Division of Vascular Surgery, The DeWitt Daughtry Family Department of Surgery, Leonard M. Miller School of Medicine, University of Miami, Miami, FL 33136, USA.
J Vasc Surg. 2011 Nov;54(5):1430-8. doi: 10.1016/j.jvs.2011.05.029. Epub 2011 Sep 8.
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.
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.
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).
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.
基质金属蛋白酶-2（MMP-2）可降解 IV 型胶原，促进血管生成和伤口愈合过程中的内皮细胞（EC）迁移。过氧化物酶体生物发生因子 2（PEX2）是 MMP-2 自催化激活的副产物，竞争性抑制新激活的 MMP-2 与 EC 表面结合和迁移。我们假设 PEX2 在肢体缺血时升高，并导致毛细血管密度降低，从而导致伤口愈合不良。
使用 Western blot 鉴定手术诱导缺血的 FVB/NJ 小鼠后肢中的 PEX2。通过计算小鼠背部受伤后暴露的肌肉面积来评估 PEX2 对愈合的影响，并每天给予人重组 PEX2（hrPEX2）注射。还将伤口注射表达 PEX2 的慢病毒（PEX2-LV），并在术后第 7 天收获并进行染色。用光学显微镜评估上皮间隙。用 Tie2-绿色荧光蛋白（GFP）（+）转基因 FVB 小鼠（EC 标记为绿色）进行伤口损伤，并通过病毒转导用 PEX2-LV 处理后，评估毛细血管密度。术后第 7 天收获伤口，液氮冷冻，切片，并用 Hoechst 染色。通过荧光显微镜评估血管密度作为每 10 个高倍视野的平均毛细血管数。使用配对 t 检验评估组间差异。
缺血后肢腓肠肌中 PEX2 在 POD2 时升高 5.5 ± 2.0 倍（P =.005），在 POD4 时升高 2.9 ± 0.69 倍（P =.004）。所有小鼠的伤口表面积（或缺乏肉芽组织和暴露的肌肉）每天都在减少，但在接受 hrPEX2 治疗的小鼠中减少了 12%至 16%（P <.004）。对照组的伤口在 POD3 时完全被肉芽组织覆盖。接受 hrPEX2 注射的伤口在 POD7 时未完全覆盖，但仍有暴露的肌肉。PEX2-LV 病毒转导后伤口的显微镜检查显示，上皮间隙的平均宽度为 1.6 ± 0.3μm，而对照组为 0.64 ± 0.3μm（P <.04）。来自 Tie2-GFP 小鼠的伤口平均有 3.8 ± 1.1 个毛细血管，而对照组有 6.9 ± 1.2 个（P <.007）。
我们的研究将升高的 PEX2 与缺血和伤口愈合不良联系起来。我们证明了缺血性小鼠后肢中存在可比的 PEX2 升高。在接受 hrPEX2 或 PEX2-LV 病毒转导的伤口中，产生的肉芽组织较少，愈合受到延迟。显微镜检查显示伤口的毛细血管较少，支持 PEX2 减少血管生成的假设。
