Jacob Grünler

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.

Isolation and functional expression of human COQ2, a gene encoding a polyprenyl transferase involved in the synthesis of CoQ

The COQ2 gene in Saccharomyces cerevisiae encodes a Coq2 (p-hydroxybenzoate:polyprenyl transferase), which is required in the biosynthetic pathway of CoQ (ubiquinone). This enzyme catalyses the prenylation of p-hydroxybenzoate with an all-trans polyprenyl group. We have isolated cDNA which we believe encodes the human homologue of COQ2 from a human muscle and liver cDNA library. The clone contained an open reading frame of length 1263 bp, which encodes a polypeptide that has sequence homology with the Coq2 homologues in yeast, bacteria and mammals. The human COQ2 gene, when expressed in yeast Coq2 null mutant cells, rescued the growth of this yeast strain in the absence of a non-fermentable carbon source and restored CoQ biosynthesis. However, the rate of CoQ biosynthesis in the rescued cells was lower when compared with that in cells rescued with the yeast COQ2 gene. CoQ formed when cells were incubated with labelled decaprenyl pyrophosphate and nonaprenyl pyrophosphate, showing that the human enzyme is active and that it participates in the biosynthesis of CoQ.

MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

Wound healing is a complex process that is characterized by an initial inflammatory phase followed by a proliferative phase. This transition is a critical regulatory point; however, the factors that mediate this process are not fully understood. Here, we evaluated microRNAs (miRs) in skin wound healing and characterized the dynamic change of the miRNome in human skin wounds. miR-132 was highly upregulated during the inflammatory phase of wound repair, predominantly expressed in epidermal keratinocytes, and peaked in the subsequent proliferative phase. TGF-β1 and TGF-β2 induced miR-132 expression in keratinocytes, and transcriptome analysis of these cells revealed that miR-132 regulates a large number of immune response- and cell cycle-related genes. In keratinocytes, miR-132 decreased the production of chemokines and the capability to attract leukocytes by suppressing the NF-κB pathway. Conversely, miR-132 increased activity of the STAT3 and ERK pathways, thereby promoting keratinocyte growth. Silencing of the miR-132 target heparin-binding EGF-like growth factor (HB-EGF) phenocopied miR-132 overexpression in keratinocytes. Using mouse and human ex vivo wound models, we found that miR-132 blockade delayed healing, which was accompanied by severe inflammation and deficient keratinocyte proliferation. Together, our results indicate that miR-132 is a critical regulator of skin wound healing that facilitates the transition from the inflammatory to the proliferative phase.

Coenzyme Q10 prevents peripheral neuropathy and attenuates neuron loss in the db-/db- mouse, a type 2 diabetes model.

Diabetic peripheral neuropathy (DPN) is the most common complication in both type 1 and type 2 diabetes. Here we studied some phenotypic features of a well-established animal model of type 2 diabetes, the leptin receptor-deficient db−/db− mouse, and also the effect of long-term (6 mo) treatment with coenzyme Q10 (CoQ10), an endogenous antioxidant. Diabetic mice at 8 mo of age exhibited loss of sensation, hypoalgesia (an increase in mechanical threshold), and decreases in mechanical hyperalgesia, cold allodynia, and sciatic nerve conduction velocity. All these changes were virtually completely absent after the 6-mo, daily CoQ10 treatment in db−/db− mice when started at 7 wk of age. There was a 33% neuronal loss in the lumbar 5 dorsal root ganglia (DRGs) of the db−/db− mouse versus controls at 8 mo of age, which was significantly attenuated by CoQ10. There was no difference in neuron number in 5/6-wk-old mice between diabetic and control mice. We observed a strong down-regulation of phospholipase C (PLC) β3 in the DRGs of diabetic mice at 8 mo of age, a key molecule in pain signaling, and this effect was also blocked by the 6-mo CoQ10 treatment. Many of the phenotypic, neurochemical regulations encountered in lumbar DRGs in standard models of peripheral nerve injury were not observed in diabetic mice at 8 mo of age. These results suggest that reactive oxygen species and reduced PLCβ3 expression may contribute to the sensory deficits in the late-stage diabetic db−/db− mouse, and that early long-term administration of the antioxidant CoQ10 may represent a promising therapeutic strategy for type 2 diabetes neuropathy.

Insulin-like growth factor-I receptor activity is essential for Kaposi's sarcoma growth and survival

Kaposis sarcoma (KS) is a highly vascular tumour and is the most common neoplasm associated with human immunodeficiency virus (HIV-1) infection. Growth factors, in particular vascular endothelial growth factor (VEGF), have been shown to play an important role in its development. The role of insulin-like growth factors (IGFs) in the pathophysiology of different tumours led us to evaluate the role of IGF system in KS. The IGF-I receptors (IGF-IR) were identified by immunohistochemistry in biopsies taken from patients with different AIDS/HIV-related KS stages and on KSIMM cells (an established KS-derived cell line). Insulin-like growth factor-I is a growth factor for KSIMM cells with a maximum increase of 3H-thymidine incorporation of 130±27.6% (P<0.05) similar to that induced by VEGF and with which it is additive (281±13%) (P<0.05). Moreover, specific blockade of the receptor (either by α IR3 antibody or by picropodophyllin, a recently described selective IGF-IR tyrosine phosphorylation inhibitor) induced KSIMM apoptosis, suggesting that IGF-IR agonists (IGF-I and -II) mediate antiapoptotic signals for these cells. We were able to identify an autocrine loop essential for KSIMM cell survival in which IGF-II is the IGF-IR agonist secreted by the cells. In conclusion, IGF-I pathway inhibition is a promising therapeutical approach for KS tumours.

Markers of innate immune activity in patients with type 1 and type 2 diabetes mellitus and the effect of the anti‐oxidant coenzyme Q10 on inflammatory activity

Major long‐term complications in patients with diabetes are related to oxidative stress, caused by the hyperglycaemia characteristic for diabetes mellitus. The anti‐oxidant coenzyme Q10 (CoQ10) has therefore been proposed as a beneficial supplement to diabetes treatment. Apart from its anti‐oxidative function, CoQ10 appears to modulate immune functions by largely unknown mechanisms. The aim of this study was therefore to investigate the effect of CoQ10 on antimicrobial peptides and natural killer (NK) cells, both innate immune components implicated in the pathogenesis of diabetes and diabetes‐associated long‐term complications such as cardiovascular disease. We determined serum levels of antimicrobial peptides and the phenotype of NK cells isolated from peripheral blood of patients with type 1 (T1DM) or type 2 diabetes mellitus (T2DM) and from healthy controls. In addition, the same parameters were determined in diabetic patients after a 12‐week period of CoQ10 supplementation. Two antimicrobial peptides, the human cathelicidin antimicrobial peptide (CAMP) and the human beta defensin 1 (hBD1), were reduced in serum from patients with T1DM. This defect was not reversible by CoQ10 supplementation. In contrast, CoQ10 reduced the levels of circulating hBD2 in these patients and induced changes in subset distribution and activation markers in peripheral NK cells. The results of the present study open up novel approaches in the prevention of long‐term complications associated to T1DM, although further investigations are needed.

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.

Effects of IGFBP-1 and IGFBP-2 and their fragments on migration and IGF-induced proliferation of human dermal fibroblasts

OBJECTIVE A family of six insulin-like growth factor (IGF) binding-proteins (IGFBP) bind to IGF-I and IGF-II with high affinity and modulate their activity. We have recently shown that a neutrophil-derived protease activity cleaved IGFBP-1, -2 and -4. IGFBP-1 and IGFBP-2 have a C-terminal Arg-Gly-Asp (RGD) sequence, and IGFBP-1 has been shown by others to stimulate migration through binding of its RGD sequence to α5β1 integrin. The aim of this study was to determine the effect of this IGFBP protease on IGF-induced proliferation and the effect of IGFBP-1 and IGFBP-2 and their proteolytic fragments on migration in normal and high glucose of human dermal fibroblasts (HDF). DESIGN We investigated the effect of intact or cleaved IGFBP-1 and -2 on proliferation in cultured HDFs and on HDF migration in normal and high glucose. RESULTS Both IGFBP-1 and IGFBP-2 and their proteolytic fragments stimulated HDF migration and the stimulatory effect was abolished by pre-treating cells with a α5β1 integrin antibody. High glucose impaired migration of HDFs; however, the addition of IGFBP-1, IGFBP-2 or fragments increased migration to levels observed in normoglycemia. IGFBP-2 inhibited IGF-II induced proliferation; however, the inhibitory effect was reduced after being cleaved. Intact native IGFBP-1 showed either potentiating or inhibitory effects on IGF-I induced proliferation depending on the confluence of cells, and proteolysis of IGFBP-1 did not change these effects. IGFBP-1 was found to increase phosphorylation of FAK and ERK1/2 and this effect was inhibited by the monoclonal integrin a5β1 ab. CONCLUSIONS IGFBP-1 and -2 and their proteolytic fragments may improve tissue repair under inflammatory conditions, through effects on proliferation and migration of HDFs in normal and high glucose.

Coenzyme Q10 and oxidative stress, the association with peripheral sensory neuropathy and cardiovascular disease in type 2 diabetes mellitus

OBJECTIVE Our study aimed to explore associations between metabolic control, oxidative stress and coenzyme Q10 (CoQ10) in relation to diabetes complications in a representative population of type 2 diabetes. RESEARCH DESIGN AND METHODS A geographic cohort of 156 subjects was recruited. Serum concentrations of CoQ10 and vitamin E were measured by HPLC. ROS was determined by free oxygen radicals testing (FORT). Glutaredoxin (Grx) activity, oxidized LDL cholesterol (oxLDLc), high sensitive CRP (hsCRP), HbA1c, urine albumin, serum creatinine, serum cystatin C, and plasma lipids were assayed with routine laboratory protocols. RESULTS Serum CoQ10 was higher than in nondiabetics. HbA1c, fP-glucose, hyperlipidemia, inflammation (hsCRP), and increased BMI were associated with signs of oxidative stress as increased levels of FORT, Grx activity and/or increased levels of oxLDLc Oxidative stress was found to be strongly correlated with prevalence of cardiovascular disease (CVD) and peripheral sensory neuropathy (PSN). In both gender groups there were positive correlations between CoQ10 and oxLDLc, and between BMI and the ratio CoQ10/chol. Grx activity was inversely correlated to oxLDLc and CoQ10. Women with CVD and PSN had higher waist index, oxLDLc, and FORT levels compared to men but lower CoQ10 levels. Men had worse kidney function and lower vitamin E. Multiple regression analysis showed increased levels of CoQ10 to be significantly correlated with increased levels of cholesterol, triglycerides, vitamin E, fB-glucose and BMI. CONCLUSIONS Hyperlipidemia, hyperglycemia and inflammation were associated with oxidative stress, which was correlated to the prevalence of diabetes complications. CoQ10 was increased in response to oxidative stress. There were gender differences in the risk factors associated with diabetes complications.

Glutaredoxin mediated redox effects of coenzyme Q10 treatment in type 1 and type 2 diabetes patients.

The possible beneficial effects of coenzyme Q10 (CoQ10) supplementation on disease progression and oxidant status in diabetes remains debated. In the present study, patients with type 1 and type 2 diabetes were treated with oral CoQ10, 100 mg twice daily for 12 weeks. We assessed total antioxidant capacity, intra- and extracellular levels of the redox regulating protein glutaredoxin 1 (Grx1), CoQ10, oxidized LDL-cholesterol, lipid profile and HbA1c. We have previously shown that extracellular Grx1 is increased in patients with type 2 diabetes compared to healthy subjects. In the present study, CoQ10 treatment significantly decreased serum Grx1 activity as well as total antioxidant capacity independent of type of diabetes, indicating an improvement to a less oxidized extracellular environment. The effect on serum Grx1 activity was more prominent in patients not on statin treatment. Conversely, intracellular Grx1 activity as well as mRNA levels increased independent of statin treatment. There was a significant improvement in oxidized LDL-cholesterol and lipid profile, with a tendency to improved metabolic control (HbA1c). Additionally, we describe for the first time that CoQ10 is a direct substrate for glutathione, and that Grx1 catalyzes this reaction, thus presenting a novel mechanism for CoQ10 reduction which could explain our findings of an increased intracellular Grx1. In conclusion, 12 weeks CoQ10 treatment significantly improved the extracellular redox balance and lipid profile, indicating that prolonged treatment may have beneficial effects also on clinical outcome in diabetes.