Phenotypic and genome-wide studies on dicarbonyls: major associations to glomerular filtration rate and gamma-glutamyltransferase activity.

BACKGROUND: The dicarbonyl compounds methylglyoxal (MG), glyoxal (GO) and 3-deoxyglucosone (3-DG) have been linked to various diseases. However, disease-independent phenotypic and genotypic association studies with phenome-wide and genome-wide reach, respectively, have not been provided. METHODS: MG, GO and 3-DG were measured by LC-MS in 1304 serum samples of two populations (KORA, n = 482; BiDirect, n = 822) and assessed for associations with genome-wide SNPs (GWAS) and with phenome-wide traits. Redundancy analysis (RDA) was used to identify major independent trait associations. FINDINGS: Mutual correlations of dicarbonyls were highly significant, being stronger between MG and GO (ρ = 0.6) than between 3-DG and MG or GO (ρ = 0.4). Significant phenotypic results included associations of all dicarbonyls with sex, waist-to-hip ratio, glomerular filtration rate (GFR), gamma-glutamyltransferase (GGT), and hypertension, of MG and GO with age and C-reactive protein, of GO and 3-DG with glucose and antidiabetics, of MG with contraceptives, of GO with ferritin, and of 3-DG with smoking. RDA revealed GFR, GGT and, in case of 3-DG, glucose as major contributors to dicarbonyl variance. GWAS did not identify genome-wide significant loci. SNPs previously associated with glyoxalase activity did not reach nominal significance. When multiple testing was restricted to the lead SNPs of GWASs on the traits selected by RDA, 3-DG was found to be associated (p = 2.3 × 10-5) with rs1741177, an eQTL of NF-κB inhibitor NFKBIA. INTERPRETATION: This large-scale, population-based study has identified numerous associations, with GFR and GGT being of pivotal importance, providing unbiased perspectives on dicarbonyls beyond the current state. FUNDING: Deutsche Forschungsgemeinschaft, Helmholtz Munich, German Centre for Cardiovascular Research (DZHK), German Federal Ministry of Research and Education (BMBF).

Non-cross-linking advanced glycation end products affect prohormone processing.

Advanced glycation end products (AGEs) are non-enzymatic post-translational modifications of amino acids and are associated with diabetic complications. One proposed pathomechanism is the impaired processing of AGE-modified proteins or peptides including prohormones. Two approaches were applied to investigate whether substrate modification with AGEs affects the processing of substrates like prohormones to the active hormones. First, we employed solid-phase peptide synthesis to generate unmodified as well as AGE-modified protease substrates. Activity of proteases towards these substrates was quantified. Second, we tested the effect of AGE-modified proinsulin on the processing to insulin. Proteases showed the expected activity towards the unmodified peptide substrates containing arginine or lysine at the C-terminal cleavage site. Indeed, modification with Nε-carboxymethyllysine (CML) or methylglyoxal-hydroimidazolone 1 (MG-H1) affected all proteases tested. Cysteine cathepsins displayed a reduction in activity by ∼50% towards CML and MG-H1 modified substrates. The specific proteases trypsin, proprotein convertases subtilisin-kexins (PCSKs) type proteases, and carboxypeptidase E (CPE) were completely inactive towards modified substrates. Proinsulin incubation with methylglyoxal at physiological concentrations for 24 h resulted in the formation of MG-modified proinsulin. The formation of insulin was reduced by up to 80% in a concentration-dependent manner. Here, we demonstrate the inhibitory effect of substrate-AGE modifications on proteases. The finding that PCSKs and CPE, which are essential for prohormone processing, are inactive towards modified substrates could point to a yet unrecognized pathomechanism resulting from AGE modification relevant for the etiopathogenesis of diabetes and the development of obesity.

Transplantation of porcine adrenal spheroids for the treatment of adrenal insufficiency.

Primary adrenal insufficiency is a life-threatening disorder, which requires lifelong hormone replacement therapy. Transplantation of xenogeneic adrenal cells is a potential alternative approach for the treatment of adrenal insufficiency. For a successful outcome of this replacement therapy, transplanted cells should provide adequate hormone secretion and respond to adrenal physiological stimuli. Here, we describe the generation and characterization of primary porcine adrenal spheroids capable of replacing the function of adrenal glands in vivo. Cells within the spheroids morphologically resembled adult adrenocortical cells and synthesized and secreted adrenal steroid hormones in a regulated manner. Moreover, the embedding of the spheroids in alginate led to the formation of cellular elongations of steroidogenic cells migrating centripetally towards the inner part of the slab, similar to zona Fasciculata cells in the intact organ. Finally, transplantation of adrenal spheroids in adrenalectomized SCID mice reversed the adrenal insufficiency phenotype, which significantly improved animals' survival. Overall, such adrenal models could be employed for disease modeling and drug testing, and represent the first step toward potential clinical trials in the future.

Gastric Bypass Resolves Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD) in Low-BMI Patients: A Prospective Cohort Study.

OBJECTIVE: Metabolic dysfunction-associated fatty liver disease (MAFLD) reflects the multifactorial pathogenesis of fatty liver disease in metabolically sick patients. The effects of metabolic surgery on MAFLD have not been investigated. This study assesses the impact of Roux-en-Y gastric bypass (RYGB) on MAFLD in a prototypical cohort outside the guidelines for obesity surgery. METHODS: Twenty patients were enrolled in this prospective, single-arm trial investigating the effects of RYGB on advanced metabolic disease (DRKS00004605). Inclusion criteria were an insulin-dependent type 2 diabetes, body mass index of 25 to 35 kg/m 2 , glucagon-stimulated C-peptide of >1.5 ng/mL, glycated hemoglobin >7%, and age 18 to 70 years. A RYGB with intraoperative liver biopsies and follow-up liver biopsies 3 years later was performed. Steatohepatitis was assessed by expert liver pathologists. Data were analyzed using the Wilcoxon rank sum test and a P value <0.05 was defined as significant. RESULTS: MAFLD completely resolved in all patients 3 years after RYGB while fibrosis improved as well. Fifty-five percent were off insulin therapy with a significant reduction in glycated hemoglobin (8.45±0.27% to 7.09±0.26%, P =0.0014). RYGB reduced systemic and hepatic nitrotyrosine levels likely through upregulation of NRF1 and its dependent antioxidative and mitochondrial genes. In addition, central metabolic regulators such as SIRT1 and FOXO1 were upregulated while de novo lipogenesis was reduced and ß-oxidation was improved in line with an improvement of insulin resistance. Lastly, gastrointestinal hormones and adipokines secretion were changed favorably. CONCLUSIONS: RYGB is a promising therapy for MAFLD even in low-body mass index patients with insulin-treated type 2 diabetes with complete histologic resolution. RYGB restores the oxidative balance, adipose tissue function, and gastrointestinal hormones.

Novel Nongenetic Murine Model of Hyperglycemia and Hyperlipidemia-Associated Aggravated Atherosclerosis.

Objective: Atherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients. Genetic mouse models require breeding efforts which are time-consuming and costly. Our aim was to establish a new nongenetic model of inducible metabolic risk factors that mimics hyperlipidemia, hyperglycemia, or both and allows the detection of phenotypic differences dependent on the metabolic stressor(s). Methods and Results: Wild-type mice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin and fed either a high-fat diet (HFD) for 12 or 20 weeks or a high-cholesterol/high-fat diet (Paigen diet, PD) for 8 weeks. To evaluate atherosclerosis, two different vascular sites (aortic sinus and the truncus of the brachiocephalic artery) were examined in the mice. Combined hyperlipidemic and hyperglycemic (HGHCi) mice fed a HFD or PD displayed characteristic features of aggravated atherosclerosis when compared to hyperlipidemia (HCi HFD or PD) mice alone. Atherosclerotic plaques of HGHCi HFD animals were larger, showed a less stable phenotype (measured by the increased necrotic core area, reduced fibrous cap thickness, and less α-SMA-positive area) and had more inflammation (increased plasma IL-1ß level, aortic pro-inflammatory gene expression, and MOMA-2-positive cells in the BCA) after 20 weeks of HFD. Differences between the HGHCi and HCi HFD models were confirmed using RNA-seq analysis of aortic tissue, revealing that significantly more genes were dysregulated in mice with combined hyperlipidemia and hyperglycemia than in the hyperlipidemia-only group. The HGHCi-associated genes were related to pathways regulating inflammation (increased Cd68, iNos, and Tnfa expression) and extracellular matrix degradation (Adamts4 and Mmp14). When comparing HFD with PD, the PD aggravated atherosclerosis to a greater extent in mice and showed plaque formation after 8 weeks. Hyperlipidemic and hyperglycemic mice fed a PD (HGHCi PD) showed less collagen (Sirius red) and increased inflammation (CD68-positive cells) within aortic plaques than hyperlipidemic mice (HCi PD). HGHCi-PD mice represent a directly inducible hyperglycemic atherosclerosis model compared with HFD-fed mice, in which atherosclerosis is severe by 8 weeks. Conclusion: We established a nongenetically inducible mouse model allowing comparative analyses of atherosclerosis in HCi and HGHCi conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice.

A laser-mediated photo-manipulative toolbox for generation and real-time monitoring of DNA lesions.

With the advancement of laser-based microscopy tools, it is now possible to explore mechano-kinetic processes occurring inside the cell. Here, we describe the advanced protocol for studying the DNA repair kinetics in real time using the laser to induce the DNA damage. This protocol can be used for inducing, testing, and studying the repair mechanisms associated with DNA double-strand breaks, interstrand cross-link repair, and single-strand break repair. For complete details on the use and execution of this protocol, please refer to Kumar et al. (2017, 2020).

Interaction between magnesium and methylglyoxal in diabetic polyneuropathy and neuronal models.

OBJECTIVE: The lack of effective treatments against diabetic sensorimotor polyneuropathy demands the search for new strategies to combat or prevent the condition. Because reduced magnesium and increased methylglyoxal levels have been implicated in the development of both type 2 diabetes and neuropathic pain, we aimed to assess the putative interplay of both molecules with diabetic sensorimotor polyneuropathy. METHODS: In a cross-sectional study, serum magnesium and plasma methylglyoxal levels were measured in recently diagnosed type 2 diabetes patients with (n = 51) and without (n = 184) diabetic sensorimotor polyneuropathy from the German Diabetes Study baseline cohort. Peripheral nerve function was assessed using nerve conduction velocity and quantitative sensory testing. Human neuroblastoma cells (SH-SY5Y) and mouse dorsal root ganglia cells were used to characterize the neurotoxic effect of methylglyoxal and/or neuroprotective effect of magnesium. RESULTS: Here, we demonstrate that serum magnesium concentration was reduced in recently diagnosed type 2 diabetes patients with diabetic sensorimotor polyneuropathy and inversely associated with plasma methylglyoxal concentration. Magnesium, methylglyoxal, and, importantly, their interaction were strongly interrelated with methylglyoxal-dependent nerve dysfunction and were predictive of changes in nerve function. Magnesium supplementation prevented methylglyoxal neurotoxicity in differentiated SH-SY5Y neuron-like cells due to reduction of intracellular methylglyoxal formation, while supplementation with the divalent cations zinc and manganese had no effect on methylglyoxal neurotoxicity. Furthermore, the downregulation of mitochondrial activity in mouse dorsal root ganglia cells and consequently the enrichment of triosephosphates, the primary source of methylglyoxal, resulted in neurite degeneration, which was completely prevented through magnesium supplementation. CONCLUSIONS: These multifaceted findings reveal a novel putative pathophysiological pathway of hypomagnesemia-induced carbonyl stress leading to neuronal damage and merit further investigations not only for diabetic sensorimotor polyneuropathy but also other neurodegenerative diseases associated with magnesium deficiency and impaired energy metabolism.

Characterization of experimental diabetic neuropathy using multicontrast magnetic resonance neurography at ultra high field strength.

In light of the limited treatment options of diabetic polyneuropathy (DPN) available, suitable animal models are essential to investigate pathophysiological mechanisms and to identify potential therapeutic targets. In vivo evaluation with current techniques, however, often provides only restricted information about disease evolution. In the study of patients with DPN, magnetic resonance neurography (MRN) has been introduced as an innovative diagnostic tool detecting characteristic lesions within peripheral nerves. We developed a novel multicontrast ultra high field MRN strategy to examine major peripheral nerve segments in diabetic mice non-invasively. It was first validated in a cross-platform approach on human nerve tissue and then applied to the popular streptozotocin(STZ)-induced mouse model of DPN. In the absence of gross morphologic alterations, a distinct MR-signature within the sciatic nerve was observed mirroring subtle changes of the nerves' fibre composition and ultrastructure, potentially indicating early re-arrangements of DPN. Interestingly, these signal alterations differed from previously reported typical nerve lesions of patients with DPN. The capacity of our approach to non-invasively assess sciatic nerve tissue structure and function within a given mouse model provides a powerful tool for direct translational comparison to human disease hallmarks not only in diabetes but also in other peripheral neuropathic conditions.

Serum uromodulin and Roux-en-Y gastric bypass: improvement of a marker reflecting nephron mass.

BACKGROUND: Early diagnosis of kidney disease in obese patients and in such patients with type 2 diabetes (T2D) can significantly improve treatment outcome. Serum uromodulin (sUMOD) may be a sensitive parameter for early detection of nephropathy. OBJECTIVES: To analyze sUMOD and traditional markers of kidney function in a cohort study of patients with and without obesity or T2D undergoing metabolic surgery compared with blood donors. SETTING: University of Heidelberg, Germany. METHODS: Patients with obesity (body mass index >35 kg/m2) without T2D (n = 10) and T2D (n = 10) and patients with nonsevere obesity (body mass index, 25-35 kg/m2) and insulin-dependent T2D (n = 16) undergoing Roux-en-Y gastric bypass (RYGB) were enrolled. The control group consisted of 190 blood donors. sUMOD was compared with established renal markers. RESULTS: Using sUMOD, impaired kidney function at baseline was present in both groups with T2D and in none of the patients with obesity without T2D. This impairment was not detectable through traditional markers. Significant improvement of sUMOD was shown in patients with obesity and T2D 12 months postoperatively (from 130.0 ± 77.5 to 239.5 ± 179.0 ng/mL; P = .004) and in patients with nonsevere obesity and T2D 6 months after RYGB (from 140.6 ± 78.0 to 298.7 ± 154.0 ng/mL; P = .017). In patients with obesity without T2D, sUMOD remained stable (P = .375). CONCLUSIONS: sUMOD may serve as a tissue-specific biomarker in incipient diabetic nephropathy. Improvement of sUMOD after RYGB seems to profoundly restore the structural integrity of nephrons in these patients at risk for diabetic nephropathy.

Metabolic surgery improves renal injury independent of weight loss: a meta-analysis.

BACKGROUND: Metabolic surgery is the most effective therapy for patients with type 2 diabetes (T2D), also improving diabetic kidney disease. Whether these effects depend on weight loss is currently unknown. OBJECTIVES: To assess the correlation between weight loss and improvement of diabetic kidney disease in patients with T2D undergoing metabolic surgery. SETTING: University of Heidelberg, Germany. METHODS: A systematic literature search was performed in December 2018 using the MEDLINE, EMBASE, Web of Science, and Cochrane Central Register of Controlled Trials databases without language restrictions or time limit. Studies reporting exact data on change in urinary albumin-creatinine ratio (uACR) or albuminuria as well as change in body mass index in patients with T2D undergoing metabolic surgery were included. Out of 2145 potentially eligible hits, 15 studies were included. Study quality was assessed using the Downs and Black score. Data were pooled using a random-effects model, and a Spearman's rank correlation was performed. RESULTS: No correlation was found between improved renal injury (change in uACR or albuminuria) and weight loss (change in body mass index) (rs = -.306, P = 0.504, and rs = -.086, P = .872), and no significant correlation was found between improved renal injury (change in uACR or albuminuria) and improved glycemic control (change in A1C) (rs = .378, P = .403, and rs = .500, P = .391. CONCLUSION: Metabolic surgery can improve diabetic kidney disease independent of weight loss and glycemic control. Other mechanisms, including modified adipokine balance, signaling pathways of fat tissue and gut hormones, or reduced systemic inflammation, contribute to improved renal injury, while weight loss seems to play a lesser role than expected.

Methylglyoxal and a spinal TRPA1-AC1-Epac cascade facilitate pain in the db/db mouse model of type 2 diabetes.

Painful diabetic neuropathy (PDN) is a devastating neurological complication of diabetes. Methylglyoxal (MG) is a reactive metabolite whose elevation in the plasma corresponds to PDN in patients and pain-like behavior in rodent models of type 1 and type 2 diabetes. Here, we addressed the MG-related spinal mechanisms of PDN in type 2 diabetes using db/db mice, an established model of type 2 diabetes, and intrathecal injection of MG in conventional C57BL/6J mice. Administration of either a MG scavenger (GERP10) or a vector overexpressing glyoxalase 1, the catabolic enzyme for MG, attenuated heat hypersensitivity in db/db mice. In C57BL/6J mice, intrathecal administration of MG produced signs of both evoked (heat and mechanical hypersensitivity) and affective (conditioned place avoidance) pain. MG-induced Ca2+ mobilization in lamina II dorsal horn neurons of C57BL/6J mice was exacerbated in db/db, suggestive of MG-evoked central sensitization. Pharmacological and/or genetic inhibition of transient receptor potential ankyrin subtype 1 (TRPA1), adenylyl cyclase type 1 (AC1), protein kinase A (PKA), or exchange protein directly activated by cyclic adenosine monophosphate (Epac) blocked MG-evoked hypersensitivity in C57BL/6J mice. Similarly, intrathecal administration of GERP10, or inhibitors of TRPA1 (HC030031), AC1 (NB001), or Epac (HJC-0197) attenuated hypersensitivity in db/db mice. We conclude that MG and sensitization of a spinal TRPA1-AC1-Epac signaling cascade facilitate PDN in db/db mice. Our results warrant clinical investigation of MG scavengers, glyoxalase inducers, and spinally-directed pharmacological inhibitors of a MG-TRPA1-AC1-Epac pathway for the treatment of PDN in type 2 diabetes.

Methylglyoxal accumulation de-regulates HoxA5 expression, thereby impairing angiogenesis in glyoxalase 1 knock-down mouse aortic endothelial cells.

Impaired angiogenesis leads to long-term complications and is a major contributor of the high morbidity in patients with Diabetes Mellitus (DM). Methylglyoxal (MGO) is a glycolysis byproduct that accumulates in DM and is detoxified by the Glyoxalase 1 (Glo1). Several studies suggest that MGO contributes to vascular complications through mechanisms that remain to be elucidated. In this study we have clarified for the first time the molecular mechanism involved in the impairment of angiogenesis induced by MGO accumulation. Angiogenesis was evaluated in mouse aortic endothelial cells isolated from Glo1-knockdown mice (Glo1KD MAECs) and their wild-type littermates (WT MAECs). Reduction in Glo1 expression led to an accumulation of MGO and MGO-modified proteins and impaired angiogenesis of Glo1KD MAECs. Both mRNA and protein levels of the anti-angiogenic HoxA5 gene were increased in Glo1KD MAECs and its silencing improved both their migration and invasion. Nuclear NF-ĸB-p65 was increased 2.5-fold in the Glo1KD as compared to WT MAECs. Interestingly, NF-ĸB-p65 binding to HoxA5 promoter was also 2-fold higher in Glo1KD MAECs and positively regulated HoxA5 expression in MAECs. Consistent with these data, both the exposure to a chemical inhibitor of Glo1 "SpBrBzGSHCp2" (GI) and to exogenous MGO led to the impairment of migration and the increase of HoxA5 mRNA and NF-ĸB-p65 protein levels in microvascular mouse coronary endothelial cells (MCECs). This study demonstrates, for the first time, that MGO accumulation increases the antiangiogenic factor HoxA5 via NF-ĸB-p65, thereby impairing the angiogenic ability of endothelial cells.

The Expression of Aldolase B in Islets Is Negatively Associated With Insulin Secretion in Humans.

Context: Reduced ß-cell mass, impaired islet function, and dedifferentiation are considered causal to development of hyperglycemia and type 2 diabetes. In human cohort studies, changes of islet cell-specific expression patterns have been associated with diabetes but not directly with in vivo insulin secretion. Objective: This study investigates alterations of islet gene expression and corresponding gene variants in the context of in vivo glycemic traits from the same patients. Methods: Fasting blood was collected before surgery, and pancreatic tissue was frozen after resection from 18 patients undergoing pancreatectomy. Islet tissue was isolated by laser capture microdissection. Islet transcriptome was analyzed using microarray and quantitative RT-PCR. Proteins were examined by immunohistochemistry and western blotting. The association of gene variants with insulin secretion was investigated with oral glucose tolerance test (OGTT)-derived insulin secretion measured in a large cohort of subjects at increased risk of type 2 diabetes and with hyperglycemic clamp in a subset. Results: Differential gene expression between islets from normoglycemic and hyperglycemic patients was prominent for the glycolytic enzyme ALDOB and the obesity-associated gene FAIM2. The mRNA levels of both genes correlated negatively with insulin secretion and positively with HbA1c. Islets of hyperglycemic patients displayed increased ALDOB immunoreactivity in insulin-positive cells, whereas α- and δ-cells were negative. Exposure of isolated islets to hyperglycemia augmented ALDOB expression. The minor allele of the ALDOB variant rs550915 associated with significantly higher levels of C-peptide and insulin during OGTT and hyperglycemic clamp, respectively. Conclusion: Our analyses suggest that increased ALDOB expression in human islets is associated with lower insulin secretion.

Purification and Characterization of the Soluble form of the Receptor for Advanced Glycation End-Products (sRAGE): A Novel Fast, Economical and Convenient Method.

The receptor for advanced glycation end-products (RAGE) is a multi-ligand receptor which belongs to the pattern recognition receptor family and can bind to various ligands such as advanced glycation end-products (AGEs), members of the S100 protein family, glycosaminoglycans, amyloid ß peptides, high-mobility group box-1 (HMGB1) and nucleic acids through its extracellular domain. The RAGE-ligand interaction leads to the activation of MAP kinase and NF-kB signaling pathways. Further ligand-induced up-regulation of RAGE is involved in various patho-physiological situations including late diabetic complications, Alzheimer disease and several other neurodegenerative diseases. A secreted soluble isoform of RAGE (sRAGE), corresponding to the extracellular domain only, has the ability to block RAGE-associated cellular activation and signaling. Further application of recombinant sRAGE has been shown to block RAGE-mediated pathophysiological conditions in various models of cancer or multiple sclerosis. These finding demonstrates sRAGE as a therapeutic tool to block RAGE-associated inflammatory signaling. In this manuscript, we describe a two-step simple, novel and convenient method for expressing and purifying scalable quantities of biologically active murine form of sRAGE by using E.coli as an expression host. The method we propose has several advantages over the current available methods particularly in terms of yield and quality of preparation. The sRAGE produced by this expression system retains all the secondary structural properties as analyzed by the ligand binding affinities. The produced protein also retains all the DNA-RAGE binding functional properties and thus can be a valuable tool for studying dynamics of this novel RAGE ligand. Moreover this method can be utilized by researchers to generate biologically active endotoxin-free sRAGE for in vivo applications to study and treat RAGE-associated pathologies.

Acetyl-CoA Carboxylase 1-Dependent Protein Acetylation Controls Breast Cancer Metastasis and Recurrence.

Breast tumor recurrence and metastasis represent the main causes of cancer-related death in women, and treatments are still lacking. Here, we define the lipogenic enzyme acetyl-CoA carboxylase (ACC) 1 as a key player in breast cancer metastasis. ACC1 phosphorylation was increased in invading cells both in murine and human breast cancer, serving as a point of convergence for leptin and transforming growth factor (TGF) ß signaling. ACC1 phosphorylation was mediated by TGFß-activated kinase (TAK) 1, and ACC1 inhibition was indispensable for the elevation of cellular acetyl-CoA, the subsequent increase in Smad2 transcription factor acetylation and activation, and ultimately epithelial-mesenchymal transition and metastasis induction. ACC1 deficiency worsened tumor recurrence upon primary tumor resection in mice, and ACC1 phosphorylation levels correlated with metastatic potential in breast and lung cancer patients. Given the demonstrated effectiveness of anti-leptin receptor antibody treatment in halting ACC1-dependent tumor invasiveness, our work defines a "metabolocentric" approach in metastatic breast cancer therapy.

Homeostatic nuclear RAGE-ATM interaction is essential for efficient DNA repair.

The integrity of genome is a prerequisite for healthy life. Indeed, defects in DNA repair have been associated with several human diseases, including tissue-fibrosis, neurodegeneration and cancer. Despite decades of extensive research, the spatio-mechanical processes of double-strand break (DSB)-repair, especially the auxiliary factor(s) that can stimulate accurate and timely repair, have remained elusive. Here, we report an ATM-kinase dependent, unforeseen function of the nuclear isoform of the Receptor for Advanced Glycation End-products (nRAGE) in DSB-repair. RAGE is phosphorylated at Serine376 and Serine389 by the ATM kinase and is recruited to the site of DNA-DSBs via an early DNA damage response. nRAGE preferentially co-localized with the MRE11 nuclease subunit of the MRN complex and orchestrates its nucleolytic activity to the ATR kinase signaling. This promotes efficient RPA2S4-S8 and CHK1S345 phosphorylation and thereby prevents cellular senescence, IPF and carcinoma formation. Accordingly, loss of RAGE causatively linked to perpetual DSBs signaling, cellular senescence and fibrosis. Importantly, in a mouse model of idiopathic pulmonary fibrosis (RAGE-/-), reconstitution of RAGE efficiently restored DSB-repair and reversed pathological anomalies. Collectively, this study identifies nRAGE as a master regulator of DSB-repair, the absence of which orchestrates persistent DSB signaling to senescence, tissue-fibrosis and oncogenesis.

Signal integration at the PI3K-p85-XBP1 hub endows coagulation protease activated protein C with insulin-like function.

Coagulation proteases have increasingly recognized functions beyond hemostasis and thrombosis. Disruption of activated protein C (aPC) or insulin signaling impair function of podocytes and ultimately cause dysfunction of the glomerular filtration barrier and diabetic kidney disease (DKD). We here show that insulin and aPC converge on a common spliced-X-box binding protein-1 (sXBP1) signaling pathway to maintain endoplasmic reticulum (ER) homeostasis. Analogous to insulin, physiological levels of aPC maintain ER proteostasis in DKD. Accordingly, genetically impaired protein C activation exacerbates maladaptive ER response, whereas genetic or pharmacological restoration of aPC maintains ER proteostasis in DKD models. Importantly, in mice with podocyte-specific deficiency of insulin receptor (INSR), aPC selectively restores the activity of the cytoprotective ER-transcription factor sXBP1 by temporally targeting INSR downstream signaling intermediates, the regulatory subunits of PI3Kinase, p85α and p85ß. Genome-wide mapping of condition-specific XBP1-transcriptional regulatory patterns confirmed that concordant unfolded protein response target genes are involved in maintenance of ER proteostasis by both insulin and aPC. Thus, aPC efficiently employs disengaged insulin signaling components to reconfigure ER signaling and restore proteostasis. These results identify ER reprogramming as a novel hormonelike function of coagulation proteases and demonstrate that targeting insulin signaling intermediates may be a feasible therapeutic approach ameliorating defective insulin signaling.

Electrical Muscle Stimulation Induces an Increase of VEGFR2 on Circulating Hematopoietic Stem Cells in Patients With Diabetes.

PURPOSE: External electric muscle stimulation (EMS) of the thigh muscles was found to reduce pain resulting from diabetic neuropathy (DN), a vascular complication of diabetes. This study investigated circulating hematopoietic stem cells (HSCs) after EMS treatment. Impaired function of HSCs and the subpopulation endothelial progenitor cells (EPCs), important for neovascularization and endothelial repair, has been associated with DN. METHODS: Twenty-four patients with painful DN were treated 3 times with EMS over a period of 1 week. Blood samples were collected before and after the first EMS treatment. Before a fourth treatment, neuropathic pain was evaluated and a third blood sample was collected. Cells were used for flow cytometry. FINDINGS: Patients with painful DN reported that the pain decreased after 3 times of 1-hour treatments with EMS (Neuropathy Symptom Score: from 8 to 6, P = 0.001; Neuropathy Disability Score: from 5.5 to 5, P = 0.027, n = 24). At the end of the study, diastolic blood pressure had decreased from 80 to 70 mm Hg (P = 0.043), and plasma adrenaline and noradrenaline metabolites metanephrine and normetanephrine were reduced (both P ≤ 0.01; n = 21). A single EMS treatment caused an immediate and transient decrease in the frequency of CD34+ HSCs in circulation (-20%; P < 0.001; n = 27). In 9 of the patients with DN, the proportion of HSCs expressing vascular endothelial growth factor receptor 2 (VEGFR2; defining the HSCs as EPCs) increased by 36% (P = 0.011) after EMS treatment. Proteins required for binding to endothelium (junctional adhesion molecule A and CD31), homing toward hypoxic tissue (C-X-C chemokine receptor type 4), and endothelial differentiation (CD31) were increased on HSCs immediately after EMS treatment. An increased frequency of VEGFR2 expression was also observed on HSCs of 6 healthy control volunteers (34%; P = 0.046) after EMS treatment, but not after sham treatment. IMPLICATIONS: Three EMS treatments decreased symptoms of pain caused by DN and reduced diastolic blood pressure and biomarkers of stress. A single EMS treatment increased molecules mediating attachment and differentiation on the surface of HSCs in circulation. We hypothesize that the EMS-induced increase in surface attachment molecules on the HSCs caused the HSCs to leave circulation and that EMS treatment improves the function of HSCs and EPCs in vivo.

STZ causes depletion of immune cells in sciatic nerve and dorsal root ganglion in experimental diabetes.

Streptozotocin (STZ) treatment, a common model for inducing diabetes in rodent models, induces thermal hyperalgesia and neuronal toxicity independently of hyperglycemia by oxidizing and activating TRPA1 and TRPV1. Following treatment with STZ, CD45+ immune cells were found to be depleted in sciatic nerve (SN) and DRG in mice, prior to hyperglycemia. Macrophages were also lost in DRG and NFκB-p65-activation was increased in SN macrophages. Immune cells were significantly reduced in both SN and DRG up to three weeks, post-treatment. Loss of PNS-resident macrophages in response to STZ-mediated toxicity may affect the regenerative capacity of the nerve in response to further injury caused by diabetes.

Osteointegration and Resorption of Intravertebral and Extravertebral Calcium Phosphate Cement.

STUDY DESIGN: Eleven patients with painful osteoporotic vertebral fractures who underwent kyphoplasty using calcium phosphate (CaP) cement were followed up for 1 week, 1, 2, and 3 years in a monocentric, nonrandomized, noncontrolled retrospective trial. OBJECTIVE: This study investigates long-term radiomorphologic features of intraosseous CaP cement implants and of extraosseous CaP cement leakages for up to 3 years after implantation by kyphoplasty. SUMMARY OF BACKGROUND DATA: Kyphoplasty is frequently used for the treatment of painful osteoporotic fractures. Of the materials available, CaP is frequently used as a filling material. Resorption of this material is frequently observed, although clinical outcome is comparable with other cements. METHODS: Kyphoplasty utilizing CaP cement was performed in 11 patients with painful osteoporotic vertebral fractures. All patients received a pharmacological antiosteoporosis treatment consisting of calcium, vitamin D, and a standard dose of oral bisphosphonates. Radiomorphologic measurements, pain, and mobility were assessed. RESULTS: Intraosseous and extraosseous CaP cement volumes decreased significantly over 3 years. However, vertebral stability as determined by a constant vertebral body height and the sagittal index was not impaired. Pain improved significantly 2 years after implantation and the mobility scores 1 year after kyphoplasty at least until the third year. CONCLUSIONS: Intravertebral CaP cement implants are resorbed slowly over time without jeopardizing stability and clinical outcomes most likely because of a slowly progressing osseous replacement. Extraosseous CaP cement material because of leakages during the kyphoplasty procedure is almost completely resorbed as early as 2 years after the leakage occurred. Therefore, CaP cement is an important alternative to PMMA-based cement materials utilized for kyphoplasty of osteoporotic vertebral fractures.