Faculty Bibliography

We previously reported that topically applied calreticulin (CRT), a calcium-binding ER chaperone protein comprising N, P, and C domains, markedly enhances diabetic murine (db/db) and porcine cutaneous wound healing. Consistent with the potent wound healing effects, we further showed, in vitro, that exogenous CRT stimulated proliferation of keratinocytes and fibroblasts, induced concentration-dependent migration of these cells and monocytes and macrophages, and upregulated protein expression of collagen, fibronectin, and TGF-beta-3 in fibroblasts. Notably, all these broad-ranging effects purport novel non-ER functions for CRT that act from outside the cell inward. The current studies address: 1) whether the ER chaperone function of CRT is required for its extracellular functions, 2) the molecular structure(s) of CRT that function in its biological activities and 3) the in vitro effects of CRT on diabetic compared to normal mouse and human fibroblasts. Using CRT null mouse embryo fibroblasts (K42) compared to wild type (K41) in proliferation and migration assays (scratch plate and chamber), we show that exogenous CRT stimulates proliferation of null K42 cells to a similar extent as K41 cells (2-fold at 10 pg/ml). However, K42 cells require 100 times more CRT for a peak migratory response (1 vs 100 ng/ml), with a 20% decreased response. We also show that the C domain stimulates fibroblast proliferation to the same extent and peak response as the entire molecule. Finally, we show that fibroblasts isolated from db/db mouse skin and human fibroblasts cultured in high glucose, to simulate type II diabetes, respond to CRT by migration and proliferation albeit with 1/3 less robust response requiring 10-fold more CRT for peak responses compared to controls. The breath of novel non-ER functions of CRT, structure-function relationships, and effects on diabetic cells in vitro underscore this molecule as a potential potent agent for the topical treatment for healing diabetic wounds

BACKGROUND: Management of severe traumatic lower extremity injuries remains a considerable challenge. Free tissue transfer is now a standard part of reconstruction for Gustilo IIIB and IIIC injuries. There is limited information on arterial injury patterns in this population. We undertook a review of our experience to gain insight on vascular injury patterns and surgical outcomes. STUDY DESIGN: A 26-year retrospective analysis was performed of all lower extremity Gustilo IIIB and IIIC injuries requiring microvascular reconstruction at New York University Medical Center. Patient demographics, Gustilo classification, angiographic findings (conventional/computed tomographic angiography/magnetic resonance angiography), recipient vessels, elapsed time from injury, flap choices, and outcomes were examined. RESULTS: Two hundred twenty-two free flaps on 191 patients were performed from September 1982 until March 2008. There were 151 males and 40 females ranging in age from 4 to 83 years (median age 33 years). Patients sustained either Gustilo IIIB (170 patients) or IIIC (21 patients) open fractures. One hundred fifty-four patients had angiograms (78.2% IIIB, 100% IIIC). Sixty-six (42.9%) had normal 3-vessel runoff and 88 (57.1%) were abnormal. Sixty-one patients (31.9%) had anterior tibial injuries, 17 patients (8.9%) had posterior tibial injuries, and 30 (15.7%) had peroneal injuries. Sixty-three complications occurred (11 early thrombosis, 33 requiring secondary procedures, and 10 requiring amputation). CONCLUSIONS: Angiography of severe lower extremity injuries requiring free flap reconstruction usually revealed arterial injury and is generally indicated. In our experience, the anterior tibial artery is most commonly injured and the posterior tibial artery is most likely to be spared and used as a recipient

AlloDerm has been used as a tissue supplement in conjunction with the pectoralis major muscle to provide full coverage over an implant in breast reconstruction. While this method of reconstruction has shown promising results there is little known on the relationship of AlloDerm size and potential immediate expansion volume. A retrospective chart review was completed evaluating all tissue expander or primary implant reconstructions using AlloDerm. Data recorded included: The type/size of implant/expander, dimensions of the AlloDerm used, initial fill volume, number of expansions and time period of expansion. Statistical analysis was completed with a linear regression model. AlloDerm was used on 49 patients (72 reconstructions). Thirty-four patients (50 reconstructions) underwent reconstruction with a tissue expander and 15 patients (22 reconstructions) underwent a single stage reconstruction with a permanent implant. The tissue expander volume filled (cc) could be predicted by 5 x surface area of AlloDerm (cm(2)) - 12 (R(2) = 0.62) and 80 x height of AlloDerm (cm) - 15 (R(2) = 0.59). The tissue expanders could be filled to an average of 75% of total size and required three to four injections in the postoperative period to reach full expansion. Obviously, a requirement for maximal implant expansion is an appropriate skin sparing mastectomy. There is a mathematical relationship between fill volume and surface area as well as height of AlloDerm used in breast reconstruction. This analysis provides a guideline for immediate implant expansion to surgeons using AlloDerm in reconstructive breast surgery

When free tissue transfer is employed for defects of the lower third of the leg, recipient anastomoses are typically performed to major vessels. The aim of this study was to assess soleal perforators located in the distal half of the leg as potential vessels for free flap recipient vessels. Six fresh cadavers (12 limbs) were dissected. Perforators of adequate size (>or=1 mm) were documented as was the location and ease of dissection. Lower extremity magnetic resonance angiograms (MRAs) of 18 extremities were retrospectively reviewed. Two free tissue transfers to lower extremity perforators were presented. Soleal perforators most reliably matched our recipient vessel requirements. Perforators were of adequate size to support free tissue transfer, easy to dissect, and were located at mid/distal fibula level. MRA evaluation confirmed these results. One free tissue reconstruction was performed for trauma (posterior tibial perforator) and one was performed for a chronic radiation wound (peroneal perforator). The soleus muscle is easily exposed and is supplied distally by perforators from both the posterior tibial and the peroneal artery systems. These perforating branches are more accessible than the major lower extremity arteries, making the exposure and anastomosis technically easier and sparing potential iatrogenic injury to critical vessels

OBJECTIVE: Progenitor cells (PCs) contribute to postnatal neovascularization and tissue repair. Here, we explore the mechanism contributing to decreased diabetic circulating PC number and propose a novel treatment to restore circulating PC number, peripheral neovascularization, and tissue healing. RESEARCH DESIGN AND METHODS: Cutaneous wounds were created on wild-type (C57BL/J6) and diabetic (Lepr(db/db)) mice. Blood and bone marrow PCs were collected at multiple time points. RESULTS: Significantly delayed wound closure in diabetic animals was associated with diminished circulating PC number (1.9-fold increase vs. 7.6-fold increase in lin(-)/sca-1(+)/ckit(+) in wild-type mice; P < 0.01), despite adequate numbers of PCs in the bone marrow at baseline (14.4 +/- 3.2% lin(-)/ckit(+)/sca1(+) vs. 13.5 +/- 2.8% in wild-type). Normal bone marrow PC mobilization in response to peripheral wounding occurred after a necessary switch in bone marrow stromal cell-derived factor-1alpha (SDF-1alpha) expression (40% reduction, P < 0.01). In contrast, a failed switch mechanism in diabetic bone marrow SDF-1alpha expression (2.8% reduction) resulted in impaired PC mobilization. Restoring the bone marrow SDF-1alpha switch (54% reduction, P < 0.01) with plerixafor (Mozobil, formerly known as AMD3100) increased circulating diabetic PC numbers (6.8 +/- 2.0-fold increase in lin(-)/ckit(+), P < 0.05) and significantly improved diabetic wound closure compared with sham-treated controls (32.9 +/- 5.0% vs. 11.9 +/- 3% at day 7, P > 0.05; 73.0 +/- 6.4% vs. 36.5 +/- 7% at day 14, P < 0.05; and 88.0 +/- 5.7% vs. 66.7 +/- 5% at day 21, P > 0.05, respectively). CONCLUSIONS: Successful ischemia-induced bone marrow PC mobilization is mediated by a switch in bone marrow SDF-1alpha levels. In diabetes, this switch fails to occur. Plerixafor represents a potential therapeutic agent for improving ischemia-mediated pathology associated with diabetes by reducing bone marrow SDF-1alpha, restoring normal PC mobilization and tissue healing

BACKGROUND/AIMS: Neovascularization involves angiogenesis and vasculogenesis mediated by cytokines and soluble chemokines. The predominant stimulus is ischemia, however, recent data suggest that ionizing radiation (IR) has angiogenic potential. In this study we evaluated whether IR increases vascularity and perfusion in vivo. METHODS: In wild-type mice, a full-thickness, pedicled skin flap was created and isolated for localized irradiation at a dose of 5 Gy. Serial Doppler analysis of the flap was performed. The skin flaps were then harvested at various time points for vascularity and histologic analysis. Blood was concurrently harvested for serum and hematopoietic progenitor cell population analysis. RESULTS: IR to an ischemic flap augmented the angiogenic cytokines SDF-1 and VEGF. Serum MMP-9 and s-kit levels, which are critical for progenitor cell mobilization, were also increased. When hematopoietic progenitor cells were evaluated by Sca1+/Flk1+ cells, a correlate 2-fold increase was seen compared to controls. When the flaps were examined, both vascularity and perfusion were increased. CONCLUSION: In this study we demonstrate that local, low-dose IR upregulates angiogenic chemokines and results in progenitor cell mobilization to the systemic circulation. There is a resultant increase in the vascularity of the irradiated flap, suggesting that the pro-angiogenic effects of IR can be harnessed locally

Diabetes is characterized by several poorly understood phenomena including dysfunctional wound healing and impaired vasculogenesis. p53, a master cell cycle regulator, is upregulated in diabetic wounds and has recently been shown to play a regulatory roles in vasculogenic pathways. We have previously described a novel method to topically silence target genes in a wound bed with small interfering (si)RNA. We hypothesized that silencing p53 results in improved diabetic wound healing and augmentation of vasculogenic mediators. Paired 4-mm stented wounds were created on diabetic db/db mice. Topically applied p53 siRNA, evenly distributed in an agarose matrix, was applied to wounds at postwound day 1 and 7 (matrix alone and nonsense siRNA served as controls). Animals were sacrificed at postwound days 10 and 24. Wound time to closure was photometrically assessed, and wounds were harvested for histology, immunohistochemistry, and immunofluorescence. Vasculogenic cytokine expression was evaluated via Western blot, reverse transcription-polymerase chain reaction, and enzyme-linked immunosorbent assay. The ANOVA/t-test was used to determine significance (p</= 0.05). Local p53 silencing resulted in faster wound healing with wound closure at 18+/-1.3 d in the treated group vs. 28+/-1.0 d in controls. The treated group demonstrated improved wound architecture at each time point while demonstrating near-complete local p53 knockdown. Moreover, treated wounds showed a 1.92-fold increase in CD31 endothelial cell staining over controls. Western blot analysis confirmed near-complete p53 knockdown in treated wounds. At day 10, VEGF secretion (enzyme-linked immunosorbent assay) was significantly increased in treated wounds (109.3+/-13.9 pg/mL) vs. controls (33.0+/-3.8 pg/mL) while reverse transcription-polymerase chain reaction demonstrated a 1.86-fold increase in SDF-1 expression in treated wounds vs. controls. This profile was reversed after the treated wounds healed and before closure of controls (day 24). Augmented vasculogenic cytokine profile and endothelial cell markers are associated with improved diabetic wound healing in topical gene therapy with p53 siRNA

The inflammatory response to ionizing radiation (IR) includes a proangiogenic effect that could be counterproductive in cancer but can be exploited for treating impaired wound healing. We demonstrate for the first time that IR stimulates hypoxia-inducible factor-1alpha (HIF-1alpha) up-regulation in endothelial cells (ECs), a HIF-1alpha-independent up-regulation of stromal cell-derived factor-1 (SDF-1), as well as endothelial migration, all of which are essential for angiogenesis. 5 Gray IR-induced EC HIF-1alpha and SDF-1 expression was greater when combined with hypoxia suggesting an additive effect. While small interfering RNA silencing of HIF-1alpha mRNA and abolition of HIF-1alpha protein induction down-regulated SDF-1 induction by hypoxia alone, it had little effect on SDF-1 induction by IR, demonstrating an independent pathway. SDF-1-mediated EC migration in hypoxic and/or radiation-treated media showed IR induced strong SDF-1-dependent migration of ECs, augmented by hypoxia. IR activates a novel pathway stimulating EC migration directly through the expression of SDF-1 independent of HIF-1alpha induction. These observations might be exploited for stimulation of wound healing or controlling tumor angiogenesis