Leila Jazayeri

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.

Using genetically modified microvascular free flaps to deliver local cancer immunotherapy with minimal systemic toxicity.

Background: Clinical use of cancer gene therapy has been prevented by the inability to deliver high levels of local transgene expression with acceptable host toxicity. The authors’ laboratory has developed an ex vivo technique to genetically modify free flaps to deliver immunotherapy locally without systemic toxicity. Methods: Superficial inferior epigastric flaps were dissected in Fischer rats, perfused with a viral vector expressing the antitumor interleukin-12 (IL-12) for 1 hour, and re-anastomosed. Beneath the flaps was a bolus of 1 × 106 beta-human chorionic gonadotropin–secreting MADB-106 tumor cells. Tumor growth was monitored using beta-human chorionic gonadotropin levels (secreted by the tumor) and size. IL-12 expression in tissue was assessed by enzyme-linked immunosorbent assay. Tumor inflammatory infiltrate was assessed using immunohistologic staining (CD8 and CD161) and enzyme-linked immunosorbent assay (interferon-&ggr;). Serum levels of liver enzymes and histologic analysis were used to assess systemic toxicity. Results: IL-12 expression was confirmed in the flap and surrounding tissue. The rate of tumor growth in the IL-12–treated group was significantly suppressed compared with the control group (p < 0.001). Liver enzyme levels remained normal, and histological evaluation of the liver, lung, and spleen revealed no evidence of inflammation in the treated group. Conclusions: Using genetically modified free flaps, the authors were able to deliver IL-12 directly into the local environment of a tumor and suppress its growth without eliciting toxic systemic effects. This technique could provide valuable adjuvant treatment after oncologic surgery for soft-tissue cancers, with the transduced flap reconstructing the defect and supplying a therapeutic agent to the resected tumor bed.

Distal digital replantation.

Background: Hand surgeons have been hesitant to perform distal digital replantation because of the technical challenges and the perception of a high cost-to-benefit ratio. Recent studies, however, have shown high survival rates and excellent functional and aesthetic results, providing renewed enthusiasm for distal replantation. Methods: The authors reviewed the literature and summarize key points regarding the surgical treatment, perioperative care, and outcomes of distal digital replantation. They describe specific techniques and considerations for surgical repair in each of four distal zones as described by Sebastin and Chung. Results: Zone 1A replantation involves an artery-only anastomosis of a longitudinal pulp artery. Venous anastomosis first becomes possible in zone 1B. Zone 1C involves periarticular amputations where arthrodesis of the distal interphalangeal joint is usually indicated. Repair of the artery, vein, and nerve is technically optimal in zone 1D, where venous anastomosis should be performed. Overall, survival rates for distal digital replantation are similar to those reported for more proximal replantation. The literature reports good outcomes regarding nail salvage, fingertip sensibility, and range of motion, with restoration of length and aesthetic appearance. Conclusions: Distal replantation performed at institutions that specialize in microsurgery and specifically tailored to the level of injury is associated with good survival, function, and patient satisfaction and superior aesthetic outcome. More prospective data are needed to evaluate the cost of treatment, psychological outcomes, and functional outcomes of distal replantation compared with revision amputation.

Diabetes increases p53-mediated apoptosis following ischemia.

Background: Diabetes impairs the ability of tissue to respond adequately to ischemia. The underlying mechanisms contributing to this impaired response remain unknown. Because increases in apoptosis have been linked to a spectrum of diabetic complications, the authors examined whether programmed cell death is involved in the pathogenesis of poor diabetic tissue responses to ischemia. Methods: Analysis for apoptosis and levels of proaptotic protein, p53, were performed on streptozocin-induced diabetic mice and wild-type controls in a murine model of soft-tissue ischemia (n = 6). In vitro, chronic hyperglycemic culture conditions were used to test inducibility and reversibility of the diabetic phenotype. Small interfering RNA was used to assess the role of p53. Results: Ischemia-induced apoptosis and p53 levels were increased significantly in diabetic dermal fibroblasts both in vivo and in vitro. Chronic hyperglycemic culture was sufficient to induce the increased apoptotic phenotype, and this was not reversible with long-term normoglycemic conditions. Blocking p53 with small interfering RNA resulted in significant protection against ischemic apoptosis. Conclusions: These findings suggest that diabetes causes an increased apoptotic response to ischemia through a p53-mediated mechanism. This increase is not reversible by exposure to low-glucose conditions. This suggests that glycemic control alone will be unable to prevent tissue necrosis in diabetic patients and suggests novel therapeutic strategies for this condition.

Two-stage Implant-based Breast Reconstruction: An Evolution of the Conceptual and Technical Approach over a Two-decade Period

BACKGROUND Over a two-decade period, the senior author (P.G.C.) has had extensive experience with two-stage implant-based breast reconstruction with total musculofascial coverage. During this period, the approach has evolved substantially. The evolution has been based on changes in breast cancer treatment, available technology and, most importantly, yearly evaluation of surgical outcomes. METHODS This article describes changes in the conceptual approach to breast reconstruction, and the resulting evolution of surgical techniques. Evolving concepts and current techniques are described as they relate to each consecutive stage of implant-based breast reconstruction. RESULTS For the first stage of breast reconstruction (i.e., placement of the tissue expander), key concepts and techniques described are the vertical mastectomy defect, the point of maximal expansion, the musculofascial pocket, and the inferior fasciotomy. For the second stage of breast reconstruction (i.e., the exchange procedure), key concepts and techniques described are implant selection, setting the inframammary fold, defining the inferolateral shape of the breast, and circumferential capsulotomy. CONCLUSION The purpose of this article is to relay the lessons learned from this long experience and to provide a conceptual and technical framework to two-stage implant-based breast reconstruction.

Quantifying migration and polarization of murine mesenchymal stem cells on different bone substitutes by confocal laser scanning microscopy

INTRODUCTION Cell migration is preceded by cell polarization. The aim of the present study was to evaluate the impact of the geometry of different bone substitutes on cell morphology and chemical responses in vitro. MATERIALS AND METHODS Cell polarization and migration were monitored temporally by using confocal laser scanning microscopy (CLSM) to follow green fluorescent protein (GFP)±mesenchymal stem cells (MSCs) on anorganic cancellous bovine bone (Bio-Oss(®)), β-tricalcium phosphate (β-TCP) (chronOS(®)) and highly porous calcium phosphate ceramics (Friedrich-Baur-Research-Institute for Biomaterials, Germany). Differentiation GFP±MSCs was observed using pro-angiogenic and pro-osteogenic biomarkers. RESULTS At the third day of culture polarized vs. non-polarized cellular sub-populations were clearly established. Biomaterials that showed more than 40% of polarized cells at the 3rd day of culture, subsequently showed an enhanced cell migration compared to biomaterials, where non-polarized cells predominated (p<0.003). This trend continued untill the 7th day of culture (p<0.003). The expression of vascular endothelial growth factor was enhanced in biomaterials where cell polarization predominated at the 7th day of culture (p=0.001). CONCLUSIONS This model opens an interesting approach to understand osteoconductivity at a cellular level. MSCs are promising in bone tissue engineering considering the strong angiogenic effect before differentiation occurs.

Discussion. Effects of adipose-derived stem cells on improving the viability of diced cartilage grafts.

T article demonstrates the benefit of stem cells on the survivability of diced cartilage grafts. The authors expose stem cells to two different preparations of diced cartilage: (1) diced cartilage in fascia and (2) diced cartilage in Surgicel (Johnson & Johnson, Somerville, N.J.). The most significant finding was that stem cells reduced cartilage resorption in the fascia group from 50 percent to 38 percent. That amounts to a 24 percent improvement in survivability. They also found that fascia prevents more cartilage resorption than Surgicel, and confirm the many studies that demonstrate the benefits of diced cartilage,1–13 some of which join the fading debate of whether fascia or Surgicel is a better envelope. However, there are a few concerning features of their particular experiment along with some positive ones. The untreated diced cartilage group had a 50 percent reduction in postoperative weight. One has to wonder why there was so much resorption. The human corollary indicates that one should expect easily almost complete survival of diced cartilage in fascia. In one of the latest studies,14 there has been no evidence of significant resorption in over 200 cases followed postoperatively for approximately 2 years. Because no benefit was seen in the Surgicel group in this study, where would the benefit be of using stem cells for diced cartilage? If resorption in the animal model they used was 50 percent at 8 weeks, one has to wonder how much higher it would be at 8 months. It is not at all clear why the control group had such a high (50 percent) resorption rate. Could it be that the removal of the specimen was incomplete? Certainly, that might be a possibility in the Surgicel group, in which the authors admitted that the specimens from were “scattered in the subcutaneous tissue.” Furthermore, the only nonhistologic endpoint is “percentage reduction in weight.” We like that this parameter actually incorporates all specimens and is not subjective. The problem, however, is that the units are not listed, and presenting the data as a percentage of reduction can be misleading. The authors do propose possible mechanisms of action by which stem cells exert their beneficial effects on tissue survivability: (1) stimulation of angiogenesis, (2) increasing collagen secretion by fibroblasts, and (3) in vivo differentiation. However, the study itself does not pinpoint the specific mechanism. It is quite possible that stem cells simply achieve their effect by reducing generalized inflammation in the diced cartilage in fascia group even though they were not capable of impacting the known strong inflammatory effects of Surgicel. It is all pure speculation in this particular study. One concerning issue with this study is that it is essentially a nonblinded histologic study. Although histology performed in a nonblinded fashion is an accepted tool with which to explore cellular explanations for certain phenomena, the problem is as follows. In this study, one slide was subjectively selected from each specimen. Each specimen was 3 cm long, and the slides were cut at 5 m; thus, there is the potential for thousands (although more likely hundreds) of slides, and so of course selecting one to then perform all of these studies inherently implies bias. To the authors’ credit, they provide convincing evidence that Surgicel does not work as well as fascia and the differences in percentage weight reduction between group II or IV (both essentially 80 percent) and group I (50 percent) is more impressive. However, a control group of substrate alone would have been ideal. An AlloDerm (LifeCell Corp., Branchburg, N.J.)/acellular dermal From the Divisions of Plastic and Reconstructive Surgery, Stanford University, and the University of California, San Francisco. Received for publication May 31, 2011; accepted June 1, 2011. Copyright ©2012 by the American Society of Plastic Surgeons