title: Elevation of hemopexin-like fragment of matrix metalloproteinase-2 tissue levels inhibits ischemic wound healing and angiogenesis.
abstract: OBJECTIVE
  Matrix metalloproteinase-2 (MMP-2) degrades type IV collagen and enables endothelial cell (EC) migration during angiogenesis and wound healing. Peroxisomal biogenesis factor 2 (PEX2), a by-product of activated MMP-2 autocatalysis, competitively inhibits newly activated MMP-2 from EC surface binding and migration. We hypothesize that PEX2 is elevated during limb ischemia and contributes to poor wound healing, with decreased capillary density.
  
  
  METHODS
  Western blot was used to identify PEX2 in the hind limbs of FVB/NJ mice with surgically induced ischemia. The PEX2 effect on healing was evaluated by calculating the area of exposed muscle after wounding the dorsum of mice and administering daily injections with human recombinant PEX2 (hrPEX2). Wounds were also injected with lentivirus-expressing PEX2 (PEX2-LV), harvested on postoperative day 7 and processed for staining. Epithelial gap was assessed with light microscopy. Capillary density was evaluated after wounding Tie2-green fluorescent protein (GFP)(+) transgenic FVB mice (ECs labeled green) and viral transduction with PEX2-LV. Wounds were harvested on postoperative day (POD) 7, frozen in liquid nitrogen, sectioned, and stained with Hoechst. Vessel density was assessed via fluorescence microscopy as the average number of capillaries/10 high-powered fields. Paired t test was used to assess differences between the groups.
  
  
  RESULTS
  PEX2 was elevated 5.5 ± 2.0-fold (P = .005) on POD 2 and 2.9 ± 0.69-fold (P = .004) on POD 4 in gastrocnemius muscles of ischemic hind limbs. The wound surface area, or lack of granulation tissue and exposed muscle, decreased daily in all mice but was greater in the hrPEX2-treated mice by 12% to 16% (P < .004). Wounds in the control group were completely covered with granulation tissue by POD 3. Wounds injected with hrPEX2 were not completely covered by POD 7 but continued to have exposed muscle. Microscopic examination of wounds after PEX2-LV viral transduction demonstrated an average epithelial gap of 1.6 ± 0.3 vs 0.64 ± 0.3 μm in control wounds (P < .04). Wounds from Tie2-GFP mice had an average number of 3.8 ± 1.1 capillaries vs 6.9 ± 1.2 in control wounds (P < .007).
  
  
  CONCLUSIONS
  Our study links elevated PEX2 to ischemia and poor wound healing. We demonstrate comparative PEX2 elevation in ischemic murine hind limbs. Less granulation tissue is produced and healing is retarded in wounds subjected to hrPEX2 or viral transduction with PEX2-LV. Microscopic examination shows the wounds exhibit fewer capillaries, supporting the hypothesis that PEX2 decreases angiogenesis.
authors: Division of Vascular Surgery, The DeWitt Daughtry Family Department of Surgery, Leonard M. Miller School of Medicine, University of Miami, Miami, FL 33136, USA.
authors: April E
authors: AE
authors: Nedeau
authors: Katherine A
authors: KA
authors: Gallagher
authors: Zhao-Jun
authors: ZJ
authors: Liu
authors: Omaida C
authors: OC
authors: Velazquez
mesh_terms: D043165:Angiostatic Proteins / Q000008:administration & dosage / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002196:Capillaries / Q000201:enzymology / Q000503:physiopathology; D004195:Disease Models, Animal; D004789:Enzyme Activation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006614:Hindlimb; D006801:Humans; D007511:Ischemia / Q000201:enzymology* / Q000473:pathology / Q000503:physiopathology; D017078:Laser-Doppler Flowmetry; D016086:Lentivirus / Q000235:genetics; D020778:Matrix Metalloproteinase 2 / Q000008:administration & dosage / Q000378:metabolism*; D008565:Membrane Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000201:enzymology / Q000473:pathology; D018919:Neovascularization, Physiologic; D010446:Peptide Fragments / Q000008:administration & dosage / Q000378:metabolism*; D000074428:Peroxisomal Biogenesis Factor 2; D011401:Promoter Regions, Genetic; D042787:Receptor, TIE-2 / Q000235:genetics; D012039:Regional Blood Flow; D013997:Time Factors; D015854:Up-Regulation; D014945:Wound Healing
chemical_list: D043165:Angiostatic Proteins; D008565:Membrane Proteins; C000614230:PEX2 protein, human; C000614231:PEX2 protein, mouse; D010446:Peptide Fragments; D000074428:Peroxisomal Biogenesis Factor 2; D049452:Green Fluorescent Proteins; D042787:Receptor, TIE-2; D020778:Matrix Metalloproteinase 2; C522362:Mmp2 protein, mouse