Cultured Human Bone Marrow–Derived CD31 Cells Are Effective for Cardiac and Vascular Repair Through Enhanced Angiogenic,Adhesion, and Anti-Inflammatory Effects

Background

Cell therapy for cardiovascular disease has been limited by low engraftment of administered cells and modest therapeutic effects. Bone marrow (BM) -derived CD31⁺ cells are a promising cell source owing to their high angiovasculogenic and paracrine activities.

Objectives

This study sought to identify culture conditions that could augment the cell adhesion, angiogenic, and anti-inflammatory activities of BM-derived CD31⁺ cells, and to determine whether these cultured CD31⁺ cells are effective for cardiac and vascular repair.

Methods

CD31⁺ cells were isolated from human BM by magnetic-activated cell sorting and cultured for 10 days under hematopoietic stem cell, mesenchymal stem cell, or endothelial cell culture conditions. These cells were characterized by adhesion, angiogenesis, and inflammatory assays. The best of the cultured cells were implanted into myocardial infarction (MI) and hindlimb ischemia (HLI) models to determine therapeutic effects and underlying mechanisms.

Results

The CD31⁺ cells cultured in endothelial cell medium (EC-CD31⁺ cells) showed the highest adhesion and angiogenic activities and lowest inflammatory properties in vitro compared with uncultured or other cultured CD31⁺ cells. When implanted into mouse MI or HLI models, EC-CD31⁺ cells improved cardiac function and repaired limb ischemia to a greater extent than uncultured CD31⁺ cells. Histologically, injected EC-CD31⁺ cells exhibited higher retention, neovascularization, and cardiomyocyte proliferation. Importantly, cell retention and endothelial transdifferentiation was sustained up to 1 year.

Conclusions

Short-term cultured EC-CD31⁺ cells have higher cell engraftment, vessel-formation, cardiomyocyte proliferation, and anti-inflammatory potential, are highly effective for both cardiac and peripheral vascular repair, and enhance survival of mice with heart failure. These cultured CD31⁺ cells may be a promising source for treating ischemic cardiovascular diseases.     

Local immune activity in acute coronary syndrome: oxLDL abrogates LPS-tolerance in mononuclear cells isolated from culprit lesion

Background

OxLDL plays a major role in the initiation and progression of atherosclerotic lesions even though further factors are needed to promote fibrous cap rupture and thrombotic occlusion of the arterial lumen. Pathogens have been implicated in this process but it remains unclear how they can cooperate with oxLDL in amplifying the destructive inflammatory response.

Objective

To phenotypically analyze culprit coronary inflammatory cells, evaluate their responsiveness to endotoxins and ascertain whether oxLDL alters the sensitivity of coronary mononuclear cells to bacterial components.

Methods

Mononuclear cells isolated from culprit and non-culprit coronary blood samples of patients with ST-segment elevation myocardial infarction (STEMI) and controls were analyzed for cell-specific surface markers and cytokines by flow-cytometry.

Results and conclusions

CD14 + cells contained elevated levels of TLR4, expressed high CD80, and produced huge amounts of inflammatory cytokines in response to LPS. Using a well-established model of endotoxin tolerance, we next showed that mononuclear cells isolated from control coronary artery, but not from culprit coronary artery, were tolerant to LPS, but pre-treatment of such cells with oxLDL abrogated LPS tolerance. Flow-cytometry analysis also showed that IL-17A, IL-21 and IFN-γ were over-produced by CD4 + and CD56 + cells isolated from the culprit coronary artery. All this data indicate that monocytes circulating in the culprit coronary artery of patients with STEMI are primed to synthesize high levels of inflammatory cytokines and suggest that oxLDL can amplify the inflammatory response of such cells to endotoxins.     

Prelamin A accumulation in endothelial cells induces premature senescence and functional impairment

Background

Defects in lamin A maturation result in premature aging syndromes and severe atherosclerosis as observed in the Hutchinson–Gilford Progeria Syndrome. In age-related atherosclerosis, several features of cellular senescence have been characterized in endothelial cells including telomere shortening and increased oxidative stress. However, to date, very little is known about lamin A alterations in these cells.

Objectives

To study lamin A-related senescence and its consequences in the activation status of primary endothelial cells.

Methods

Healthy primary endothelial cells and progenitors issued from human umbilical vein or cord blood were used. Lamin A defects were induced by protease inhibitor (Atazanavir) treatment for 48 h.

Results

We show that protease inhibitor treatment leads to the accumulation of farnesylated prelamin A, inducing nuclear shape abnormalities and premature senescence in both differentiated and progenitor endothelial cells. ICAM-1-dependent activation and monocytes adhesion was increased in mature endothelial cells. In parallel, the ability to generate microvascular networks in matrigel was decreased for endothelial progenitors. The effects of protease inhibitor treatment on nuclear shapes were reversed when cells were treated in combination with Pravastatin and Zoledronate in both mature and progenitor endothelial cells. Reversion was also demonstrated with a morpholino antisense-oligonucleotide targeting lamin A-specific splice site.

Discussion

This study shows that protease inhibitor treatment reproduces premature senescence due to lamin A defects in primary endothelial cells and progenitors after 48 h exposure. The cells used were non-aged as extracted from cord blood or umbilical vein, allowing one to consider that other senescence pathways were not activated and that the observed alterations were specific of prelamin A accumulation. Both mature endothelial cells and precursors were sensitive to prelamin accumulation and thus, could be used in the future as a valuable model to test different approaches aimed at specifically reversing lamin A-related cells senescence.     

Lacidipine improves endothelial repair capacity of endothelial progenitor cells from patients with essential hypertension

Background

Endothelial progenitor cells (EPCs) play a critical role in maintaining the integrity of vascular endothelium following arterial injury. Lacidipine has a beneficial effect on endothelium of hypertensive patients, but limited data are available on EPCs-mediated endothelial protection. This study tests the hypothesis that lacidipine treatment can improve endothelial repair capacity of EPCs from hypertensive patients through increasing CXC chemokine receptor four (CXCR4) signaling.

Methods

In vivo reendothelialization capacity of EPCs from hypertensive patients with or without in vitro lacidipine treatment was examined in a nude mouse model of carotid artery injury. Expression of CXCR4 and alteration in migration and adhesion functions of EPCs were evaluated.

Results

Basal CXCR4 expression was markedly reduced in EPCs from hypertensive patients compared with normal subjects. In parallel, the phosphorylation of Janus kinase-2 (JAK-2) of EPCs, a CXCR4 downstream signaling, was also significantly decreased. Lacidipine promoted CXCR4/JAK-2 signaling expression of in vitro EPCs. Transplantation of EPCs pretreated with lacidipine significantly accelerated in vivo reendothelialization. The enhanced in vitro function and in vivo reendothelialization capacity of EPCs were inhibited by shRNA-mediated knockdown of CXCR4 expression or pretreatment with JAK-2 inhibitor AG490, respectively. In hypertensive patients, lacidipine treatment for 4 weeks also resulted in an upregulation of CXCR4/JAK-2 signaling of EPCs, which was associated with augmented EPCs-mediated reendothelialization and improved endothelial function.

Conclusion

Deterioration of CXCR4 signaling may lead to impaired EPCs-mediated reendothelialization of hypertensive patients. Lacidipine-modified EPCs via a partially CXCR4 signaling contribute to enhanced endothelial repair capacity in hypertension.     

Polymeric stent materials dysregulate macrophage and endothelial cell functions: Implications for coronary artery stent

Background

Biodegradable polymers have been applied as bulk or coating materials for coronary artery stents. The degradation of polymers, however, could induce endothelial dysfunction and aggravate neointimal formation. Here we use polymeric microparticles to simulate and demonstrate the effects of degraded stent materials on phagocytic activity, cell death and dysfunction of macrophages and endothelial cells.

Methods

Microparticles made of low molecular weight polyesters were incubated with human macrophages and coronary artery endothelial cells (ECs). Microparticle-induced phagocytosis, cytotoxicity, apoptosis, cytokine release and surface marker expression were determined by immunostaining or ELISA. Elastase expression was analyzed by ELISA and the elastase-mediated polymer degradation was assessed by mass spectrometry.

Results

We demonstrated that poly(D,L-lactic acid) (PLLA) and polycaprolactone (PCL) microparticles induced cytotoxicity in macrophages and ECs, partially through cell apoptosis. The particle treatment alleviated EC phagocytosis, as opposed to macrophages, but enhanced the expression of vascular cell adhesion molecule (VCAM)-1 along with decreased nitric oxide production, indicating that ECs were activated and lost their capacity to maintain homeostasis. The activation of both cell types induced the release of elastase or elastase-like protease, which further accelerated polymer degradation.

Conclusions

This study revealed that low molecule weight PLLA and PCL microparticles increased cytotoxicity and dysregulated endothelial cell function, which in turn enhanced elastase release and polymer degradation. These indicate that polymer or polymer-coated stents impose a risk of endothelial dysfunction after deployment which can potentially lead to delayed endothelialization, neointimal hyperplasia and late thrombosis.     

Antineutrophil cytoplasmic antibodies stabilize adhesion and promote migration of flowing neutrophils on endothelial cells

Objective

Recruitment of neutrophils to sites of inflammation requires coordinated regulation of their capture, activation, and migration on vascular endothelium. This study examines whether exposure of neutrophils to antineutrophil cytoplasmic antibodies (ANCAs) can disrupt this sequence of events.

Methods

Isolated human neutrophils were perfused in the presence or absence of ANCA‐positive IgG over endothelial cells that had been activated with either 2 units/ml or 100 units/ml of tumor necrosis factor α (TNFα) for 4 hours.

Results

When endothelial cells were activated with 100 units/ml of TNFα, neutrophils were captured from flow, a small proportion of adherent cells rolled, and the majority transmigrated through the endothelial cell monolayer. When neutrophils were treated with ANCA IgG immediately before, 5 minutes before, or 15 minutes before perfusion, none rolled on contact with the endothelium, but the majority still transmigrated. When endothelial cells were activated with 2 units/ml of TNFα, the majority of untreated adherent neutrophils rolled, a few transmigrated, and the number that attached decreased with time during washout. In contrast, when neutrophils were pretreated with ANCA IgG just before perfusion, adhesion was stabilized, and the number of neutrophils that transmigrated was increased 10‐fold. Priming of the neutrophils with TNFα before the addition of ANCA further increased the stability of neutrophil binding, but did not significantly increase transmigration.

Conclusion

Rather than frustrating the transmigration process, ANCAs promoted the migration of neutrophils through the endothelium. That the effect was evident at a relatively low level of endothelial activation suggests that ANCAs may potentiate the early vasculitic lesion and promote tissue damage and recruitment of other proinflammatory cells.

     

Reduction of AMPK activity and altered MAPKs signalling in peripheral blood mononuclear cells in response to acute glucose ingestion following a short-term high fat diet in young healthy men

Background

Peripheral blood mononuclear cells (PBMCs) are known to respond to systematic changes in nutrient availability. The impact of a short-term high fat diet (HFD), with and without acute glucose ingestion, on the energy-sensing enzyme 5’ AMP-activated protein kinase (AMPK) as well as mitochondrial oxidative phosphorylation (OXPHOS) proteins in PBMCs is currently unknown.

Methods

Nine healthy, lean young males participated in a 7 day HFD intervention, designed to induce transient glucose intolerance. The phosphorylation status and total protein content of AMPK and inflammatory mitogen-activated protein kinases (MAPKs), and total OXPHOS protein in PBMCs, along with circulating cytokines, were assessed in the fasted state and following an oral glucose tolerance test (OGTT) before and after the HFD.

Results

One week of HFD resulted in relative glucose intolerance. The HFD resulted in a reduction of AMPK phosphorylation under fasting basal conditions and following the OGTT (both P < 0.05), while there were no differences in OXPHOS protein expression. Although the short-term HFD had no effect on basal phosphorylation of p38, JNK or ERK1/2, the activation of MAPKs signalling in response to glucose ingestion was attenuated post-HFD as compared to pre-HFD (P < 0.05 for all). Circulating cytokines were not significantly affected by the HFD.

Conclusions

We conclude that impaired glucose tolerance in response to 7 day HFD resulted in decreased AMPK activity and impaired glucose-stimulated MAPK activation following glucose ingestion in vivo in PBMCs from young, lean subjects. Further studies are warranted to explore how dietary manipulations impact interplay between AMPK and inflammatory signalling, along with immune function, in PBMCs.     

Lymphotoxin β–mediated stimulation of synoviocytes in rheumatoid arthritis

Objective

Lymphotoxin β (LTβ), a cytokine produced by T cells and B cells, plays a central role in the normal development of lymph nodes and is critical in the formation of ectopic germinal center reactions in rheumatoid synovitis. Because resident fibroblast‐like synoviocytes (FLS) express receptors for LTβ, we examined the consequences of FLS activation by LTβ.

Methods

FLS from patients with rheumatoid arthritis were isolated and examined for the expression of LTβ receptor. FLS were incubated with LTα1β2 and assayed for the production of cytokines and chemokines and the up‐regulation of adhesion molecules.

Results

Exposure of FLS to recombinant LTα1β2 resulted in the production of multiple inflammatory cytokines and metalloproteinases, implicating FLS as amplifiers of the inflammatory process in the inflamed joint. Additionally, LTα1β2 was found to up‐regulate the expression of cell adhesion molecules, rendering FLS to efficient adhesion substrates for T cells. LTα1β2 also induced production of the chemokines CCL2 and CCL5, which elicited transmigration activity of T cells. Upon stimulation with LTα1β2, FLS did not acquire characteristics of follicular dendritic cells.

Conclusion

These data document that FLS are involved in multiple stages of the inflammatory process, including the recruitment and retention of lymphocytes in the synovial microenvironment. We propose that the heterotypic interaction between LTβ‐producing lymphocytes and responding FLS contributes to the establishment of complex lymphoid microstructures, and that this may be one element that defines susceptibility of the synovial membrane to lymphoid organogenesis.

     

Two α-dicarbonyls downregulate migration,invasion, and adhesion of liver cancer cells in a p53-dependent manner

Background

Hepatocellular carcinoma accounts for more than 600,000 deaths per year due to it being a highly invasive tumor. The α-dicarbonyl, methylglyoxal demonstrates efficacy at reducing tumor burden, however the anti-cancerous activities of 3-deoxyglucosone, have never been studied.

Aims

To determine the anti-cancerous potential of methylglyoxal and 3-deoxyglucosone on liver tumor cells.

Methods

The in vitro effects of methylglyoxal and 3-deoxyglucosone were studied by investigating migration, invasion, and adhesion of Huh-7, HepG2, and Hep3B cells.

Results

3-Deoxyglucosone inhibited migration of Huh-7 and HepG2 cells. Methylglyoxal decreased migration of HepG2 cells. Additionally, 3-deoxyglucosone and methylglyoxal impaired invasion, and adhesion of Huh-7 and HepG2 cells. In Hep3B cells, a p53 null cell line, 3-deoxyglucosone and methylglyoxal had no effect on migration, invasion, or adhesion. However, both compounds inhibited invasion of wild-type p53 transfected Hep3B cells. Silencing of p53 in Huh-7 and HepG2 cells abrogated the effects of the α-dicarbonyls on cell invasion. 3DG and MG did not alter p53 total protein but promoted nuclear translocation of p53.

Conclusions

These studies suggest that 3-deoxyglucosone and methylglyoxal impair invasion, migration, and adhesion of hepatocellular carcinoma. The effects of both compounds on cell invasion are dependent on p53 and imply that α-dicarbonyls could be efficacious in the treatment of p53-expressing invasive liver tumors.     

The preventive effect of uncarboxylated osteocalcin against free fatty acid-induced endothelial apoptosis through the activation of phosphatidylinositol 3-kinase/Akt signaling pathway

Objective

Increasing evidence suggests that osteocalcin (OC), one of the osteoblast-specific proteins, has been associated with atherosclerosis, but results are conflicting. The aim of this study was to elucidate the independent effect of uncarboxylated osteocalcin (ucOC), an active form of osteocalcin which has been suggested to have an insulin sensitizing effect, on vascular endothelial cells.

Materials and Methods

We used human aortic endothelial cells and treated them with ucOC. Linoleic acid (LA) was used as a representative free fatty acid. Apoptosis was evaluated using various methods including a terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling analysis kit and Western blotting for cleaved caspase 3, cleaved poly (ADP-ribose) polymerase and Bcl-xL. The phosphorylations of Akt and endothelial nitric oxide synthase (eNOS) as well as the level of NO were measured to confirm the effect of ucOC on insulin signaling pathway.

Results

Pretreatment of ucOC (30 ng/ml) prevented LA-induced apoptosis in insulin-stimulated endothelial cells; effects were abolished by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin. Treatment of ucOC (ranged from 0.3 to 30 ng/ml) significantly increased the phosphorylation of Akt and eNOS and nitric oxide secretion from endothelial cells in a PI3-kinase dependent manner.

Conclusions

Our study is the first to demonstrate the independent effect of ucOC on vascular endothelial cells. Our results further suggest that ucOC could have beneficial effects on atherosclerosis.     

Berberine attenuates lipopolysaccharide-induced extracelluar matrix accumulation and inflammation in rat mesangial cells: Involvement of NF-κB signaling pathway

Background

Our previous studies demonstrated that berberine could improve the renal function in rats and mice with diabetic nephropathy (DN) and inhibit extracellular matrix (ECM) component, fibronectin (FN) expression in rat mesangial cells (MCs) cultured under high glucose. However, the molecular mechanisms have not been fully elucidated.

Objective

To explore the potential mechanisms of berberine in the treatment of DN, we investigated the effects of berberine on lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation and its downstream inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase (iNOS) and fibronectin (FN) protein expression in rat MCs.

Method

Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The activation of NF-κB was detected by Western blot and confocal microscopy. The protein levels of ICAM-1, TGF-β1, iNOS and FN in rat MCs were detected by Western blot.

Results

Our results revealed that berberine significantly suppressed LPS-induced cell proliferation and inhibited LPS-induced NF-κB nuclear translocation in MCs, as well as protein expression of ICAM-1, TGF-β1, iNOS and FN.

Conclusion

Berberine significantly repressed LPS-induced cell proliferation and FN expression in rat MCs through inhibiting the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators. The ameliorative effects of berberine on DN might be associated with this inhibition effect on NF-κB signaling pathway which was independent of its hypoglycemic effect.     

Participation of Mac-1, LFA-1 and VLA-4 integrins in the in vitro adhesion of sickle cell disease neutrophils to endothelial layers, and reversal of adhesion by simvastatin

Background

Pharmacological approaches to inhibit increased leukocyte adhesive interactions in sickle cell disease may represent important strategies for the prevention of vaso-occlusion in patients with this disorder. We investigated, in vitro, the adhesion molecules involved in endothelial-sickle cell disease neutrophil interactions and the effect of simvastatin on sickle cell disease neutrophil adhesion to tumor necrosis factor-α-activated endothelial monolayers (human umbilical vein endothelial cells), and neutrophil chemotaxis.

Design and Methods

Sickle cell disease patients in steady state and not on hydroxyurea were included in the study. Endothelial cells treated, or not, with tumor necrosis factor-α and simvastatin were used for neutrophil adhesion assays. Neutrophils treated with simvastatin were submitted to interleukin 8-stimulated chemotaxis assays.

Results

Sickle cell disease neutrophils showed greater adhesion to endothelial cells than control neutrophils. Adhesion of control neutrophils to endothelial cells was mediated by Mac-1 under basal conditions and by the Mac-1 and LFA-1 integrins under inflammatory conditions. In contrast, adhesion of sickle cell disease neutrophils to endothelium, under both basal and tumor necrosis factor-α-stimulated conditions, was mediated by Mac-1 and LFA-1 integrins and also by VLA-4. Under stimulated inflammatory conditions, simvastatin significantly reduced sickle cell disease neutrophil adhesion, and this effect was reversed by inhibition of nitric oxide synthase. Furthermore, intercellular adhesion molecule-1 expression was significantly abrogated on tumor necrosis factor-α-stimulated endothelium incubated with simvastatin, and statin treatment inhibited the interleukin-8-stimulated migration of both control and sickle cell disease neutrophils.

Conclusions

The integrins Mac-1, LFA-1 and, interestingly, VLA-4 mediate the adhesion of sickle cell disease leukocytes to activated endothelial cell layers, in vitro. Our data indicate that simvastatin may be able to reduce endothelial activation and consequent leukocyte adhesion in this in vitro model; future experiments and clinical trials may determine whether simvastatin therapy could be employed in patients with sickle cell disease, with beneficial effects on vaso-occlusion.     

Amniotic mesenchymal stem cells with robust chemotactic properties are effective in the treatment of a myocardial infarction model

Background

We previously reported that amniotic mesenchymal stem cells (AMMs) possess high angio-vasulogenic properties. In this study, we investigated the chemotactic abilities of AMMs for improved cardiac function and regenerative angiogenesis.

Methods

The expressions of chemotactic and angiogenic genes were determined by qRT-PCR. Myocardial infarction (MI) was induced in NOD/SCID mice and cells were directly transplanted into the border regions of ischemic heart tissue. Immunohistochemical analysis was also conducted.

Results

AMMs significantly expressed the representative chemotactic factor GCP-2, NAP-2 as well as angiogenic factor Hif-1a. AMMs also highly expressed the chemokine receptors CCR2, CCR3 and CCR5. AMM transplantation improved left ventricular function, capillary density, angiogenic cytokine levels, angiopoetin (Ang)-1 and vascular endothelial growth factor (VEGF-A) levels in affected tissue. Immunohistochemical assaying also revealed increased engraftment and endothelial phenotypes.

Conclusion

Our findings suggest that due to elevated survival and related chemotactic potential, AMMs are a promising stem cell source for the treatment of ischemic cardiovascular disease.     

Cardiovascular disease in patients with inflammatory rheumatic disease is associated with up‐regulation of markers of inflammation in cardiac microvessels and cardiomyocytes

Objective

Various inflammatory rheumatic diseases (IRDs) are associated with increased mortality due to cardiovascular disease. The aim of this study was to investigate heart biopsy specimens obtained from patients undergoing coronary artery bypass grafting and compare markers of inflammation and endothelial cell activation in the cardiac and skeletal muscle of patients with and those without IRD.

Methods

Paired biopsy specimens of cardiac and skeletal muscle were obtained from 22 consecutive patients with IRD and 8 patients without IRD, all of whom were undergoing coronary artery bypass grafting. The biopsy specimens were evaluated in a blinded manner by conventional microscopy and digital image analysis for cell markers (CD3, CD4, CD8, CD68, CD163, and CD31), HLA (HLA–ABC, HLA–DR, and HLA–DQ), adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1), and proinflammatory cytokines (interleukin‐1α, interleukin‐1β, and tumor necrosis factor).

Results

Patients with IRD had significantly higher expression of adhesion molecules, proinflammatory cytokines, and all classes of HLA on cardiomyocytes and endothelial cells but no increase on mononuclear cells in the myocardium compared with patients without IRD. Furthermore, cardiac muscle from patients with IRD displayed significantly higher local expression of inflammation and activation of cardiac microvessels compared with skeletal muscle from the same patients.

Conclusion

Patients with cardiovascular disease had increased expression of adhesion molecules, HLA, and proinflammatory cytokines in heart tissue, indicating local inflammation involving microvessels and cardiomyocytes that could play a role in the pathogenesis of cardiovascular disease. The more pronounced changes in patients with IRD compared with patients without IRD might contribute to the increased risk of cardiovascular disease and premature death in patients with IRD.

     

Aggregation of mononuclear and red blood cells through an ��4��1-Lu/basal cell adhesion molecule interaction in sickle cell disease

Background

Abnormal interactions between red blood cells, leukocytes and endothelial cells play a critical role in the occurrence of the painful vaso-occlusive crises associated with sickle cell disease. We investigated the interaction between circulating leukocytes and red blood cells which could lead to aggregate formation, enhancing the incidence of vaso-occlusive crises.

Design and Methods

Blood samples from patients with sickle cell disease (n=25) and healthy subjects (n=5) were analyzed by imaging and classical flow cytometry after density gradient separation. The identity of the cells in the peripheral blood mononuclear cell layer was determined using antibodies directed specifically against white (anti-CD45) or red (anti-glycophorin A) blood cells.

Results

Aggregates between red blood cells and peripheral blood mononuclear cells were visualized in whole blood from patients with sickle cell disease. The aggregation rate was 10-fold higher in these patients than in control subjects. Both mature red blood cells and reticulocytes were involved in these aggregates through their interaction with mononuclear cells, mainly with monocytes. The size of the aggregates was variable, with one mononuclear cell binding to one, two or several red blood cells. Erythroid Lu/basal cell adhesion molecule and α₄β₁ integrin were involved in aggregate formation. The aggregation rate was lower in patients treated with hydroxycarbamide than in untreated patients.

Conclusions

Our study gives visual evidence of the existence of circulating red blood cell-peripheral blood mononuclear cell aggregates in patients with sickle cell disease and shows that these aggregates are decreased during hydroxycarbamide treatment. Our results strongly suggest that erythroid Lu/basal cell adhesion molecule proteins are implicated in these aggregates through their interaction with α₄β₁ integrin on peripheral blood mononuclear cells.     

The non-alcoholic fraction of beer increases stromal cell derived factor 1 and the number of circulating endothelial progenitor cells in high cardiovascular risk subjects: A randomized clinical trial

Rationale

Moderate alcohol consumption is associated with a decrease in cardiovascular risk, but fermented beverages seem to confer greater cardiovascular protection due to their polyphenolic content. Circulating endothelial progenitor cells (EPC) are bone-marrow-derived stem cells with the ability to repair and maintain endothelial integrity and function and are considered as a surrogate marker of vascular function and cumulative cardiovascular risk. Nevertheless, no study has been carried out on the effects of moderate beer consumption on the number of circulating EPC in high cardiovascular risk patients.

Objective

To compare the effects of moderate consumption of beer, non-alcoholic beer and gin on the number of circulating EPC and EPC-mobilizing factors.

Methods

In this crossover trial, 33 men at high cardiovascular risk were randomized to receive beer (30 g alcohol/d), the equivalent amount of polyphenols in the form of non-alcoholic beer, or gin (30 g alcohol/d) for 4 weeks. Diet and physical exercise were carefully monitored.

Results

The number of circulating EPC and EPC-mobilizing factors were determined at baseline and after each intervention. After the beer and non-alcoholic beer interventions, the number of circulating EPC significantly increased by 8 and 5 units, respectively, while no significant differences were observed after the gin period. In correlation, stromal cell derived factor 1 increased significantly after the non-alcoholic and the beer interventions.

Conclusions

The non-alcoholic fraction of beer increases the number of circulating EPC in peripheral blood from high cardiovascular risk subjects.

Clinical trial registration

http://www.controlled-trials.com/ISRCTN95345245 ISRCTN95345245     

Gallic acid protects against endothelial injury by restoring the depletion of DNA methyltransferase 1 and inhibiting proteasome activities

Background

The aim of this study was to investigate the protective effects of gallic acid, a common phenolic compound naturally present in food and nutraceuticals, on endothelial cell death and the mechanisms involved.

Methods

Endothelial cell death was induced by the combination of homocysteine, adenosine and tumour necrosis factor (TNF) in human vascular endothelial cells (EAhy926 and HBEC-5i cells). The protective effects of gallic acid were evaluated against cytotoxicity, apoptosis and microparticle release. Underlying mechanisms were further investigated focusing on the involvement of DNA methyltransferase 1 (DNMT1) and proteasome activities.

Results

Our results showed that gallic acid dose-dependently arrested cytotoxicity in EAhy926 and HBEC-5i cells induced by the combination. Gallic acid showed anti-apoptotic effects and reduced the formation of microparticles. Notably, gallic acid reversed DNMT1 depletions at the protein level. The cytoprotective and anti-apoptotic effects of gallic acid were counteracted by the pre-treatment with DNMT1 inhibitor, 5-aza-2-deoxycytidine (5-aza-dC). Treatment with gallic acid led to the accumulation of ubiquitinated protein aggregates and the reduction in chymotrypsin-like proteasome activities indicating proteasome inhibition.

Conclusion

Our results demonstrate for the first time that gallic acid is capable of protecting endothelial cells from injury induced by the combination of homocysteine, adenosine and TNF, at least in part, by restoring the depletion of DNMT1 and inhibiting proteasome activities.