005) and decreased CD31+ vascular density (P<0.005). Gender-mismatched bone marrow transplantation demonstrated significantly fewer chimeric vessels in aged mice (P<0.05), which confirmed a deficit in bone marrow–mediated vasculogenesis. Age had no effect on total EPC number in mice or humans. Reciprocal bone marrow transplantations confirmed that impaired neovascularization resulted from defects in the response of aged tissue to hypoxia and not from intrinsic defects in EPC function. We demonstrate that aging decreased hypoxia-inducible factor 1α stabilization in ischemic tissues because of increased prolyl hydroxylase–mediated hydroxylation (P<0.05) and proteasomal degradation. This resulted in a diminished hypoxia response, including decreased stromal cell–derived factor 1 (P<0.005) and vascular endothelial growth factor (P<0.0004). This effect can be reversed with the iron chelator deferoxamine, which results in hypoxia-inducible factor 1α stabilization and increased tissue survival. Conclusions— Aging impairs EPC trafficking to sites of ischemia through a failure of aged tissues to normally activate the hypoxia-inducible factor 1α–mediated hypoxia response.

Pulsed electromagnetic fields accelerate normal and diabetic wound healing by increasing endogenous FGF-2 release.

Background: Chronic wounds, particularly in diabetics, result in significant morbidity and mortality and have a profound economic impact. The authors demonstrate that pulsed electromagnetic fields significantly improve both diabetic and normal wound healing in 66 mice through up-regulation of fibroblast growth factor (FGF)-2 and are able to prevent tissue necrosis in diabetic tissue after an ischemic insult. Methods: Db/db and C57BL6 mice were wounded and exposed to pulsed electromagnetic fields. Gross closure, cell proliferation, and vascularity were assessed. Cultured medium from human umbilical vein endothelial cells exposed to pulsed electromagnetic fields was analyzed for FGF-2 and applied topically to wounds. Skin flaps were created on streptozocin-induced diabetic mice and exposed to pulsed electromagnetic fields. Percentage necrosis, oxygen tension, and vascularity were determined. Results: Pulsed electromagnetic fields accelerated wound closure in diabetic and normal mice. Cell proliferation and CD31 density were significantly increased in pulsed electromagnetic field–treated groups. Cultured medium from human umbilical vein endothelial cells in pulsed electromagnetic fields exhibited a three-fold increase in FGF-2, which facilitated healing when applied to wounds. Skin on diabetic mice exposed to pulsed electromagnetic fields did not exhibit tissue necrosis and demonstrated oxygen tensions and vascularity comparable to those in normal animals. Conclusions: This study demonstrates that pulsed electromagnetic fields are able to accelerate wound healing under diabetic and normal conditions by up-regulation of FGF-2–mediated angiogenesis. They also prevented tissue necrosis in response to a standardized ischemic insult, suggesting that noninvasive angiogenic stimulation by pulsed electromagnetic fields may be useful to prevent ulcer formation, necrosis, and amputation in diabetic patients.

IFATS Collection: Adipose Stromal Cells Adopt a Proangiogenic Phenotype Under the Influence of Hypoxia

Evolving evidence suggests a possible role for adipose stromal cells (ASCs) in adult neovascularization, although the specific cues that stimulate their angiogenic behavior are poorly understood. We evaluated the effect of hypoxia, a central mediator of new blood vessel development within ischemic tissue, on proneovascular ASC functions. Murine ASCs were exposed to normoxia (21% oxygen) or hypoxia (5%, 1% oxygen) for varying lengths of time. Vascular endothelial growth factor (VEGF) secretion by ASCs increased as an inverse function of oxygen tension, with progressively higher VEGF expression at 21%, 5%, and 1% oxygen, respectively. Greater VEGF levels were also associated with longer periods in culture. ASCs were able to migrate towards stromal cell‐derived factor (SDF)‐1, a chemokine expressed by ischemic tissue, with hypoxia augmenting ASC expression of the SDF‐1 receptor (CXCR4) and potentiating ASC migration. In vivo, ASCs demonstrated the capacity to proliferate in response to a hypoxic insult remote from their resident niche, and this was supported by in vitro studies showing increasing ASC proliferation with greater degrees of hypoxia. Hypoxia did not significantly alter the expression of endothelial surface markers by ASCs. However, these cells did assume an endothelial phenotype as evidenced by their ability to tubularize when seeded with differentiated endothelial cells on Matrigel. Taken together, these data suggest that ASCs upregulate their proneovascular activity in response to hypoxia, and may harbor the capacity to home to ischemic tissue and function cooperatively with existing vasculature to promote angiogenesis. STEM CELLS 2009;27:266–274

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.

Hypoxia, hormones, and endothelial progenitor cells in hemangioma.

Hemangiomas are the most common tumor of infancy, and although the natural history of these lesions is well described, their etiology remains unknown. One current theory attributes the development of hemangiomas to placentally-derived cells; however, conclusive evidence to support a placental origin is lacking. While placental tissue and hemangiomas do share molecular similarities, it is possible that these parallels are the result of analogous responses of endothelial cells and vascular progenitors to similar environmental cues. Specifically, both tissue types consist of actively proliferating cells that exist within a low oxygen, high estrogen environment. The hypoxic environment leads to an upregulation of hypoxia inducible factor-1alpha (HIF-1alpha) responsive chemokines such as stromal cell derived factor-1alpha (SDF-1alpha) and vascular endothelial growth factor (VEGF), both of which are known to promote the recruitment and proliferation of endothelial progenitor cells. Increased hormone levels in the postpartum period further potentiate the growth of these lesions. In this model, increased stabilization of HIF-1 in concert with increased levels of estrogen create a milieu that promotes new blood vessel development, ultimately contributing to the pathogenesis of infantile hemangiomas.

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.

Aging and diabetes impair the neovascular potential of adipose-derived stromal cells.

Background: Aging and diabetes are major risk factors for poor wound healing and tissue regeneration that reflect an impaired ability to respond to ischemic insults. The authors explored the intrinsic neovascular potential of adipose-derived stromal cells in the setting of advanced age and in type 1 and type 2 diabetes. Methods: Adipose-derived stromal cells isolated from young, aged, streptozotocin-induced, and db/db diabetic mice were exposed to normoxia and hypoxia in vitro. Vascular endothelial growth factor (VEGF) expression, proliferation, and tubulization were measured. Conditioned media harvested from adipose-derived stromal cell cultures were assessed for their ability to stimulate human umbilical vein endothelial cell proliferation (n = 3 and n = 3). Results: Young adipose-derived stromal cells demonstrated significantly higher levels of VEGF production, proliferation, and tubulogenesis than those derived from aged, streptozotocin-induced, and db/db mice in both normoxia and hypoxia. Although aged and diabetic adipose-derived stromal cells retained the ability to up-regulate VEGF secretion, proliferation, and tubulogenesis in response to hypoxia, the response was blunted compared with young controls. Conditioned media derived from these cells cultured in normoxia in vitro also had a significantly greater ability to increase human umbilical vein endothelial cell proliferation compared with media harvested from aged, streptozotocin-induced, and db/db adipose-derived stromal cells. This effect was magnified in conditioned media harvested from hypoxic adipose-derived stromal cell cultures. Conclusions: This study demonstrates that aging and type 1 and type 2 diabetes impair intrinsic adipose-derived stromal cell function; however, these cells may still be a suitable source of angiogenic cells that can potentially improve neovascularization of ischemic tissues.

Comparative healing of surgical incisions created by the PEAK PlasmaBlade, conventional electrosurgery, and a scalpel.

Background: The PEAK PlasmaBlade is a new electrosurgical device that uses pulsed radiofrequency to generate a plasma-mediated discharge along the exposed rim of an insulated blade, creating an effective cutting edge while the blade stays near body temperature. Methods: Full-thickness incisions were made on the dorsums of pigs with the PlasmaBlade, a conventional electrosurgical device, and a scalpel, and blood loss was quantified. Wounds were harvested at designated time points, tested for wound tensile strength, and examined histologically for scar formation and tissue damage. Results: Bleeding was reduced significantly (59 percent) in PlasmaBlade incisions compared with scalpel incisions, and acute thermal damage from the PlasmaBlade (66 ± 5 &mgr;m) was significantly less than both cut and coagulation mode electrosurgical incisions (456 ± 35 &mgr;m and 615 ± 22 &mgr;m, respectively). Histologic scoring for injury and wound strength was equivalent between the PlasmaBlade and scalpel incisions. By 6 weeks, the healed PlasmaBlade and scalpel incisions were approximately three times stronger, and scar cosmetic appearance was significantly better compared with electrosurgical incisions. Conclusions: The PlasmaBlade is a promising new surgical instrument that provides atraumatic, scalpel-like cutting precision and electrosurgical-like hemostasis, resulting in minimal bleeding, tissue injury, and scar formation.