HypoxamiR-210 accelerates wound healing in diabetic mice by improving cellular metabolism

The study addresses why wound healing is impaired in diabetes, focusing on hypoxia-adaptive pathways and metabolic regulation. Wound healing demands high energy in a hypoxic environment, necessitating coordination between mitochondrial respiration and glycolysis. In diabetes, hyperglycemia represses HIF-1 signaling, impairing cellular responses (angiogenesis, regeneration) and complicating energy balance. miR-210, a hypoxia-induced microRNA under HIF-1 control, regulates genes involved in metabolism, angiogenesis, proliferation/apoptosis, and tissue regeneration. Prior work in ischemic (non-diabetic) wounds suggested miR-210 inhibits keratinocyte proliferation and that anti-miR-210 can accelerate closure; however, its regulation and function in diabetic wounds had not been explored. The authors hypothesize that hyperglycemia blunts hypoxia-induced miR-210 in diabetes, contributing to impaired healing, and that restoring miR-210 locally could correct metabolic imbalance and improve diabetic wound healing.

Background literature establishes HIF-1 as a master regulator of oxygen homeostasis with broad downstream targets, with impaired HIF-1 signaling in diabetes contributing to poor wound healing; pharmacologic HIF-1 activation improves angiogenesis and tissue regeneration. miR-210 is a hypoxamiR directly induced by HIF-1 via an HRE, modulating approximately 30 target genes linked to metabolism and regeneration. In ischemic wounds, elevated miR-210 at wound edges can inhibit keratinocyte proliferation, and anti-miR-210 accelerated closure, indicating context dependency. Reports on diabetic tissues show variable miR-210 levels (elevated in serum/urine or heart, reduced in islets) without adjustment for tissue oxygenation. The literature underscores the need to clarify miR-210’s role in diabetic wound context, where both hypoxia and hyperglycemia co-exist and HIF-1 is repressed.

• Human subjects: Wound biopsies were obtained from 8 patients with diabetic foot ulcers (DFU) and 7 patients with venous ulcers (VU) after informed consent; ethical approvals were secured.
• Animal model: db/db diabetic mice and normoglycemic wild-type (WT) controls. Two 6-mm full-thickness dorsal wounds per mouse were created. Mice were randomized to receive intradermal injections of miR-210 mimic (0.125 nmol) or negative control mimic at four wound-edge sites on day 0 and day 6. In some experiments, DMOG (2 mM, 100 µL) or vehicle was applied locally every other day. Wounds were photographed every other day; wound area was quantified by ImageJ and expressed as percentage of initial area. Wounds were harvested on day 8 for analyses. Sample sizes per figure are provided and analyses were blinded.
• Cell culture: Primary human dermal fibroblasts (HDF), human dermal microvascular endothelial cells (HDMEC), and keratinocytes were exposed to normoxia (21% O2) or hypoxia (1% O2) for indicated times. High glucose (30 mM) versus normal glucose (5.5 mM) conditions were used. HDF were transfected with stabilized miR-210 mimic or control mimic using Lipofectamine RNAiMAX.
• Molecular assays: miR-210 expression assessed by in situ hybridization (LNA probes) and quantitative RT-PCR (TaqMan/SYBR). mRNA levels of miR-210 mitochondrial targets (ISCU, SDHD, ALDH5A1, NDUFA4, COX10) quantified by qRT-PCR.
• Histology and immunostaining: Formalin-fixed paraffin-embedded sections analyzed by H&E for granulation tissue, Masson-Goldner Trichrome for collagen deposition, and immunofluorescence for CD31 (angiogenesis), Ki67 (proliferation), CD11b (inflammation), and HIF-1α. Image analysis quantified positive cells or stained areas.
• Metabolic assays: Ex vivo wound granulation tissue OCR measured using Seahorse XF Analyzer. Lactate levels (glycolysis) quantified colorimetrically and normalized to protein. ROS measured in wounds via 4-hydroxynonenal (4-HNE) ELISA. In HDF: OCR (mitochondrial respiration), ECAR (glycolysis), and real-time ATP production rates (oxidative phosphorylation vs glycolysis) measured by Seahorse assays; ROS measured by EPR with CMH spin probe.
• Functional assay: HDF migration assessed by scratch assay under hypoxia/high glucose with miR-210 or control mimic; glycolysis inhibitors (2-deoxy-D-glucose 15 mM, syrosingopine 10 µM, sodium oxamate 45 mM) applied to test dependence on glycolysis.
• Statistics: GraphPad Prism; normality by Kolmogorov–Smirnov; outliers by Grubbs. Two-sided Student’s t test or nonparametric equivalents for two groups; one-way or two-way ANOVA with Bonferroni post hoc for multiple groups. P<0.05 significant. Data shown as mean ± s.e.m.

• High glucose blunts hypoxia-induced miR-210 in key skin cell types (HDF, HDMEC, keratinocytes): hypoxia increased miR-210 over 6–24 h, but 30 mM glucose significantly reduced this induction (one-way ANOVA, P<0.05).
• miR-210 is suppressed in diabetic wounds: In mice, miR-210 increased in WT wounds vs WT skin (ISH and qRT-PCR), but was significantly lower in db/db wounds despite greater hypoxia. In humans, miR-210 was reduced in DFU vs VU (ISH and qRT-PCR; Student’s t test, P<0.05). DMOG restored HIF-1α and increased miR-210 levels in db/db wounds (ANOVA, P<0.05).
• Local miR-210 reconstitution accelerates healing specifically in diabetic mice: Intradermal miR-210 mimic significantly increased miR-210 levels in wounds and improved wound closure kinetics in db/db mice without affecting WT mice (two-way ANOVA, P<0.05). Distribution was local; systemic levels unchanged.
• Histological improvements in db/db wounds with miR-210: Increased granulation tissue, collagen deposition (Masson trichrome), angiogenesis (CD31+), proliferation (Ki67+), and reduced inflammation (CD11b+) vs control mimic (ANOVA, P<0.05).
• Metabolic reprogramming in vivo: miR-210 reduced expression of mitochondrial targets ISCU and SDHD in db/db wounds (qRT-PCR). It decreased elevated OCR in db/db wounds to WT levels (Seahorse), increased lactate production (indicative of enhanced glycolysis), and reduced ROS (4-HNE) (all ANOVA, P<0.05). Total ATP did not increase significantly.
• Metabolic and functional effects in HDF under diabetic-like conditions: miR-210 mimic decreased ISCU and SDHD mRNA; reduced OCR; increased ECAR; shifted ATP production toward glycolysis; decreased ROS (paired tests, P<0.05). miR-210 enhanced HDF migration under hypoxia/high glucose, which was abrogated by glycolysis inhibitors (2-DG, syrosingopine, oxamate), indicating glycolysis-dependent migration.

The findings support that hyperglycemia suppresses HIF-1-driven miR-210 induction during hypoxia, contributing to impaired diabetic wound healing. Restoring miR-210 reprograms cellular metabolism from excessive mitochondrial respiration toward glycolysis, reducing ROS and meeting energy demands in the hypoxic wound milieu. This metabolic shift enhances essential reparative processes—angiogenesis, proliferation, collagen deposition, and resolution of inflammation—thereby accelerating closure in diabetic, but not normoglycemic, wounds where miR-210 levels and hypoxic responses are sufficient. Mechanistically, repression of ISCU and SDHD by miR-210 likely reduces TCA/ETC activity and OCR, while increased glycolysis boosts lactate and supports cell migration, aligning with the role of glycolysis in angiogenesis and tissue repair. The specificity to diabetes underscores the context-dependent actions of hypoxamiR-210 and reconciles reports of both pro- and anti-healing effects under different ischemic contexts and dosing paradigms.

Hyperglycemia blunts hypoxia-induced miR-210 in diabetic wounds, leading to elevated OCR, reduced glycolysis, and increased ROS that impair healing. Local reconstitution of miR-210 restores metabolic balance by downregulating ISCU/SDHD, decreasing OCR, enhancing glycolysis, lowering ROS, and improving key reparative processes, thereby accelerating wound healing specifically in diabetic mice. These results identify miR-210 as a focused hypoxic effector with therapeutic potential for diabetic foot ulcers. Future research should optimize dosing and delivery, evaluate long-term safety and efficacy, assess effects on additional cell types, and translate to clinical trials in patients with DFU.

• The in vivo efficacy was demonstrated in a single mouse model (db/db) with assessments up to day 8; long-term outcomes, scarring, and recurrence were not evaluated.
• Human data were limited to cross-sectional expression comparisons (DFU vs VU) without interventional validation.
• Potential off-target effects and optimal dosing/timing of miR-210 mimic were not comprehensively assessed; dose–response and safety were not reported.
• Affinity and distribution studies indicate local action, but detailed pharmacokinetics and biodistribution were limited.
• The study focused largely on fibroblasts for in vitro functional assays; effects on keratinocytes, endothelial cells, and immune cells were not functionally dissected in vitro in the diabetic context.
• ATP levels did not increase, suggesting complex energy dynamics that merit further analysis.