Daniel J. Ceradini

Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction 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.

Decreasing Intracellular Superoxide Corrects Defective Ischemia-induced New Vessel Formation in Diabetic Mice

Tissue ischemia promotes vasculogenesis through chemokine-induced recruitment of bone marrow-derived endothelial progenitor cells (EPCs). Diabetes significantly impairs this process. Because hyperglycemia increases reactive oxygen species in a number of cell types, and because many of the defects responsible for impaired vasculogenesis involve HIF1-regulated genes, we hypothesized that HIF1 function is impaired in diabetes because of reactive oxygen species-induced modification of HIF1α by the glyoxalase 1 (GLO1) substrate methylglyoxal. Decreasing superoxide in diabetic mice by either transgenic expression of manganese superoxide dismutase or by administration of an superoxide dismutase mimetic corrected post-ischemic defects in neovascularization, oxygen delivery, and chemokine expression, and normalized tissue survival. In hypoxic fibroblasts cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the EPC mobilizing chemokine stromal cell-derived factor-1 (SDF-1) and of vascular epidermal growth factor, which modulates growth and differentiation of recruited EPCs. In hypoxic EPCs cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the SDF-1 receptor CXCR4, and endothelial nitric-oxide synthase, an enzyme essential for EPC mobilization. HIF1α modification by methylglyoxal reduced heterodimer formation and HIF1α binding to all relevant promoters. These results provide a basis for the rational design of new therapeutics to normalize impaired ischemia-induced vasculogenesis in patients with diabetes.

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.

Diabetes impairs endothelial progenitor cell-mediated blood vessel formation in response to hypoxia.

Background: Diabetics suffer from vascular dysfunction with increased risks of coronary artery disease and peripheral vascular disease secondary to an impaired ability to respond to tissue ischemia. Because endothelial progenitor cells are known to home to sites of ischemia and participate in new blood vessel growth, the authors examined the effects of diabetes on human endothelial progenitor cell function and peripheral tissue signaling in hypoxia, and determined whether these cells might be a useful cell-based therapy for diabetic vascular complications. Methods: Circulating human endothelial progenitor cells from type 2 diabetic patients and controls were isolated and subjected to in vitro adhesion, migration, and proliferation assays (n = 5). Cell mobilization and recruitment were studied in vivo in diabetic and nondiabetic environments (n = 6). Exogenous human diabetic and normal cells were analyzed for therapeutic efficacy in a murine ischemia model (n = 6). Results: Adhesion, migration, and proliferation of human diabetic endothelial progenitor cells in response to hypoxia was significantly reduced compared with controls. In diabetic mice, cell mobilization from the bone marrow and recruitment into ischemic tissue was significantly reduced compared with controls. Normal cells injected systemically as replacement therapy in a diabetic mouse increased but did not normalize ischemic tissue survival. Conclusions: These findings suggest that diabetes causes defects in both the endothelial progenitor cell and peripheral tissue responses to hypoxia. These changes in endothelial progenitor cell function and signaling offer a novel explanation for the poor clinical outcome of type 2 diabetics following ischemic events. Based on these findings, it is unlikely that endothelial progenitor cell–based cellular therapies will be able to prevent diabetic complications.

Skin Graft Vascularization Involves Precisely Regulated Regression and Replacement of Endothelial Cells through Both Angiogenesis and Vasculogenesis

Background: Long-term survival of a skin graft is dependent on eventual revascularization. The authors’ aim in the present study was to determine whether skin graft vascularization occurs by (1) simple reconnection of vessels, (2) ingrowth of recipient vasculature, (3) outgrowth of donor-derived vessels, and/or (4) recruitment of bone marrow–derived endothelial progenitor cells. Methods: Full-thickness skin grafts (1 × 1 cm) were transferred between wild-type FVB/N mice (n = 20) and transgenic tie2/lacZ mice (n = 20), where lacZ expression is controlled by the endothelial specific tie2 promoter, allowing differentiation of recipient and donor endothelial cells. The contribution of endothelial progenitor cells to skin graft neovascularization was determined using a bone marrow transplant model where tie2/lacZ bone marrow was transplanted into wild-type mice (n = 20). Results: Vascular regression in the graft was observed at the periphery starting on day 3 and moving centrally through day 21, sparing graft vessels in the absolute center of the graft. At the same time, vascular ingrowth occurred from the wound bed to replace the regressing vessels. Furthermore, bone marrow–derived endothelial progenitor cells contributed to these new vessels starting as early as day 7. Surprisingly, the contribution of bone marrow–derived vessels to the overall process was approximately 15 to 20 percent of new endothelial cells. Conclusions: Replacement of the donor graft vasculature by endothelial and endothelial progenitor cells from the recipient along preexisting channels is the predominant mechanism for skin graft revascularization. This mechanism is likely similar for all nonvascularized free grafts and suggests novel strategies for optimizing the vascularization of tissue constructs engineered in vitro.

Bone morphogenic protein-2 gene therapy for mandibular distraction osteogenesis

Abstract: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.

Jaw in a day: total maxillofacial reconstruction using digital technology.

Background: Tumors of the mandible are complex, often requiring replacement of bone, soft tissue, and teeth. The fibula flap has become a routine procedure in large tumors of the jaw, providing bone and soft tissue at the time of the resection. In current practice, dental reconstruction is delayed for 3 to 6 months, leaving the patient without teeth in the interim. This can be disfiguring and anxiety provoking for the patient. Methods: In this article, the authors present three patients with benign tumors of the mandible who underwent virtually guided resection, fibula reconstruction, and insertion of an implant-retained dental prosthesis in one operation. In addition, the authors report their early experience using this technique in the maxilla. Results: The authors present a case series of three patients with benign mandibular tumors and one patient with a benign maxillary tumor who underwent total reconstruction using computer-aided design and computer-aided manufacturing technology in a single stage. Conclusions: In the right situation, total mandibular reconstruction is possible in a single stage. This is demonstrated by the successful outcomes of these patients. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Increased circulating AC133+ CD34+ endothelial progenitor cells in children with hemangioma.

UNLABELLED Hemangioma is the most common soft-tissue tumor of infancy. Despite the frequency of these vascular tumors, the origin of hemangioma-endothelial cells is unknown. Circulating endothelial progenitor cells (EPCs) have recently been identified as vascular stem cells with the capacity to contribute to postnatal vascular development. We have attempted to determine whether circulating EPCs are increased in hemangioma patients and thereby provide insight into the role of EPCs in hemangioma growth. METHODS AND RESULTS Peripheral blood mononuclear cells (PBMCs) were isolated from hemangioma patients undergoing surgical resection (N = 5) and from age-matched controls (N = 5) undergoing strabismus correction surgery. PBMCs were stained with fluorescent-labeled antibodies for AC133, CD34, and VEGFR2/KDR. Fluorescent-labeled isotype antibodies served as negative controls. Histologic sections of surgical specimens were stained with the specific hemangioma markers Glut1, CD32, and merosin, to confirm the diagnosis of common hemangioma of infancy. EPCs harvested from healthy adult volunteers were stained with Glut1, CD32, and merosin, to assess whether cultured EPCs express known hemangioma markers. Hemangioma patients had a 15-fold increase in the number of circulating CD34 AC133 dual-staining cells relative to controls (0.78+/-0.14% vs.0.052+/-0.017%, respectively). Similarly, the number of PBMCs that stained positively for both CD34 and KDR was also increased in hemangioma patients (0.49+/-0.074% vs. 0.19+/-0.041% in controls). Cultured EPCs stained positively for the known hemangioma markers Glut1, CD32, merosin. CONCLUSIONS This is the first study to suggest a role for EPCs in the pathogenesis of hemangioma. Our results imply that increased levels of circulating EPCs may contribute to the formation of this vascular tumor.

Topical vascular endothelial growth factor reverses delayed wound healing secondary to angiogenesis inhibitor administration.

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.

Functional outcomes of virtually planned free fibula flap reconstruction of the mandible.

Background: The free fibula osteocutaneous flap has become the criterion standard for reconstruction of complex mandibular defects. The authors present their institutional experience with optimization of flap contouring and inset using virtual planning and prefabricated cutting jigs. Methods: All free fibula–based mandible reconstructions performed at the authors’ institution using virtual planning technology between 2009 and 2012 were retrospectively analyzed. The authors evaluated a variety of patient and procedural variables and outcomes. A series of cases performed before virtual planning was reviewed for comparison purposes. Results: Fifty-four reconstructions were performed in 52 patients. Patients were divided evenly between a private university-affiliated medical center and a large county hospital. The most common indications were malignancy (43 percent), ameloblastoma (26 percent), and osteonecrosis/osteomyelitis (23 percent). Thirty percent of patients had irradiation of the recipient site and 38 percent had previous surgery. Sixty-three percent of patients received dental implants, with 47 percent achieving functional dentition. Twenty-five percent of patients had immediate dental implant placement, and 9 percent had immediate dental restoration. Postoperative imaging demonstrated excellent precision and accuracy of flap positioning. Comparison with cases performed before virtual planning demonstrated increased complexity of flap design along with reduced operative time in the virtually planned group. Conclusions: Preoperative virtual planning along with use of prefabricated cutting jigs allows for precise contouring and positioning of microvascular fibula free flaps in mandibular reconstruction. Using this technique, the authors have achieved unprecedented rates of dental rehabilitation along with reduced operative times. The authors believe that virtual planning technologies are an emerging criterion standard in mandible reconstruction. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.