Gian Paolo Fadini

Number and Function of Endothelial Progenitor Cells as a Marker of Severity for Diabetic Vasculopathy

Objective—Peripheral arterial disease (PAD) is a threatening complication of diabetes. As endothelial progenitor cells (EPCs) are involved in neovasculogenesis and maintenance of vascular homeostasis, their impairment may have a role in the pathogenesis of diabetic vasculopathy. This study aimed to establish whether number and function of EPCs correlate with PAD severity in type 2 diabetic patients. Methods and Results—EPCs were defined by the expression of CD34, CD133 and KDR, and quantified by flow cytometry in 127 diabetic patients with and without PAD. PAD severity has been assessed as carotid atherosclerosis and clinical stage of leg atherosclerosis obliterans. Diabetic patients with PAD displayed a significant 53% reduction in circulating EPCs versus non-PAD patients, and EPC levels were negatively correlated with the degree of carotid stenosis and the stage of leg claudication. Moreover, the clonogenic and adhesion capacity of cultured EPCs were significantly lower in diabetic patients with PAD versus patients without. Conclusions—This study demonstrates that EPC decrease is related to PAD severity and that EPC function is altered in diabetic subjects with PAD, strengthening the pathogenetic role of EPC dysregulation in diabetic vasculopathy. EPC count may be considered a novel biological marker of peripheral atherosclerosis in diabetes.

The Oral Dipeptidyl Peptidase-4 Inhibitor Sitagliptin Increases Circulating Endothelial Progenitor Cells in Patients With Type 2 Diabetes: Possible role of stromal-derived factor-1α

OBJECTIVE Vasculoprotective endothelial progenitor cells (EPCs) are regulated by stromal-derived factor-1α (SDF-1α) and are reduced in type 2 diabetes. Because SDF-1α 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-1α, 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-1α and a decrease in MCP-1. CONCLUSIONS Sitagliptin increases circulating EPCs in type 2 diabetic patients with concomitant upregulation of SDF-1α. This ancillary effect of DPP-4 inhibition might have potential favorable cardiovascular implications.

Technical notes on endothelial progenitor cells : Ways to escape from the knowledge plateau

In the last 10 years an increasing interest has been devoted to the study of endothelial progenitor cells (EPCs), a subtype of immature cells involved in endothelial repair and neoangiogenesis. EPCs have been discovered as a novel integrated part of the cardiovascular system, which plays a comprehensive role in tissue homeostasis. Consistently, alterations and/or reduction of the circulating EPC pool have been associated with different manifestations of cardiovascular disorders and atherosclerosis. This is why, the extent of the EPC pool is now considered a mirror of vascular health, while EPC reduction has become a surrogate biomarker of cardiovascular risk and of the ongoing vascular damage. Unfortunately, the methods used to study EPCs still lack standardization, and this is significantly decelerating progress in the field. In this review, we focus on some aspects related to the two methods used to assess circulating EPCs: flow cytometry and cell culture. We uncover the many traps hidden in the choice of the right protocol, and suggest the best solutions on the basis of evidence and background theories.

Autologous stem cell therapy for peripheral arterial disease Meta-analysis and systematic review of the literature

BACKGROUND Peripheral arterial disease (PAD) is a common cause of disability and mortality. Up to one third of patients are not susceptible to traditional revascularization and may benefit from stem cell therapies. OBJECTIVE In this meta-analysis, we sought to determine whether autologous cell therapy is effective in the treatment of PAD. METHODS We searched the English literature in Medline, Excerpta Medica and the Cochrane database for trials of autologous cell therapy in patients with PAD published before 31 January 2009. We included controlled and non-controlled, randomized and non-randomized trials using autologous bone marrow or granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood cells to treat PAD. We also collected data from trials of G-CSF monotherapy, as a control treatment. RESULTS In a meta-analysis of 37 trials, autologous cell therapy was effective in improving surrogate indexes of ischemia, subjective symptoms and hard endpoints (ulcer healing and amputation). On the contrary, G-CSF monotherapy was not associated with significant improvement in the same endpoints. Patients with thromboangiitis obliterans showed some larger benefits than patients with atherosclerotic PAD. The intramuscular route of administration and the use of bone marrow cells seemed somehow more effective than intrarterial administration and the use of mobilized peripheral blood cells. The procedures were well tolerated and generally safe. CONCLUSION This meta-analysis indicates that intramuscular autologous bone marrow cell therapy is a feasible, relatively safe and potentially effective therapeutic strategy for PAD patients, who are not candidate for traditional revascularization. Larger, placebo-controlled, randomized multicenter trials need to be planned and conducted to confirm these findings.

Peripheral Blood CD34+KDR+ Endothelial Progenitor Cells Are Determinants of Subclinical Atherosclerosis in a Middle-Aged General Population

Background and Purpose— Disruption of the endothelial layer is the first step in the atherogenic process. Experimental studies have shown that endothelial progenitor cells (EPCs) are involved in endothelial homeostasis and repair. Conversely, EPC depletion has been demonstrated in the setting of established atherosclerotic diseases. With this background, we evaluated whether variations in the number of EPCs are associated with subclinical atherosclerosis in healthy subjects. Methods— Carotid intima-media thickness (IMT), high-sensitive C-reactive protein (hsCRP), levels of circulating EPCs, and cardiovascular risk were compared in 137 healthy subjects. Six subpopulations of progenitor cells were determined by flow cytometry on the basis of the surface expression of CD34, CD133, and KDR antigens: CD34+, CD133+, CD34+CD133+, CD34+KDR+, CD133+KDR+, and CD34+CD133+KDR+. Results— Among different antigenic profiles of EPCs, only CD34+KDR+ cells were significantly reduced in subjects with increased IMT. Specifically, CD34+KDR+ cells were inversely correlated with IMT, even after adjustment for hsCRP and 10-year Framingham risk and independently of other cardiovascular parameters. Conclusions— Depletion of CD34+KDR+ EPCs is an independent predictor of early subclinical atherosclerosis in healthy subjects and may provide additional information beyond classic risk factors and inflammatory markers.

Downregulation of the Longevity-Associated Protein Sirtuin 1 in Insulin Resistance and Metabolic Syndrome: Potential Biochemical Mechanisms

OBJECTIVE Sirtuins (SIRTs) are NAD+-dependent deacetylases that regulate metabolism and life span. We used peripheral blood mononuclear cells (PBMCs) to determine ex vivo whether insulin resistance/metabolic syndrome influences SIRTs. We also assessed the potential mechanisms linking metabolic alterations to SIRTs in human monocytes (THP-1) in vitro. RESEARCH DESIGN AND METHODS SIRT1-SIRT7 gene and protein expression was determined in PBMCs of 54 subjects (41 with normal glucose tolerance and 13 with metabolic syndrome). Insulin sensitivity was assessed by the minimal model analysis. Subclinical atherosclerosis was assessed by carotid intima-media thickness (IMT). In THP-1 cells exposed to high glucose or fatty acids in vitro, we explored SIRT1 expression, p53 acetylation, Jun NH2-terminal kinase (JNK) activation, NAD+ levels, and nicotinamide phosphoribosyltransferase (NAMPT) expression. The effects of SIRT1 induction by resveratrol and of SIRT1 gene silencing were also assessed. RESULTS In vivo, insulin resistance and metabolic syndrome were associated with low PBMC SIRT1 gene and protein expression. SIRT1 gene expression was negatively correlated with carotid IMT. In THP-1 cells, high glucose and palmitate reduced SIRT1 and NAMPT expression and reduced the levels of intracellular NAD+ through oxidative stress. No effect was observed in cells exposed to linoleate or insulin. High glucose and palmitate increased p53 acetylation and JNK phosphorylation; these effects were abolished in siRNA SIRT1–treated cells. Glucose- and palmitate-mediated effects on NAMPT and SIRT1 were prevented by resveratrol in vitro. CONCLUSIONS Insulin resistance and subclinical atherosclerosis are associated with SIRT1 downregulation in monocytes. Glucotoxicity and lypotoxicity play a relevant role in quenching SIRT1 expression.

Endothelial Dysfunction in Diabetes: The role of reparatory mechanisms

Type 2 diabetes is characterized by a two- to fourfold increased risk of cardiovascular disease. This is generally attributed to the adverse effects of hyperglycemia and oxidative stress on vascular biology. It has also been shown that patients with prediabetic conditions, such as impaired fasting glucose and impaired glucose tolerance, are at increased risk of cardiovascular disease as well (1). This result suggests that abnormalities in carbohydrate metabolism form a continuum that progressively worsens cardiovascular health; the first step of the adverse sequence of events that leads to the atherosclerotic process is thought to be endothelial dysfunction (2). Vascular endothelial cells play a major role in maintaining cardiovascular homeostasis. In addition to providing a physical barrier between the vessel wall and lumen, the endothelium secretes a number of mediators that regulate platelet aggregation, coagulation, fibrinolysis, and vascular tone. The term “endothelial dysfunction” refers to a condition in which the endothelium loses its physiological properties: the tendency to promote vasodilation, fibrinolysis, and anti-aggregation. Endothelial cells secrete several mediators that can alternatively mediate either vasoconstriction, such as endothelin-1 and thromboxane A2, or vasodilation, such as nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor. NO is the major contributor to endothelium-dependent relaxation in conduit arteries, whereas the contribution of endothelium-derived hyperpolarizing factor predominates in smaller resistance vessels. In patients with diabetes, endothelial dysfunction appears to be a consistent finding; indeed, there is general agreement that hyperglycemia and diabetes lead to an impairment of NO production and activity. The endothelium has a limited intrinsic capacity of self-repair, being built up by terminally differentiated cells with a low proliferative potential. That is why endothelial repair is accomplished through the contribution of circulating cells, namely endothelial progenitor cells (EPCs), in physiological and pathological conditions. In this review, we will outline the mechanisms of endothelial dysfunction …

Rosiglitazone Reduces Glucose-Induced Oxidative Stress Mediated by NAD(P)H Oxidase via AMPK-Dependent Mechanism

Objective—Hyperglycemia is the main determinant of long-term diabetic complications, mainly through induction of oxidative stress. NAD(P)H oxidase is a major source of glucose-induced oxidative stress. In this study, we tested the hypothesis that rosiglitazone (RSG) is able to quench oxidative stress initiated by high glucose through prevention of NAD(P)H oxidase activation. Methods and Results—Intracellular ROS were measured using the fluoroprobe TEMPO-9-AC in HUVECs exposed to control (5 mmol/L) and moderately high (10 mmol/L) glucose concentrations. NAD(P)H oxidase and AMPK activities were determined by Western blot. We found that 10 mmol/L glucose increased significantly ROS production in comparison with 5 mmol/L glucose, and that this effect was completely abolished by RSG. Interestingly, inhibition of AMPK, but not PPAR&ggr;, prevented this effect of RSG. AMPK phosphorylation by RSG was necessary for its ability to hamper NAD(P)H oxidase activation, which was indispensable for glucose-induced oxidative stress. Downstream of AMPK activation, RSG exerts antioxidative effects by inhibiting PKC. Conclusions—This study demonstrates that RSG activates AMPK which, in turn, prevents hyperactivity of NAD(P)H oxidase induced by high glucose, possibly through PKC inhibition. Therefore, RSG protects endothelial cells against glucose-induced oxidative stress with an AMPK-dependent and a PPAR&ggr;-independent mechanism.

Cardiovascular effects of DPP-4 inhibition: Beyond GLP-1

Dipeptydil-peptidase-4 (DPP-4) inhibitors are available as oral anti-hyperglycemic drugs for the treatment of type 2 diabetes. Their metabolic effect is mediated through sparing incretin hormones (such as glucagon-like peptide-1, GLP-1) from the rapid degradation by DPP-4. In turn, GLP-1 improves meal-stimulated insulin secretion by pancreatic β-cells thus reducing hyperglycemia. It has been shown that GLP-1 signaling is also active in the cardiovascular system, where it may exert beneficial effects. However, DPP-4 has several non-incretin substrates, and its immunomodulatory activity is known from decades. DPP-4 physiologically cleaves cytokines, chemokines and neuropeptides involved in inflammation, immunity, and vascular function. Owing to these off-target mechanisms, DPP-4 inhibitors hold promise for cardiovascular protection, but may also face unexpected side effects. Herein, we review available data on the cardiovascular effects of DPP-4 inhibitors, with a special interest in GLP-1-independent mechanisms. The modulation of endothelial progenitor cells, inflammatory pathway and ischemic response emerges as the major cardiovascular target of DPP-4 inhibitors.

Time Course and Mechanisms of Circulating Progenitor Cell Reduction in the Natural History of Type 2 Diabetes

OBJECTIVE Reduction of bone marrow–derived circulating progenitor cells has been proposed as a novel mechanism of cardiovascular disease in type 2 diabetes. The present study was designed to describe the extent and potential mechanisms of progenitor cell reduction during the natural history of type 2 diabetes. RESEARCH DESIGN AND METHODS We identified 425 individuals, divided into seven categories according to carbohydrate metabolism status (normal glucose tolerance [NGT], impaired fasting glucose, impaired glucose tolerance [IGT], and newly diagnosed type 2 diabetes) and diabetes duration (0–9, 10–19, and ≥20 years). These categories were examined as ideally describing the natural history of type 2 diabetes development and progression. We measured CD34+ and CD34+KDR+ progenitor cells by flow cytometry. We also evaluated progenitor cells in 20 coupled bone marrow and peripheral blood samples and examined progenitor cell apoptosis in 34 subjects. RESULTS In comparison to NGT, CD34+ cells were significantly reduced in IGT and had a first nadir in newly diagnosed type 2 diabetes and a second nadir after 20 years of diabetes. Statistical adjustment for possible confounders confirmed that CD34+ cell counts are deeply reduced at time of diagnosis, that they partially recover during the subsequent 0–19 years, and that they dip again after ≥20 years. A similar, but less consistent, trend was detected for CD34+KDR+ cells. Peripheral blood CD34+ cells were directly correlated with bone marrow CD34+ cells and inversely correlated with CD34+ cell apoptosis. CONCLUSIONS Circulating progenitor cell reduction marks the clinical onset of type 2 diabetes. Both defective mobilization and increased apoptosis may account for this phenomenon. While a partial recovery occurs during subsequent years, bone marrow reserve seems exhausted in the long term.