A polygenic risk score to help discriminate primary adrenal insufficiency of different etiologies.

BACKGROUND: Autoimmune Addison's disease (AAD) is the most common cause of primary adrenal insufficiency (PAI). Despite its exceptionally high heritability, tools to estimate disease susceptibility in individual patients are lacking. We hypothesized that polygenic risk score (PRS) for AAD could help investigate PAI pathogenesis in pediatric patients. METHODS: We here constructed and evaluated a PRS for AAD in 1223 seropositive cases and 4097 controls. To test its clinical utility, we reevaluated 18 pediatric patients, whose whole genome we also sequenced. We next explored the individual PRS in more than 120 seronegative patients with idiopathic PAI. RESULTS: The genetic susceptibility to AAD-quantified using PRS-was on average 1.5 standard deviations (SD) higher in patients compared with healthy controls (p < 2e - 16), and 1.2 SD higher in the young patients compared with the old (p = 3e - 4). Using the novel PRS, we searched for pediatric patients with strikingly low AAD susceptibility and identified cases of monogenic PAI, previously misdiagnosed as AAD. By stratifying seronegative adult patients by autoimmune comorbidities and disease duration we could delineate subgroups of PRS suggesting various disease etiologies. CONCLUSIONS: The PRS performed well for case-control differentiation and susceptibility estimation in individual patients. Remarkably, a PRS for AAD holds promise as a means to detect disease etiologies other than autoimmunity.

Self-management and hospitalization in 615 Swedish patients with Addison's disease during the coronavirus disease 2019 pandemic: a retrospective study.

OBJECTIVE: Autoimmune Addison's disease (AAD) entails a chronic adrenal insufficiency and is associated with an increased risk of severe infections. It is, however, unknown how patients with AAD were affected by the coronavirus disease 2019 (COVID-19) pandemic of 2020-2021. This study was aimed at investigating the incidence of COVID-19 in patients with AAD in Sweden, the self-adjustment of medications during the disease, impact on social aspects, and treatment during hospitalization. Additionally, we investigated if there were any possible risk factors for infection and hospitalization. DESIGN AND METHODS: Questionnaires were sent out from April to October 2021 to 813 adult patients with AAD in the Swedish Addison Registry. The questionnaires included 55 questions inquiring about COVID-19 sickness, hospital care, medications, and comorbidities, focusing on the pre-vaccine phase. RESULTS: Among the 615 included patients with AAD, COVID-19 was reported in 17% of which 8.5% required hospital care. Glucocorticoid treatment in hospitalized patients varied. For outpatients, 85% increased their glucocorticoid dosage during sickness. Older age (P = .002) and hypertension (P = .014) were associated with an increased risk of hospital care, while younger age (P < .001) and less worry about infection (P = .030) were correlated with a higher risk of COVID-19. CONCLUSIONS: In the largest study to date examining AAD during the COVID-19 pandemic, we observed that although one-fifth of the cohort contracted COVID-19, few patients required hospital care. A majority of the patients applied general recommended sick rules despite reporting limited communication with healthcare during the pandemic.

Repression of hypoxia-inducible factor-1 contributes to increased mitochondrial reactive oxygen species production in diabetes.

Background: Excessive production of mitochondrial reactive oxygen species (ROS) is a central mechanism for the development of diabetes complications. Recently, hypoxia has been identified to play an additional pathogenic role in diabetes. In this study, we hypothesized that ROS overproduction was secondary to the impaired responses to hypoxia due to the inhibition of hypoxia-inducible factor-1 (HIF-1) by hyperglycemia. Methods: The ROS levels were analyzed in the blood of healthy subjects and individuals with type 1 diabetes after exposure to hypoxia. The relation between HIF-1, glucose levels, ROS production and its functional consequences were analyzed in renal mIMCD-3 cells and in kidneys of mouse models of diabetes. Results: Exposure to hypoxia increased circulating ROS in subjects with diabetes, but not in subjects without diabetes. High glucose concentrations repressed HIF-1 both in hypoxic cells and in kidneys of animals with diabetes, through a HIF prolyl-hydroxylase (PHD)-dependent mechanism. The impaired HIF-1 signaling contributed to excess production of mitochondrial ROS through increased mitochondrial respiration that was mediated by Pyruvate dehydrogenase kinase 1 (PDK1). The restoration of HIF-1 function attenuated ROS overproduction despite persistent hyperglycemia, and conferred protection against apoptosis and renal injury in diabetes. Conclusions: We conclude that the repression of HIF-1 plays a central role in mitochondrial ROS overproduction in diabetes and is a potential therapeutic target for diabetic complications. These findings are timely since the first PHD inhibitor that can activate HIF-1 has been newly approved for clinical use. Funding: This work was supported by grants from the Swedish Research Council, Stockholm County Research Council, Stockholm Regional Research Foundation, Bert von Kantzows Foundation, Swedish Society of Medicine, Kung Gustaf V:s och Drottning Victorias Frimurarestifelse, Karolinska Institute's Research Foundations, Strategic Research Programme in Diabetes, and Erling-Persson Family Foundation for S-B.C.; grants from the Swedish Research Council and Swedish Heart and Lung Foundation for T.A.S.; and ERC consolidator grant for M.M.

GWAS for autoimmune Addison's disease identifies multiple risk loci and highlights AIRE in disease susceptibility.

Autoimmune Addison's disease (AAD) is characterized by the autoimmune destruction of the adrenal cortex. Low prevalence and complex inheritance have long hindered successful genetic studies. We here report the first genome-wide association study on AAD, which identifies nine independent risk loci (P < 5 × 10-8). In addition to loci implicated in lymphocyte function and development shared with other autoimmune diseases such as HLA, BACH2, PTPN22 and CTLA4, we associate two protein-coding alterations in Autoimmune Regulator (AIRE) with AAD. The strongest, p.R471C (rs74203920, OR = 3.4 (2.7-4.3), P = 9.0 × 10-25) introduces an additional cysteine residue in the zinc-finger motif of the second PHD domain of the AIRE protein. This unbiased elucidation of the genetic contribution to development of AAD points to the importance of central immunological tolerance, and explains 35-41% of heritability (h2).

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

Wound healing is a high energy demanding process that needs a good coordination of the mitochondria with glycolysis in the characteristic highly hypoxic environment. In diabetes, hyperglycemia impairs the adaptive responses to hypoxia with profound negative effects on different cellular compartments of wound healing. miR-210 is a hypoxia-induced microRNA that regulates cellular metabolism and processes important for wound healing. Here, we show that hyperglycemia blunted the hypoxia-dependent induction of miR-210 both in vitro and in human and mouse diabetic wounds. The impaired regulation of miR-210 in diabetic wounds is pathogenic, since local miR-210 administration accelerated wound healing specifically in diabetic but not in non-diabetic mice. miR-210 reconstitution restores the metabolic balance in diabetic wounds by reducing oxygen consumption rate and ROS production and by activating glycolysis with positive consequences on cellular migration. In conclusion, miR-210 accelerates wound healing specifically in diabetes through improvement of the cellular metabolism.

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.

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.

Perlecan heparan sulfate deficiency impairs pulmonary vascular development and attenuates hypoxic pulmonary hypertension.

AIMS: Excessive vascular cell proliferation is an important component of pulmonary hypertension (PH). Perlecan is the major heparan sulfate (HS) proteoglycan in the vascular extracellular matrix. It binds growth factors, including FGF2, and either restricts or promotes cell proliferation. In this study, we have explored the effects of perlecan HS deficiency on pulmonary vascular development and in hypoxia-induced PH. METHODS AND RESULTS: In normoxia, Hspg2(Δ3/Δ3) mice, deficient in perlecan HS, had reduced pericytes and muscularization of intra-acinar vessels. Pulmonary angiography revealed a peripheral perfusion defect. Despite these abnormalities, right ventricular systolic pressure (RVSP) and myocardial mass remained normal. After 4 weeks of hypoxia, increases in the proportion of muscularized vessels, RVSP, and right ventricular hypertrophy were significantly less in Hspg2(Δ3/Δ3) compared with wild type. The early phase of hypoxia induced a significantly lower increase in fibroblast growth factor receptor-1 (FGFR1) protein level and receptor phosphorylation, and reduced pulmonary artery smooth muscle cell (PASMC) proliferation in Hspg2(Δ3/Δ3). At 4 weeks, FGF2 mRNA and protein were also significantly reduced in Hspg2(Δ3/Δ3) lungs. Ligand and carbohydrate engagement assay showed that perlecan HS is required for HS-FGF2-FGFR1 ternary complex formation. In vitro, proliferation assays showed that PASMC proliferation is reduced by selective FGFR1 inhibition. PASMC adhesion to fibronectin was higher in Hspg2(Δ3/Δ3) compared with wild type. CONCLUSIONS: Perlecan HS chains are important for normal vascular arborization and recruitment of pericytes to pulmonary vessels. Perlecan HS deficiency also attenuates hypoxia-induced PH, where the underlying mechanisms involve impaired FGF2/FGFR1 interaction, inhibition of PASMC growth, and altered cell-matrix interactions.

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.

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.

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

BACKGROUND: 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. METHODS: The mitochondrial DNA damage was evaluated by long-fragment polymerase chain reaction in human dermal fibroblasts exposed to high glucose level and hypoxia (an additional source of reactive oxygen species) or in organs from diabetic animals (db/db mice) at different ages. Reactive oxygen species production was assessed in vitro by fluorescence and in vivo by nitrosylation of the proteins. The antioxidant enzymes were assessed by enzyme activity and by quantitative real-time polymerase chain reaction while the mitochondrial repair activity (base excision repair) was determined by using abasic site-containing oligonucleotides as substrates. RESULTS: Hyperglycaemia, when combined with hypoxia, is able to induce mitochondrial DNA damage in human dermal fibroblasts. The deleterious effect is mediated by mitochondrial reactive oxygen species, being abolished when the mitochondria electron transport is blocked. The accumulation of mitochondrial DNA damage in vivo is, however, decreased in 'old' diabetic animals (db/db) despite higher reactive oxygen species levels. This mitochondrial DNA protection might be conferred by an increased base excision repair activity. CONCLUSION: Increased base excision repair activity in tissues affected by the chronic complications of diabetes is a potential mechanism that can overcome mitochondrial DNA damage induced by hyperglycaemia-related reactive oxygen species overproduction.

Stabilization of HIF-1alpha 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) 1alpha is the critical transcription factor that regulates adaptive responses to hypoxia. HIF-1alpha 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-1alpha 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-1alpha 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-1alpha 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-1alpha is essential for the development of diabetic wounds, and we provide evidence that stabilization of HIF-1alpha is critical to reverse the pathological process.

Hypoxia-inducible factor-1alpha and hypoxia-inducible factor-2alpha are expressed in kaposi sarcoma and modulated by insulin-like growth factor-I.

PURPOSE: Neoangiogenesis is essential for tumor development. Hypoxia-inducible factor (HIF), a transcriptional factor composed of two subunits (alpha and beta), plays a key role in this process, activating proangiogenic factors such as vascular endothelial growth factor (VEGF). The HIF alpha subunits are critically regulated by oxygen and are also modulated by growth factors. Kaposi sarcoma (KS) is a highly vascular tumor that releases large amounts of VEGF and for which we have recently described an essential role for the insulin-like growth factor (IGF) system. We therefore investigated the expression of HIF alpha subunits in biopsies from KS tumors and their modulation by IGF-I in KSIMM, a KS cell line. RESULTS: Both HIF-1alpha and HIF-2alpha were expressed in KS biopsies in all tumoral stages. HIF-1alpha immunopositivity increased through the tumor development with highest expression in the late nodular stages. In KSIMM cells, IGF-I induced accumulation of both HIF alpha subunits. The induction suggests a translation mechanism as documented by cycloheximide chase experiment coupled with constant RNA levels as evaluated by quantitative real-time PCR. IGF-I-induced HIF alpha accumulation was followed by an increase in HIF function as assessed both by reporter gene assay and by induction of endogenous target gene expression (VEGF-A). Specific blockade of IGF-I receptor with alphaIR3 antibody or with picropodophyllin, a specific IGF-IR tyrosine kinase inhibitor, diminishes the basal and IGF-I-dependent induction of both HIF alpha congeners. CONCLUSION: These novel findings show the coupling between the IGF and HIF signaling in KS and suggest a coordinated contribution by these pathways to the characteristic vascular phenotype of this tumor.

Intraarticular corticosteroids decrease synovial RANKL expression in inflammatory arthritis.

OBJECTIVE: Intraarticular corticosteroids are frequently used as successful adjuvant therapy for inflammatory arthritides, but little is known about their effects on molecules that regulate bone biology. We undertook this study to investigate the effect of intraarticular corticosteroids on the synovial expression of RANKL and osteoprotegerin (OPG). METHODS: We evaluated RANKL, OPG, and surface marker expression by immunohistochemical methods in synovial knee biopsy samples obtained from 13 patients with inflammatory arthritis before and 2 weeks following intraarticular injection of triamcinolone hexacetonide. We further investigated the effect of dexamethasone (DEX) on RANKL expression by lymphocytes from rheumatoid arthritis synovial fluids (RA SF), using flow cytometric analysis. Finally, we evaluated the in vitro effect of DEX on RANKL and OPG expression in osteoblast-like cells, by Western blotting. RESULTS: Intraarticular corticosteroids induced a decrease in the number of synovial T cells without influencing the number of macrophages, evaluated as both CD68+ and CD163+ cells. This change was paralleled by a decrease of synovial RANKL expression with a concomitant reduction of the RANKL:OPG ratio. DEX down-regulated RANKL expression on lymphocytes derived from RA SF. Moreover, in vitro pretreatment of osteoblast-like cells with tumor necrosis factor favored an antiresorptive effect of DEX treatment through a similar down-regulation of RANKL expression. CONCLUSION: The decrease in inflammation attributed to intraarticular corticosteroids is accompanied by down-modulation of bone destruction markers. These findings offer a rationale for the beneficial effect of corticosteroids on joint erosion in arthritis.

Anti-tumor necrosis factor therapy increases synovial osteoprotegerin expression in rheumatoid arthritis.

OBJECTIVE: Treatment of rheumatoid arthritis (RA) with tumor necrosis factor (TNF)-blocking agents, including etanercept and infliximab, has resulted in reductions in the radiographic progression of RA. However, the exact mechanism by which this protection occurs has not been determined. In order to add to such knowledge, we investigated the effect of anti-TNF therapy on the expression of osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) in synovial tissue. METHODS: The expression of OPG and RANKL in synovial biopsy specimens was evaluated by immunohistochemistry. Serial synovial biopsy specimens were obtained from 18 patients with RA, before and after treatment with etanercept (9 patients) or infliximab (9 patients). Biopsy specimens were evaluated by double-blind semiquantitative analysis and image analysis. The in vitro effect of TNF antagonists on the RANKL/OPG expression in osteoblasts and endothelial cells was evaluated by Western blotting. Statistical analysis was performed using Wilcoxon's signed rank test, followed by the Bonferroni correction for multiple comparisons of paired samples. The results of in vitro experiments were evaluated by one-way analysis of variance, with Tukey's post hoc test. RESULTS: Treatment with both infliximab and etanercept increased the expression of OPG in synovial tissue. After 8 weeks of treatment, neither infliximab nor etanercept influenced RANKL expression. In both groups of patients, the RANKL:OPG ratio decreased following therapy. In vitro, both of the TNF antagonists mimicked the in vivo effect, inducing a decrease in the RANKL:OPG ratio in TNF-primed osteoblasts and endothelial cells. CONCLUSION: Therapy with TNF antagonists in RA modulates the OPG/RANKL system, a potential mechanism that could explain the retardation of radiographic damage observed following anti-TNF therapy.