Appropriate glycemic management protects the germline but not the uterine environment in hyperglycemia.

Emerging evidence indicates that parental diseases can impact the health of subsequent generations through epigenetic inheritance. Recently, it was shown that maternal diabetes alters the metaphase II oocyte transcriptome, causing metabolic dysfunction in offspring. However, type 1 diabetes (T1D) mouse models frequently utilized in previous studies may be subject to several confounding factors due to severe hyperglycemia. This limits clinical translatability given improvements in glycemic control for T1D subjects. Here, we optimize a T1D mouse model to investigate the effects of appropriately managed maternal glycemic levels on oocytes and intrauterine development. We show that diabetic mice with appropriate glycemic control exhibit better long-term health, including maintenance of the oocyte transcriptome and chromatin accessibility. We further show that human oocytes undergoing in vitro maturation challenged with mildly increased levels of glucose, reflecting appropriate glycemic management, also retain their transcriptome. However, fetal growth and placental function are affected in mice despite appropriate glycemic control, suggesting the uterine environment rather than the germline as a pathological factor in developmental programming in appropriately managed diabetes.

Identification of early risk factors for anti-citrullinated-protein-antibody positive rheumatoid arthritis-a prospective cohort study.

OBJECTIVE: Individuals positive for anti-cyclic-peptide-antibodies (anti-CCP) and musculoskeletal complaints (MSK-C) are at risk for developing rheumatoid arthritis (RA). In this study we aimed to investigate factors involved in arthritis progression. METHODS: Anti-CCP2-positive individuals with MSK-C referred to a rheumatologist were recruited. Individuals lacked arthritis at clinical and ultrasound examination and were followed for ≥three years or until clinical arthritis diagnosis. Blood samples from inclusion were analyzed for; nine anti-citrullinated-protein-antibody (ACPA) reactivities (citrullinated α-1-enolase, fibrinogen, filaggrin, histone, vimentin and tenascin peptides); 92 inflammation-associated proteins; and HLA-shared epitope alleles. Cox regression was applied to the data to identify independent predictors in a model. RESULTS: 267 individuals were included with median follow up of 49 months (IQR: 22-60). 101 (38%) developed arthritis after median 14 months (IQR: 6-27). The analysis identified that presence of at least one ACPA reactivity (HR 8.0, 95% CI 2.9-22), ultrasound detected tenosynovitis (HR 3.4, 95% CI 2.0-6.0), IL6 levels (HR 1.5, 95% CI 1.2-1.8) and IL15-Rα levels (HR 0.6, 95% CI 0.4-0.9) are significant independent predictors for arthritis progression in a prediction model (Harrell's C 0.76 [SE 0.02], AUC 0.82 [95% CI 0.76-0.89], cross-validated AUC 0.70 [95% CI 0.56-0.85]). CONCLUSION: We propose a high-Risk-RA phase characterized by presence of ACPA reactivity, tenosynovitis, IL6, and IL15-Rα and suggest that these factors need to be further investigated for their biological effects and clinical values, to identify individuals at particular low risk and high risk for arthritis progression.

Triggering of a Dll4-Notch1 loop impairs wound healing in diabetes.

Diabetic foot ulcerations (DFUs) represent a major medical, social, and economic problem. Therapeutic options are restricted due to a poor understanding of the pathogenic mechanisms. The Notch pathway plays a pivotal role in cell differentiation, proliferation, and angiogenesis, processes that are profoundly disturbed in diabetic wounds. Notch signaling is activated upon interactions between membrane-bound Notch receptors (Notch 1-4) and ligands (Jagged 1-2 and Delta-like 1, 3, 4), resulting in cell-context-dependent outputs. Here, we report that Notch1 signaling is activated by hyperglycemia in diabetic skin and specifically impairs wound healing in diabetes. Local inhibition of Notch1 signaling in experimental wounds markedly improves healing exclusively in diabetic, but not in nondiabetic, animals. Mechanistically, high glucose levels activate a specific positive Delta-like 4 (Dll4)-Notch1 feedback loop. Using loss-of-function genetic approaches, we demonstrate that Notch1 inactivation in keratinocytes is sufficient to cancel the repressive effects of the Dll4-Notch1 loop on wound healing in diabetes, thus making Notch1 signaling an attractive locally therapeutic target for the treatment of DFUs.

Hypoxia and hypoxia-inducible factors in diabetes and its complications.

Hypoxia-inducible factors (HIFs) are the key regulators of oxygen homeostasis in response to hypoxia. In diabetes, multiple tissues are hypoxic but adaptive responses to hypoxia are impaired due to insufficient activation of HIF signalling, which results from inhibition of HIF-1α stability and function due to hyperglycaemia and elevated fatty acid levels. In this review, we will summarise and discuss current findings about the regulation of HIF signalling in diabetes and the pathogenic roles of hypoxia and dysregulated HIF signalling in the development of diabetes and its complications. The therapeutic potential of targeting HIF signalling for the prevention and treatment of diabetes and related complications is also discussed.

Anticitrullinated protein antibodies facilitate migration of synovial tissue-derived fibroblasts.

OBJECTIVES: Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology. METHODS: Fibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting. RESULTS: Challenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis. CONCLUSION: We propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration.

A Notch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells.

Hes1 is a Notch target gene that plays a major role during embryonic development. Previous studies have shown that HIF-1α can interact with the Notch intracellular domain and enhance Notch target gene expression. In this study, we have identified a Notch-independent mechanism that regulates the responsiveness of the Hes1 gene to hypoxia. Using P19 cells we show that silencing the Notch DNA binding partner CSL does not prevent hypoxia-dependent upregulation of Hes1 expression. In contrast to CSL, knockdown of HIF-1α or Arnt expression prevents Hes1 induction in hypoxia. Deletion analysis of the Hes1 promoter identified a minimal region near the transcription start site that is still responsive to hypoxia. In addition, we show that mutating the GA-binding protein (GABP) motif significantly reduced Hes1 promoter-responsiveness to hypoxia or to HIF-1 overexpression whereas mutation of the hypoxia-responsive element (HRE) present in this region had no effect. Chromatin immunoprecipitation assays demonstrated that HIF-1α binds to the proximal region of the Hes1 promoter in a Notch-independent manner. Using the same experimental approach, the presence of GABPα and GABPß1 was also observed in the same region of the promoter. Loss- and gain-of-function studies demonstrated that Hes1 gene expression is upregulated by hypoxia in a GABP-dependent manner. Finally, co-immunoprecipitation assays demonstrated that HIF-1α but not HIF-2α is able to interact with either GABPα or GABPß1. These results suggest a Notch-independent mechanism where HIF-1 and GABP contribute to the upregulation of Hes1 gene expression in response to hypoxia.

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss.

OBJECTIVES: Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. METHODS: Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. RESULTS: Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. CONCLUSIONS: We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Disturbed hypoxic responses as a pathogenic mechanism of diabetic foot ulcers.

Diabetic foot ulceration (DFU) is a chronic complication of diabetes that is characterized by impaired wound healing in the lower extremities. DFU remains a major clinical challenge because of poor understanding of its pathogenic mechanisms. Impaired wound healing in diabetes is characterized by decreased angiogenesis, reduced bone marrow-derived endothelial progenitor cell (EPC) recruitment, and decreased fibroblast and keratinocyte proliferation and migration. Recently, increasing evidence has suggested that increased hypoxic conditions and impaired cellular responses to hypoxia are essential pathogenic factors of delayed wound healing in DFU. Hypoxia-inducible factor-1 (HIF-1, a heterodimer of HIF-1α and HIF-1ß) is a master regulator of oxygen homeostasis that mediates the adaptive cellular responses to hypoxia by regulating the expression of genes involved in angiogenesis, metabolic changes, proliferation, migration, and cell survival. However, HIF-1 signalling is inhibited in diabetes as a result of hyperglycaemia-induced HIF-1α destabilization and functional repression. Increasing HIF-1α expression and activity using various approaches promotes angiogenesis, EPC recruitment, and granulation, thereby improving wound healing in experimental diabetes. The mechanisms underlying HIF-1α regulation in diabetes and the therapeutic strategies targeting HIF-1 signalling for the treatment of diabetic wounds are discussed in this review. Further investigations of the pathways involved in HIF-1α regulation in diabetes are required to advance our understanding of the mechanisms underlying impaired wound healing in diabetes and to provide a foundation for developing novel therapeutic approaches to treat DFU.

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) ß3 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 PLCß3 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.

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 H(3) -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.

Hyperoxia inhibits glucose-induced insulin secretion and mitochondrial metabolism in rat pancreatic islets.

Isolated pancreatic islets containing the insulin-producing beta cells are devoid of circulation. They may therefore experience hypoxia with possible negative effects on beta cell function and survival. We investigated (1) whether hyperoxia in vitro would be beneficial by counteracting putative effects of lost circulation and, further, (2) whether previous hyperoxia would attenuate the impact of subsequently induced severe hypoxia. Islets from Sprague-Dawley rats were exposed to 95% O2 for 18 h. This hyperoxic exposure diminished glucose-induced insulin secretion by 47% and inhibited oxygen consumption by 39-41%. Mitochondrial complexes I-III were decreased by 29-37%. Negative effects on insulin secretion and complexes III and IV waned after a 22 h period of normoxia following hyperoxia whereas complexes I and II were still diminished, ROS production was increased and rates of apoptosis tended to be increased (P=0.07). The effects of previous hyperoxia on susceptibility to damage by subsequent hypoxia were tested after 5.5h of 0.8% O2. Previous hyperoxia did not affect hypoxia-induced enhancement of HIF-1 alpha but modestly and significantly attenuated hypoxia-induced decreases in insulin contents. We conclude that hyperoxia exerts largely negative effects on beta cells, effects which are functional and possibly also toxic. A paradoxical positive finding (attenuation of hypoxia-induced effects) could be secondary to a protective effect of the hyperoxia-induced reduction of oxidative metabolism.

Rare coding variants in NOX4 link high ROS levels to psoriatic arthritis mutilans.

Psoriatic arthritis mutilans (PAM) is the rarest and most severe form of psoriatic arthritis, characterized by erosions of the small joints and osteolysis leading to joint disruption. Despite its severity, the underlying mechanisms are unknown, and no susceptibility genes have hitherto been identified. We aimed to investigate the genetic basis of PAM by performing massive parallel sequencing in sixty-one patients from the PAM Nordic cohort. We found rare variants in the NADPH oxidase 4 (NOX4) in four patients. In silico predictions show that the identified variants are potentially damaging. NOXs are the only enzymes producing reactive oxygen species (ROS). NOX4 is specifically involved in the differentiation of osteoclasts, the cells implicated in bone resorption. Functional follow-up studies using cell culture, zebrafish models, and measurement of ROS in patients uncovered that these NOX4 variants increase ROS levels both in vitro and in vivo. We propose NOX4 as the first candidate susceptibility gene for PAM. Our study links high levels of ROS caused by NOX4 variants to the development of PAM, offering a potential therapeutic target.

Influence of liver-X-receptor on tissue cholesterol, coenzyme Q and dolichol content.

The organ content of the mevalonate pathway lipids was investigated in liver-X-receptor (LXR) α, ß and double knock-out mice. An extensive or moderate increase of total cholesterol in the double KO mice was found in all organs elicited by the increase of the esterified form. In LXRα and double KO mice, coenzyme Q (CoQ) was decreased in liver and increased in spleen, thymus and lung, while dolichol was increased in all organs investigated. This effect was confirmed using LXR- agonist GW 3965. Analysis of CoQ distribution in organelles showed that the modifications are present in all cellular compartments and that the increase of the lipid in mitochondria was the result of a net increase of CoQ without changing the number of mitochondria. It appears that LXR influences not only cellular cholesterol homeostasis but also the metabolism of CoQ and dolichol, in an indirect manner.

Effect of Isocaloric Meals on Postprandial Glycemic and Metabolic Markers in Type 1 Diabetes-A Randomized Crossover Trial.

The aim of this study was to assess the effect of four isocaloric meals with different macronutrient compositions on postprandial blood glucose, lipids, and glucagon in adults with type 1 diabetes (T1D). Seventeen subjects tested four isocaloric meals in a randomized crossover design. The meal compositions were as follows: high-carbohydrate (HC); high-carbohydrate with extra fiber (HC-fiber); low-carbohydrate high-protein (HP); and low-carbohydrate high-fat (HF). Blood glucose and lipid measurements were collected up to 4 h and glucagon up to 3 h postprandially. Mean postprandial glucose excursions were lower after the HP compared to the HC (p = 0.036) and HC-fiber meals (p = 0.002). There were no differences in mean glucose excursions after the HF meal compared to the HC and HP meals. The HF meal resulted in higher triglyceride excursions compared to the HP meal (p < 0.001) but not compared to the HC or HC-fiber meals. Glucagon excursions were higher at 180 min after the HP meal compared to the HC and HF meals. In conclusion, the low-carbohydrate HP meal showed the most favorable glycemic and metabolic effects during a 4 h postprandial period in subjects with T1D.

Fast-Acting Insulin Aspart in Patients with Type 1 Diabetes in Real-World Clinical Practice: A Noninterventional, Retrospective Chart and Database Study.

INTRODUCTION: This study utilized continuous glucose monitoring data to analyze the effects of switching to treatment with fast-acting insulin aspart (faster aspart) in adults with type 1 diabetes (T1D) in clinical practice. METHODS: A noninterventional database review was conducted in Sweden among adults with T1D using multiple daily injection (MDI) regimens who had switched to treatment with faster aspart as part of basal-bolus treatment. Glycemic data were retrospectively collected during the 26 weeks before switching (baseline) and up to 32 weeks after switching (follow-up) to assess changes in time in glycemic range (TIR; 70-180 mg/dL), mean sensor glucose, glycated hemoglobin (HbA1c) levels, coefficient of variation, time in hyperglycemia (level 1, > 180 to ≤ 250 mg/dL; level 2, > 250 mg/dL), and time in hypoglycemia (level 1, ≥ 54 to < 70 mg/dL; level 2, < 54 mg/dL) (ClinicalTrials.gov Identifier NCT03895515). RESULTS: Overall, 178 participants were included in the study cohort. The analysis population included 82 individuals (mean age 48.5 years) with adequate glucose sensor data. From baseline to follow-up, statistically significant improvements were reported for TIR (mean increase 3.3%-points [approximately 48 min/day]; p = 0.006) with clinically relevant improvement (≥ 5%) in 43% of participants. Statistically significant improvements from baseline were observed for mean sensor glucose levels, HbA1c levels, and time in hyperglycemia (levels 1 and 2), with no statistically significant changes in time spent in hypoglycemia. CONCLUSIONS: Switching to faster aspart was associated with improvements in glycemic control without increasing hypoglycemia in adults with T1D using MDI in this real-world setting.

Comparing the Blood Response to Hyperbaric Oxygen with High-Intensity Interval Training-A Crossover Study in Healthy Volunteers.

High-intensity interval training (HIIT) and hyperbaric oxygen therapy (HBOT) induce reactive oxygen species (ROS) formation and have immunomodulatory effects. The lack of readily available biomarkers for assessing the dose-response relationship is a challenge in the clinical use of HBOT, motivating this feasibility study to evaluate the methods and variability. The overall hypothesis was that a short session of hyperbaric oxygen (HBO2) would have measurable effects on immune cells in the same physiological range as shown in HIIT; and that the individual response to these interventions can be monitored in venous blood and/or peripheral blood mononuclear cells (PBMCs). Ten healthy volunteers performed two interventions; a 28 min HIIT session and 28 min HBO2 in a crossover design. We evaluated bulk RNA sequencing data from PBMCs, with a separate analysis of mRNA and microRNA. Blood gases, peripheral venous oxygen saturation (SpvO2), and ROS levels were measured in peripheral venous blood. We observed an overlap in the gene expression changes in 166 genes in response to HIIT and HBO2, mostly involved in hypoxic or inflammatory pathways. Both interventions were followed by downregulation of several NF-κB signaling genes in response to both HBO2 and HIIT, while several interferon α/γ signaling genes were upregulated. Only 12 microRNA were significantly changed in HBO2 and 6 in HIIT, without overlap between interventions. ROS levels were elevated in blood at 30 min and 60 min compared to the baseline during HIIT, but not during/after HBO2. In conclusion, HBOT changed the gene expression in a number of pathways measurable in PBMC. The correlation of these changes with the dose and individual response to treatment warrants further investigation.

COVID-19-Induced Acute Respiratory Distress Syndrome Treated with Hyperbaric Oxygen: Interim Safety Report from a Randomized Clinical Trial (COVID-19-HBO).

BACKGROUND: A few prospective trials and case series have suggested that hyperbaric oxygen therapy (HBOT) may be efficacious for the treatment of severe COVID-19, but safety is a concern for critically ill patients. We present an interim analysis of the safety of HBOT via a randomized controlled trial (COVID-19-HBO). METHODS: A randomized controlled, open-label, clinical trial was conducted in compliance with good clinical practice to explore the safety and efficacy of HBOT for severe COVID-19 in critically ill patients with moderate acute respiratory distress syndrome (ARDS). Between 3 June 2020, and 17 May 2021, 31 patients with severe COVID-19 and moderate-to-severe ARDS, a ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2) < 26.7 kPa (200 mmHg), and at least two defined risk factors for intensive care unit (ICU) admission and/or mortality were enrolled in the trial and randomized 1:1 to best practice, or HBOT in addition to best practice. The subjects allocated to HBOT received a maximum of five treatments at 2.4 atmospheres absolute (ATA) for 80 min over seven days. The subjects were followed up for 30 days. The safety endpoints were analyzed. RESULTS: Adverse events (AEs) were common. Hypoxia was the most common adverse event reported. There was no statistically significant difference between the groups. Numerically, serious adverse events (SAEs) and barotrauma were more frequent in the control group, and the differences between groups were in favor of the HBOT in PaO2/FiO2 (PFI) and the national early warning score (NEWS); statistically, however, the differences were not significant at day 7, and no difference was observed for the total oxygen burden and cumulative pulmonary oxygen toxicity dose (CPTD). CONCLUSION: HBOT appears to be safe as an intervention for critically ill patients with moderate-to-severe ARDS induced by COVID-19. CLINICAL TRIAL REGISTRATION: NCT04327505 (31 March 2020) and EudraCT 2020-001349-37 (24 April 2020).

Hypoxic erythrocytes mediate cardioprotection through activation of soluble guanylate cyclase and release of cyclic GMP.

Red blood cells (RBCs) mediate cardioprotection via nitric oxide-like bioactivity, but the signaling and the identity of any mediator released by the RBCs remains unknown. We investigated whether RBCs exposed to hypoxia release a cardioprotective mediator and explored the nature of this mediator. Perfusion of isolated hearts subjected to ischemia-reperfusion with extracellular supernatant from mouse RBCs exposed to hypoxia resulted in improved postischemic cardiac function and reduced infarct size. Hypoxia increased extracellular export of cyclic guanosine monophosphate (cGMP) from mouse RBCs, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on RBC-soluble guanylate cyclase and cGMP transport and was sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice to increase nitric oxide bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. In a placebo-controlled clinical trial, a clear cardioprotective, soluble guanylate cyclase-dependent effect was induced by RBCs collected from patients randomized to 5 weeks nitrate-rich diet. It is concluded that RBCs generate and export cGMP as a response to hypoxia, mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention, suggesting preventive and therapeutic opportunities in ischemic heart disease.

Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes.

Diabetes mellitus (DM) is a progressive disorder with severe late complications. Normal wound healing involves a series of complex and well-orchestrated molecular events dictated by multiple factors. In diabetes, wound healing is grossly impaired due to defective, and dysregulated cellular and molecular events at all phases of wound healing resulting in chronic wounds that fail to heal. Carnosine, a dipeptide of alanine and histidine and an endogenous antioxidant is documented to accelerate healing of wounds and ulcers. However, not much is known about its role in wound healing in diabetes. Therefore, we studied the effect of carnosine in wound healing in db/db mice, a mice model of Type 2 DM. Six millimeter circular wounds were made in db/db mice and analyzed for wound healing every other day. Carnosine (100 mg/kg) was injected (I.P.) every day and also applied locally. Treatment with carnosine enhanced wound healing significantly, and wound tissue analysis showed increased expression of growth factors and cytokines genes involved in wound healing. In vitro studies with human dermal fibroblasts and microvascular-endothelial cells showed that carnosine increases cell viability in presence of high glucose. These effects, in addition to its known role as an antioxidant and a precursor for histamine synthesis, provide evidence for a possible therapeutic use of carnosine in diabetic wound healing.