Crosstalk of human coronary perivascular adipose-derived stem cells with vascular cells: role of tissue factor.

The coronary perivascular adipose tissue (cPVAT) has been associated to the burden of cardiovascular risk factors and to the underlying vessel atherosclerotic plaque severity. Although the "outside to inside" hypothesis of PVAT-derived-adipokine regulation of vessel function is currently accepted, whether the resident mesenchymal stem cells (ASCs) in PVAT have a regulatory role on the underlying vascular arterial smooth muscle cells (VSMCs) is not known. Here, we investigated the interactions between resident PVAT-ASCs and VSMCs. ASCs were obtained from PVAT overlying the left anterior descending (LAD) coronary artery of hearts removed at heart transplant operations. PVAT was obtained both from patients with non-ischemic and ischemic heart disease as the cause of heart transplant. ASCs were isolated from PVAT, phenotypically characterized by flow cytometry, functionally tested for proliferation, and differentiation. Crosstalk between ASCs and VSMCs was investigated by co-culture studies. ASCs were detected in the adventitia of the LAD-PVAT showing differentiation capacity and angiogenic potential. ASCs obtained from PVAT of non-ischemic and ischemic hearts showed different tissue factor (TF) expression levels, different VSMCs recruitment capacity through the axis ERK1/2-ETS1 signaling and different angiogenic potential. Induced upregulation of TF in ASCs isolated from ischemic PVAT rescued their angiogenic capacity in subcutaneously implanted plugs in mice, whereas silencing TF in ASCs decreased the proangiogenic capacity of non-ischemic ASCs. The results indicate for the first time a novel mechanism of regulation of VSMCs by PVAT-ASCs in angiogenesis, mediated by TF expression in ASCs. Regulation of TF in ASCs may become a therapeutic intervention to increase cardiac protection.

Endothelium-Released Microvesicles Transport miR-126 That Induces Proangiogenic Reprogramming in Monocytes.

We have recently shown that in ischemic tissue, the hypoxic endothelial cells (EC) release extracellular microvesicles (EMVs) that are rich in tissue factor (TF). These TF-EMVs induce monocyte (Mo) homing to the ischemic zone, their differentiation into EC-like cells, and the formation of new blood vessels increasing tissue perfusion. In addition to membrane proteins, EMVs contain noncoding RNAs that can modulate cellular signaling pathways in the recipient cells. Here, we have investigated whether miRNA contained into secreted EMVs may be transferred into Mo where they could modulate EC-like cell differentiation and angiogenic responses. Our results indicated that EMVs released from activated ECs contain high levels of miR-126 and that the levels are directly proportional to TF expression in EMVs. Interestingly, miR-126 is transferred to Mo when they are incubated with TF-EMVs. Increased levels of miR-126 in Mo do not promote EC-like cell differentiation but regulate angiogenesis by targeting several components of the VEGF pathway, as SPRED1 and PI3KR2. Our findings reveal that activated ECs secrete EMVs carrying miR-126, which can modulate Mo reprogramming of angiogenic genes.

Ischaemic tissue released microvesicles induce monocyte reprogramming and increase tissue repair by a tissue factor-dependent mechanism.

AIMS: Despite increasing evidence that monocytes may acquire endothelial features, it remains unclear how monocytes participate in angiogenesis after ischaemic damage. We investigated whether ischaemic cells can release microvesicles (MVs) and promote neovascularization in a model of peripheral artery disease (PAD). METHODS AND RESULTS: To model PAD, we used an in vivo experimental model of hind-limb ischaemia (HLI) in mice. MVs were isolated from the ischaemic muscle and from peripheral blood at different times after unilateral femoral artery ligation. MVs were phenotypically characterized to identify cell origin. HLI in mice induced the release of MVs with a much higher content of tissue factor (TF) than non-HLI control mice both in the MVs isolated from the affected limb muscle area and from blood. MVs were mainly released from endothelial cells (ECs) and induced Mo differentiation to endothelial cell-like (ECL) cells. Differentiation to ECL cells encompassed highly strict hierarchical transcription factor activation, initiated by ETS1 activation. MVs secreted by microvascular ECs over-expressing TF (upTF-EMVs), were injected in the ischaemic hind-limb in parallel with control EMVs (from random siRNA-treated cells) or EMVs released by silenced TF ECs. In animals treated with upTF-EMVs in the ischaemic zone, there was a highly significant increase in functional new vessels formation (seen by magnetic resonance angiography), a concomitant increase in the pool of circulating Ly6Clow Mo expressing vascular EC markers, and a significantly higher number of Mo/macrophages surrounding and integrating the newly formed collaterals. CONCLUSION: Ischaemia-activated ECs release EMVs rich in TF that induce monocyte differentiation into ECL cells and the formation of new vessels in the ischaemic zone. TF by this mechanism of formation of new blood microvessels can contribute to ischaemic tissue repair.

Alternative C3 Complement System: Lipids and Atherosclerosis.

Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin αMß2 receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.

Triglyceride-induced cardiac lipotoxicity is mitigated by Silybum marianum.

BACKGROUND AND AIMS: Silybum marianum (SM) is an herbal product with cytoprotective and antioxidant properties. We have previously demonstrated that SM ameliorates ventricular remodeling and improves cardiac performance. Here, we evaluated whether SM could exert beneficial effects against cardiac lipotoxicity in a pig model of closed-chest myocardial infarction (MI). METHODS: Study 1 investigated the effect of SM administration on lipid profile and any potential SM-related adverse effects. Animals received SM or placebo during 10 days and were afterward sacrificed. Study 2 evaluated the effectiveness of SM daily administration in reducing cardiac lipotoxicity in animals subjected to a 1.5h myocardial infarction (MI), who were subsequently reperfused for 2.5h and euthanized or kept under study for three weeks and then sacrificed. RESULTS: Animals administered a 10-day SM regime presented a sharp decline in plasma triglyceride levels vs. controls, with no other modifications in lipid profile. The decrease in triglyceride concentration was accompanied by a marked reduction in triglyceride intestinal absorption and glycoprotein-P expression. Three weeks post-MI the triglyceride content in the ischemic myocardium of the SM-treated animals was significantly lower than in the ischemic myocardium of placebo-controls. This effect was associated with an enhanced cardiac expression of PPARγ and triglyceride clearance receptors. This long-term SM-administration induced a lower expression of lipid receptors in subcutaneous adipose tissue. No SM-related side-effects were registered. CONCLUSION: SM administration reduces plasma triglyceride levels through attenuation of triglyceride intestinal absorption and modulates cardiac lipotoxicity in the ischemic myocardium, likely contributing to improve ventricular remodeling.

Molecular mapping of platelet hyperreactivity in diabetes: the stress proteins complex HSPA8/Hsp90/CSK2α and platelet aggregation in diabetic and normal platelets.

The molecular understanding of the pathophysiological changes elicited by diabetes in platelets may help in further elucidating the involvement of this pseudo-cell in the increased risk of developing cardiovascular disease and thrombosis in diabetic subjects. We aimed to investigate the differential characteristics of platelets from diabetic patients and nondiabetic controls to unveil the molecular mechanisms behind the increased platelet reactivity in diabetes. We compared platelets from diabetic and control subjects by 2 dimensional-electrophoresis followed by mass spectrometry. Changes in selected differential proteins were validated by immunoprecipitation assays and western blot. Platelet aggregation was measured by light transmittance aggregometry induced by collagen and ADP, and dynamic coagulation analysis of whole blood was measured by thromboelastometry. We observed significant differences in proteins related to platelet aggregation, cell migration, and cell homeostasis. Subjects with diabetes showed higher platelet aggregation and thrombogenicity and higher contents of the stress-related protein complex HSPA8/Hsp90/CSK2α than nondiabetic subjects. Changes in the chaperones HSPA8 and Hsp90, and in CSK2α protein contents correlated with changes in platelet aggregation and blood coagulation activity. In conclusion, the complex HSPA8/Hsp90/CSK2α is involved in diabetes-related platelet hyperreactivity. The role of the HSPA8/Hsp90/CSK2α complex may become a molecular target for the development of future preventive and therapeutic strategies for platelet dysfunction associated with diabetes and its complications.

Antiplatelet Activity of Isorhamnetin via Mitochondrial Regulation.

With the diet, we ingest nutrients capable of modulating platelet function, which plays a crucial role in developing cardiovascular events, one of the leading causes of mortality worldwide. Studies that demonstrate the antiplatelet and antithrombotic potential of bioactive compounds are vital to maintaining good cardiovascular health. In this work, we evaluate the flavonol isorhamnetin's antiplatelet effect on human platelets, using collagen, thrombin receptor activator peptide 6 (TRAP-6), and phorbol myristate acetate (PMA) as agonists. Isorhamnetin induced a significant inhibition on collagen- and TRAP-6-induced platelet aggregation, with half-maximum inhibitory concentration (IC50) values of 8.1 ± 2.6 and 16.1 ± 11.1 µM, respectively; while it did not show cytotoxic effect. Isorhamnetin reduced adenosine triphosphate levels (ATP) in platelets stimulated by collagen and TRAP-6. We also evidenced that isorhamnetin's antiplatelet activity was related to the inhibition of mitochondrial function without effect on reactive oxygen species (ROS) levels. Additionally, we investigated isorhamnetin's effect on thrombus formation in vitro under flow conditions on the damaged vessel wall. In this context, we demonstrate that isorhamnetin at 20 µM induced a significant inhibition on platelet deposition, confirming its antithrombotic effect. Our findings corroborate the antiplatelet and antithrombotic potential of isorhamnetin present in many foods of daily consumption.

Sex Differences and Emerging New Risk Factors for Atherosclerosis and Its Thrombotic Complications.

Cardiovascular diseases (CVD) remain the world's leading cause of death and disability in both men and women, but with different prognostics and outcomes between sexes. Although the burden of CVD is generally related to the conventional risk factors, the relevance of non-traditional risk factors is increasingly being recognized to explain the so-called "residual risk". Men and women share many similarities regarding classical cardiovascular risk factors but have different disease pathophysiology, clinical presentations, prevalence, and outcomes of CVDs. How sex-specificities regarding the effects of non-traditional risk factors may contribute to the evolution of atherosclerosis and its clinical manifestations in males and females remain largely underanalyzed. The present review summarizes the current knowledge for sex differences in atherosclerotic plaque composition and clinical evolution in association with risk factors, such as inflammation, lipoprotein(a), hemostasis, intraplaque calcification, and depression. We further discuss the potential sex-differential impact of chronic infectious diseases, gut microbiome and, epigenetic gene expression regulation for atherosclerosis and the effect of female-specific disorders in CVD.

PCSK9 and LRP5 in macrophage lipid internalization and inflammation.

AIMS: Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. Low-density lipoprotein receptors (LDLR) play a critical role in regulating blood cholesterol levels by binding to and clearing LDLs from the circulation. The disruption of the interaction between proprotein convertase subtilisin/kexin 9 (PCSK9) and LDLR reduces blood cholesterol levels. It is not well known whether other members of the LDLR superfamily may be targets of PCSK9. The aim of this work was to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target and to study the role of PCSK9 and LRP5 in foam cell formation and lipid accumulation. METHODS AND RESULTS: Primary cultures of human inflammatory cells (monocytes and macrophages) were silenced for LRP5 or PCSK9 and challenged with LDLs. We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages show less intracellular CE accumulation. In macrophages, internalization of LRP5-bound LDL is already highly evident after 5 h of LDL incubation and lasts up to 24 h; however, in the absence of both LRP5 and PCSK9, there is a strong reduction of CE accumulation indicating a role for both proteins in lipid uptake. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally, PCSK9 participates in TLR4/NFkB signalling; a decreased TLR4 protein expression levels and a decreased nuclear translocation of NFκB were detected in PCSK9 silenced cells after lipid loading, indicating a downregulation of the TLR4/NFκB pathway. CONCLUSION: Our results show that both LRP5 and PCSK9 participate in lipid uptake in macrophages. In the absence of LRP5, there is a reduced release of PCSK9 indicating that LRP5 also participates in the mechanism of release of soluble PCSK9. Furthermore, PCSK9 up-regulates TLR4/NFκB favouring inflammation.

HDL (High-Density Lipoprotein) Remodeling and Magnetic Resonance Imaging-Assessed Atherosclerotic Plaque Burden: Study in a Preclinical Experimental Model.

OBJECTIVE: HDL (high-density lipoprotein) role in atherosclerosis is controversial. Clinical trials with CETP (cholesterylester transfer protein)-inhibitors have not provided benefit. We have shown that HDL remodeling in hypercholesterolemia reduces HDL cardioprotective potential. We aimed to assess whether hypercholesterolemia affects HDL-induced atherosclerotic plaque regression. Approach and Results: Atherosclerosis was induced in New Zealand White rabbits for 3-months by combining a high-fat-diet and double-balloon aortic denudation. Then, animals underwent magnetic resonance imaging (basal plaque) and randomized to receive 4 IV infusions (1 infusion/wk) of HDL isolated from normocholesterolemic (NC-HDL; 75 mg/kg; n=10), hypercholesterolemic (HC-HDL; 75 mg/Kg; n=10), or vehicle (n=10) rabbits. Then, animals underwent a second magnetic resonance imaging (end plaque). Blood, aorta, and liver samples were obtained for analyses. Follow-up magnetic resonance imaging revealed that NC-HDL administration regressed atherosclerotic lesions by 4.3%, whereas, conversely, the administration of HC-HDLs induced a further 6.5% progression (P<0.05 versus basal). Plaque characterization showed that HC-HDL administered animals had a 2-fold higher lipid and cholesterol content versus those infused NC-HDL and vehicle (P<0.05). No differences were observed among groups in CD31 levels, nor in infiltrated macrophages or smooth muscle cells. Plaques from HC-HDL administered animals exhibited higher Casp3 (caspase 3) content (P<0.05 versus vehicle and NC-HDL) whereas plaques from NC-HDL infused animals showed lower expression of Casp3, Cox1 (cyclooxygenase 1), inducible nitric oxide synthase, and MMP (metalloproteinase) activity (P<0.05 versus HC-HDL and vehicle). HDLs isolated from animals administered HC-HDL displayed lower antioxidant potential and cholesterol efflux capacity as compared with HDLs isolated from NC-HDL-infused animal and vehicle or donor HDL (P<0.05). There were no differences in HDL-ApoA1 content, ABCA1 (ATP-binding cassette transporter A1) vascular expression, and SRB1 (scavenger receptor B1) and ABCA1 liver expression. CONCLUSIONS: HDL particles isolated from a hypercholesterolemic milieu lose their ability to regress and stabilize atherosclerotic lesions. Our data suggest that HDL remodeling in patients with co-morbidities may lead to the loss of HDL atheroprotective functions.

Maintenance therapy with ex vivo expanded lymphokine-activated killer cells and rituximab in patients with follicular lymphoma is safe and may delay disease progression.

Anti-cluster of differentiation 20 (CD20) monoclonal antibodies (mAbs) have shown promise in follicular lymphoma (FL) as post-induction therapy, by enhancing antibody-dependent cellular cytotoxicity (ADCC). However, cytotoxic cells are reduced after this treatment. We hypothesised that ex vivo expanded lymphokine-activated killer (LAK) cells administered to FL-remission patients are safe and improve anti-CD20 efficacy. This open, prospective, phase II, single-arm study assessed safety and efficacy of ex vivo expanded LAK cells in 20 FL-remission patients following rituximab maintenance. Mononuclear cells were obtained in odd rituximab cycles and stimulated with interleukin 2 (IL-2) for 8 weeks, after which >5 × 108 LAK cells were injected. Patients were followed-up for 5 years. At the end of maintenance, peripheral blood cells phenotype had not changed markedly. Natural killer, LAK and ADCC activities of mononuclear cells increased significantly after recombinant human IL-2 (rhIL-2) stimulation in all cycles. Rituximab significantly enhanced cytotoxic activity. No patients discontinued treatment. There were no treatment-related serious adverse events. Three patients had progressed by the end of follow-up. After a median (interquartile range) follow-up of 59.4 (43.8-70.9) months, 85% of patients remained progression free. No deaths occurred. Quality-of-life improved throughout the study. Post-induction LAK cells with rituximab seem safe in the long term. Larger studies are warranted to confirm efficacy.

Role of Autophagy in Von Willebrand Factor Secretion by Endothelial Cells and in the In Vivo Thrombin-Antithrombin Complex Formation Promoted by the HIV-1 Matrix Protein p17.

Although the advent of combined antiretroviral therapy has substantially improved the survival of HIV-1-infected individuals, non-AIDS-related diseases are becoming increasingly prevalent in HIV-1-infected patients. Persistent abnormalities in coagulation appear to contribute to excess risk for a broad spectrum of non-AIDS defining complications. Alterations in coagulation biology in the context of HIV infection seem to be largely a consequence of a chronically inflammatory microenvironment leading to endothelial cell (EC) dysfunction. A possible direct role of HIV-1 proteins in sustaining EC dysfunction has been postulated but not yet investigated. The HIV-1 matrix protein p17 (p17) is secreted from HIV-1-infected cells and is known to sustain inflammatory processes by activating ECs. The aim of this study was to investigate the possibility that p17-driven stimulation of human ECs is associated with increased production of critical coagulation factors. Here we show the involvement of autophagy in the p17-induced accumulation and secretion of von Willebrand factor (vWF) by ECs. In vivo experiments confirmed the capability of p17 to exert a potent pro-coagulant activity soon after its intravenous administration.

High-density lipoprotein remodelled in hypercholesterolaemic blood induce epigenetically driven down-regulation of endothelial HIF-1α expression in a preclinical animal model.

AIMS: High-density lipoproteins (HDLs) are circulating micelles that transport proteins, lipids, and miRNAs. HDL-transported miRNAs (HDL-miRNAs) have lately received attention but their effects on vascular cells are not fully understood. Additionally, whether cardiovascular risk factors affect HDL-miRNAs levels and miRNA transfer to recipient cells remains equally poorly known. Here, we have investigated the changes induced by hypercholesterolaemia on HDL-miRNA levels and its effect on recipient endothelial cells (ECs). METHODS AND RESULTS: Pigs were kept on a high-fat diet (HC; n = 10) or a normocholesterolaemic chow (NC; n = 10) for 10 days reaching cholesterol levels of 321.0 (229.7-378.5) mg/dL and 74.0 (62.5-80.2) mg/dL, respectively. HDL particles were isolated, purified, and quantified. HDL-miRNA profiling (n = 149 miRNAs) of HC- and NC-HDLs was performed by multipanel qPCR. Cell cultures of porcine aortic ECs were used to determine whether HDL-miRNAs were delivered to ECs. Potential target genes modulated by miRNAs were identified by bioinformatics and candidate miRNAs were validated by molecular analysis. In vivo effects in the coronary arteries of normocholesterolaemic swine administered HC- or NC-HDLs were analysed. Among the HDL-miRNAs, four were found in different amounts in HC- and NC-HDL (P < 0.05). miR-126-5p and -3p and miR-30b-5p (2.7×, 1.7×, and 1.3×, respectively) were found in higher levels and miR-103a-3p and miR-let-7g-5p (-1.6×, -1.4×, respectively) in lower levels in HC-HDL. miR-126-5p and -3p were transferred from HC-HDL to EC (2.5×; P < 0.05), but not from NC-HDL, by a SRB1-mediated mechanism. Bioinformatics revealed that HIF1α was the miR-126 target gene with the highest predictive value, which was accordingly found to be markedly reduced in HC-HDL-treated ECs and in miR126 mimic transfected ECs. In vivo validation confirmed that HIF1α was diminished in the coronary endothelial layer of NC pigs administered HC-HDL vs. those administered NC-HDL (P < 0.05). CONCLUSION: Hypercholesterolaemia induces changes in the miRNA content of HDL enhancing miR126 and its delivery to ECs with the consequent down-regulation of its target gene HIF1α.

MicroRNA-145 Regulates the Differentiation of Adipose Stem Cells Toward Microvascular Endothelial Cells and Promotes Angiogenesis.

RATIONALE: Adipose-derived stem cells (ASCs) are a potential adult mesenchymal stem cell source for restoring endothelial function in ischemic tissues. However, the mechanism that promotes ASCs differentiation toward endothelial cells (ECs) is not known. OBJECTIVE: To investigate the mechanisms of ASCs differentiation into ECs. METHODS AND RESULTS: ASCs were isolated from clinical lipoaspirates and cultured with DMEM or endothelial cell-conditioned medium. Endothelial cell-conditioned medium induced downregulation of miR-145 in ASCs and promoted endothelial differentiation. We identified bFGF (basic fibroblast growth factor) released by ECs as inducer of ASCs differentiation through receptor-induced AKT (protein kinase B) signaling and phosphorylation of FOXO1 (forkhead box protein O1) suppressing its transcriptional activity and decreasing miR-145 expression. Blocking bFGF-receptor or PI3K/AKT signaling in ASCs increased miR-145 levels. Modulation of miR-145 in ASCs, using a miR-145 inhibitor, regulated their differentiation into ECs: increasing proliferation, migration, inducing expression of EC markers (VE-cadherin, VEGFR2 [vascular endothelial growth factor receptor 2], or VWF [von Willebrand Factor]), and tube-like formation. Furthermore, in vivo, downregulation of miR-145 in ASCs enhanced angiogenesis in subcutaneously implanted plugs in mice. In a murine hindlimb ischemia model injection of ASCs with downregulated miR-145 induced collateral flow and capillary formation evidenced by magnetic resonance angiography. Next, we identified ETS1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1) as the target of miR-145. Upregulation of miR-145 in ASCs, by mimic miR-145, suppressed ETS1 expression and consequently abolished EC differentiation and the angiogenic properties of endothelial cell-conditioned medium-preconditioned ASCs; whereas, overexpression of ETS1 reversed the abrogated antiangiogenic capacity of miR-145. ETS1 overexpression induced similar results to those obtained with miR-145 knockdown. CONCLUSIONS: bFGF released by ECs induces ASCs differentiation toward ECs through miR-145-regulated expression of ETS1. Downregulation of miR-145 in ASCs induce vascular network formation in ischemic muscle.

Silybum marianum provides cardioprotection and limits adverse remodeling post-myocardial infarction by mitigating oxidative stress and reactive fibrosis.

AIMS: Milk thistle (Silybum marianum; SM) is an herb commonly used for hepatoprotection with antioxidant and antifibrotic properties. We investigated in pigs the cardiac effects of SM intake during the acute phase of myocardial infarction (MI) and remodeling period post-MI. METHODS: Study-1 tested the effect of SM use on the acute phase of MI. Hence, animals were distributed to a control group or to receive SM prior infarction (1.5 h ischemia). Animals were sacrificed after 2.5 h of reperfusion. Study-2 tested the effect of SM use in the cardiac remodeling phase. Accordingly, animals received for 10 d diet ±â€¯SM prior MI and followed the same regime for 3 weeks and then sacrificed. Study-3 tested the effect of SM in a non-infarcted heart; therefore, animals received for 10 d diet ±â€¯SM and then sacrificed. RESULTS: Animals taking SM before MI showed a reduction in cardiac damage (decreased oxidative damage, ROS production and xanthine oxidase levels; preserved mitochondrial function; and increased myocardial salvage; p < 0.05) versus controls. Animals that remained on chronic SM intake post-MI improved left ventricular remodeling. This was associated with the attenuation of the TGFß1/TßRs/SMAD2/3 signaling, lower myofibroblast transdifferentiation and collagen content in the border zone (p < 0.05 vs. all other groups). Cardiac contractility improved in animals taking SM (p < 0.05 vs. post-MI-control). No changes in cardiac function or fibrosis were detected in animals on SM but without MI. CONCLUSION: Intake of SM protects the heart against the deleterious effects of an MI and favors cardiac healing. These benefits may be attributed to the antioxidant and antifibrotic properties of SM.

C-Reactive Protein in Atherothrombosis and Angiogenesis.

C-reactive protein (CRP) is a short pentraxin mainly found as a pentamer in the circulation, or as non-soluble monomers CRP (mCRP) in tissues, exerting different functions. This review is focused on discussing the role of CRP in cardiovascular disease, including recent advances on the implication of CRP and its forms specifically on the pathogenesis of atherothrombosis and angiogenesis. Besides its role in the humoral innate immune response, CRP contributes to cardiovascular disease progression by recognizing and binding multiple intrinsic ligands. mCRP is not present in the healthy vessel wall but it becomes detectable in the early stages of atherogenesis and accumulates during the progression of atherosclerosis. CRP inhibits endothelial nitric oxide production and contributes to plaque instability by increasing endothelial cell adhesion molecules expression, by promoting monocyte recruitment into the atheromatous plaque and by enzymatically binding to modified low-density lipoprotein. CRP also contributes to thrombosis, but depending on its form it elicits different actions. Pentameric CRP has no involvement in thrombogenesis, whereas mCRP induces platelet activation and thrombus growth. In addition, mCRP has apparently contradictory pro-angiogenic and anti-angiogenic effects determining tissue remodeling in the atherosclerotic plaque and in infarcted tissues. Overall, CRP contributes to cardiovascular disease by several mechanisms that deserve an in-depth analysis.

Effectiveness of a motivational intervention on overweight/obese patients in the primary healthcare: a cluster randomized trial.

BACKGROUND: Overweight and obesity are common health problems which increase the risk of developing several serious health conditions. The main difficulty in the management of weight-loss lies in its maintenance, once it is achieved. The aim of this study was to investigate whether a motivational intervention, together with current clinical practice, was more efficient than a traditional intervention, in the treatment of overweight and obesity and whether this intervention reduces cardiovascular risk factors associated with overweight and obesity. METHODS: Multi-centre cluster randomized trial with a 24-month follow-up included 864 overweight/obese patients randomly assigned. Motivational intervention group (400 patients), delivered by a nurse trained by an expert psychologist, in 32 sessions, 1 to 12 fortnightly, and 13 to 32, monthly, on top of their standard programmed diet and exercise. The control group (446 patients), received the usual follow-up. RESULTS: Weight reduction was statistically significant in the second year with a mean reduction of 1.0 Kg in the control group and 2.5 Kg in the intervention group (p = 0. 02). While 18.1% of patients in the control group reduced their weight by more than 5%, this percentage rose to 26.9% in the intervention group, which is statistically significant (p = 0.04). Patients in the motivational intervention group had significantly greater improvements in triglycerides and APOB/APOA1ratio. CONCLUSIONS: The results highlight the importance of the group motivational interview in the treatment of overweight /obese patients in primary care, and in the improvement of their associated cardiovascular risks factors. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01006213 October 30, 2009.

Inflammation and hemostasis in older octogenarians: implication in 5-year survival.

Social changes and medical advances have increased longevity, but the conditions governing healthy vs unhealthy cardiovascular (CV) aging are not fully known. Factors beyond classical CV risk factors may have an important unrecognized value. We sought to identify proteins differentially expressed in healthy octogenarians (HOs) without a history of cardiovascular disease (CVD) and preserved functional and cognitive state compared with octogenarians with a history of CVD and cognitive decline (UHOs) using a systems biology approach, and investigated how these proteins relate to CV mortality at 5-year follow-up. Plasmas obtained from older octogenarians (87 ± 0 years) were analyzed by 2-DE + MS and bioinformatic pathway analysis in HOs (N = 38) and UHOs with cognitive (MEC<25) and functional (Barthel<90) decline and a previous ischemic event (acute myocardial infarction and/or stroke; N = 27). Results were validated by ELISA in HOs and UHOs and in an additional group of older octogenarians without cognitive impairment but with a previous CVD manifestation (HO-CVD; N = 35). UHOs showed a coordinated change in several inflammation-related proteins (AMBP, RBP4, and ITIH4; P < 0.05), together with a significant increase in the major inducer of the acute-phase reaction, interleukin-6 (P = 0.03). UHOs also showed a coordinated increase in hemostatic proteins that was associated with an impairment of fibrinolysis and an increased 5-year CV mortality (P = 0.003). The combination of inflammation (ITIH4 and interleukin-6) and hemostatic markers (D-dimer, A2AP, and coagulation factor XIII) was able to discriminate the presence of an unhealthy phenotype in the elderly (AUC = 0.750; P = 0.001). Unhealthy older octogenarians show increased levels of several plasma proteins of inflammation and coagulation. In older octogenarians, the increase in hemostatic markers indicated an increase in 5-year CV mortality at follow-up.

Allogenic adipose-derived stem cell therapy overcomes ischemia-induced microvessel rarefaction in the myocardium: systems biology study.

BACKGROUND: Myocardial microvascular loss after myocardial infarction (MI) remains a therapeutic challenge. Autologous stem cell therapy was considered as an alternative; however, it has shown modest benefits due to the impairing effects of cardiovascular risk factors on stem cells. Allogenic adipose-derived stem cells (ASCs) may overcome such limitations, and because of their low immunogenicity and paracrine potential may be good candidates for cell therapy. In the present study we investigated the effects of allogenic ASCs and their released products on cardiac rarefaction post MI. METHODS: Pig subcutaneous adipose tissue ASCs were isolated, expanded and GFP-labeled. ASC angiogenic function was assessed by the in-vivo chick chorioallantoic membrane (CAM) model. Pigs underwent MI induction and 7 days after were randomized to receive: allogenic ASCs (intracoronary infusion); conditioned media (CM; intravenous infusion); ASCs + CM; or PBS/placebo (control). Cardiac damage and function were monitored by 3-T cardiac magnetic resonance imaging upon infusion (baseline CMR) and 1 and 3 weeks thereafter. We assessed in the myocardium: microvessel density; angiogenic markers (CD105, CD31, TF, VEGFR2, VEGFR1, vWF, eNOS, CD62); collagen deposition; and reparative fibrosis (TGFß/TßRII/collagen). Differential proteomics of ASCs and CM was performed to characterize the ASC protein signature. RESULTS: CAM indicated a significant ASC proangiogenic capacity. In pigs after MI, only PBS/placebo animals displayed an impaired cardiac function 3 weeks after infusion (p < 0.05 vs baseline). Administration of ASCs + CM significantly enhanced neovessel formation and favored cardiac repair post MI (p < 0.05 vs the other groups). Molecular markers of angiogenesis were significantly upregulated both at transcriptional and protein levels (p < 0.05). The in-silico bioinformatics analysis of the ASC and CM proteome (interactome) indicated activation of a coordinated protein network involved in the formation of microvessels and the resolution of rarefaction. CONCLUSION: Coadministration of allogenic ASCs and their CM synergistically contribute to the neovascularization of the infarcted myocardium through a coordinated upregulation of the proangiogenic protein interactome.