Circulating biomarkers and cardiac function over 3 years after chemotherapy with anthracyclines: the ICOS-ONE trial.

AIMS: A multicentre trial, ICOS-ONE, showed increases above the upper limit of normality of cardiac troponin (cTn) in 27% of patients within 12 months after the end of cancer chemotherapy (CT) with anthracyclines, whether cardiac protection with enalapril was started at study entry in all (prevention arm) or only upon first occurrence on supra-normal cTn (troponin-triggered arm). The aims of the present post hoc analysis were (i) to assess whether anthracycline-based treatment could induce cardiotoxicity over 36 month follow-up and (ii) to describe the time course of three cardiovascular biomarkers (i.e. troponin I cTnI-Ultra, B-type natriuretic peptide BNP, and pentraxin 3 PTX3) and of left ventricular (LV) function up to 36 months. METHODS AND RESULTS: Eligible patients were those prescribed first-in-life CT, without evidence of cardiovascular disease, normal cTn, LV ejection fraction (EF) >50%, not on renin-angiotensin aldosterone system antagonists. Patients underwent echocardiography and blood sampling at 24 and 36 months. No differences were observed in biomarker concentration between the two study arms, 'prevention' vs. 'troponin-triggered'. During additional follow-up 13 more deaths occurred, leading to a total of 23 (9.5%), all due to a non-cardiovascular cause. No new occurrences of LV-dysfunction were reported. Two additional patients were admitted to the hospital for cardiovascular causes, both for acute pulmonary embolism. No first onset of raised cTnI-Ultra was reported in the extended follow-up. BNP remained within normal range: at 36 months was 23.4 ng/L, higher (N.S.) than at baseline, 17.6 ng/L. PTX3 peaked at 5.2 ng/mL 1 month after CT and returned to baseline values thereafter. cTnI-Ultra peaked at 26 ng/L 1 month after CT and returned to 3 ng/L until the last measurement at 36 months. All echocardiographic variables remained stable during follow-up with a median LVEF of 63% and left atrial volume index about 24 mL/m2 . CONCLUSIONS: First-in-life CT with median cumulative dose of anthracyclines of 180 mg/m2 does not seem to cause clinically significant cardiac injury, as assessed by circulating biomarkers and echocardiography, in patients aged 51 years (median), without pre-existing cardiac disease. This may suggest either a 100% efficacy of enalapril (given as preventive or troponin-triggered) or a reassuringly low absolute cardiovascular risk in this cohort of patients, which may not require intensive cardiologic follow-up.

Circulating microRNA-132 levels improve risk prediction for heart failure hospitalization in patients with chronic heart failure.

AIMS: Non-coding microRNAs (miRNAs) are critically involved in cardiovascular pathophysiology. Since they are measurable in most body fluids, they have been proposed as circulating biomarkers. We examined the prognostic value of a specific candidate miRNA in a large cohort of patients with chronic heart failure (HF) enrolled in a multicentre clinical trial. METHODS AND RESULTS: Plasma levels of miR-132 were measured using miRNA-specific PCR-based technologies at randomization in 953 patients with chronic, symptomatic HF from the GISSI-Heart Failure trial. The association with fatal (all-cause and cardiovascular death) and non-fatal events (time to first admission to hospital for cardiovascular reasons or worsening of HF) and the incremental risk prediction were estimated in adjusted models. Higher circulating miR-132 levels were independently associated with younger age, better renal filtration, ischaemic aetiology of HF, more severe HF symptoms, higher diastolic blood pressure, higher cholesterol, and male sex. After extensive adjustment for demographic, clinical, and echocardiographic risk factors and baseline NT-proBNP concentrations, miR-132 remained associated only with HF hospitalizations (hazard ratio 0.79, 95% confidence interval 0.66-0.95, P = 0.01) and improved its risk prediction with the continuous net reclassification index (cNRI 0.205, P = 0.001). CONCLUSION: In well characterized patients with chronic HF, circulating miR-132 levels rise with the severity of HF. Lower circulating miR-132 levels improved risk prediction for HF readmission beyond traditional risk factors, but not for mortality. MiR-132 may be helpful to intensify strategies aimed at reducing re-hospitalization, which has a substantial health and economic burden in HF.

Effects of dipeptidyl peptidase-4 (DPP-4) inhibition on angiogenesis and hypoxic injury in type 2 diabetes.

AIMS: We examined whether, in diabetic Ob/Ob mice, the dipeptidyl peptidase-4 (DPP-4) inhibitor (PKF275-055), an antihyperglycemic drug, that inhibits the biological inactivation of SDF-1 (stromal cell-derived factor-1), may increase endothelial progenitor cells (EPCs) mobilization and incorporation, which, in turn, may regenerate capillaries and reduce myocardial ischemia induced by strenuous exercise. MAIN METHODS: Half of sixteen control and Ob/Ob mice and eight Ob/Ob mice treated with PKF275-055 for four weeks underwent a forced swim protocol. Oral glucose tolerance, circulating EPCs, capillary ultrastructure and density, hypoxic areas and SDF-1 localization in myocardium were measured. KEY FINDINGS: Ob/Ob mice were glucose intolerant, had a significant low number of circulating EPCs and myocardial capillaries compared to lean controls. The DPP-4 inhibitor significantly improved their glucose tolerance, doubled the number of circulating EPCs, stimulated the formation of functional vessels and SDF-1 localization in the endothelium of myocardial capillaries and arterioles. Cardiac hypoxia after forced swim in Ob/Ob mice was significantly reduced when they were treated with the DPP-4 inhibitor. SIGNIFICANCE: DPP-4 inhibition may re-establish an adequate capillary network in the myocardium of diabetic Ob/Ob mice by the mobilization and SDF-1-mediated incorporation of EPCs and, consequently, reducing the susceptibility to myocardial ischemic injury provoked by strenuous exercise.

An opto-structural method to estimate the stress-strain field induced by cell contraction on substrates of controlled stiffness in vitro.

PURPOSE: Mechanical properties of the extra-cellular matrix (ECM) such as stiffness mediate cell signaling, proliferation, migration, and differentiation. Within this context, we developed a method to estimate in vitro the stress-strain field induced by contraction of cardiovascular progenitor cells on substrates of controlled stiffness. METHODS: Two alginate-agarose hydrogels were polymerized and mechanically characterized under compression. The hydrogels showed different levels of stiffness, mimicking either normal or pathologic ECM of the cardiac tissue, with an average compressive equilibrium modulus of 3 and 25 kPa, respectively. To estimate substrate deformation induced by the adhering cells, fluorescent microspheres were included under the surface layer of the hydrogels as displacement trackers. The hydrogels were polymerized in multiwell plates and seeded with cells that were allowed to adhere for 24 hours. On the softer substrate, images of the substrate surface and the cells were acquired using time-lapse fluorescence microscopy. Image processing enabled tracking the microsphere movements and mapping local substrate deformation because of tensile stresses produced by the cells. The resulting tensile stresses could then be calculated from measured stiffness. RESULTS AND CONCLUSIONS: The substrate strains ranged between a maximum contraction of -26.5% to a maximum stretching of 19.8%. The calculated stresses ranged between a maximum compression of -0.53 kPa to a maximum tension of 0.4 kPa (nN/µm²). These results may help to interpret experimental findings, showing important differences in cell morphology and expression of phenotypic markers, induced by culturing cells on substrates with different mechanical properties.

Levels of circulating pro-angiogenic cells predict cardiovascular outcomes in patients with chronic heart failure.

BACKGROUND: Circulating pro-angiogenic cells (PACs) contribute to vascular and myocardial regeneration. A low level of PACs is associated with worse outcome in patients with coronary heart disease. However, little is known about PACs in heart failure (HF). METHODS AND RESULTS: Blood was sampled at baseline in 111 patients with HF, 67 from 5 Italian Centers and 44 from Frankfurt, Germany. In cultured mononuclear cells from peripheral blood, PACs were counted as double-stained by tetramethylindocarbocyanine-labeled acetylated LDL and fluorescein-5-isothiocyanate-labeled lectin. Mean age of the patients was 62 years, 12 were females, 66 had ischemic etiology, 26 were in New York Heart Association Class >II. Cutoffs for PACs were assessed by receiver operating characteristic curves, to identify the optimal cutoffs for PAC level in predicting outcomes. Mean level of PACs was 35+/-29 (mean+/-SD) cells/mm(2), 2- to 3-fold lower than in age-matched healthy volunteers, but unrelated to severity of HF, age, or sex. Over 2.5 years, 12 cardiovascular deaths and 47 first hospitalizations for cardiovascular reasons were recorded. After adjustment for demographic and clinical variables, elevated creatinine and natriuretic peptides, and PACs

Effects of exercise training on endothelial progenitor cells in patients with chronic heart failure.

BACKGROUND: The enhancement of circulating endothelial progenitor cells (EPCs) obtained by exercise training can be beneficial to patients with cardiac disease. Changes in the levels and differentiation of CD34(pos)/KDR(pos) EPCs, as well as the plasma concentration of vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF)-1 EPC-mobilizing cytokines, were evaluated in patients with chronic heart failure after 8 weeks of supervised aerobic training (SAT) and 8 weeks of subsequent discontinued SAT (DSAT). METHODS AND RESULTS: The levels of circulating EPC and EPC differentiation potential of 22 patients who underwent SAT were studied by fluorescence-activated cell sorter analysis and colony forming-unit assay, respectively. The plasma levels of VEGF and SDF-1 were measured by enzyme-linked immunosorbent assay. In response to SAT, the levels of both EPC and VEGF/SDF-1 markedly increased (P < .001 vs baseline) but returned to the baseline levels after DSAT. A similar change was observed with the EPC clonogenic potential, but on DSAT the baseline level was incompletely attained. CONCLUSIONS: In response to SAT, patients with chronic heart failure show enhanced EPC levels and clonogenic potential that is mirrored by increased plasma VEGF and SDF-1 levels. DSAT can interfere with the maintenance of training-acquired VEGF/SDF-1-related EPC levels and clonogenic potential.

Mesoangioblasts, vessel-associated multipotent stem cells, repair the infarcted heart by multiple cellular mechanisms: a comparison with bone marrow progenitors, fibroblasts, and endothelial cells.

OBJECTIVE: To test the potential of mesoangioblasts (Mabs) in reducing postischemic injury in comparison with bone marrow progenitor cells (BMPCs), fibroblasts (Fbs), and embryonic stem cell-derived endothelial cells (ECs), and to identify putative cellular protective mechanisms. METHODS AND RESULTS: Cells were injected percutaneously in the left ventricular (LV) chamber of C57BL/6 mice, 3 to 6 hours after coronary ligation, and detected in the hearts 2 days and 6 weeks later. Echocardiographic examinations were performed at 6 weeks. LV dilation was reduced and LV shortening fraction was improved with Mabs and BMPCs but not with ECs and Fbs. Donor cell colonization of the host myocardium was modest and predominantly in the smooth muscle layer of vessels. Capillary density was higher in the peripheral infarct area and apoptotic cardiomyocytes were fewer with Mabs and BMPCs. Mabs and BMPCs, but not Fbs or ECs, promoted survival of cultured cardiocytes under low-oxygen in culture. This activity was present in Mab-conditioned medium and could be replaced by a combination of basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-1, and hepatocyte growth factor (HGF), all of which are produced by these cells. Conditioned medium from Mabs, but not from Fbs, stimulated proliferation of smooth muscle cells in vitro. CONCLUSIONS: Mabs appear as effective as BMPCs in reducing postinfarction LV dysfunction, likely through production of antiapoptotic and angiogenic factors.

Expression of junctional adhesion molecule-A prevents spontaneous and random motility.

Junctional adhesion molecule-A (JAM-A) is a cell-surface glycoprotein that localizes to intercellular junctions and associates with intracellular proteins via PSD95-Dlg-ZO1-binding residues. To define the functional consequences of JAM-A expression, we have produced endothelial cells from JAM-A-deficient mice. We report here that the absence of JAM-A enhanced spontaneous and random motility. In turn, the enhanced motility of JAM-A-negative cells was abrogated either on transfection of exogenous JAM-A or on treatment with inhibitors of glycogen synthase kinase-3beta (GSK-3beta). In addition, in JAM-A-positive cells, motility was enhanced on inactivation of protein kinase Czeta (PKCzeta), which is an inhibitor of GSK-3beta. Although these findings suggested that JAM-A might inhibit GSK-3beta, we found that expression per se of JAM-A did not change the levels of inactive GSK-3beta. Thus, JAM-A expression may regulate effectors of motility that are also downstream of the PKCzeta/GSK-3beta axis. In support of this view, we found that JAM-A absence increased the number of actin-containing protrusions, reduced the stability of microtubules and impaired the formation of focal adhesions. Notably, all the functional consequences of JAM-A absence were reversed either on treatment with GSK-3beta inhibitors or on transfection of full-length JAM-A, but not on transfection of a JAM-A deletion mutant devoid of the PSD95-Dlg-ZO1-binding residues. Thus, by regulating cytoskeletal and adhesive structures, JAM-A expression prevents cell motility, probably in a PSD95-Dlg-ZO1-dependent manner.

Skeletal myogenic progenitors in the endothelium of lung and yolk sac.

We previously showed that clonable skeletal myogenic cells can be derived from the embryonic aorta but become very rare in the more mature and structured fetal aorta. The aim of this study was to investigate whether, during fetal and postnatal development, these myogenic progenitors progressively disappear or may rather associate with the microvascular district, being thus distributed to virtually all tissues. To test this hypothesis, we used F1 embryos (or mice) from a transgenic line expressing a striated muscle-specific reporter gene (LacZ) crossed with a transgenic line expressing a different endothelial-specific reporter genes (GFP). Endothelial cells were isolated from yolk sac (at E11) and lung (at E11, E17, P1, P10, and P60), two organs embryologically unrelated to paraxial mesoderm, rich in vessels, and devoid of skeletal muscle. Endothelial cells, purified by magnetic bead selection (CD31/PECAM-1(+)) or cell sorting (Tie2-GFP(+)) were then challenged for their skeletal myogenic potential in vitro and in vivo. The results demonstrated that both yolk sac and lung contain progenitor cells, which express endothelial markers and are endowed with a skeletal myogenic potential that they reveal when in the presence of differentiating myoblasts, in vitro, and regenerating muscle, in vivo. The number (or potency to generate skeletal muscle) of these vessels associated cells decreases rapidly with age and is very low in mature animals, possibly correlating with reduced regenerative capacity of adult mammalian tissues.

The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility.

Using the Cre/loxP system we conditionally inactivated beta-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured beta-catenin -/- endothelial cells showed a different organization of intercellular junctions with a decrease in alpha-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell-cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of beta-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages.

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, beta-catenin, and the phosphatase DEP-1/CD148.

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin-null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin-null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.

Vascular endothelial growth factor induces SHC association with vascular endothelial cadherin: a potential feedback mechanism to control vascular endothelial growth factor receptor-2 signaling.

Vascular endothelial (VE)-cadherin is endothelium specific, mediates homophilic adhesion, and is clustered at intercellular junctions. VE-cadherin is required for normal development of the vasculature in the embryo and for angiogenesis in the adult. Here, we report that VE-cadherin is associated with VE growth factor (VEGF) receptor-2 (VEGFR-2) on the exposure of endothelial cells to VEGF. The binding parallels receptor phosphorylation on tyrosine residues, which is maximal at 5 minutes and then declines within 30 minutes. Tyrosine phosphorylation of VE-cadherin was maximal at 30 minutes after the addition of the growth factor. At this time point, the protein could be coimmunoprecipitated with the adaptor protein Shc. Pull-down experiments with different Shc domains and mutants of the VE-cadherin cytoplasmic tail have shown that Shc binds to the carboxy-terminal domain of the VE-cadherin tail through its Src homology 2 domain (SH2). We found that Shc phosphorylation lasts longer in endothelial cells carrying a targeted null mutation in the VE-cadherin gene than in VE-cadherin-positive cells. These data suggest that VE-cadherin expression exerts a negative effect on Shc phosphorylation by VEGFR-2. We speculate that VE-cadherin binding to Shc promotes its dephosphorylation through associated phosphatases.

VE-cadherin regulates endothelial actin activating Rac and increasing membrane association of Tiam.

Previously published reports support the concept that, besides promoting homotypic intercellular adhesion, cadherins may transfer intracellular signals. However, the signaling pathways triggered by cadherin clustering and their biological significance are still poorly understood. We report herein that transfection of VE-cadherin (VEC) cDNA in VEC null endothelial cells induces actin rearrangement and increases the number of vinculin positive adhesion plaques. VEC expression augments the level of active Rac but decreases active Rho. Microinjection of a dominant negative Rac mutant altered stress fiber organization, whereas inhibition of Rho was ineffective. VEC expression increased protein and mRNA levels of the Rac-specific guanosine exchange factor Tiam-1 and induced its localization at intercellular junctions. In addition, in the presence of VEC, the amounts of Tiam, Rac, and the Rac effector PAK as well as the level of PAK phosphorylation were found increased in the membrane/cytoskeletal fraction. These observations are consistent with a role of VEC in localizing Rac and its signaling partners in the same membrane compartment, facilitating their reciprocal interaction. Through this mechanism VEC may influence the constitutive organization of the actin cytoskeleton.