Role of HLA matching and donor specific antibody development in long-term survival, acute rejection and cardiac allograft vasculopathy.

Although there are different data supporting benefits of HLA matching in kidney transplantation, its role in heart transplantation is still unclear. HLA mismatch (MM) between donor and recipient can lead to the development of donor-specific antibodies (DSA) which produces negative events on the outcome of heart transplantation. Moreover, DSAs are involved in the development of antibody-mediated rejection (AMR) and are associated with an increase in cardiac allograft vasculopathy (CAV). In this study it is analyzed retrospectively the influence of HLA matching and anti-HLA antibodies on overall survival, AMR and CAV in heart transplantation. For this retrospective study are recruited heart transplanted patients at the Cardiac Transplantation Centre of Naples between 2000 and 2019. Among the 155 heart transplant patients, the mean number of HLA-A, B, -DR MM (0 to 6) between donor and recipient was 4.5 ± 1.1. The results show a negative association between MM HLA-DR and survival (p = 0.01). Comparison of patients with 0-1 MM at each locus to all others with 2 MM, for both HLA class I and class II, has not showed significant differences in the development of CAV. Our analysis detected DSA in 38.1% of patients. The production of de novo DSA reveals that there is not an influence on survival (p = 0.72) and/or AMR (p = 0.39). Instead, there is an association between the production of DSA class II and the probability of CAV development (p = 0.03). Mean fluorescence intensity (MFI) values were significantly higher in CAV-positive patients that CAV-negative patients (p = 0.02). Prospective studies are needed to evaluate HLA class II matching as an additional parameter for heart allocation, especially considering the increment of waiting list time.

Reduced levels of hepcidin associated with lower ferritin concentration and increased number of previous donations in periodic blood donors: A pilot study.

BACKGROUND: Hepcidin is one of the major negative regulators of iron balance. Periodic blood donors are highly susceptible to iron deficiency. Our goal was to evaluate the possible association between serum hepcidin levels and iron homeostasis parameters in periodic blood donors. MATERIALS AND METHODS: We enrolled a total of n = 39 periodic healthy blood donors (n = 24 M and n = 15 F). A solid phase enzyme-linked immunosorbent assay (ELISA) was performed to measure endogenous hepcidin-25 levels in serum biospecimens collected from each study participant. Statistical analysis evaluated possible associations between hepcidin levels and ferritin, transferrin, total iron binding capacity (TIBC), unsaturated iron binding capacity (UIBC), transferrin saturation (TSAT), and number of previous donations. RESULTS: Reduced serum hepcidin levels significantly correlated with lower ferritin concentration (r = 0.56, IC 95%: 0.51-0.60, p < 0.01). A multiple linear regression analysis showed that hepcidin levels were independently and negatively correlated with ferritin (p < 0.01). In addition, the number of previous blood donations was significantly associated with reduced hepcidin levels, independently of the other covariates (p < 0.01). CONCLUSION: Reduced serum hepcidin levels were significantly associated with reduced levels of ferritin and with increased number of previous donations suggesting its possible clinical role as non-invasive "point-of-care" in predicting iron deficiency among periodic blood donors.

De novo DNA methylation induced by circulating extracellular vesicles from acute coronary syndrome patients.

BACKGROUND AND AIMS: DNA methylation is associated with gene silencing, but its clinical role in cardiovascular diseases (CVDs) remains to be elucidated. We hypothesized that extracellular vesicles (EVs) may carry epigenetic changes, showing themselves as a potentially valuable non-invasive diagnostic liquid biopsy. We isolated and characterized circulating EVs of acute coronary syndrome (ACS) patients and assessed their role on DNA methylation in epigenetic modifications. METHODS: EVs were recovered from plasma of 19 ACS patients and 50 healthy subjects (HS). Flow cytometry, qRT-PCR, and Western blot (WB) were performed to evaluate both intra-vesicular and intra-cellular signals. ShinyGO, PANTHER, and STRING tools were used to perform GO and PPI network analyses. RESULTS: ACS-derived EVs showed increased levels of DNA methyltransferases (DNMTs) (p<0.001) and Ten-eleven translocation (TET) genes reduction. Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B, were significantly increased in plasma ACS-EVs. DNA methylation analysis on PBMCs from healthy donors treated with HS- and ACS-derived EVs showed an important role of DNMTs carried by EVs. PPI network analysis evidenced that ACS-EVs induced changes in PBMC methylome. In the most enriched subnetwork, the hub gene SRC was connected to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, BAIAP2 genes that were showed to have many molecular effects on various cell types into onset of several CVDs. Modulation in gene expression after ACS-EVs treatment was confirmed for SRC, NOTCH1, FOXO3, RXRA, DGKG and BAIAP2 (p<0.05). CONCLUSIONS: Our data showed an important role for ACS-derived EVs in gene expression modulation through de novo DNA methylation signals, and modulating signalling pathways in target cells.

Clinical epigenetics and acute/chronic rejection in solid organ transplantation: An update.

The lack of a precise stratification algorithm for predicting patients at high risk of graft rejection challenges the current solid organ transplantation (SOT) clinical setting. In fact, the established biomarkers for transplantation outcomes are unable to accurately predict the onset time and severity of graft rejection (acute or chronic) as well as the individual response to immunosuppressive drugs. Thus, identifying novel molecular pathways underlying early immunological responses which can damage transplant integrity is needed to reach precision medicine and personalized therapy of SOT. Direct epigenetic-sensitive mechanisms, mainly DNA methylation and histone modifications, may play a relevant role for immune activation and long-term effects (e.g., activation of fibrotic processes) which may be translated in new non-invasive biomarkers and drug targets. In particular, the measure of DNA methylation by using the blood-based "epigenetic clock" system may be an added value to the donor eligibility criteria providing an estimation of the heart biological age as well as a predictive biomarkers. Besides, monitoring of DNA methylation changes may aid to predict acute vs chronic graft damage in kidney transplantation (KT) patients. For example, hypermethylation of genes belonging to the Notch and Wnt pathways showed a higher predictive value for chronic injury occurring at 12 months post-KT with respect to established clinical parameters. Detecting higher circulating cell-free DNA (cfDNA) fragments carrying hepatocyte-specific unmethylated loci in the inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), insulin like growth factor 2 receptor (IGF2R), and vitronectin (VTN) genes may be useful to predict acute graft injury after liver transplantation (LT) in serum samples. Furthermore, hypomethylation in the forkhead box P3 (FOXP3) gene may serve as a marker of infiltrating natural Treg percentage in the graft providing the ability to predict acute rejection events after heart transplantation (HTx). We aim to update on the possible clinical relevance of DNA methylation changes regulating immune-related pathways underlying acute or chronic graft rejection in KT, LT, and HTx which might be useful to prevent, monitor, and treat solid organ rejection at personalized level.

Integrated analysis of DNA methylation profile of HLA-G gene and imaging in coronary heart disease: Pilot study.

AIMS: Immune endothelial inflammation, underlying coronary heart disease (CHD) related phenotypes, could provide new insight into the pathobiology of the disease. We investigated DNA methylation level of the unique CpG island of HLA-G gene in CHD patients and evaluated the correlation with cardiac computed tomography angiography (CCTA) features. METHODS: Thirty-two patients that underwent CCTA for suspected CHD were enrolled for this study. Obstructive CHD group included fourteen patients, in which there was a stenosis greater than or equal to 50% in one or more of the major coronary arteries detected; whereas subjects with Calcium (Ca) Score = 0, uninjured coronaries and with no obstructive CHD (no critical stenosis, NCS) were considered as control subjects (n = 18). For both groups, DNA methylation profile of the whole 5'UTR-CpG island of HLA-G was measured. The plasma soluble HLA-G (sHLA-G) levels were detected in all subjects by specific ELISA assay. Statistical analysis was performed using R software. RESULTS: For the first time, our study reported that 1) a significant hypomethylation characterized three specific fragments (B, C and F) of the 5'UTR-CpG island (p = 0.05) of HLA-G gene in CHD patients compared to control group; 2) the hypomethylation level of one specific fragment of 161bp (+616/+777) positively correlated with coronary Ca score, a relevant parameter of CCTA (p<0.05) between two groups evaluated and was predictive for disease severity. CONCLUSIONS: Reduced levels of circulating HLA-G molecules could derive from epigenetic marks. Epigenetics phenomena induce hypomethylation of specific regions into 5'UTR-CpG island of HLA-G gene in CHD patients with obstructive non critical stenosis vs coronary stenosis individuals.

Non-nutritional sweeteners effects on endothelial vascular function.

AIM: Hyperglycemia status induces endothelial dysfunction, although the underlying pathogenic mechanisms are not fully understood. There are several studies connecting sugar/sweetened beverages to the cardiovascular disease. Currently, many sweeteners have been extensively introduced into lifestyle to normalize blood glucose levels without altering the sweet taste. However, there is growing concern for their impact on metabolic health. METHODS: Human endothelial cells were treated with Glucose, Fructose, Aspartame, Rebaudioside A, Stevioside, or Steviol. Morphological characteristics, in vitro angiogenesis and array gene expression were analyzed. RESULTS: High-glucose and fructose concentrations significantly decreased cell features such as angiogenic capability. Interestingly, non-caloric sweeteners did not significantly modified all cell characteristics and they did not compromised cell angiogenic ability. Array gene expression analysis revealed that the chemokine fractalkine (CX3CL1) and the enzyme transferase (HPRT1) were always significantly upregulated and downregulated respectively, after glucose and fructose treatments (P > .05), whereas they were non-differentially expressed with all the other sweeteners. Interestingly, both genes are considered as cardiovascular disease risk biomarkers. Specifically, upregulation of CX3CL1/CX3CR1 occurs in the human placenta and serum levels of the ligand are associated with markers of insulin resistance in GDM. CONCLUSIONS: Differently from glucose and fructose, steviol glycosides do not damage endothelial cells. Prospective preclinical studies and clinical trials are warranted to confirm the long-term safety of such compounds.

Blood transfusions and adverse acute events: a retrospective study from 214 transfusion-dependent pediatric patients comparing transfused blood components by apheresis or by whole blood.

INTRODUCTION: Blood transfusion is a lifesaving procedure for patients affected by hematological diseases or hemorrhage risk. AIM: This retrospective study was aimed to evaluate clinical safety of pediatric transfusions by comparing the frequency of adverse events caused by apheretic blood components vs whole blood. METHODS: From 2011 to 2015, 214 patients (blood malignancy patients, n = 144 and thalassemic patients, n = 70) received 12 531 units of blood components. The adverse acute reactions occurred during patient hospitalization were reported to the Hemovigilance system and assessed by fitting a logistic mixed-effect model. RESULTS: A total of 33 (0.3%) adverse acute events occurred. Odds ratio (OR) of adverse events from apheresis vs whole blood transfusion adjusted by patient classification was not statistically significant (OR [95% CI], 0.75 [0.23-2.47]). CONCLUSION: Our findings showed no significant differences in the prevalence of adverse acute events between blood component collected by apheresis vs whole blood in our study center.

Genetic background, epigenetic factors and dietary interventions which influence human longevity.

Longevity is mainly conditioned by genetic, epigenetic and environmental factors. Different genetic modifications seem to be positively associated to longevity, including SNPs in SIRT1, APOE, FOXO3A, ACE, ATM, NOS1 and NOS2 gene. Epigenetic changes as DNA hyper- and hypo-methylation influence significantly human longevity by activating/deactivating different genes involved in physiological mechanisms. Several studies have confirmed that centenarians have a lower DNA methylation content compared to young subjects, which showed more homogeneously methylated DNA region. Also the up-regulation of miR-21 seems to be more associated with longevity in different populations of long-lived subjects, suggesting its role as potential epigenetic biomarkers. A non-pharmacological treatment that seems to contrast age-related diseases and promote longevity is represented by dietary intervention. It has been evaluated the effects of dietary restriction of both single nutrients or total calories to extend lifespan. However, in daily practice it is very difficult to guarantee adherence/compliance of the subjects to dietary restriction and at the same time avoid dangerous nutritional deficiencies. As consequence, the attention has focused on a variety of substances both drugs and natural compounds able to mime the beneficial effects of caloric restriction, including resveratrol, quercetin, rapamycin, metformin and 2-deoxy-D-glucose.

Genetic and epigenetic-sensitive regulatory network in immune response: a putative link between HLA-G and diabetes.

INTRODUCTION: Human leukocyte antigen-G (HLA-G) gene encodes for a tolerogenic molecule constitutively expressed in human pancreas and upregulated upon inflammatory signals. The 14 bp INS/DEL polymorphism in the 3'UTR of HLA-G may influence the susceptibility for diabetes and coronary heart diseases (CHD), thus suggesting a novel candidate gene. DNA hypomethylation at HLA-G promoter may be a putative useful clinical biomarker for CHD onset. Upregulation of soluble HLA-G isoform (sHLA-G) was detected in prediabetic and diabetic subjects, suggesting a putative role in metabolic dysfunctions. AREAS COVERED: We conducted a scoping literature review of genetic and epigenetic-sensitive mechanisms regulating HLA-G in diabetes. English-language manuscripts published between 1997 and 2019, were identified through PubMed, Google Scholar, and Web of Science database searches. After selecting 14 original articles representing case-control studies, we summarized and critically evaluated their main findings. EXPERT COMMENTARY: Although epigenetic modifications are involved in the onset of hyperglycemic conditions evolving into diabetes and CHD, it is still difficult to obtain simple and useful clinical biomarkers. Inflammatory-induced KDM6A/INF-ß/HLA-G axis might be a part of the epigenetic network leading to overexpression of HLA-G at pancreatic level. Network medicine may show whether HLA-G is involved in diabetes and CHD.

Effect of Single Sensitization Event on Human Leukocyte Antigen Alloimmunization in Kidney Transplant Candidates: A Single-Center Experience.

OBJECTIVES: Human leukocyte antigen alloimmunization is caused by exposure to HLA antigens through transfusion, pregnancy, or transplant. Our study objective was to present the rate of positivity of anti-HLA antibody considering the effects of a single sensitization event in kidney transplant candidates at our center. MATERIALS AND METHODS: Our study reviewed 606 kidney transplant candidates. Patient sera were analyzed using Luminex xMAP technology. Panel reactive antibody positivity rates and antibody strengths in patients were analyzed according to a single sensitization event. RESULTS: Our findings showed 246 patients (40.6%) with a panel reactive antibody > 0, of which 97 (39.4%) were sensitized from a single event, 119 (48.4%) were sensitized by multiple events, and 30 (12.2%) had no known sensitizing event. Considering patients sensitized by a single event with a panel reactive antibody > 0, we found that 25.8% had received transplant only, 49.5% had previous pregnancy only, and 24.7% had received transfusion only. The strength of antibodies was significantly higher in patients with previous transplant procedures than in those with transfusion for HLA-A (P < .01), HLA-B (P < .05), HLA-C (P < .05), HLA-DR (P < .001), HLA-DQ (P < .05), and HLA-DP (P < .05). Similarly, we observed significantly higher median fluorescence intensity values for HLA-A, -DR, -DQ, and -DP loci in patients with a previous transplant procedure versus pregnancy. The strength of antibodies against HLA-DR was significantly higher in patients with a previous pregnancy compared with those with transfusion (P < .01). CONCLUSIONS: This study documents the profile of HLA alloimmunization in kidney transplant candidates. In particular, transplant procedures appear to have a greater immunologic impact, followed by pregnancy and transfusion. Our data confirm and are in accordance with those of several studies in which the sensitization events were associated with higher prevalence of anti-HLA antibodies.

Erythrocyte genotyping for transfusion-dependent patients at the Azienda Universitaria Policlinico of Naples.

BACKGROUND AND OBJECTIVES: Although minor erythrocyte antigens are not considered clinically significant in sporadic transfusions, they may be relevant for multi-transfusion patients. When serological assay is not conceivable, molecular genotyping allows predicting the red blood cell phenotype, extending the typing until minor blood groups. The aim of this study was to evaluate the utility of blood group genotyping and compare the molecular typing of erythrocyte antigens with the established serological methods. MATERIALS AND METHODS: We selected 225 blood donors and 50 transfusion-dependent patients at the Division of Immunohematology of the Second University of Naples. Blood samples were analyzed with NEO Immucor automated system and genotyped for 38 red blood cell antigens and phenotypic variants with the kit HEA BeadChip™. The comparative study was conducted for RhCE and Kell antigens whose typing is available with both methods. RESULTS: We observed a good correlation between serological and molecular methods for donors that were concordant for 99.5% (224/225) and discordant for 0.5% (1/225). Patients resulted concordant only for 46.0% (23/50) and discordant for 54.0% (27/50); discrepancies were 46.0% (23/50) and 8.0% (4/50) for RhCE and Kell systems respectively. Through molecular genotyping we also identified polymorphisms in RhCE, Kell, Duffy, Colton, Lutheran and Scianna loci in donors and patients. CONCLUSIONS: Blood group genotyping is particularly useful for poly-transfused patients. Molecular analysis confirms and extends serological test data and then allows us to obtain a better match. This molecular assay can be used in the future to prevent alloimmunization in transfusion-dependent patients.

Epigenetic reprogramming in atherosclerosis.

Recent data support the involvement of epigenetic alterations in the pathogenesis of atherosclerosis. The most widely investigated epigenetic mechanism is DNA methylation although also histone code changes occur during the diverse steps of atherosclerosis, such as endothelial cell proliferation, vascular smooth muscle cell (SMC) differentiation, and inflammatory pathway activation. In this review, we focus on the main genes that are epigenetically modified during the atherogenic process, particularly nitric oxide synthase (NOS), estrogen receptors (ERs), collagen type XV alpha 1 (COL15A1), vascular endothelial growth factor receptor (VEGFR), and ten-eleven translocation (TET), which are involved in endothelial dysfunction; gamma interferon (IFN-γ), forkhead box p3 (FOXP3), and tumor necrosis factor-α (TNF-α), associated with atherosclerotic inflammatory process; and p66shc, lectin-like oxLDL receptor (LOX1), and apolipoprotein E (APOE) genes, which are regulated by high cholesterol and homocysteine (Hcy) levels. Furthermore, we also discuss the role of non-coding RNAs (ncRNA) in atherosclerosis. NcRNAs are involved in epigenetic regulation of endothelial function, SMC proliferation, cholesterol synthesis, lipid metabolism, and inflammatory response.

Association between human leukocyte antigen class I and II alleles and hepatitis C virus infection in high-risk hemodialysis patients awaiting kidney transplantation.

Recent evidences have shown that several host genetic factors influence susceptibility or protection to hepatitis C virus (HCV) infection. There are controversial data regarding the associations of human leukocyte antigens (HLA) and the clearance or progression of HCV. The aim of this study was to investigate whether particular HLA molecules were associated with HCV infection in recipients awaiting kidney transplantation considered at high-risk to infection due to protracted hemodialysis treatment. To this purpose, 301 kidney recipients with HCV infection and 1103 uninfected recipients were examined for HLA class I and II molecules. In our case-control study, HLA-A(*)26 is positively associated with HCV infection while HLA-A(*)29, -B(*)40 and -DRB1(*)01 are negatively associated with HCV infection. Multiple logistic regression analysis demonstrated that age (OR = 1.02; 95% CI = 1.01-1.04; p < 0.00), HLA-A(*)26, -A(*)29, -B(*)40 and -DRB1(*)01 [(OR = 1.54; 95% CI = 1.03-2.30; p = 0.03); (OR = 0.50; 95% CI = 0.26-0.99; p = 0.05); (OR = 0.42; 95% CI = 0.23-0, 7; p = 0.01); (OR = 0.62; 95% CI = 0.41-0, 94; p = 0.03); respectively] are independent predictors of HCV infection. Our results suggest that particular HLA molecules, as host genetic factors, may have a relationship with susceptibility or protection to HCV infection also in recipients awaiting kidney transplantation.

Anti-HLA-A, -B, -DR, -DQB1 and -DQA1 antibodies reactive epitope determination with HLAMatchmaker in multipare awaiting list for heart transplant.

Human leukocyte antigen (HLA) antibodies represent a significant risk factor for transplant failure. It is very important to characterize anti-HLA antibodies as epitopes rather than antigens so that this knowledge can be applied clinically. The aim of the study was to investigate the extra reactivity patterns in sensitized multipare. Here, we have used the HLAMatchmaker program, a theoretical algorithm, to explain these unexpected antibody reactivity patterns in multipare awaiting for heart transplant. The patient was sensitized during pregnancy by alleles HLA-A(*)24:02, HLA-DRB1(*)07:01, HLA-DRB4(*)01:01, DQB1(*)02:02 and DQA1(*)02:01 mismatches with development of respective antibodies. However, the patient' sera were shown an unexpected reactivity not directed toward HLA mismatches of daughters: A(∗)23:01, A(*)24:03 and B(*)15:12 for class I and DRB4(*)01:03, DRB1(*)09:02, DRB1(*)09:01, DQB1(*)03:01, DQB1(*)03:03, DQB1(*)03:02, DQB1(*)04:02, DQB1(*)04:01 and DQB1(*)02:01 for class II. By HLAMatchmaker analysis we found that these antibodies reacted with eplet shared by antigens in single allele Luminex panels. These eplets were: 62EE, 66GKH, 70KAH, 71HS, 127K, 113YH, 144KR, 150AAH, 151AHV, 163TG and 167DG for class I and 4Q, 74RRAE, 71RRA, 98KN, 120N, and 135G, 25FT, 34HE, 41ER, 47EK2, 48LF for class II. Thus, HLAMatchmaker software together with to solid phase techniques could open new horizons for a more precise characterization of the HLA-antibodies.

Evidence of Bacteroides fragilis protection from Bartonella henselae-induced damage.

Bartonella henselae is able to internalize endothelial progenitor cells (EPCs), which are resistant to the infection of other common pathogens. Bacteroides fragilis is a gram-negative anaerobe belonging to the gut microflora. It protects from experimental colitis induced by Helicobacter hepaticus through the polysaccharide A (PSA). The aim of our study was to establish: 1) whether B. fragilis colonization could protect from B. henselae infection; if this event may have beneficial effects on EPCs, vascular system and tissues. Our in vitro results establish for the first time that B. fragilis can internalize EPCs and competes with B. henselae during coinfection. We observed a marked activation of the inflammatory response by Real-time PCR and ELISA in coinfected cells compared to B. henselae-infected cells (63 vs 23 up-regulated genes), and after EPCs infection with mutant B. fragilis ΔPSA (≅90% up-regulated genes) compared to B. fragilis. Interestingly, in a mouse model of coinfection, morphological and ultrastructural analyses by hematoxylin-eosin staining and electron microscopy on murine tissues revealed that damages induced by B. henselae can be prevented in the coinfection with B. fragilis but not with its mutant B. fragilis ΔPSA. Moreover, immunohistochemistry analysis with anti-Bartonella showed that the number of positive cells per field decreased of at least 50% in the liver (20±4 vs 50±8), aorta (5±1 vs 10±2) and spleen (25±3 vs 40±6) sections of mice coinfected compared to mice infected only with B. henselae. This decrease was less evident in the coinfection with ΔPSA strain (35±6 in the liver, 5±1 in the aorta and 30±5 in the spleen). Finally, B. fragilis colonization was also able to restore the EPC decrease observed in mice infected with B. henselae (0.65 vs 0.06 media). Thus, our data establish that B. fragilis colonization is able to prevent B. henselae damages through PSA.

Effects of ACE inhibition on circulating endothelial progenitor cells, vascular damage, and oxidative stress in hypertensive patients.

PURPOSE: The pathogenic role of angiotensin-converting enzyme (ACE) inhibition in hypertensive patients regarding endothelial progenitor-cell (EPC) function is still poorly understood. The aim of the study was to evaluate EPC number, function, and relationship to carotid intima media thickness (IMT) progression. METHODS: We studied 36 newly diagnosed mildly hypertensive patients free of cardiovascular disease and related risk factors without prior or concurrent therapy with ACE inhibitors. Patients were randomized to receive enalapril 20 mg/day (n = 18) or zofenopril 30 mg/day (n = 18). EPC number and migrating capacity, plasma nitrite and nitrate (NOx), and isoprostane concentrations were evaluated. Carotid IMT was determined by ultrasonography at baseline and after 1 and 5 years of follow-up. RESULTS: EPC number increased during the follow-up, with no statistical differences between treatment groups. There was an inverse correlation between circulating EPCs and IMT increase over time. Plasma NOx decreased during the study without evident differences between treatment groups. Isoprostanes decreased more markedly in zofenopril-treated patients. Multiple linear regression model demonstrated that carotid IMT was significantly inversely correlated with EPC but not with migratory cells after adjusting for confounders. CONCLUSIONS: The study demonstrated that EPC levels increased during the follow-up in both groups of newly diagnosed hypertensive patients treated with ACE inhibitors. These drugs prevented progression of vascular damage, with an inverse correlation between circulating EPC levels and IMT values.

Impairment of circulating endothelial progenitors in Down syndrome.

BACKGROUND: Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome. METHODS: Circulating endothelial progenitors of Down syndrome affected individuals were isolated, in vitro cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of CXCL12 gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis. RESULTS: We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells. CONCLUSIONS: Our data provide evidences for a reduced number and altered morphology of endothelial progenitor cells in Down syndrome, also showing the higher susceptibility to oxidative stress and to pathogen infection compared to euploid cells, thereby confirming the angiogenesis and immune response deficit observed in Down syndrome individuals.

Modification of the detrimental effect of TNF-α on human endothelial progenitor cells by fasudil and Y27632.

Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor-α (TNF-α) reduced their number and biological activity. Yet, signal transduction events linked to TNF-α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in- flammation. Results demonstrated that incubation with fasudil starting from 50 µM but not Y27632 determined a dose-dependent improvement of EPC number during exposure to TNF-α (P < 0.05 vs. TNF-α alone). Analysis of the signal transduction pathway activated by TNF-α revealed that the increased expression of p-p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF-α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF-α) as well as the inhibitor of Erk1/2-specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF-α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D-DIGE and MALDI-TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin-related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway.

Dual role of parathyroid hormone in endothelial progenitor cells and marrow stromal mesenchymal stem cells.

Hematopoietic stem cells derive regulatory information also from parathyroid hormone (PTH). To explore the possibility that PTH may have a role in regulation of other stem cells residing in bone marrow, such as mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) we assessed the effect of this hormone on the in vitro behavior of MSCs and EPCs. We evidenced that MSCs were much more responsive to PTH than EPCs. PTH increased the proliferation rate of MSCs with a diminution of senescence and apoptosis. Taken together, our results may suggest a protective effect of PTH on MSCs that reduces stress phenomena and preserve genome integrity. At the opposite, PTH did not modify the fate of EPCs in culture.