Roberta Cappellari

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.

An unbalanced monocyte polarisation in peripheral blood and bone marrow of patients with type 2 diabetes has an impact on microangiopathy

Aim/hypothesisMonocytes/macrophages play important roles in adipose and vascular tissues and can be polarised as inflammatory M1 or anti-inflammatory M2. We sought to analyse monocyte polarisation status in type 2 diabetes, which is characterised by chronic inflammation.MethodsWe enrolled 60 individuals without diabetes and 53 patients with type 2 diabetes. We quantified standard monocyte subsets defined by cluster of differentiation (CD)14 and CD16. In addition, based on the phenotype of polarised macrophages in vitro, we characterised and quantified more definite M1 (CD68+CCR2+) and M2 (CX3CR1+CD206+/CD163+) monocytes. We also analysed bone marrow (BM) samples and the effects of granulocyte-colony stimulating factor (G-CSF) stimulation in diabetic and control individuals.ResultsWe found no alterations in standard monocyte subsets (classical, intermediate and non-classical) when comparing groups. For validation of M1 and M2 phenotypes, we observed that M2 were enriched in non-classical monocytes and had lower TNF-α content, higher LDL scavenging and lower transendothelial migratory capacity than M1. Diabetic patients displayed an imbalanced M1/M2 ratio compared with the control group, attributable to a reduction in M2. The M1/M2 ratio was directly correlated with waist circumference and HbA1c and, among diabetic patients, M2 reduction and M1/M2 increase were associated with microangiopathy. A decrease in M2 was also found in the BM from diabetic patients, with a relative M2 excess compared with the bloodstream. BM stimulation with G-CSF mobilised M2 macrophages in diabetic but not in healthy individuals.Conclusions/interpretationWe show that type 2 diabetes markedly reduces anti-inflammatory M2 monocytes through a dysregulation in bone-marrow function. This defect may have a negative impact on microangiopathy.

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.

Pro-inflammatory monocyte-macrophage polarization imbalance in human hypercholesterolemia and atherosclerosis

Monocyte-macrophages (MoMas) play a major role in atherosclerosis. In mice, hypercholesterolemia increases pro-inflammatory monocytes that promote plaque growth, but whether this is true also in humans in unknown. We herein analyzed monocyte subsets and MoMa phenotypes in familiar (FH, n = 22) and non-familiar (NFH, n = 20) hypercholesterolemic compared with normocholesterolemic (CTRL, n = 20) patients. We found that FH and NFH had higher circulating pro-inflammatory CD68(+)CCR2(+) M1 MoMas than CTRL, while anti-inflammatory CX3CR1(+)CD163(+)/CD206(+) M2 MoMas were reduced only in NFH. As a result, the M1/M2 polarization balance was increased in FH and, more markedly in NFH. M1 MoMas and the M1/M2 polarization ratio were directly correlated to pre-treatment LDL cholesterol levels and strongly associated with the presence of atherosclerotic plaques. In conclusion, we show for the first time that human hypercholesterolemia is associated with a pro-inflammatory imbalance of circulating monocytic cells, which can predispose to the development of atherosclerosis.

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.

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.

Monocyte–macrophage polarization balance in pre-diabetic individuals

Pre-diabetes is characterized by increased cardiovascular risk and chronic inflammation. The activation of monocyte–macrophages plays major roles in vascular biology. Herein, we aimed to analyze monocyte–macrophage polarization status in subjects with IFG and/or IGT compared with normal glucose tolerant (NGT) individuals. We enrolled 87 middle-aged individuals with low prevalence of cardiovascular disease. Based on OGTT, they were divided into 49 NGT and 38 pre-diabetic (IFG and/or IGT). Using flow cytometry analysis of peripheral blood cells, we quantified traditional monocyte subsets based on CD14 and CD16 expression as well as novel monocyte–macrophage pro-inflammatory CD68+CCR2+ M1 and anti-inflammatory CX3CR1+CD163+/CD206+ M2 phenotypes. The M1/M2 ratio was taken to represent the polarization balance. There were no differences in traditional classical (CD14++CD16−), intermediate (CD14++CD16+) and nonclassical (CD14+CD16+) monocytes between groups. Rather, compared to NGT, pre-diabetic subjects showed a significant increase in pro-inflammatory M1 cells and percent expression of the oxLDL scavenger receptor CD68, without changes in anti-inflammatory M2 cells. M1 levels and CD68 expression were directly correlated with HbA1c. We show for the first time that otherwise healthy pre-diabetic subjects have excess M1 inflammatory cells in peripheral blood, which may contribute to cardiovascular risk.

Alternative Activation of Human Macrophages Is Rescued by Estrogen Treatment In Vitro and Impaired by Menopausal Status

CONTEXT AND OBJECTIVE During their reproductive years, women are generally protected from cardiovascular disease events by their estrogen-replete status. Our starting hypothesis was that lower estrogen levels after menopause are associated with macrophage activation profiles skewed toward proinflammatory phenotypes. Research Design and Setting: This was an in vitro and ex vivo study in human blood-derived macrophages. SUBJECTS We obtained blood from 12 healthy male donors for the in vitro study and from 5 premenopausal and 8 postmenopausal women for the ex vivo study. OUTCOME We measured macrophage immunophenotypes in the resting state and after activation with M1-associated (lipopolysaccharide [LPS]/interferon-γ [IFN-γ]) or M2-associated (IL-4/IL-13) stimuli and expression of estrogen receptors (ERs) and other transcription factors. RESULTS Unpolarized macrophages expressed both ERα and ERβ, and ERα but not ERβ levels were decreased by M1 stimuli. LPS/IFN-γ also induced down-regulation of CD163 and CD206, markers of alternative activation, and increased cell-bound TNF-α and IL-10. These effects were prevented by 17β-estradiol treatment through impaired nuclear factor-κB liberation. In agreement with a role for 17β-estradiol in attenuating the inflammatory response, M1/M2 subpopulations in monocytes and unstimulated macrophages from premenopausal and postmenopausal donors were similar. In contrast, M2 activation appeared to be blunted in macrophages from postmenopausal women, leading to an increased M1/M2 response ratio. CONCLUSIONS Estrogen treatment prevented LPS/IFN-γ action on human M2 macrophage markers and cytokine production, whereas menopausal estrogen loss was associated with an impaired response to alternative activation, suggesting that these mechanisms affect the cardiovascular risk profile in relation to menopausal status.

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.