Peroxisome proliferator-activated receptors-mediated diabetic wound healing regulates endothelial cells' mitochondrial function via sonic hedgehog signaling - PubMed (original) (raw)
. 2025 Sep 10:13:tkaf063.
doi: 10.1093/burnst/tkaf063. eCollection 2025.
Fugang Xiao 1, Qin Li 1, Mengling Yang 1, Linrui Dai 1, Shiyan Yu 1, Xiaoshi Zhang 1, Xiaoyan Jiang 1, Seungkuk Ahn 2, Wenxin Wang 2, David G Armstrong 3, Hongyan Wang 1, Guangbin Huang 4, Wuquan Deng 1 5
Affiliations
- PMID: 41216186
- PMCID: PMC12597028
- DOI: 10.1093/burnst/tkaf063
Peroxisome proliferator-activated receptors-mediated diabetic wound healing regulates endothelial cells' mitochondrial function via sonic hedgehog signaling
Shunli Rui et al. Burns Trauma. 2025.
Abstract
Background: Diabetic foot ulcer (DFU) is a common and debilitating complication of diabetes, often leading to delayed wound healing. The peroxisome proliferator-activated receptors (PPARs) play a crucial role in regulating cellular metabolism and promoting angiogenesis. This study aims to elucidate the mechanisms through which the activation of PPARs enhances wound healing, particularly under diabetic conditions, as these mechanisms remain inadequately understood.
Methods: Differentially expressed genes in DFU wounds and normal skin tissues were identified using the GEO database. PPAR expression in DFU neovascularization was validated by quantitative reverse transcription polymerase chain reaction, immunofluorescence, and western blotting. In vivo, diabetic mice treated with PPAR agonists (chiglitazar) underwent wound healing assessment, including collagen deposition and angiogenesis. In vitro, advanced glycation end-products (AGEs)-induced endothelial cell models were used to evaluate PPAR activation effects on cell migration, tube formation, and mitochondrial function. Whole transcriptome sequencing and mitochondrial analysis were performed to explore the underlying mechanisms, particularly the sonic hedgehog (SHH)-mitochondrial axis.
Results: PPAR expression was significantly downregulated in DFU tissues (p < 0.05), and PPAR activation in diabetic mice enhanced wound healing, collagen deposition, granulation tissue proliferation, and angiogenesis (p < 0.05). In vitro, PPAR activation protected endothelial cells, promoting vascular endothelial growth factor-A (VEGF-A) and CD31 expression, reducing apoptosis, and enhancing cell migration and tube formation (p < 0.05). Mechanistically, PPARs activated mitochondrial oxidative phosphorylation and membrane function through the SHH signaling pathway. SHH gene silencing reversed the effects of PPAR activation on mitochondrial function and angiogenesis.
Conclusions: PPAR signaling plays a critical role in DFU healing, with its inhibition linked to vascular dysfunction. Activation of the PPARs/SHH-mitochondrial axis significantly enhances endothelial cell metabolism and angiogenesis. This study provides insights into the molecular mechanisms of diabetic wound healing and supports the clinical potential of PPAR agonists for DFU treatment.
Keywords: Diabetic wound healing; Endothelial function; Oxidative phosphorylation; Peroxisome proliferator-activated receptors; Sonic hedgehog.
© The Author(s) 2025. Published by Oxford University Press.
Conflict of interest statement
None declared.
Figures
Graphical Abstract
Figure 1
Dysregulation of PPARs expression in DFU. (a) Heat map of DEGs (adj. p < 0.01 based on sequencing results). (b) Volcano map of DEGs (adj. p < 0.01 based on sequencing results). (c) KEGG pathway enrichment of DEGs. (d) A qRT-PCR analysis was conducted to evaluate the relative mRNA expression levels of PPARs in the skin, comparing healthy individuals to patients diagnosed with T2DM, n = 4. (e, f, g, h, i, j) Representative immunofluorescence images and analyses of PPARs (labeled with Alexa Fluor 555) expression levels in skin neovascularization (CD31, labeled with Alexa Fluor 488), comparing normal individuals to patients with T2DM. The cell nucleus was stained with DAPI (blue), n = 3 (scale bar: 100 μm). (k, l) The relative expression levels of PPARs in skin tissue were compared between normal individuals and patients with T2DM using western blot analysis, n = 4. The results are expressed as mean ± SD. *p < 0.5, **p < 0.01, ***p < 0.01; ns, not significant. DFU diabetic foot ulcer, SD standard deviation
Figure 2
Activating PPAR signaling enhances wound healing in diabetic mice. (a) A systematic evaluation protocol for the assessment of skin wound healing. (b, c) Images and mode patterns depicting wound closure in the NC, Ros (10 mg/kg), Chi-10 (10 mg/kg), and Chi-30 (30 mg/kg) groups at days 0, 3, 5, 7, and 9 post-operation. (d) Wound closure rates were quantified using ImageJ software by calculating the percentage of closure relative to the day 0 wound size, and comparisons were made between the Ros, Chi-10, and Chi-30 groups against the NC group, n = 10. (e, f, g) Quantitative assessment of neo-epithelium gap width and re-epithelization degree, marked by horizontal black lines, n = 3 (scale bar: 1000 μm). (h, i) Cutaneous wound sections were subjected to Masson’s trichrome staining, n = 3 (scale bar: 1000 μm). The results are expressed as mean ± SD. *P < .05, **P < .01, ***P < .001; ns, not significant. PPAR peroxisome proliferator-activated receptor, Ros rosiglitazone, SD standard deviation
Figure 3
PPAR signaling activation enhances angiogenesis in vivo. (a, b, c) Representative immunofluorescence images and analyses for detection of PPARs (labeled with Alexa Fluor 555) expression levels in neovascularization (CD31, labeled with Alexa Fluor 488) of skin wounds on day 9 of the diabetic wound healing model after Chi intervention, n = 3 (scale bar: 100 μm). (d) The relative level of PPAR expression in skin tissue during the diabetic wound healing model after Chi intervention was detected by western blot, n = 4. (e) The relative level of CD31 and VEGF-A expression in skin tissue during the diabetic wound healing model after Chi intervention was detected by western blot, n = 4. (f) Images and quantification of immunofluorescence staining for CD31 and α-SMA to reflect the degree of neovascularization, n = 3 (scale bar: 400 μm). (g) Images and quantification of immunofluorescence staining for VEGF-A in skin wounds on day 9 after Chi intervention, n = 3 (scale bar: 400 μm). (h) Representative immunofluorescence images and analyses for Ki67 in skin wounds on day 9 after Chi intervention, n = 3 (scale bar: 400 μm). The results are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. PPAR peroxisome proliferator-activated receptor, Ros rosiglitazone, SD standard deviation, VEGF-A vascular endothelial growth factor A
Figure 4
Enhancing the functionality of HUVECs by activating PPARs signaling in vitro. Cells were pretreated with 200 μg/ml AGEs for 24 h, followed by exposure to 1 μM Chi for an additional 48 h. (a, b, c, d) Cell scratch assays were observed in living cells at 0, 8, 16, and 24 h, n = 4 (scale bar: 500 μm). (e, f) Transwell assays were conducted for evaluation of HUVEC migration, n = 4 (scale bar: 100 μm). (g, h) Tube formation assays were conducted to measure segment length and evaluate HUVECs’ tube formation capability, n = 3 (scale bar: 100 μm). (i, j) HUVEC cell apoptosis were measured by flow cytometry, n = 3. (k, l) Representative immunofluorescence images and analyses for CD31 in HUVECs after intervention, n = 3 (scale bar: 200 μm). (m, n) Representative immunofluorescence images and analyses for VEGF-A in HUVECs after intervention, n = 3 (scale bar: 200 μm). (o, p, q) The relative levels of CD31 and VEGF-A in HUVECs after intervention were measured by western blot, n = 4. The results are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. PPAR peroxisome proliferator-activated receptor, HUVECs human umbilical vein endothelial cells, Ros rosiglitazone, AGEs advanced glycation end-products, VEGF-A vascular endothelial growth factor A
Figure 5
Activation of PPAR signaling enhances OXPHOS. Cells were pretreated with 200 μg/ml AGEs for 24 h, then exposed to 1 μM Chi for 48 h. (a) Heat map of DEGs (adj. _P_-value <.01 based on sequencing results). (b) KEGG pathway enrichment of mitochondrial DEGs. (c) The morphology of mitochondrial was viewed by transmission electron microscope. (d, e) MitoTracker™ Red CMXRos was measured by flow cytometry after intervention, n = 3. (f) The relative levels of mitochondrial OXPHOS in HUVECs after intervention were measured by western blot, n = 3. The results are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. PPAR peroxisome proliferator-activated receptor, HUVECs human umbilical vein endothelial cells, OXPHOS oxidative phosphorylation, AGEs advanced glycation end-products, SD standard deviation
Figure 6
PPAR signaling promotes HUVEC function via SHH expression. (a) Volcano map of DEGs (adj. p < 0.01 based on sequencing results). (b, c, d) Cells were transfected with SHH small interfering RNA (siSHH) for 24 h, treated with 200 μg/ml AGEs for 24 h, and then exposed to 500 μmol SAG for 24 h. Western blot was used to detect the relative levels of SHH and PTCH1 protein in HUVECs after Chi intervention in the presence or absence of siSHH, n = 4. (e, f) Tube formation assays were used to assess segment length and HUVECs’ tube formation ability, n = 3 (scale bar: 100 μm). (g, h) Transwell assays were conducted for evaluation of HUVEC migration, n = 3 (scale bar: 100 μm). (i, j) MitoTracker™ Red CMXRos was measured by flow cytometry after siSHH and SAG intervention, n = 3. (k, l, m) The relative levels of CD31 and VEGF-A in HUVECs after siSHH and SAG intervention were measured by western blot, n = 4. The results are expressed as mean ± SD. *p < 0.05, **p< 0.01, ***p < 0.001; ns, not significant. PPAR peroxisome proliferator-activated receptor, HUVECs human umbilical vein endothelial cells, OXPHOS oxidative phosphorylation, AGEs advanced glycation end-products, SD standard deviation, VEGF-A vascular endothelial growth factor A, SHH sonic hedgehog signaling
Figure 7
SHH mediates PPAR signaling to promote OXPHOS and regulate HUVEC function. (a) Cells were transfected with siSHH for 24 h, treated with 200 μg/ml AGEs for 24 h, and then exposed to 500 μM SAG for 24 h. The relative levels of mitochondrial OXPHOS in HUVECs, n = 3. (b) OCR and maximal respiration response of HUVECs were measured with Seahorse after SAG intervention, n = 3. (c, d) Flow cytometry was performed using mitochondrial ATP fluorescent probes after SAG intervention, n = 3. (e, f) Tube formation assays were used to assess segment length and HUVECs’ tube formation ability after Chi intervention, n = 3 (scale bar: 100 μm). (g, h) Transwell assays were conducted for evaluation of HUVEC migration after Chi intervention, n = 3 (scale bar: 100 μm). (i, j) MitoTracker™ Red CMXRos was measured by flow cytometry after Chi intervention, n = 3. (k) The relative levels of mitochondrial OXPHOS in HUVECs after Chi intervention were measured by western blot, n = 3. (l) OCR and maximal respiration response of HUVECs were measured with Seahorse after Chi intervention, n = 3. (m, n) Flow cytometry was performed using mitochondrial ATP fluorescent probes after Chi intervention, n = 3. (o, p, q) The relative levels of CD31 and VEGF-A in HUVECs after Chi intervention were measured by western blot, n = 4. The results are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. PPAR peroxisome proliferator-activated receptor, HUVECs human umbilical vein endothelial cells, OXPHOS oxidative phosphorylation, AGEs advanced glycation end-products, SD standard deviation, VEGF-A vascular endothelial growth factor A, SHH sonic hedgehog signaling, OCR oxygen consumption rate
Figure 8
Schematic of the molecular mechanism (created with
BioRender.com
)
References
- Boyko EJ, Zelnick LR, Braffett BH, Pop-Busui R, Cowie CC, Lorenzi GM. et al. Risk of foot ulcer and lower-extremity amputation among participants in the diabetes control and complications trial/epidemiology of diabetes interventions and complications study. Diabetes Care 2022;45:357–64. 10.2337/dc21-1816. -DOI -PMC -PubMed
LinkOut - more resources
Full Text Sources