Interplay between gut microbiota and p66Shc affects obesity-associated insulin resistance.

The 66 kDa isoform of the mammalian Shc gene promotes adipogenesis, and p66Shc-/- mice accumulate less body weight than wild-type (WT) mice. As the metabolic consequences of the leaner phenotype of p66Shc-/- mice is debated, we hypothesized that gut microbiota may be involved. We confirmed that p66Shc-/- mice gained less weight than WT mice when on a high-fat diet (HFD), but they were not protected from insulin resistance and glucose intolerance. p66Shc deletion significantly modified the composition of gut microbiota and their modification after an HFD. This was associated with changes in gene expression of Il-1b and regenerating islet-derived protein 3 γ ( Reg3g) in the gut and in systemic trimethylamine N-oxide and branched chain amino acid levels, despite there being no difference in intestinal structure and permeability. Depleting gut microbiota at the end of HFD rendered both strains more glucose tolerant but improved insulin sensitivity only in p66Shc-/- mice. Microbiota-depleted WT mice cohoused with microbiota-competent p66Shc-/- mice became significantly more insulin resistant than WT mice cohoused with WT mice, despite no difference in weight gain. These findings reconcile previous inconsistent observations on the metabolic phenotype of p66Shc-/- mice and illustrate the complex microbiome-host-genotype interplay under metabolic stress.-Ciciliot, S., Albiero, M., Campanaro, S., Poncina, N., Tedesco, S., Scattolini, V., Dalla Costa, F., Cignarella, A., Vettore, M., Di Gangi, I. M., Bogialli, S., Avogaro, A., Fadini, G. P. Interplay between gut microbiota and p66Shc affects obesity-associated insulin resistance.

p66Shc gene expression in peripheral blood mononuclear cells and progression of diabetic complications.

BACKGROUND: The risk of diabetic complications is modified by genetic and epigenetic factors. p66Shc drives the hyperglycaemic cell damage and its deletion prevents experimental diabetic complications. We herein tested whether p66Shc expression in peripheral blood mononuclear cells (PBMCs) predicts adverse outcomes in people with diabetes. METHODS: In a cohort of 100 patients with diabetes (16 type 1 and 84 type 2), we quantified baseline p66Shc expression in PBMCs by quantitative PCR. Patients were extensively characterized for demographics, anthropometrics, biochemical data, prevalence of complications, and medications. With a pseudo-prospective design, we retrieved cardiovascular death, major adverse cardiovascular events (MACE), and new occurrence of micro- or macroangiopathy during follow-up. RESULTS: At baseline, patients were on average 60 year old, with 10-year diabetes duration, and overall poor glycaemic control (HbA1c 7.8%). Patients with high versus low p66Shc expression (based on median value) had very similar baseline characteristics. Average p66Shc expression did not differ by presence/absence of complications. During a median 5.6-year follow-up, the primary endpoint of cardiovascular death or MACE occurred in 22 patients, but no relation was detected between cardiovascular outcomes and p66Shc expression. In patients who developed new complications at follow-up, baseline p66Shc was significantly higher, especially for macroangiopathy. The incidence of new macroangiopathy was > 3-times higher in patients with high versus those with low baseline p66Shc expression. CONCLUSIONS: p66Shc expression in PBMCs was not associated with prevalent diabetic complications but predicted new onset of complications, especially macroangiopathy, although no relation with hard cardiovascular endpoints was detected.

p66Shc deletion or deficiency protects from obesity but not metabolic dysfunction in mice and humans.

AIMS/HYPOTHESIS: Oxygen radicals generated by p66Shc drive adipogenesis, but contradictory data exist on the role of p66Shc in the development of obesity and the metabolic syndrome. We herein explored the relationships among p66Shc, adipose tissue remodelling and glucose metabolism using mouse models and human adipose tissue samples. METHODS: In wild-type (WT), leptin-deficient (ob/ob), p66Shc(-/-) and p66Shc(-/-) ob/ob mice up to 30 weeks of age, we analysed body weight, subcutaneous and visceral adipose tissue histopathology, glucose tolerance and insulin sensitivity, and liver and muscle fat accumulation. A group of mice on a high fat diet (HFD) was also analysed. A parallel study was conducted on adipose tissue collected from patients undergoing elective surgery. RESULTS: We found that p66Shc(-/-) mice were slightly leaner than WT mice, and p66Shc(-/-) ob/ob mice became less obese than ob/ob mice. Despite their lower body weight, p66Shc(-/-) mice accumulated ectopic fat in the liver and muscles, and were glucose intolerant and insulin resistant. Features of adverse adipose tissue remodelling induced by obesity, including adipocyte enlargement, apoptosis, inflammation and perfusion were modestly and transiently improved by p66Shc (also known as Shc1) deletion. After 12 weeks of the HFD, p66Shc(-/-) mice were leaner than but equally glucose intolerant and insulin resistant compared with WT mice. In 77 patients, we found a direct correlation between BMI and p66Shc protein levels. Patients with low p66Shc levels were less obese, but were not protected from other metabolic syndrome features (diabetes, dyslipidaemia and hypertension). CONCLUSIONS/INTERPRETATION: In mice and humans, reduced p66Shc levels protect from obesity, but not from ectopic fat accumulation, glucose intolerance and insulin resistance.

The molecular signature of impaired diabetic wound healing identifies serpinB3 as a healing biomarker.

AIMS/HYPOTHESIS: Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The mechanisms underlying delaying wound healing in diabetes are incompletely understood and tools to identify such pathways are eagerly awaited. METHODS: Wound biopsies were obtained from 75 patients with diabetic foot ulcers. Matched subgroups of rapidly healing (RH, n = 17) and non-healing (NH, n = 11) patients were selected. Proteomic analysis was performed by labelling with isobaric tag for relative and absolute quantification and mass spectrometry. Differentially expressed proteins were analysed in NH vs RH for identification of pathogenic pathways. Individual sample gene/protein validation and in vivo validation of candidate pathways in mouse models were carried out. RESULTS: Pathway analyses were conducted on 92/286 proteins that were differentially expressed in NH vs RH. The following pathways were enriched in NH vs RH patients: apoptosis, protease inhibitors, epithelial differentiation, serine endopeptidase activity, coagulation and regulation of defence response. SerpinB3 was strongly upregulated in RH vs NH wounds, validated as protein and mRNA in individual samples. To test the relevance of serpinB3 in vivo, we used a transgenic mouse model with α1-antitrypsin promoter-driven overexpression of human SERPINB3. In this model, wound healing was unaffected by SERPINB3 overexpression in non-diabetic or diabetic mice with or without hindlimb ischaemia. In an independent validation cohort of 47 patients, high serpinB3 protein content was confirmed as a biomarker of healing improvement. CONCLUSIONS/INTERPRETATION: We provide a benchmark for the unbiased discovery of novel molecular targets and biomarkers of impaired diabetic wound healing. High serpinB3 protein content was found to be a biomarker of successful healing in diabetic patients.

The dipeptidyl peptidase-4 inhibitor saxagliptin improves function of circulating pro-angiogenic cells from type 2 diabetic patients.

BACKGROUND: Type 2 diabetes (T2D) is associated with reduction and dysfunction of circulating pro-angiogenic cells (PACs). DPP-4 inhibitors, a class of oral agents for T2D, might possess pleiotropic vasculoprotective activities. Herein, we tested whether DPP-4 inhibition with Saxagliptin affects the function of circulating PACs from T2D and healthy subjects. METHODS: PACs were isolated from T2D (n = 20) and healthy (n = 20) subjects. Gene expression, clonogenesis, proliferation, adhesion, migration and tubulisation were assessed in vitro by incubating PACs with or without Saxagliptin and SDF-1α. Stimulation of angiogenesis by circulating cells from T2D patients treated with Saxagliptin or other non-incretinergic drugs was assessed in vivo using animal models. RESULTS: Soluble DPP-4 activity was predominant over cellular activity and was successfully inhibited by Saxagliptin. At baseline, T2D compared to healthy PACs contained less acLDL(+)Lectin(+) cells, and showed altered expression of genes related to adhesion and cell cycle regulation. This was reflected by impaired adhesion and clonogenesis/proliferative response of T2D PACs. Saxagliptin + SDF-1α improved adhesion and tube sustaining capacity of PACs from T2D patients. CD14+ PACs were more responsive to Saxagliptin than CD14- PACs. While Saxagliptin modestly reduced angiogenesis by mature endothelial cells, circulating PACs-progeny cells from T2D patients on Saxagliptin treatment displayed higher growth factor-inducible in vivo angiogenetic activity, compared to cells from T2D patients on non-incretinergic regimen. CONCLUSIONS: Saxagliptin reverses PACs dysfunction associated with T2D in vitro and improves inducible angiogenesis by circulating cells in vivo. These data add knowledge to the potential pleiotropic cardiovascular effects of DPP-4 inhibition.

Circulating myeloid calcifying cells have antiangiogenic activity via thrombospondin-1 overexpression.

Myeloid calcifying cells (MCCs) represent a subpopulation of human monocytes with procalcific potential and are characterized by coexpression of osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, an in-depth proteomic investigation of MCCs based on fluorescence-activated cell sorting, protein extraction and digestion, isobaric tag for relative and absolute quantitation labeling, fractionation, and analysis on matrix-assisted laser desorption/ionization-time of flight/time of flight and LTQ Orbitrap mass spectrometers identified and quantified more than 700 proteins and revealed pathways activated in OC(+)BAP(+) MCCs compared with those in OC(-)BAP(-) cells. Among proteins referable to angiogenesis, the thrombospondin-1 pathway was markedly up-regulated in MCCs vs. control cells. Up-regulation of the thrombospondin-1 pathway was confirmed by a genome-wide transcriptional analysis. Using in vitro and in vivo angiogenesis assays, we found that freshly isolated MCCs and cultured MCCs display an antiangiogenic function by means of both paracrine activity (conditioned medium) and altered spatial localization in cocultures with endothelial cells. Thrombospondin-1 inhibition by antibody-mediated neutralization or gene knockdown restored the angiogenic activity of OC(+)BAP(+) MCCs toward normal values and abolished the antiangiogenic effects of MCC conditioned medium. These data indicate that circulating MCCs exert antiangiogenic activity by virtue of their overexpression of thrombospondin-1. The study highlights the successful identification and validation of a pathogenic pathway by a gold standard proteomic/transcriptomic analysis of blood cells.

Myeloid calcifying cells promote atherosclerotic calcification via paracrine activity and allograft inflammatory factor-1 overexpression.

Several cell types contribute to atherosclerotic calcification. Myeloid calcifying cells (MCCs) are monocytes expressing osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, we tested whether MCCs promote atherosclerotic calcification in vivo. We show that the murine spleen contains OC(+)BAP(+) cells with a phenotype similar to human MCCs, a high expression of adhesion molecules and CD11b, and capacity to calcify in vitro and in vivo. Injection of GFP(+) OC(+)BAP(+) cells into 8- or 40-week ApoE(-/-) mice led to more extensive calcifications in atherosclerotic areas after 24 or 4 weeks, respectively, compared to control OC(-)BAP(-) cells. Despite that OC(+)BAP(+) cells had a selective transendothelial migration capacity, tracking of the GFP signal revealed that presence of injected cells within atherosclerotic areas was an extremely rare event and so GFP mRNA was undetectable by qPCR of lesion extracts. By converse, injected OC(+)BAP(+) cells persisted in the bloodstream and bone marrow up to 24 weeks, suggesting a paracrine effect. Indeed, OC(+)BAP(+) cell-conditioned medium (CM) promoted calcification by cultured vascular smooth muscle cells (VSMC) more than CM from OC(-)BAP(-) cells. A genomic and proteomic investigation of MCCs identified allograft inflammatory factor (AIF)-1 as a potential candidate of this paracrine activity. AIF-1 stimulated VSMC calcification in vitro and monocyte-specific (CD11b-driven) AIF-1 overexpression in ApoE(-/-) mice increased calcium content in atherosclerotic areas. In conclusion, we show that murine OC(+)BAP(+) cells correspond to human MCCs and promote atherosclerotic calcification in ApoE(-/-) mice, through paracrine activity and modulation of resident cells by AIF-1 overexpression.

Stem cell compartmentalization in diabetes and high cardiovascular risk reveals the role of DPP-4 in diabetic stem cell mobilopathy.

Bone marrow (BM) derived stem and progenitor cells contribute to cardiovascular homeostasis and are affected by cardiovascular risk factors. We devised a clinical data-driven approach to test candidate stem cell mobilizing mechanisms in pre-clinical models. We found that PB and BM CD34+ cell counts were directly correlated, and that most circulating CD34+ cells were viable, non-proliferating and derived from the BM. Thus, we analyzed PB and BM CD34+ cell levels as a two-compartment model in 72 patients with or without cardiovascular disease. Self-organizing maps showed that disturbed compartmentalization of CD34+ cells was associated with aging and cardiovascular risk factors especially diabetes. High activity of DPP-4, a regulator of the mobilizing chemokine SDF-1α, was associated with altered stem cell compartmentalization. For validation of these findings, we assessed the role of DPP-4 in the BM mobilization response of diabetic rats. Diabetes differentially affected DPP-4 activity in PB and BM and impaired stem/progenitor cell mobilization after ischemia or G-CSF administration. DPP-4 activity in the BM was required for the mobilizing effect of G-CSF, while in PB it blunted ischemia-induced mobilization. Indeed, DPP-4 deficiency restored ischemia (but not G-CSF)-induced stem cell mobilization and improved vascular recovery in diabetic animals. In conclusion, the analysis of stem cell compartmentalization in humans led us to discover mechanisms of BM unresponsiveness in diabetes determined by tissue-specific DPP-4 dysregulation.

NETosis Delays Diabetic Wound Healing in Mice and Humans.

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing.

Diabetes modifies the relationships among carotid plaque calcification, composition and inflammation.

BACKGROUND AND AIMS: Diabetes is traditionally associated with vascular calcification, but the molecular mechanisms are largely unknown. We herein explored the relationships among carotid plaque calcification, composition and gene expression, and how these are modified by diabetes. METHODS: We collected carotid endoarterectomy specimen from 59 patients, of whom 23 had diabetes. We analysed histology with pentachromic staining, calcification with Alizarin red and Von Kossa's staining, chemical calcium extraction and quantification, as well as gene expression by quantitative PCR. RESULTS: We detected no differences in the extent of plaque calcification and in plaque composition between diabetic and non-diabetic patients. In non-diabetic plaques, calcium content was directly correlated with the area occupied by muscle/fibrinoid tissue and inversely correlated with collagen, but such correlations were not seen in plaques from diabetic patients. While consistent correlations were found between calcium content and RUNX2 (direct), as well as Osteopontin (inverse), diabetes modified the association between plaque calcification and inflammatory gene expression. Only in diabetic plaques, calcium content was inversely correlated with MCP1 and IL1b, whereas the direct correlation with TNF-alpha expression seen in non-diabetic plaques was lost in diabetes. CONCLUSIONS: Though plaque composition and calcification were not quantitatively affected, diabetes modified the relationships between plaque calcium, composition and inflammation. These results suggest that the mechanisms and the clinical significance of atherosclerotic calcification in diabetic may be different than in non-diabetic patients.

Bone Marrow Macrophages Contribute to Diabetic Stem Cell Mobilopathy by Producing Oncostatin M.

Diabetes affects bone marrow (BM) structure and impairs mobilization of stem cells (SCs) into peripheral blood (PB). This amplifies multiorgan complications because BMSCs promote vascular repair. Because diabetes skews macrophage phenotypes and BM macrophages (BMMΦ) prevent SC mobilization, we hypothesized that excess BMMΦ contribute to diabetic SC mobilopathy. We show that patients with diabetes have increased M1 macrophages, whereas diabetic mice have increased CD169(+) BMMΦ with SC-retaining activity. Depletion of BMMΦ restored SC mobilization in diabetic mice. We found that CD169 labels M1 macrophages and that conditioned medium (CM) from M1 macrophages, but not from M0 and M2 macrophages, induced chemokine (C-X-C motif) ligand 12 (CXCL12) expression by mesenchymal stem/stromal cells. In silico data mining and in vitro validation identified oncostatin M (OSM) as the soluble mediator contained in M1 CM that induces CXCL12 expression via a mitogen-activated protein kinase kinase-p38-signal transducer and activator of a transcription 3-dependent pathway. In diabetic mice, OSM neutralization prevented CXCL12 induction and improved granulocyte-colony stimulating factor and ischemia-induced mobilization, SC homing to ischemic muscles, and vascular recovery. In patients with diabetes, BM plasma OSM levels were higher and correlated with the BM-to-PB SC ratio. In conclusion, BMMΦ prevent SC mobilization by OSM secretion, and OSM antagonism is a strategy to restore BM function in diabetes, which can translate into protection mediated by BMSCs.

Diabetes Limits Stem Cell Mobilization Following G-CSF but Not Plerixafor.

Previous studies suggest that diabetes impairs hematopoietic stem cell (HSC) mobilization in response to granulocyte colony-stimulating factor (G-CSF). In this study, we tested whether the CXCR4 antagonist plerixafor, differently from G-CSF, is effective in mobilizing HSCs in patients with diabetes. In a prospective study, individuals with and without diabetes (n = 10/group) were administered plerixafor to compare CD34(+) HSC mobilization; plerixafor was equally able to mobilize CD34(+) HSCs in the two groups, whereas in historical data, G-CSF was less effective in patients with diabetes. In a retrospective autologous transplantation study conducted on 706 patients, diabetes was associated with poorer mobilization in patients who received G-CSF with/without chemotherapy, whereas it was not in patients who received G-CSF plus plerixafor. Similarly in an allogeneic transplantation study (n = 335), diabetes was associated with poorer mobilization in patients who received G-CSF. Patients with diabetes who received G-CSF without plerixafor had a lower probability of reaching >50/µL CD34(+) HSCs, independent from confounding variables. In conclusion, diabetes negatively impacted HSC mobilization after G-CSF with or without chemotherapy but had no effect on mobilization induced by G-CSF with plerixafor. This finding has major implications for the care of patients with diabetes undergoing stem cell mobilization and transplantation and for the vascular regenerative potential of bone marrow stem cells.

NETosis is induced by high glucose and associated with type 2 diabetes.

AIMS: The role of neutrophils in diabetes and its complications is unclear. Upon challenge with microbes and inflammatory triggers, neutrophils release enzymes and nuclear material, forming neutrophils extracellular traps (NETs) and thereby dying by NETosis. We herein tested NET formation and NETosis products in high glucose and in the setting of type 2 diabetes (T2D). METHODS: NETosis was assessed in vitro in cells exposed to 0, 5, 25 mM glucose and 25 mM mannitol, DMSO and PMA using immunofluorescence staining for elastase, DNA and chromatin. Single-cell morphometric analysis was used to detect enter of elastase in the nucleus and extrusion of nuclear material. Release of NETs was quantified by staining with Hoechst 33342. In 38 T2D and 38 age- and sex-matched non-diabetic individuals, we determined plasma elastase, mono- and oligonucleosomes and double-strand (ds) DNA, as circulating NETosis products. RESULTS: NETosis was accurately reproduced in vitro: high (25 mM) glucose increased NETosis rate and release of NETs compared with 5 mM glucose and 25 mM mannitol. T2D patients showed increased plasma elastase, mono- and oligonucleosomes and dsDNA compared with non-diabetic control individuals. A positive correlation was found between HbA1c and mono- and oligonucleosomes, whereas dsDNA was correlated with the presence of nephropathy and cardiovascular disease. Serum IL-6 concentrations were higher in T2D compared with CTRL and correlated with serum dsDNA levels. CONCLUSIONS: High glucose and hyperglycemia increase release of NETs and circulating markers of NETosis, respectively. This finding provides a link among neutrophils, inflammation and tissue damage in diabetes.

Diabetes causes bone marrow autonomic neuropathy and impairs stem cell mobilization via dysregulated p66Shc and Sirt1.

Diabetes compromises the bone marrow (BM) microenvironment and reduces the number of circulating CD34(+) cells. Diabetic autonomic neuropathy (DAN) may impact the BM, because the sympathetic nervous system is prominently involved in BM stem cell trafficking. We hypothesize that neuropathy of the BM affects stem cell mobilization and vascular recovery after ischemia in patients with diabetes. We report that, in patients, cardiovascular DAN was associated with fewer circulating CD34(+) cells. Experimental diabetes (streptozotocin-induced and ob/ob mice) or chemical sympathectomy in mice resulted in BM autonomic neuropathy, impaired Lin(-)cKit(+)Sca1(+) (LKS) cell and endothelial progenitor cell (EPC; CD34(+)Flk1(+)) mobilization, and vascular recovery after ischemia. DAN increased the expression of the 66-kDa protein from the src homology and collagen homology domain (p66Shc) and reduced the expression of sirtuin 1 (Sirt1) in mice and humans. p66Shc knockout (KO) in diabetic mice prevented DAN in the BM, and rescued defective LKS cell and EPC mobilization. Hematopoietic Sirt1 KO mimicked the diabetic mobilization defect, whereas hematopoietic Sirt1 overexpression in diabetes rescued defective mobilization and vascular repair. Through p66Shc and Sirt1, diabetes and sympathectomy elevated the expression of various adhesion molecules, including CD62L. CD62L KO partially rescued the defective stem/progenitor cell mobilization. In conclusion, autonomic neuropathy in the BM impairs stem cell mobilization in diabetes with dysregulation of the life-span regulators p66Shc and Sirt1.

Diabetes impairs stem cell and proangiogenic cell mobilization in humans.

OBJECTIVE: Diabetes mellitus (DM) increases cardiovascular risk, at least in part, through shortage of vascular regenerative cells derived from the bone marrow (BM). In experimental models, DM causes morphological and functional BM alterations, but information on BM function in human DM is missing. Herein, we sought to assay mobilization of stem and proangiogenic cells in subjects with and without DM. RESEARCH DESIGN AND METHODS: In a prospective trial (NCT01102699), we tested BM responsiveness to 5 µg/kg human recombinant granulocyte colony-stimulating factor (hrG-CSF) in 24 individuals with DM (10 type 1 and 14 type 2) and 14 individuals without DM. Before and 24 h after hrG-CSF, we quantified circulating stem/progenitor cells and total and differential white blood cell counts. We also evaluated in vivo the proangiogenic capacity of peripheral blood mononuclear cells using the Matrigel plug assay. RESULTS: In response to hrG-CSF, levels of CD34(+) cells and other progenitor cell phenotypes increased in subjects without DM. Patients with DM had significantly impaired mobilization of CD34(+), CD133(+), and CD34(+)CD133(+) hematopoietic stem cells and CD133(+)KDR(+) endothelial progenitors, independently of potential confounders. The in vivo angiogenic capacity of peripheral blood mononuclear cells significantly increased after hrG-CSF in control subjects without DM, but not in patients with DM. DM was also associated with the inability to upregulate CD26/DPP-4 on CD34(+) cells, which is required for the mobilizing effect of granulocyte colony-stimulating factor. CONCLUSIONS: Stem and proangiogenic cell mobilization in response to hrG-CSF is impaired in DM, possibly because of maladaptive CD26/DPP-4 regulation. These alterations may hamper tissue repair and favor the development of cardiovascular complications.