Ilenia Baesso

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.

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.

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.

Gender Differences in Endothelial Progenitor Cells and Cardiovascular Risk Profile The Role of Female Estrogens

Objective—Endothelial progenitor cells (EPCs) participate in vascular homeostasis and angiogenesis. The aim of the present study was to explore EPC number and function in relation to cardiovascular risk, gender, and reproductive state. Methods and Results—As measured by flow-cytometry in 210 healthy subjects, CD34+KDR+ EPCs were higher in fertile women than in men, but were not different between postmenopausal women and age-matched men. These gender gradients mirrored differences in cardiovascular profile, carotid intima-media thickness, and brachial artery flow-mediated dilation. Moreover, EPCs and soluble c-kit ligand varied in phase with menstrual cycle in ovulatory women, suggesting cyclic bone marrow mobilization. Experimentally, hysterectomy in rats was followed by an increase in circulating EPCs. EPCs cultured from female healthy donors were more clonogenic and adherent than male EPCs. Treatment with 17&bgr;-estradiol stimulated EPC proliferation and adhesion, via estrogen receptors. Finally, we show that the proangiogenic potential of female EPCs was higher than that of male EPCs in vivo. Conclusions—EPCs are mobilized cyclically in fertile women, likely to provide a pool of cells for endometrial homeostasis. The resulting higher EPC levels in women than in men reflect the cardiovascular profile and could represent one mechanism of protection in the fertile female population.

T Cells in the Myenteric Plexus of Achalasia Patients Show a Skewed TCR Repertoire and React to HSV-1 Antigens

OBJECTIVE:The loss of myenteric neurons in the lower esophageal sphincter (LES) characterizes achalasia, an esophageal motor disorder. Because the presence of lymphocytic infiltrates suggests an immuno-mediated mechanism ongoing at the sites of disease, we investigated the T-cell receptor (TCR) repertoire and the ability to recognize human herpes virus type 1 (HSV-1) antigens of LES-infiltrating T lymphocytes in achalasia patients.METHODS:Fifty-nine patients with idiopathic achalasia and 38 heart-beating cadaveric multiorgan donors (controls) were studied. By flow cytometry evaluation and CDR3 length spectratyping analysis, the lymphocytes of 18 patients and 15 controls were analyzed, whereas 41 patients and 23 controls were employed for functional assays.RESULTS:Achalasia patients were characterized by a significantly higher esophagus lymphocytic infiltrate than controls (24.71%± 3.11 and 9.54%± 1.34, respectively; P < 0.05), mainly represented by CD3+CD8+ T cells. The characterization of TCR beta chain repertoire of CD3+ cells showed the expression of a limited number of TCR beta variable (BV) gene families (from two to five out of 26), with highly restricted spectratypes, suggesting a disease-associated oligoclonal selection of T cells. Furthermore, lymphocytes from achalasia LES specifically responded to exposure to HSV-1 antigens in vitro as showed by increased proliferation and Th-1 type cytokines release.CONCLUSIONS:These data suggest that the oligoclonal lymphocytic infiltrate within the LES of achalasia patients may represent the trace of an immune-inflammatory reaction triggered by HSV-1 antigens and that the Th1-type cytokines released by the activated lymphocytes may contribute to establish the neuronal damage accounting for the clinical features of idiopathic achalasia.

Modulation of immune response by the acute and chronic exposure to high altitude

PURPOSE The chronic exposure at high altitude (HA) represents an ideal model for evaluating the in vivo effects of hypobaric hypoxia. Taking advantage of the EV-K2-CNR Pyramid, this study was designed to evaluate whether acute and chronic hypoxia differently modulates the in vivo immune responses. METHODS The study includes 13 healthy female moderately active volunteers participating to the Italian HA project EV-K2-CNR. Peripheral blood lymphocytes, collected at sea level and at HA in the Pyramid Laboratory of CNR, Nepal (5050 m), were immunologically characterized by flow cytometry and a series of molecular and functional analyses. RESULTS Flow cytometric analyses showed that: a) CD3+ T lymphocytes significantly decreased during both acute and chronic exposure to HA, b) T-cell fall was totally due to CD4+ T-cell reduction, c) B lymphocytes were not influenced by the exposure to HA, and d) natural killer (NK) cells significantly increased during acute and chronic exposure. The evaluation of the Th1/Th2 pattern demonstrated a significant decrease of the expression of the Th1 cytokine interferon-gamma (IFN-gamma) by circulating T cells during acute and chronic exposure to HA. The expression by T cells of CXCR3, a chemokine receptor typically expressed by Th1/Tc1 cells, paralleled the decrease of IFN-gamma. On the contrary, the expression of IL-4 was not conditioned by the exposure to HA. Finally, functional studies showed a significant reduction of the proliferative activity in response to mitogen (PHA) both in acute and chronic HA exposure. Despite the increased number of NK cells, NK cytotoxic activity was not influenced by the HA exposure. CONCLUSIONS Our results indicate that the in vivo exposure to HA leads to an impairment of the homeostatic regulation of Th1/Th2 immune balance that potentially could favor long-term immunological alterations and increase the risk of infections.

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) activates the MAPK pathway in human neutrophils

Infection by Helicobacter pylori causes an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by the infiltration of neutrophils andmononuclear inflammatory cells. The neutrophil‐activating protein of Helicobacter pylori (HP‐NAP) is a virulence factor that activates neutrophils, monocytes, and mast cells. However, the mechanism by which HP‐NAP activates these cells is not fully understood. Here, we show that HP‐NAP induces extracellular regulated kinase (ERK) and p38‐mitogen‐activated protein kinase (MAPK) activation in human neutrophils; c‐Jun N‐terminal kinase is not activated by HP‐NAP. A MAPK/ERK kinase inhibitor and a p38‐MAPK inhibitor suppress HP‐NAP‐mediated neutrophil oxidative burst, adhesion, andchemotaxis, but not actin polymerization. Pertussis toxin (PTX) inhibits all these neutrophil functions and the MAPK activation caused by HP‐NAP. These results demonstrate that HP‐NAP activates neutrophils through a PTX‐sensitive pathway and that ERK and p38‐MAPK are involved in many neutrophil functions stimulated by HP‐NAP.

Reduced endothelial progenitor cells and brachial artery flow-mediated dilation as evidence of endothelial dysfunction in ocular hypertension and primary open-angle glaucoma.

Purpose:  This study aimed to assess vascular endothelial function in patients with ocular hypertension (OHT) or primary open‐angle glaucoma (POAG) by measuring: (a) endothelium‐dependent flow‐mediated vasodilation (FMD) of the brachial artery, and (b) circulating endothelial progenitor cells, which are believed to support the integrity of the vascular endothelium.

Multiple myeloma plasma cells show different chemokine receptor profiles at sites of disease activity

Chemokines and their receptors play a pivotal role in the regulation of B‐lymphocyte trafficking. This study was aimed at investigating the pattern of chemokine receptor expression, including CCR1 to CCR3, CCR5 to CCR7, CXCR1 to CXCR5, and the migration ability of multiple myeloma (MM) plasma cells (PC). PC were recovered from the bone marrow (BM) of 29 MM patients, extramedullary sites of 10 patients and the BM of five controls. Flow cytometry analysis showed that the receptors mainly expressed on malignant BM PC were represented by CXCR4 (70% of patients), CCR1 (25%), CCR2 (25%), CCR5 (17%) and CXCR3 (20%), while other receptors were commonly lacking. The analysis performed on extramedullary (peripheral blood and pleural effusion) malignant PC demonstrated that the most represented receptors were CXCR4 (100%), CCR2 (66%) and CXCR1 (60%). The migratory capability of malignant PC at resting conditions identified three groups of patients with different migration (low, intermediate and high). As CXCR4 was the relevant chemokine receptor expressed by MM PC, its ligand CXCL12 induced their migration. These data suggest that malignant PC from MM display different chemokine receptor profiles and that CXCR4 is fully functional and might play a role in the spreading of the disease.

Epithelial CXCR3-B regulates chemokines bioavailability in normal, but not in Sjögren's syndrome, salivary glands

Expression of CXCR3-targeting chemokines have been demonstrated in several diseases, suggesting a critical role for CXCR3 in recruiting activated T cells to sites of immune-mediated inflammation. Sjögren’s syndrome (SS) is an autoimmune disease characterized by a mononuclear cell infiltrate of activated T cells around the duct in the salivary gland. Analysis of minor salivary gland biopsy specimens from 20 healthy subjects and 18 patients with primary SS demonstrated that CXCR3, in particular, the B form of this receptor, is constitutively expressed by human salivary gland epithelial cells. Salivary gland epithelial cell cultures demonstrated that CXCR3 participate in removing relevant amount of agonists from the supernatant of exposed cells without mediating calcium flux or chemotaxis while retaining the ability to undergo internalization. Although in normal salivary gland epithelial cells, CXCR3 behaves as a chemokine-scavenging receptor, its role in SS cells is functionally impaired. The impairment of this scavenging function might favor chemotaxis, leading to heightened immigration of CXCR3-positive T lymphocytes. These findings suggest that epithelial CXCR3 may be involved in postsecretion regulation of chemokine bioavailability. They also support a critical role for CXCR3 in the pathogenesis of SS and identify its agonists as potential therapeutic targets.