Pharmacologic PPAR-γ Activation Reprograms Bone Marrow Macrophages and Partially Rescues HSPC Mobilization in Human and Murine Diabetes.

Mobilization of hematopoietic stem/progenitor cells (HSPC) from the bone marrow (BM) is impaired in diabetes. Excess oncostatin M (OSM) produced by M1 macrophages in the diabetic BM signals through p66Shc to induce Cxcl12 in stromal cells and retain HSPC. BM adipocytes are another source of CXCL12 that blunts mobilization. We tested a strategy of pharmacologic macrophage reprogramming to rescue HSPC mobilization. In vitro, PPAR-γ activation with pioglitazone switched macrophages from M1 to M2, reduced Osm expression, and prevented transcellular induction of Cxcl12 In diabetic mice, pioglitazone treatment downregulated Osm, p66Shc, and Cxcl12 in the hematopoietic BM, restored the effects of granulocyte-colony stimulation factor (G-CSF), and partially rescued HSPC mobilization, but it increased BM adipocytes. Osm deletion recapitulated the effects of pioglitazone on adipogenesis, which was p66Shc independent, and double knockout of Osm and p66Shc completely rescued HSPC mobilization. In the absence of OSM, BM adipocytes produced less CXCL12, being arguably devoid of HSPC-retaining activity, whereas pioglitazone failed to downregulate Cxcl12 in BM adipocytes. In patients with diabetes on pioglitazone therapy, HSPC mobilization after G-CSF was partially rescued. In summary, pioglitazone reprogrammed BM macrophages and suppressed OSM signaling, but sustained Cxcl12 expression by BM adipocytes could limit full recovery of HSPC mobilization.

Diabetes-Associated Myelopoiesis Drives Stem Cell Mobilopathy Through an OSM-p66Shc Signaling Pathway.

Diabetes impairs the mobilization of hematopoietic stem/progenitor cells (HSPCs) from the bone marrow (BM), which can worsen the outcomes of HSPC transplantation and of diabetic complications. In this study, we examined the oncostatin M (OSM)-p66Shc pathway as a mechanistic link between HSPC mobilopathy and excessive myelopoiesis. We found that streptozotocin-induced diabetes in mice skewed hematopoiesis toward the myeloid lineage via hematopoietic-intrinsic p66Shc. The overexpression of Osm resulting from myelopoiesis prevented HSPC mobilization after granulocyte colony-stimulating factor (G-CSF) stimulation. The intimate link between myelopoiesis and impaired HSPC mobilization after G-CSF stimulation was confirmed in human diabetes. Using cross-transplantation experiments, we found that deletion of p66Shc in the hematopoietic or nonhematopoietic system partially rescued defective HSPC mobilization in diabetes. Additionally, p66Shc mediated the diabetes-induced BM microvasculature remodeling. Ubiquitous or hematopoietic restricted Osm deletion phenocopied p66Shc deletion in preventing diabetes-associated myelopoiesis and mobilopathy. Mechanistically, we discovered that OSM couples myelopoiesis to mobilopathy by inducing Cxcl12 in BM stromal cells via nonmitochondrial p66Shc. Altogether, these data indicate that cell-autonomous activation of the OSM-p66Shc pathway leads to diabetes-associated myelopoiesis, whereas its transcellular hematostromal activation links myelopoiesis to mobilopathy. Targeting the OSM-p66Shc pathway is a novel strategy to disconnect mobilopathy from myelopoiesis and restore normal HSPC mobilization.

Acute Effects of Linagliptin on Progenitor Cells, Monocyte Phenotypes, and Soluble Mediators in Type 2 Diabetes.

CONTEXT: Circulating cells, including endothelial progenitor cells (EPCs) and monocyte subtypes, are involved in diabetic complications. Modulation of these cells may mediate additional benefits of glucose-lowering medications. OBJECTIVE: We assessed whether the dipeptidyl peptidase-4 (DPP-4) inhibitor linagliptin acutely modifies EPCs and monocyte subsets in patients with type 2 diabetes. DESIGN: This was a randomized, crossover, placebo-controlled trial. SETTING: The study was conducted at a tertiary referral diabetes outpatient clinic. PATIENTS: Forty-six type 2 diabetes patients with (n = 18) or without (n = 28) chronic kidney disease (CKD) participated in the study. INTERVENTION: Intervention included a 4-day treatment with linagliptin 5 mg or placebo during two arms separated by a 2-week washout. MAIN OUTCOME MEASURES: Before and after each treatment, we determined the levels of circulating progenitor cells (CD34, CD133, KDR) and monocyte subtypes (CD14/CD16, chemokine and scavenger receptors) and the concentrations of soluble mediators. RESULTS: Compared with placebo, linagliptin increased CD34(+)CD133(+) progenitor cells (placebo subtracted effect 40.4 ± 18.7/10(6); P = .036), CD34(+)KDR(+) EPCs (placebo subtracted effect 22.1 ± 10.2/10(6); P = .036), and CX3CR1(bright) monocytes (placebo subtracted effect 1.7 ± 0.8%; P = .032). Linagliptin abated DPP-4 activity by greater than 50%, significantly increased active glucagon-like peptide-1 and stromal cell-derived factor-1α, and reduced monocyte chemotactic protein-1, CCL22, and IL-12. Patients with CKD, as compared with those without, had lower baseline CD133(+) and CD34(+)CD133(+) cells and had borderline reduced CD34(+) and CD34(+)KDR(+) cells. The effects of linagliptin on progenitor cells and monocyte subtypes were similar in patients with or without CKD. Fasting plasma glucose, triglycerides and free fatty acids were unaffected. CONCLUSIONS: DPP-4 inhibition with linagliptin acutely increases putative vasculoregenerative and antiinflammatory cells. Direct effects of DPP-4 inhibition may be important to lower vascular risk in diabetes, especially in the presence of CKD.

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.

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.

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.

Widespread increase in myeloid calcifying cells contributes to ectopic vascular calcification in type 2 diabetes.

RATIONALE: Acquisition of a procalcific phenotype by resident or circulating cells is important for calcification of atherosclerotic plaques, which is common in diabetes. OBJECTIVE: We aim to identify and characterize circulating calcifying cells, and to delineate a pathophysiological role for these cells in type 2 diabetes. METHODS AND RESULTS: We demonstrate for the first time that a distinct subpopulation of circulating cells expressing osteocalcin and bone alkaline phosphatase (OC(+)BAP(+)) has procalcific activity in vitro and in vivo. The study of naïve patients with chronic myeloid leukemia indicated that OC(+)BAP(+) cells have a myeloid origin. Myeloid calcifying OC(+)BAP(+) cells (MCCs) could be differentiated from peripheral blood mononuclear cells, and generation of MCCs was closely associated with expression of the osteogenic transcription factor Runx2. In gender-mismatched bone marrow-transplanted humans, circulating MCCs had a much longer half-life compared with OC(-)BAP(-) cells, suggesting they belong to a stable cell repertoire. The percentage of MCCs was higher in peripheral blood and bone marrow of type 2 diabetic patients compared with controls but was lowered toward normal levels by optimization of glycemic control. Furthermore, diabetic carotid endoarterectomy specimens showed higher degree of calcification and amounts of cells expressing OC and BAP in the α-smooth muscle actin-negative areas surrounding calcified nodules, where CD68(+) macrophages colocalize. High glucose increased calcification by MCCs in vitro, and hypoxia may regulate MCC generation in vitro and in vivo. CONCLUSIONS: These data identify a novel type of blood-derived procalcific cells potentially involved in atherosclerotic calcification of diabetic patients.

The oral dipeptidyl peptidase-4 inhibitor sitagliptin increases circulating endothelial progenitor cells in patients with type 2 diabetes: possible role of stromal-derived factor-1alpha.

OBJECTIVE: Vasculoprotective endothelial progenitor cells (EPCs) are regulated by stromal-derived factor-1alpha (SDF-1alpha) and are reduced in type 2 diabetes. Because SDF-1alpha is a substrate of dipeptidyl-peptidase-4 (DPP-4), we investigated whether the DPP-4 inhibitor sitagliptin modulates EPC levels in type 2 diabetic patients. RESEARCH DESIGN AND METHODS: This was a controlled, nonrandomized clinical trial comparing 4-week sitagliptin (n = 16) versus no additional treatment (n = 16) in addition to metformin and/or secretagogues in type 2 diabetic patients. We determined circulating EPC levels and plasma concentrations of SDF-1alpha, monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and nitrites/nitrates. RESULTS: There was no difference in clinical baseline data between the sitagliptin and control arms. After 4 weeks, as compared with control subjects, patients receiving sitagliptin showed a significant increase in EPCs and SDF-1alpha and a decrease in MCP-1. CONCLUSIONS: Sitagliptin increases circulating EPCs in type 2 diabetic patients with concomitant upregulation of SDF-1alpha. This ancillary effect of DPP-4 inhibition might have potential favorable cardiovascular implications.

Pharmacologic targeting of endothelial progenitor cells.

The endothelium entered the field of regenerative medicine with the discovery of endothelial progenitor cells (EPCs). These cells participate in endothelial homeostasis and are actively involved in physiological and pathological neovascularization. Despite the unresolved discussion about the very phenotype of these cells, great efforts have been devoted to study the role of EPCs in cardiovascular diseases. EPCs are very rare in peripheral circulation and are further reduced in association with cardiovascular diseases. Therefore, finding therapies to improve EPCs could unleash the way to promising cell-based therapies. Ex-vivo expansion of EPCs is a critical step to augment EPCs number, but it still needs several improvements. An alternative strategy to partially overcome these limitations is to target EPCs with available drugs. In this review we will discuss how disparate pharmacological compounds have been used to improve EPCs number and functions.