Faculty Bibliography

The prevention of new blood vessel growth is an increasingly attractive strategy to limit tumor growth. However, it remains unclear whether anti-angiogenesis approaches will impair wound healing, a process thought to be angiogenesis dependent. Results of previous studies differ as to whether angiogenesis inhibitors delay wound healing. We evaluated whether endostatin at tumor-inhibiting doses delayed excisional wound closure. C57/BL6J mice were treated with endostatin or phosphate-buffered solution 3 days prior to the creation of two full-thickness wounds on the dorsum. Endostatin was administered daily until wound closure was complete. A third group received endostatin, but also had daily topical vascular endothelial growth factor applied locally to the wound. Wound area was measured daily and the wounds were analyzed for granulation tissue formation, epithelial gap, and wound vascularity. Endostatin-treated mice showed a significant delay in wound healing. Granulation tissue formation and wound vascularity were significantly decreased, but reepithelialization was not effected. Topical vascular endothelial growth factor application to wounds in endostatin-treated mice resulted in increased granulation tissue formation, increased wound vascularity, and wound closure approaching that of control mice. This study shows that the angiogenesis inhibitor endostatin delays wound healing and that topical vascular endothelial growth factor is effective in counteracting this effect

Ischemia is a known stimulus for vascular growth. Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are believed to contribute to new blood vessel growth, but the mechanism for this contribution is unknown. To elucidate how BM cells are able to form new blood vessels, a novel murine model of soft tissue ischemia was developed in lethally irradiated mice with BM reconstituted from either tie2/lacZ or ROSA/green fluorescent protein (GFP) mice (n = 24). BM-derived EPCs were recruited to ischemic tissue within 72 hours, and the extent of recruitment was directly proportional to the degree of tissue ischemia. At 7 days, there were persistently elevated levels of vascular endothelial growth factor (VEGF) (2.5-fold) and circulating VEGF receptor-2/CD11(-) (flk-1(+)/CD11(-)) cells (18-fold) which correlated with increased numbers of BM-derived EPCs within ischemic tissue. The cells were initially located extravascularly as proliferative clusters. By day 14, these clusters coalesced into vascular cords, which became functional vessels by day 21. In vitro examination of human EPCs from healthy volunteers (n = 10) confirmed that EPC proliferation, adhesion, and chemotaxis were all significantly stimulated in hypoxic conditions. We conclude that BM-derived cells produce new blood vessels via localized recruitment, proliferation, and differentiation of circulating cells in a sequence of events markedly different from existing paradigms of angiogenesis

The identification of bone marrow-derived endothelial progenitor cells has altered our understanding of new blood vessel growth and tissue regeneration. Previously, new blood vessel growth in the adult was thought to only occur through angiogenesis, the sprouting of new vessels from existing structures. However, it has become clear that circulating bone marrow-derived cells can form new blood vessels through a process of postnatal vasculogenesis, with endothelial progenitor cells selectively recruited to injured or ischemic tissue. How this process occurs has remained unclear. One common element in the different environments where vasculogenesis is believed to occur is the presence of a hypoxic stimulus. We have identified the chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 as critical mediators for the ischemia-specific recruitment of circulating progenitor cells. We have found that the endothelial expression of SDF-1 acts as a signal indicating the presence of tissue ischemia, and that its expression is directly regulated by hypoxia-inducible factor-1. Stromal cell-derived factor 1 is the only chemokine family member known to be regulated in this manner. Later events, including proliferation, patterning, and assembly of recruited progenitors into functional blood vessels, are also influenced by tissue oxygen tension and hypoxia. Interestingly, both SDF-1 and hypoxia are present in the bone marrow niche, suggesting that hypoxia may be a fundamental requirement for progenitor cell trafficking and function. As such, ischemic tissue may represent a conditional stem cell niche, with recruitment and retention of circulating progenitors regulated by hypoxia through differential expression of SDF-1

The trafficking of circulating stem and progenitor cells to areas of tissue damage is poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) mediates homing of stem cells to bone marrow by binding to CXCR4 on circulating cells. SDF-1 and CXCR4 are expressed in complementary patterns during embryonic organogenesis and guide primordial stem cells to sites of rapid vascular expansion. However, the regulation of SDF-1 and its physiological role in peripheral tissue repair remain incompletely understood. Here we show that SDF-1 gene expression is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of SDF-1 in ischemic tissue in direct proportion to reduced oxygen tension. HIF-1-induced SDF-1 expression increases the adhesion, migration and homing of circulating CXCR4-positive progenitor cells to ischemic tissue. Blockade of SDF-1 in ischemic tissue or CXCR4 on circulating cells prevents progenitor cell recruitment to sites of injury. Discrete regions of hypoxia in the bone marrow compartment also show increased SDF-1 expression and progenitor cell tropism. These data show that the recruitment of CXCR4-positive progenitor cells to regenerating tissues is mediated by hypoxic gradients via HIF-1-induced expression of SDF-1

Gene therapy is a promising modality for the treatment of soft tissue malignancies. Our laboratory has developed a novel technique of gene transfer using microvascular free flaps that addresses many of the current barriers preventing gene therapy from achieving widespread clinical use. Our previous work has demonstrated our ability to transduce free flaps with an adenovirus encoding the reporter gene lacZ. In this current study, we show that microvascular free flaps can be transduced with an adenovirus encoding the angiogenesis inhibitor endostatin with high levels of local flap expression. These transduced free flaps were able to serve as 'biologic pumps' and were able to secrete endostatin into the serum as demonstrated by enzyme-linked immunosorbent assay. This form of 'biologic brachytherapy' could provide a novel approach for the continuous delivery of therapeutic genes to a localized area while avoiding many of the practical obstacles currently limiting gene therapy

Distraction osteogenesis (DO) requires a long consolidation period and has a low but real failure rate. Bone morphogenic proteins (BMPs) accelerate bone deposition in fractures and critical-sized bone defects, but their effects on mandibular DO are unknown. We investigated the effect of local delivery of adenovirus containing the gene for BMP-2 (Adbmp-2) on mandibular DO in a rat model. Rats (n = 54) were distracted to 3 mm over 6 days. At the start of consolidation (POD 10), Adbmp-2 or adenovirus containing the lacZgene (AdlacZ) was injected directly into the distraction zone. After 1, 2, and 4 weeks of consolidation, mandibles were evaluated for amount of bone deposition. Adbmp-2-treated specimens demonstrated an increased amount of new bone formation by radiographic, histologic, and histomorphometric analysis. This study demonstrates that local, adenovirally-mediated delivery of BMP-2 can increase bone deposition during DO, potentially shortening consolidation and enhancing DO in poorly healing mandibles, such as occurs postirradiation