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. PEX2 is a byproduct of activated MMP-2 autocatalysis and competitively inhibits newly activated MMP-2 from EC surface binding and migration. We hypothesize that PEX2 is elevated during limb ischemia, contributing to poor wound healing by interfering with angiogenesis. We aim to identify elevated PEX2 in ischemic murine hindlimb muscle and demonstrate poor healing with decreased capillary density. Methods Western blot was used to identify PEX2 in hindlimbs of FVB/NJ mice with surgically induced ischemia. The PEX2 effect on healing was evaluated by calculating area of exposed muscle after wounding the dorsum of mice and performing daily injections with recombinant PEX2 (hrPEX2). Additionally, wounds were injected with lentivirus expressing PEX2 (PEX2-LV), harvested on post operative day 7 (POD 7), fixed and sectioned for staining with hematoxylin and eosin (H&E). Epithelial gap was assessed with light microscopy. Capillary density was evaluated after wounding Tie2-GFP+ transgenic FVB mice (ECs labeled green) and viral transduction with PEX2-LV. Wounds were harvested on POD 7, frozen in liquid nitrogen, sectioned and stained with Hoechst. Vessel density was assessed via fluorescence microscopy as average number of capillaries per ten high powered fields (HPF). Paired Student’s t-test was used to assess differences between the groups. Results PEX2 was elevated 5.5-fold (±2.0, P= .005) on POD 2 and 2.9-fold (±0.69, P= .004) on POD 4 in gastrocnemius muscles of ischemic hindlimbs. The wound surface area, or lack of granulation tissue and exposed muscle, decreased daily in all mice, but was greater in the hrPEX mice by 12% to 16% (P< .004). Wounds in the control group were completely covered with granulation tissue by POD 3, whereas 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±0.3μm versus 0.64±0.3 μm in control wounds (P< .04). Wounds from Tie-2-GFP mice had an average number of 3.8±1.1 capillaries versus 6.9±1.2 in control wounds (P< .007). Conclusion Our study is the first report linking elevated PEX2 to ischemia and poor wound healing. We demonstrate comparative PEX2 elevation in ischemic murine hindlimbs. Wounds subjected to hrPEX2 or viral transduction with PEX2-LV produce less granulation tissue and retarded healing. Microscopic evaluation of the wounds exhibit fewer capillaries, supporting the hypothesis that PEX2 decreases angiogenesis.

Proteome analysis reveals antiangiogenic environments in chronic wounds of diabetes mellitus type 2 patients

“…Further, more peptides representing the hemopexin‐like region of MMP2 were detected in the chronic wounds. Autocatalytic cleavage of this region generates an antiangiogenic peptide , which inhibits cell migration and cell adhesion of heparin‐binding growth factor and vitronectin. Assumedly, elevated expression level of MMP2 should in turn generate high concentration of free hemopexin in PHW and thus decrease angiogenic stimulation.…”

In�vitro study on human umbilical cord mesenchymal stem cells transfected with lentivirus‑mediated hNIS‑EGFP dual reporter gene and co‑labeled with superparamagnetic iron oxide

“…As it is an endogenous gene, it is expressed in different degrees by several organs and tissues, such as the thyroid, gastric mucosa, mammary glands and salivary glands, which affects the monitoring of hNIS gene expression ( 30 ). In addition, in non-thyroid tissues and cells, iodine is released quickly because of a lack of thyroid specific proteins that hold iodine, therefore radioactive iodine does not accumulate ( 31 – 33 ). Although this reduces radiation damages to tissues, the signal detection is also affected.…”

“…The shortening of T2 is more marked, manifesting as the decrease in T2 signals ( 45 ). SPIO is negatively charged, and so in the present study, SPIO was coated with positively charged polylysine to reduce electrostatic interaction ( 29 , 32 , 33 , 46 , 47 ). Thus, SPIO was able to bind with cell surface receptors via electrostatic interaction and enter into cells via endocytosis.…”

Effects of TRAIL and taurolidine on apoptosis and proliferation in human rhabdomyosarcoma, leiomyosarcoma and epithelioid cell sarcoma

“…GADD45 proteins co-operate in the activation of S and G2-M checkpoints following the exposure of cells to UV irradiation and other genotoxic stresses, thereby inducing growth arrest and apoptosis (55). The mechanisms by which GADD45 proteins function in negative growth control is not fully understood, although upregulation of these proteins was reported to be associated with increased apoptosis and p53-independent cell cycle arrest in a variety of soft tissue sarcomas (56). In a recent immunohistochemical study, high expression of GADD45 was associated with reduced invasiveness of chondrosarcomas, suggesting its potential diagnostic value in the histological grading of malignant chondrogenic tumours (57).…”