Vivekananda Gupta Sunkari

Stabilization of HIF-1α is critical to improve wound healing in diabetic mice

Relative hypoxia is essential in wound healing since it normally plays a pivotal role in regulation of all the critical processes involved in tissue repair. Hypoxia-inducible factor (HIF) 1α is the critical transcription factor that regulates adaptive responses to hypoxia. HIF-1α stability and function is regulated by oxygen-dependent soluble hydroxylases targeting critical proline and asparaginyl residues. Here we show that hyperglycemia complexly affects both HIF-1α stability and activation, resulting in suppression of expression of HIF-1 target genes essential for wound healing both in vitro and in vivo. However, by blocking HIF-1α hydroxylation through chemical inhibition, it is possible to reverse this negative effect of hyperglycemia and to improve the wound healing process (i.e., granulation, vascularization, epidermal regeneration, and recruitment of endothelial precursors). Local adenovirus-mediated transfer of two stable HIF constructs demonstrated that stabilization of HIF-1α is necessary and sufficient for promoting wound healing in a diabetic environment. Our findings outline the necessity to develop specific hydroxylase inhibitors as therapeutic agents for chronic diabetes wounds. In conclusion, we demonstrate that impaired regulation of HIF-1α is essential for the development of diabetic wounds, and we provide evidence that stabilization of HIF-1α is critical to reverse the pathological process.

Epigenetic DNA methylation in the promoters of the Igf1 receptor and insulin receptor genes in db/db mice

We have investigated promoter methylation of the Insr, Igf1 and Igf1r genes in skeletal and cardiac muscles of normal and diabetic db/db mice. No differences in Insr promoter methylation were found in the heart and skeletal muscles and no methylation was detected in the Igf1 promoter in skeletal muscle. In skeletal muscle, db/db males exhibited a 7.4-fold increase in Igf1r promoter methylation, which was accompanied by a 1.8-fold decrease in Igf1r mRNA levels, compared with controls. More than 50% of the detected methylation events were concentrated within an 18 bp sequence that includes one of the Sp1 binding sites. We conclude that the methylation level and pattern of the Igf1r promoter in skeletal muscle is related to gender and the diabetic state.

Hyperbaric oxygen therapy activates hypoxia-inducible factor 1 (HIF-1), which contributes to improved wound healing in diabetic mice

Hyperbaric oxygen (HBO) therapy has been used as an adjunctive therapy for diabetic foot ulcers, although its mechanism of action is not completely understood. Recently, it has been shown that HBO mobilizes the endothelial progenitor cells (EPCs) from bone marrow that eventually will aggregate in the wound. However, the gathering of the EPCs in diabetic wounds is impaired because of the decreased levels of local stromal‐derived factor‐1α (SDF‐1α). Therefore, we investigated the influence of HBO on hypoxia‐inducible factor 1 (HIF‐1), which is a central regulator of SDF‐1α and is down‐regulated in diabetic wounds. The effects of HBO on HIF‐1α function were studied in human dermal fibroblasts, SKRC7 cells, and HIF‐1α knock‐out and wild‐type mouse embryonic fibroblasts using appropriate techniques (Western blot, quantitative polymerase chain reaction, and luciferase hypoxia‐responsive element reporter assay). Cellular proliferation was assessed using H3‐thymidine incorporation assay. The effect of HIF in combination with HBOT was tested by inoculating stable HIF‐1α‐expressing adenovirus (Adv‐HIF) into experimental wounds in db/db mice exposed to HBO. HBO activates HIF‐1α at several levels by increasing both HIF‐1α stability (by a non‐canonical mechanism) and activity (as shown both by induction of relevant target genes and by a specific reporter assay). HIF‐1α induction has important biological relevance because the induction of fibroblast proliferation in HBO disappears when HIF‐1α is knocked down. Moreover, the local transfer of stable HIF‐1α‐expressing adenovirus (Adv‐HIF) into experimental wounds in diabetic (db/db mice) animals has an additive effect on HBO‐mediated improvements in wound healing. In conclusion, HBO stabilizes and activates HIF‐1, which contributes to increased cellular proliferation. In diabetic animals, the local transfer of active HIF further improves the effects of HBO on wound healing.

Impact of the Hypoxia-Inducible Factor-1 α (HIF1A) Pro582Ser Polymorphism on Diabetes Nephropathy

OBJECTIVE Hypoxia plays a major pathogenic role in diabetic nephropathy (DN). We have investigated in this study the effect of hypoxia-inducible factor 1 α subunit (HIF1A) genetic polymorphisms on the development of DN. RESEARCH DESIGN AND METHODS In 1,165 American type 1 diabetic patients with and without DN selected from the Genetics of Kidneys in Diabetes (GoKinD) study, the HIF1A genetic polymorphisms were genotyped with TaqMan allelic discrimination. The regulation of HIF-1α in the kidneys of diabetic mice was appreciated by immunohistochemistry, and the effect HIF1A Pro582Ser polymorphism on HIF-1α sensitivity to glucose was evaluated in vitro. RESULTS We identified a protective association between HIF1A Pro582Ser polymorphism and DN in male subjects. We also provided mechanistic insights that HIF-1α is repressed in the medulla of diabetic mice despite hypoxia and that Pro582Ser polymorphism confers less sensitivity to the inhibitory effect of glucose during a hypoxic challenge. CONCLUSIONS The current study demonstrates for the first time that HIF1A Pro582Ser polymorphism has an effect on DN, possibly by conferring a relative resistance to the repressive effect of glucose on HIF-1α.

Effects of a low-intensity electromagnetic field on fibroblast migration and proliferation

The aim of this study was to test if an extremely weak 1 GHz electromagnetic field (EMF), known to be in resonance with clusters of water molecules, has biological effects on human fibroblasts. We demonstrated that in an in vitro model of wound healing, this EMF can activate fibroblast migration. [3H]thymidine incorporation experiments demonstrated that the EMF could also activate fibroblast proliferation. Activation of the expression of human fibroblast growth factor 1 (HFGF1) after EMF exposure showed that molecular wound healing pathways are activated in response to this water-resonant EMF.

Stability of mitochondrial DNA against reactive oxygen species (ROS) generated in diabetes

Increased production of reactive oxygen species (ROS) in mitochondria has been proposed as the pathogenic mechanism for chronic complications of diabetes. Mitochondrial DNA (mtDNA) is more vulnerable to reactive oxygen species. However, there are few data on the mitochondrial DNA damage in diabetes and these are available only from patients with different duration of the disease and tissues not relevant to the chronic complications of diabetes. We therefore proposed to study the stability of mitochondrial DNA under controlled experimental conditions, to understand its contribution to chronic complications of diabetes.

Selective blockade of estrogen receptor beta improves wound healing in diabetes

Diabetic ulcer is a major complication of diabetes with a high burden on society resources. Despite concerted efforts in improving diabetes care, delayed wound healing in diabetes remains a common, dreaded complication [1]. The pathogenic mechanisms behind impaired wound healing in diabetes are poorly understood, and therefore, no specific targeted therapy is available. Aging also impairs the wound healing capacity by mechanisms that are still unraveled but can be partially explained by a decline in the production of sex steroid hormones since the defect is improved by topically or systemically delivered estrogen replacement therapy [2, 3]. A predominance of diabetic foot ulcers in males suggest that estrogens might be beneficial for wound healing in diabetes which is sustained experimentally in diabetic animals [4]. Two types of estrogen receptors (ERa and ERb) mediate the biological functions of estrogens. Recent data show that estrogen accelerates wound healing in normoglycemia via ERb [5]. However, there are no data concerning the relative contribution of ERs in wound healing in diabetes.

Mono-epoxy-tocotrienol-α enhances wound healing in diabetic mice and stimulates in vitro angiogenesis and cell migration.

Diabetes mellitus is characterized by hyperglycemia and capillary hypoxia that causes excessive production of free radicals and impaired antioxidant defense, resulting in oxidative stress and diabetes complications such as impaired wound healing. We have previously shown that modified forms of tocotrienols possess beneficial effects on the biosynthesis of the mevalonate pathway lipids including increase in mitochondrial CoQ. The aim of this study is to investigate the effects of mono-epoxy-tocotrienol-α on in vitro and in vivo wound healing models as well as its effects on mitochondrial function. Gene profiling analysis and gene expression studies on HepG2 cells and human dermal fibroblasts were performed by microarray and qPCR, respectively. In vitro wound healing using human fibroblasts was studied by scratch assay and in vitro angiogenesis using human dermal microvascular endothelial cells was studied by the tube formation assay. In vivo wound healing was performed in the diabetic db/db mouse model. For the study of mitochondrial functions and oxygen consumption rate Seahorse XF-24 was employed. In vitro, significant increase in wound closure and cell migration (p<0.05) both in normal and high glucose and in endothelial tube formation (angiogenesis) (p<0.005) were observed. Microarray profiling analysis showed a 20-fold increase of KIF26A gene expression and 11-fold decrease of lanosterol synthase expression. Expression analysis by qPCR showed significant increase of the growth factors VEGFA and PDGFB. The epoxidated compound induced a significantly higher basal and reserve mitochondrial capacity in both HDF and HepG2 cells. Additionally, in vivo wound healing in db/db mice, demonstrated a small but significant enhancement on wound healing upon local application of the compound compared to treatment with vehicle alone. Mono-epoxy-tocotrienol-α seems to possess beneficial effects on wound healing by increasing the expression of genes involved in cell growth, motility and angiogenes as well as on mitochondrial function.

Deficiency of liver-derived insulin-like growth factor-I (IGF-I) does not interfere with the skin wound healing rate

Objective IGF-I is a growth factor, which is expressed in virtually all tissues. The circulating IGF-I is however derived mainly from the liver. IGF-I promotes wound healing and its levels are decreased in wounds with low regenerative potential such as diabetic wounds. However, the contribution of circulating IGF-I to wound healing is unknown. Here we investigated the role of systemic IGF-I on wound healing rate in mice with deficiency of liver-derived IGF-I (LI-IGF-I-/- mice) during normal (normoglycemic) and impaired wound healing (diabetes). Methods LI-IGF-I-/- mice with complete inactivation of the IGF-I gene in the hepatocytes were generated using the Cre/loxP recombination system. This resulted in a 75% reduction of circulating IGF-I. Diabetes was induced with streptozocin in both LI-IGF-I-/- and control mice. Wounds were made on the dorsum of the mice, and the wound healing rate and histology were evaluated. Serum IGF-I and GH were measured by RIA and ELISA respectively. The expression of IGF-I, IGF-II and the IGF-I receptor in the skin were evaluated by qRT-PCR. The local IGF-I protein expression in different cell types of the wounds during wound healing process was analyzed using immunohistochemistry. Results The wound healing rate was similar in LI-IGF-I-/- mice to that in controls. Diabetes significantly delayed the wound healing rate in both LI-IGF-I-/- and control mice. However, no significant difference was observed between diabetic animals with normal or reduced hepatic IGF-I production. The gene expression of IGF-I, IGF-II and IGF-I receptor in skin was not different between any group of animals tested. Local IGF-I levels in the wounds were similar between of LI-IGF-I-/- and WT mice although a transient reduction of IGF-I expression in leukocytes in the wounds of LI-IGF-I-/- was observed seven days post wounding. Conclusion Deficiency in the liver-derived IGF-I does not affect wound healing in mice, neither in normoglycemic conditions nor in diabetes.

Hypoxia and inflammation synergistically promote bone destruction

Objective Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by synovial inflammation and consecutive local hypoxia, leading to cartilage and bone destruction. Hypoxia promotes osteoclasts formation in vitro but its role in mediating bone destruction in the presence of chronic inflammation has not previously been investigated. The authors aimed to investigate the effect of hypoxia on the RANKL/OPG system and bone destruction in the presence of pro inflammatory stimuli. Methods The authors investigated the in vitro effect of hypoxia on RANKL/OPG expression in osteoblast-like (Saos2) cells. Cells were cultured in normoxic (21% pO2) or hypoxic (0.5% pO2) conditions with or without tumour necrosis factor α (TNFα). Expression of RANKL and OPG mRNA was detected by rtPCR. Cellular and soluble forms of RANKL and OPG proteins were determined by western blot and ELISA respectively. Hypoxia effect on bone resorption was evaluated in a dentine pit formation assay using peripheral blood mononuclear cells from RA patients. Statistical analysis was performed using one-way analysis of variance. Results Exposure to hypoxia induced a significant increase in cellular RANKL mRNA and protein expression, with minimal changes of the cellular OPG. In contrast soluble OPG levels significantly decreased following hypoxia exposure with no changes in the soluble levels of RANKL. Concomitant exposure to both hypoxia and TNFα had an additive effect resulting in a further increase of the RANKL/OPG ratio. Small interfering RNA against HIF 2α but not HIF 1α was able to abolish hypoxia effect on cellular RANKL expression. Hypoxia mimicking by prolyl hydroxylases inhibitors acted synergistically with TNF in inducing pit formation and resorption of synthetic osteologic bone discs. Conclusions TNF and hypoxia act synergistically to promote bone destruction potentially through a HIF2α dependent mechanism. These findings add on the current understanding of bone destruction in the setting of chronic inflammation.