Angiopoietin-1 improves endothelial progenitor cell-dependent neovascularization in diabetic wounds.
BACKGROUND
The diabetic phenotype of wound healing is in part characterized by impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Angiopoietin-1 (Ang-1) is a potent mobilizer of EPCs from the bone marrow (BM). A suggested mechanism for EPC mobilization from the BM is mediated by matrix metalloproteinase 9 (MMP-9) and stem cell factor (SCF). Taken together, we hypothesized that overexpression of Ang-1 in diabetic wounds will recruit EPCs and improve neovascularization and wound healing.


METHODS
An endothelial lineage BM-labeled murine model of diabetes was developed to track BM-derived EPCs. FVBN mice were lethally irradiated and then reconstituted with BM from syngeneic Tie2/LacZ donor mice. Diabetes was induced with streptozotocin. Dorsal wounds in BM-transplanted mice were treated with Ad-Ang-1, Ad-GFP, or phosphate-buffered saline. At day 7 after injury, wounds were harvested and analyzed. A similar experiment was conducted in EPC mobilization deficient MMP-9 -/- mice to determine whether the effects of Ang-1 were EPC-dependent.


RESULTS
Overexpression of Ang-1 resulted in greatly improved re-epithelialization, neovascularization, and EPC recruitment in diabetic BM-transplanted wounds at day 7. Ang-1 treatment resulted in increased serum levels of proMMP-9 and SCF but had no effect on vascular endothelial growth factor levels. According to our FACS results, peripheral blood EPC (CD34(+)/Cd133(+)/Flk1(+)) counts at day 3 after wounding showed impaired EPC mobilization in MMP-9 -/- mice compared with those of wild-type controls. EPC mobilization was rescued by SCF administration, validating this model for EPC-mobilization-deficient mechanistic studies. In MMP-9 -/- mice, Ad-Ang-1 accelerated re-epithelialization in a similar manner, but had no effect on neovascularization.


CONCLUSIONS
Our results show that Ang-1 administration results in improved neovascularization which is dependent on EPC recruitment and has direct effects on wound re-epithelialization. These data may represent a novel strategy to correct the phenotype of impaired diabetic neovascularization and may improve diabetic wound healing.
Laboratory for Regenerative Wound Healing, Division of Pediatric, General, Thoracic and Fetal Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
Swathi
S
Balaji
Laboratory for Regenerative Wound Healing, Division of Pediatric, General, Thoracic and Fetal Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
Nate
N
Han
Laboratory for Regenerative Wound Healing, Division of Pediatric, General, Thoracic and Fetal Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
Chad
C
Moles
Laboratory for Regenerative Wound Healing, Division of Pediatric, General, Thoracic and Fetal Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
Aimen F
AF
Shaaban
Division of Infectious Diseases, Department of Medicine, Stanford University School of Medicine, Stanford, CA.
Paul L
PL
Bollyky
Laboratory for Regenerative Wound Healing, Division of Pediatric, General, Thoracic and Fetal Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH; Center for Children's Surgery, Children's Hospital Colorado and the University of Colorado School of Medicine, Aurora, CO.
Timothy M
TM
Crombleholme
Laboratory for Regenerative Wound Healing, Division of Pediatric, General, Thoracic and Fetal Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH. Electronic address: sgkeswan@texaschildrens.org.
Sundeep G
SG
Keswani
D043925:Angiogenesis Inducing Agents / Q000008:administration & dosage / Q000494:pharmacology*; D042683:Angiopoietin-1 / Q000008:administration & dosage / Q000494:pharmacology*; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism / Q000473:pathology*; D066026:Endothelial Progenitor Cells / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D051379:Mice; D018345:Mice, Knockout; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D016896:Treatment Outcome; D014945:Wound Healing / Q000187:drug effects*
D043925:Angiogenesis Inducing Agents; D042683:Angiopoietin-1; D015415:Biomarkers