GDF11 promotes wound healing in diabetic mice via stimulating HIF-1ɑ-VEGF/SDF-1ɑ-mediated endothelial progenitor cell mobilization and neovascularization

Abstract

Non-healing diabetic wounds (DW) are a serious clinical problem that remained poorly understood. We recently found that topical application of growth differentiation factor 11 (GDF11) accelerated skin wound healing in both Type 1 DM (T1DM) and genetically engineered Type 2 diabetic db/db (T2DM) mice. In the present study, we elucidated the cellular and molecular mechanisms underlying the action of GDF11 on healing of small skin wound. Single round-shape full-thickness wound of 5-mm diameter with muscle and bone exposed was made on mouse dorsum using a sterile punch biopsy 7 days following the onset of DM. Recombinant human GDF11 (rGDF11, 50 ng/mL, 10 μL) was topically applied onto the wound area twice a day until epidermal closure (maximum 14 days). Digital images of wound were obtained once a day from D0 to D14 post-wounding. We showed that topical application of GDF11 accelerated the healing of full-thickness skin wounds in both type 1 and type 2 diabetic mice, even after GDF8 (a muscle growth factor) had been silenced. At the cellular level, GDF11 significantly facilitated neovascularization to enhance regeneration of skin tissues by stimulating mobilization, migration and homing of endothelial progenitor cells (EPCs) to the wounded area. At the molecular level, GDF11 greatly increased HIF-1ɑ expression to enhance the activities of VEGF and SDF-1ɑ, thereby neovascularization. We found that endogenous GDF11 level was robustly decreased in skin tissue of diabetic wounds. The specific antibody against GDF11 or silence of GDF11 by siRNA in healthy mice mimicked the non-healing property of diabetic wound. Thus, we demonstrate that GDF11 promotes diabetic wound healing via stimulating endothelial progenitor cells mobilization and neovascularization mediated by HIF-1ɑ-VEGF/SDF-1ɑ pathway. Our results support the potential of GDF11 as a therapeutic agent for non-healing DW.

Keywords: GDF11; HIF-1ɑ; VEGF/SDF-1α; diabetic wound; endothelial progenitor cell; neovascularization.

Fig. 1. GDF11 induces neovascularization in diabetic wounds as revealed by histopathological analyses in STZ-induced T1DM mice.

a Typical examples of hematoxylin and eosin (H&E) staining showing the enhanced granulation tissue deposition (indicated by vessels, fibers and inflammatory cells) and neovascularization (black arrows in the right panels), and simultaneously decreased distance between epithelial tips (as indicated by the shortened distance between the blue arrows) induced by topical administration of rGDF11 (50 ng/mL) in the wounded skin area on day 5 following wound creation. Note the increased number of vessels with rGDF11 treatment, as indicated by red circular structures pointed by dark arrows. The distance between epithelial tips was defined by blue arrows, and area of granulation was outlined by red lines and labeled with “G”. Magnification: ×30 for left panels and 365× for right panels. Similar results were consistently observed in another two groups of animals. *P < 0.05, **P < 0.01 vs. Ctl; ^#P < 0.05, vs. T1DM; n = 3. b Typical examples of CD31 staining showing the enhanced neovascularization induced by topical administration of rGDF11 (50 ng/mL) in the wounded skin area on day 7 following wound creation. Note the increased number of vessels following rGDF11 treatment, as indicated by brown circular structures pointed by dark (upper panels) and yellow (lower panels) arrows. Magnification: ×200. Similar results were consistently observed in another two experiments. **P < 0.01 vs. Ctl; ^##P < 0.01 vs. T1DM; n = 3–4. (Mean ± SEM; ANOVA followed by Dunnett’s test for comparisons among multiple groups, and Student’s t-test for comparisons between two groups).

Fig. 2. DF11 induces neovascularization in diabetic wounds as revealed by MicroPET/CT analysis and chick embryo chorioallantoic membrane (CAM) assay in STZ-induced T1DM mice.

a, b Representative MicroPET/CT images and photographs of the wounded skin area (day 7) showing the enhanced neovascularization induced by topical application of rGDF11 (50 ng/mL). Note the markedly increased number of vessels with rGDF11 treatment. **P < 0.01 vs. Ctl, ^#P < 0.05 vs. T1DM; n = 4. c Left panel: typical examples of the chick embryo chorioallantoic membrane (CAM) examination showing the enhanced neovascularization induced by topical administration of rGDF11 (50 ng/mL) in the wounded skin area; right panel: averaged relative blood vessel density. *P < 0.05 vs. Ctl or BSA; n = 3. d Left panel: Representative images (×100 magnification) showing the effect of rGDF11 (50 ng/mL; incubation for 24 h) on the tube formation in HUVEC cells with Matrigel assay; right panel: statistical data on the relative number of tubes quantified using ImageJ software. *P < 0.05 vs. Ctl or BSA; n = 3. (Mean ± SEM; ANOVA followed by Dunnett’s test for comparisons among multiple groups, and Student’s t-test for comparisons between two groups).

Fig. 3. GDF11 promotes mobilization, migration and homing of epithelial progenitor cells (EPCs) and tube formation.

a Immunofluorescent localization (left panel; magnification ×600) and quantification (right panel) of CD34⁺/KDR⁺ EPCs (mean intensities from merged signals) in the wounded area (the fifth day after creation of a dermal wound) as an indication of EPC homing to the lesion. Shown are the ratios of EPCs over total cells. Note that the number of CD34⁺/KDR⁺ cells was considerably lower in T1DM mice than in non-DM control counterparts and treatment with rGDF11 restored the number of EPCs in the wounded area. *P < 0.05 vs. Ctl, ^#P < 0.05 vs. T1DM; n = 5–9 for each group. b Transwell assay for the effect of rGDF11 on migration of EPCs isolated from healthy mice. Note that the ability of EPC migration was substantially enhanced by rGDF11. BSA was used as a vehicle control. Magnification: ×100. **P < 0.01 vs. Ctl or BSA; n = 11–22 for each group. c Matrigel assay for the effect of rGDF11 on tube formation of EPCs. Compared with the control groups, rGDF11-treated cells exhibited larger tube area and longer tube length. Magnification: ×100. **P < 0.01 vs. Ctl or BSA; n = 8–15. (Mean ± SEM; ANOVA followed by Dunnett’s test for comparisons among multiple groups, and Student’s t-test for comparisons between two groups).

Fig. 4. Abnormal downregulation of neovascularization-related genes/proteins HIF-1α, VEGF, and SDF-1α, and restoration of their expression by GDF11.

a Expression downregulation of mRNA levels of HIF-1α, VEGF, and SDF-1α in the wounds of T1DM mice and restoration of their expression by rGDF11. *P < 0.05 vs. Ctl, ^##P < 0.01 vs^. T1DM; n = 4–6 for each group. b Expression downregulation of protein levels of HIF-1α, VEGF, and SDF-1α in the wounds of T1DM mice and restoration of their expression by rGDF11. *P < 0.05 & **P < 0.01 vs. Ctl, ^##P < 0.01 vs. T1DM; n = 4–6 for each group. (Mean ± SEM; ANOVA followed by Dunnett’s test for comparisons among multiple groups, and Student’s t-test for comparisons between two groups).

Fig. 5. Verification of the role of HIF-1α in mediating the diabetic wound healing-promoting action of GDF11.

a Photographs and mean data of dermal wounds showing the counteracting action of HIF-1α inhibitor PX-478 (20 µM; px) to the accelerating effects of GDF11 on wound healing. *P < 0.05 & **P < 0.01 vs. Ctl; ^#P < 0.05 & ^##P < 0.01 vs. T1DM; ^§P < 0.05 & ^§§P < 0.01 vs. T1DM-rGDF11; n = 5–7 for each group. b Immunofluorescence staining of EPCs showing the counteracting action of HIF-1α inhibitor PX-478 (px) to the promoting effects of GDF11 on EPC homing to the lesion. Mean data from the merged signals are expressed as the ratios of EPCs over total cells. Magnification: ×600. *P < 0.05 vs. Ctl, ^##P < 0.01 vs. T1DM, ^§P < 0.05 vs. T1DM-rGDF11; n = 5–9 for each group. c Flow cytometry analysis demonstrating the counteracting action of HIF-1α inhibitor PX-478 (px) to the enhancing effects of GDF11 on EPC homing of wounded skin in day 5. **P < 0.01 vs. Ctl, ^###P < 0.001 vs. T1DM, ^&&P < 0.05 vs. T1DM + rGDF11; n = 3 for each group. d MicroPET/CT images of the skin wound depicting the counteracting action of HIF-1α inhibitor PX-478 (20 µM; px) to the beneficial effects of GDF11 on the growth of vessels. e Transwell assay demonstrating the counteracting action of HIF-1α inhibitor PX-478 (px) to the enhancing effects of GDF11 on EPC migration. Magnification: ×100. **P < 0.01 vs. Ctl, ^##P < 0.01 vs. T1DM; n = 8–22 for each group. f Matrigel assay demonstrating the counteracting action of HIF-1α inhibitor PX-478 (px) to the enhancing effects of GDF11 on tube formation. Magnification: ×100. **P < 0.01 vs. Ctl, ^##P < 0.01 vs. T1DM; n = 7–12 for each group. (Mean ± SEM; ANOVA followed by Dunnett’s test for comparisons among multiple groups, and Student’s t-test for comparisons between two groups).

Fig. 6. Downregulation of GDF11 in diabetic skin wounds delays the healing process.

a Downregulation of endogenous GDF11 (eGDF11) at the mRNA level around the wounded area of T1DM mice relative to non-DM control mice from day 1 to day 14 after creation of skin wound. **P < 0.01 vs. non-DM control; n = 8–11 for each group. b Downregulation of endogenous GDF11 (eGDF11) at the protein level around the wounded area of T1DM mice. Left panel:.Western blot results. Right panel: verification of specificity of the anti-GDF11 antibody using the purified recombinant GDF11 and GDF8. No cross-reaction was noted between GDF11 and GDF8; that is, the anti-GDF11 antibody recognized only rGDF11 without picking up GDF8. *P < 0.05 vs. control; n = 3 for each group. c Upper panel: representative photographs showing the time-dependent closure of wounds in otherwise healthy and the wound healing-retarding effect of GDF11 antibody. Lower left panel: averaged raw values of remaining wound area as a function of time (day). Lower middle panel: averaged normalized values of remaining wound area as a function of time (day). The data collected at varying time points were normalized to those at 0 time point. Lower right panel: averaged values of wound closure as a function of time (day), expressed as percentage of wound closure. *P < 0.05 vs. Ctl; n = 6–12 for each group. d Verification of the efficacy of GDF11 antibody in inhibiting GDF11 function as indicated by the failure of Smad2/3 activation (decreases in p-Smad/2/3) in HUVECs. **P < 0.01 vs. Ctl, ^##P < 0.01 vs. rGDF11; n = 4 for each group. (Mean ± SEM; ANOVA followed by Dunnett’s test for comparisons among multiple groups, and Student’s t-test for comparisons between two groups).

Fig. 7. Downregulation of GDF11 in diabetic skin wounds delays the healing process.

a Upper panels: representative photographs showing the time-dependent closure of wounds in otherwise healthy mice and the wound healing-retarding effect upon silencing GDF11 by AAV8-sgGDF11 (“sg” represents small guide RNA for silencing GDF11 expression). Lower panels: averaged raw values of remaining wound area as a function of time (day; left panels); averaged normalized values of remaining wound area as a function of time (day; middle panel). The data collected at varying time points were normalized to those at 0 time point; averaged values of wound closure as a function of time (day; right panels), expressed as percentage of wound closure. *P < 0.05 & **P < 0.01 vs. Ctl; n = 4 for each group. b Silencing of eGDF11 reduced the mRNA (left) levels of HIF-1α and VEGF in HUVECs (left two panels). *P < 0.05 & **P < 0.01 vs. Ctl & NC (siNC); n = 3–5 for each group. Silencing of eGDF11 reduced the protein (right) levels of HIF-1α and VEGF in HUVECs (right two panels). *P < 0.05 vs. Ctl & NC (siNC); n = 3 for each group. c Silencing of eGDF11 weakened the ability of migration of EPCs isolated from healthy mice, as revealed by Transwell assay. Magnification: ×100. **P < 0.01 vs. Ctl & NC (siNC); n = 10–13 for each group. d Silencing of eGDF11 weakened tube formation of EPCs isolated from healthy mice, as revealed by Matrigel assay. Magnification: ×100. **P < 0.01 vs. Ctl & NC (siNC); n = 8–12 for each group. (Mean ± SEM; ANOVA followed by Dunnett’s test for comparisons among multiple groups, and Student’s t-test for comparisons between two groups).