Impact of the large-scale deployment of artemether/lumefantrine on the malaria disease burden in Africa: case studies of South Africa,Zambia and Ethiopia

Background

Azathioprine triggers suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Eryptosis may accelerate the clearance of Plasmodium -infected erythrocytes. The present study thus explored whether azathioprine influences eryptosis of Plasmodium -infected erythrocytes, development of parasitaemia and thus the course of malaria.

Methods

Human erythrocytes were infected in vitro with Plasmodium falciparum (P. falciparum) (strain BinH) in the absence and presence of azathioprine (0.001 – 10 μM), parasitaemia determined utilizing Syto16, phosphatidylserine exposure estimated from annexin V-binding and cell volume from forward scatter in FACS analysis. Mice were infected with Plasmodium berghei (P. berghei) ANKA by injecting parasitized murine erythrocytes (1 × 10⁶) intraperitoneally. Where indicated azathioprine (5 mg/kg b.w.) was administered subcutaneously from the eighth day of infection.

Results

In vitro infection of human erythrocytes with P. falciparum increased annexin V-binding and initially decreased forward scatter, effects significantly augmented by azathioprine. At higher concentrations azathioprine significantly decreased intraerythrocytic DNA/RNA content (≥ 1 μM) and in vitro parasitaemia (≥ 1 μM). Administration of azathioprine significantly decreased the parasitaemia of circulating erythrocytes and increased the survival of P. berghei -infected mice (from 0% to 77% 22 days after infection).

Conclusion

Azathioprine inhibits intraerythrocytic growth of P. falciparum, enhances suicidal death of infected erythrocytes, decreases parasitaemia and fosters host survival during malaria.     

The stem cell factor antibody enhances the chemotherapeutic effect of adriamycin on chemoresistant breast cancer cells

Background

The outcome of chemotherapy in breast cancer is strongly influenced by multidrug resistance (MDR). Several surrogate markers of chemoresistance have been identified including - CD24 (cluster differentiation 24) expression, stem cell growth factor (SCF), B-cell lymphocyte protein 2 (Bcl-2) and annexin V. The present study aimed to examine the expression of CD24 in the sensitive breast cancer cell line MCF-7 (Michigan Foudation-7) and MCF-7/adriamycin resistant (MCF-7/AdrRes) cells, and, if minimal effective doses of the anthracycline drug adriamycin (0.579???M and 88.2???M) would be enhanced by the antibody to SCF (anti-SCF).

Methods

CD24 expression was analysed by flow cytometry. Both Bcl-2 and annexin V protein expression were quantitatively assessed by the enzyme-linked immunosorbent assay (ELISA).

Results

In MCF-7/AdrRes cells the expression of CD24 was significantly higher compared to MCF-7 cells, 86.6% and 16.3% (p?Conclusion Adding anti-SCF to the chemotherapeutic regime of adriamycin may strongly enhance its chemotherapeutic effect in the treatment of patients with breast cancer.     

Circulating Endothelial Cells and Microparticles as Prognostic Markers in Advanced Non-Small Cell Lung Cancer

Background

Circulating endothelial cells and microparticles have prognostic value in cancer, and might be predictors of response to chemotherapy and antiangiogenic treatments. We have investigated the prognostic value of circulating endothelial cells and microparticles in patients treated for advanced non-small cell lung cancer.

Methodology/Principal Findings

Peripheral blood samples were obtained from 60 patients before first line, platinum-based chemotherapy +/− bevacizumab, and after the third cycle of treatment. Blood samples from 60 healthy volunteers were also obtained as controls. Circulating endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Phosphatidylserine-positive microparticles were evaluated by flow cytometry. Microparticle-mediated procoagulant activity was measured by the endogen thrombin generation assay. Results: pre- and posttreatment levels of markers were higher in patients than in controls (p<0.0001). Elevated levels of microparticles were associated with longer survival. Elevated pretreatment levels of circulating endothelial cells were associated with shorter survival.

Conclusions/Significance

Circulating levels of microparticles and circulating endothelial cells correlate with prognosis, and could be useful as prognostic markers in patients with advanced non-small cell lung cancer.     

PERK eIF2 alpha kinase is required to regulate the viability of the exocrine pancreas in mice

Background

The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Angiopoietins 1 and 2 (Ang1 and Ang2) are regulators of both angiogenesis and lymphangiogenesis through the Tek/Tie-2 receptor tyrosine kinase. The response of endothelial cells to stimulation with either Ang1 or Ang2 is thought to be dependent upon the origin of the endothelial cells. In this study, we examined the effects of the angiopoietins on lymphatic, venous and arterial primary endothelial cells (bmLEC, bmVEC and bmAEC, respectively), which were isolated and cultured from bovine mesenteric vessels.

Results

BmLEC, bmVEC and bmAEC cell populations all express Tie-2 and were shown to express the appropriate cellular markers Prox-1, VEGFR3, and Neuropilin-1 that define the particular origin of each preparation. We showed that while bmLECs responded slightly more readily to angiopoietin-2 (Ang2) stimulation, bmVECs and bmAECs were more sensitive to Ang1 stimulation. Furthermore, exposure of bmLECs to Ang2 induced marginally higher levels of proliferation and survival than did exposure to Ang1. However, exposure to Ang1 resulted in higher levels of migration in bmLECs than did to Ang2.

Conclusion

Our results suggest that although both Ang1 and Ang2 can activate the Tie-2 receptor in bmLECs, Ang1 and Ang2 may have distinct roles in mesenteric lymphatic endothelial cells.     

CD40/CD40L interaction induces E-selectin dependent leukocyte adhesion to human endothelial cells and inhibits endothelial cell migration

Background

CD40 is a receptor expressed on a wide range of cells such as leukocytes and endothelial cells (EC). As a member of the tumor necrosis factor (TNF) superfamily the activation of CD40 by CD40-ligand (CD40L) plays a crucial role for the development and progression of a variety of inflammatory processes including atherosclerosis. The aim of the present study was to investigate the effect of CD40/CD40L interaction on leukocyte adhesion to the endothelium and on endothelial cell migration.

Methods and results

Human umbilical vein endothelial cells (HUVEC) were stimulated with either stable transfectants of mouse myeloma cells expressing the CD40L or wild type cells (4 h). Subsequently adhesion of leukocytes expressing Sialyl Lewis X, the counterpart for E-selectin (HL60 cells), was measured under shear stress (2–2.6 dyne/cm²) using a flow chamber adhesion assay. Stimulation of CD40 led to a significant increase of E-selectin dependent adhesion of leukocytes to the endothelium. Incubation of cells with either the CD40L blocking antibody TRAP-1 or the E-selectin blocking antibody BBA2 during CD40 stimulation completely abolished adhesion of leukocytes to HUVEC. Similar results were found in human cardiac microvasculature endothelial cells (HCMEC). In contrast stimulation of CD40 had no effect on adhesion of l-selectin expressing NALM6-L cells. Furthermore, CD40/CD40L interaction abrogated VEGF-induced migration of HUVEC compared to non-stimulated controls. In comparison experiments, stimulation of endothelial cells with VEGF led to a significant phosphorylation of ERK1/2, Akt, and eNOS. Stimulation of endothelial CD40 had no effect on VEGF-induced phosphorylation of ERK1/2. However, VEGF-induced activation of Akt and eNOS was reduced to baseline levels when endothelial CD40 was stimulated.

Conclusion

CD40/CD40L interaction induces E-selectin dependent adhesion of leukocytes to human endothelial cells and reduces endothelial cell migration by inhibiting the Akt/eNOS signaling pathway.     

Microparticle miRNAs as Biomarkers of Vascular Function and Inflammation Response to Aerobic Exercise in Obesity?

Objective

This study aimed to explore the role of nine microRNAs (miRNAs) in microparticles (MPs) on the efficacy of aerobic exercise in the regulation of inflammation and vascular function in obesity.

Methods

Sedentary women with normal weight (n = 6, BMI < 25 kg/m²) and women with obesity (n = 9, BMI > 30 kg/m²) were recruited at F. Hached Hospital (Sousse, Tunisia) and enrolled in an 8‐week aerobic program. Vascular function was assessed using laser Doppler flowmetry/iontophoresis, circulating MPs by flow cytometry, miRNAs by real‐time polymerase chain reaction, and inflammation by ELISA, before and after exercise.

Results

Women with obesity presented with high prevalence of cardiovascular risk factors and a higher circulating MP level compared with healthy subjects. The MP miRNA profile was significantly different in the two groups. Exercise reduced BMI and inflammation in both groups and significantly improved endothelial‐dependent response (acetylcholine cutaneous vascular conductance) for healthy subjects, with a trend for women with obesity. Circulating MP level was increased after exercise, and miRNA expression was differentially modulated in both populations. Pearson analysis revealed a correlation between MPs miR‐124a and miR150 and adiponectin, TNFα, or IL‐6 levels.

Conclusions

The relation between MPs and miRNA profile, inflammation, vascular function, and exercise is of particular interest for defining “miRNA biomarker signature” in patients with cardiovascular disease who are potentially susceptible to respond to exercise.

     

Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury

Background

Bone marrow-derived progenitors for both epithelial and endothelial cells have been observed in the lung. Besides mature endothelial cells (EC) that compose the adult vasculature, endothelial progenitor cells (EPC) are supposed to be released from the bone marrow into the peripheral blood after stimulation by distinct inflammatory injuries. Homing of ex vivo generated bone marrow-derived EPC into the injured lung has not been investigated so far. We therefore tested the hypothesis whether homing of EPC in damaged lung tissue occurs after intravenous administration.

Methods

Ex vivo generated, characterized and cultivated rat bone marrow-derived EPC were investigated for proliferation and vasculogenic properties in vitro. EPC were tested for their homing in a left-sided rat lung transplant model mimicking a severe acute lung injury. EPC were transplanted into the host animal by peripheral administration into the femoral vein (10⁶ cells). Rats were sacrificed 1, 4 or 9 days after lung transplantation and homing of EPC was evaluated by fluorescence microscopy. EPC were tested further for their involvement in vasculogenesis processes occurring in subcutaneously applied Matrigel in transplanted animals.

Results

We demonstrate the integration of intravenously injected EPC into the tissue of the transplanted left lung suffering from acute lung injury. EPC were localized in vessel walls as well as in destructed lung tissue. Virtually no cells were found in the right lung or in other organs. However, few EPC were found in subcutaneous Matrigel in transplanted rats.

Conclusion

Transplanted EPC may play an important role in reestablishing the endothelial integrity in vessels after severe injury or at inflamatory sites and might further contribute to vascular repair or wound healing processes in severely damaged tissue. Therapeutic applications of EPC transplantation may ensue.     

Circulating Endothelial Cells and Procoagulant Microparticles in Patients with Glioblastoma: Prognostic Value

Aim

Circulating endothelial cells and microparticles are prognostic factors in cancer. However, their prognostic and predictive value in patients with glioblastoma is unclear. The objective of this study was to investigate the potential prognostic value of circulating endothelial cells and microparticles in patients with newly diagnosed glioblastoma treated with standard radiotherapy and concomitant temozolomide. In addition, we have analyzed the methylation status of the MGMT promoter.

Methods

Peripheral blood samples were obtained before and at the end of the concomitant treatment. Blood samples from healthy volunteers were also obtained as controls. Endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Microparticles were quantified by flow cytometry. Microparticle-mediated procoagulant activity was measured by endogen thrombin generation and by phospholipid-dependent clotting time. Methylation status of MGMT promoter was determined by multiplex ligation-dependent probe amplification.

Results

Pretreatment levels of circulating endothelial cells and microparticles were higher in patients than in controls (p<0.001). After treatment, levels of microparticles and thrombin generation decreased, and phospholipid-dependent clotting time increased significantly. A high pretreatment endothelial cell count, corresponding to the 99^th percentile in controls, was associated with poor overall survival. MGMT promoter methylation was present in 27% of tumor samples and was associated to a higher overall survival (66 weeks vs 30 weeks, p<0.004).

Conclusion

Levels of circulating endothelial cells may have prognostic value in patients with glioblastoma.     

Metabolic fingerprints of human primary endothelial and fibroblast cells

Introduction

Human primary cells originating from different locations within the body could differ greatly in their metabolic phenotypes, influencing both how they act during physiological/pathological processes and how susceptible/resistant they are to a variety of disease risk factors. A novel way to monitor cellular metabolism is through cell energetics assays, so we explored this approach with human primary cell types, as models of sclerotic disorders.

Objectives

In order to better understand pathophysiological processes at the cellular level, our goals were to measure metabolic pathway activities of endothelial cells and fibroblasts, and determine their metabolic phenotype profiles.

Methods

Biolog Phenotype MicroArray? technology was used for the first time to characterize metabolic phenotypes of diverse primary cells. These colorimetric assays enable detection of utilization of 367 specific biochemical substrates by human endothelial cells from the coronary artery (HCAEC), umbilical vein (HUVEC) and normal, healthy lung fibroblasts (NHLF).

Results

Adenosine, inosine, d-mannose and dextrin were strongly utilized by all three cell types, comparable to glucose. Substrates metabolized solely by HCAEC were mannan, pectin, gelatin and prevalently tricarballylic acid. HUVEC did not show any uniquely metabolized substrates whereas NHLF exhibited strong utilization of sugars and carboxylic acids along with amino acids and peptides.

Conclusion

Taken together, we show for the first time that this simple energetics assay platform enables metabolic characterization of primary cells and that each of the three human cell types examined gives a unique and distinguishable profile.

     

Production of a therapeutic protein by fusing it with two fragments of the carboxyl-terminal peptide of human chorionic gonadotropin β-subunit in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objective

To produce a therapeutic protein (endostatin) by fusion with two fragments of the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin β-subunit in Pichia pastoris.

Results

Two CTP sequences were fused to the C-terminal of human endostatin, and the fusion protein (endo-CTP) was expressed by P. pastoris. Endo-CTP inhibited proliferation of endothelial cells with an IC₅₀ of 7 μg ml^?1, and 30 % of cells were annexin V-positive after treatment with 20 μg endo-CTP ml^?1 for 48 h. Migration of endothelial cells was inhibited by endo-CTP in a concentration-dependent manner. The half-life of endo-CTP in Sprague–Dawley rats was much longer than that of its commercial counterpart (Endostar).

Conclusion

A long-acting endostatin can be produced using CTP technology.

     

Polysialic acid is released by human umbilical vein endothelial cells (HUVEC) in vitro

Background

Sialic acids represent common terminal residues on numerous mammalian glycoconjugates, thereby influencing e.g. lumen formation in developing blood vessels. Interestingly, besides monosialylated also polysialylated glycoconjugates are produced by endothelial cells. Polysialic acid (polySia) is formed in several organs during embryonal and postnatal development influencing, for instance, cell migration processes. Furthermore, the function of cytokines like basic fibroblast growth factor (bFGF) is modulated by polySia.

Results

In this study, we demonstrated that human umbilical vein endothelial cells (HUVEC) also secrete polysialylated glycoconjugates. Furthermore, an interaction between polySia and vascular endothelial growth factor (VEGF) was observed. VEGF modulates like bFGF the migration of HUVEC. Since both growth factors interact with polySia, we examined, if polySia modulates the migration of HUVEC. To this end scratch assays were performed showing that the migration of HUVEC is stimulated, when polySia was degraded.

Conclusions

Since polySia can interact with bFGF as well as VEGF and the degradation of polySia resulted in an increased cell migration capacity in the applied scratch assay, we propose that polySia may trap these growth factors influencing their biological activity. Thus, polySia might also contribute to the fine regulation of physiological processes in endothelial cells.

     

Indian hedgehog gene transfer is a chondrogenic inducer of human mesenchymal stem cells

Introduction

Anti-endothelial cell antibodies (AECAs) are thought to be critical for vasculitides in collagen diseases, but most were directed against molecules localized within the cell and not expressed on the cell surface. To clarify the pathogenic roles of AECAs, we constructed a retroviral vector system for identification of autoantigens expressed on the endothelial cell surface.

Methods

AECA activity in sera from patients with collagen diseases was measured with flow cytometry by using human umbilical vein endothelial cells (HUVECs). A cDNA library of HUVECs was retrovirally transfected into a rat myeloma cell line, from which AECA-positive clones were sorted with flow cytometry. cDNA of the cells was analyzed to identify an autoantigen, and then the clinical characteristics and the functional significance of the autoantibody were evaluated.

Results

Two distinct AECA-positive clones were isolated by using serum immunoglobulin G (IgG) from a patient with systemic lupus erythematosus (SLE). Both clones were identical to cDNA of fibronectin leucine-rich transmembrane protein 2 (FLRT2). HUVECs expressed FLRT2 and the prototype AECA IgG bound specifically to FLRT2-transfected cells. Anti-FLRT2 antibody activity accounted for 21.4% of AECAs in SLE. Furthermore, anti-FLRT2 antibody induced complement-dependent cytotoxicity against FLRT2-expressing cells.

Conclusions

We identified the membrane protein FLRT2 as a novel autoantigen of AECAs in SLE patients by using the retroviral vector system. Anti-FLRT2 antibody has the potential to induce direct endothelial cell cytotoxicity in about 10% of SLE patients and could be a novel molecular target for intervention. Identification of such a cell-surface target for AECAs may reveal a comprehensive mechanism of vascular injury in collagen diseases.     

Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

Background

Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles.

Results

Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that < 10% of nuclei were damaged in nanoparticle-transfected samples, compared to > 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles.

Conclusions

We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.     

SRJ23, a new semisynthetic andrographolide derivative: in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis in prostate cancer cells

Purpose

3,19-(3-Chloro-4-fluorobenzylidene)andrographolide (SRJ23), a new semisynthetic derivative of andrographolide (AGP), exhibited selectivity against prostate cancer cells in the US National Cancer Institute (NCI) in vitro anti-cancer screen. Herein, we report the in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis induced by SRJ23.

Methods

3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used in assessing in vitro growth inhibition of compounds against prostate cancer (PC-3, DU-145 and LNCaP) and mouse macrophage (RAW 264.7) cell lines. Flow cytometry was utilised to analyse cell cycle distribution, whereas fluorescence microscopy was performed to determine morphological cell death. DNA fragmentation and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry were done to confirm apoptosis induced by SRJ23. Quantitation of cell cycle and apoptotic regulatory proteins were determined by immunoblotting.

Results

AGP and SRJ23 selectively inhibited the growth of prostate cancer cells compared with RAW 264.7 cells at low micromolar concentrations; however, SRJ23 was more potent. Mechanistically, SRJ23-treated PC-3 cells displayed down-regulation of cyclin-dependent kinase (CDK) 1 without affecting levels of CDK4 and cyclin D1. However, SRJ23 induced down-regulation of CDK4 and cyclin D1 but without affecting CDK1 in DU145 and LNCaP cell lines. DNA histogram analysis revealed that the SRJ23 induced G2/M in PC-3 cells but G1 arrest in DU-145 and LNCaP cells. Morphologically, both compounds induced predominantly apoptosis, which was further confirmed by DNA fragmentation and annexin V-FITC staining. The DNA fragmentation was inhibited in the presence of caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was associated with an increase in caspase 8 expression and activation. This thought to have induced cleavage of Bid into t-Bid. Additionally, increased expression and activation of caspase 9 and Bax proteins were apparent, with a concomitant down-regulation of Bcl-2 protein. Similar apoptosis cascade of events was observed in SRJ23-treated DU145 and LNCaP cell lines.

Conclusion

SRJ23 inhibited the growth of prostate cancer cells by inducing G₂/M and G1 arrest via down-regulation of CDK1, and CDK4 and cyclin, respectively, and initiated caspase-8-mediated mitochondrial apoptosis. Taken together, these data support the potential of this compound as a new anti-prostate cancer agent.     

Effects of High Intensity Training and High Volume Training on Endothelial Microparticles and Angiogenic Growth Factors

Aims

Endothelial microparticles (EMP) are complex vesicular structures shed from activated or apoptotic endothelial cells. As endurance exercise affects the endothelium, the objective of the study was to examine levels of EMP and angiogenic growth factors following different endurance exercise protocols.

Methods

12 subjects performed 3 different endurance exercise protocols: 1. High volume training (HVT; 130 min at 55% peak power output (PPO); 2. 4×4 min at 95% PPO; 3. 4×30 sec all-out. EMPs were quantified using flow cytometry after staining platelet-poor-plasma. Events positive for Annexin-V and CD31, and negative for CD42b, were classified as EMPs. Vascular endothelial growth factor (VEGF), migratory inhibiting factor (MIF) and hepatocyte growth factor (HGF) were determined by ELISA technique. For all these measurements venous blood samples were taken pre, 0′, 30′, 60′ and 180′ after each intervention. Furthermore, in vitro experiments were performed to explore the effect of collected sera on target endothelial functions and MP uptake capacities.

Results

VEGF and HGF significantly increased after HIT interventions. All three interventions caused a significant decrease in EMP levels post exercise compared to pre values. The sera taken after exercise increased the uptake of EMP in target endothelial cells compared to sera taken under resting conditions, which was shown to be phosphatidylserin-dependent. Increased EMP uptake was associated with an improved protection of target cells against apoptosis. Sera taken prior and after exercise promoted target endothelial cell migration, which was abrogated after inhibition of VEGF.

Conclusion

Physical exercise leads to decreased EMP levels and promotes a phosphatidylserin-dependent uptake of EMP into target endothelial cells, which is associated with a protection of target cells against apoptosis.     

Mononuclear-cell-derived microparticles attenuate endothelial inflammation by transfer of miR-142-3p in a CD39 dependent manner

Plasma microparticles (MP) bear functional active ectonucleotidases of the CD39 family with implications in vascular inflammation. MP appear to be able to fuse with cells and transfer genetic information. Here, we tested whether levels of different immunomodulatory microRNAs (miRs) in plasma MP are modulated by CD39 after experimental hepatectomy. We further investigated whether horizontal transfer of miR-142-3p between mononuclear (MNC) and endothelial cells via MP is regulated by purinergic signaling. Partial hepatectomy was performed in C57BL/6 wild type and Cd39 null mice. MP were collected via ultracentrifugation. MNC were stimulated with nucleotides and nucleosides, in vitro, and tested for miR-142-3p levels. Fusion of MNC-derived MP and endothelial cells with subsequent transfer of miR-142-3p was imaged by flow cytometry and confocal microscopy. Endothelial inflammation and apoptosis were quantified after transfection with miR-142-3p. Significantly lower miR-142-3p levels were observed in plasma MP of Cd39 null mice after partial hepatectomy, when compared to C57BL/6 wild types (p?<?0.05). In contrast to extracellular nucleotides, anti-inflammatory adenosine significantly increased miR-142-3p levels in MNC-derived MP, in vitro (p?<?0.05). MNC-derived MP are able to transfer miR-142-3p to endothelial cells by fusion. Transfection of endothelial cells with miR-142-3p decreased TNF-α levels (p?<?0.05) and endothelial apoptosis (p?<?0.05). MiR-142-3p levels in MNC-derived MP are modulated by nucleoside signaling and might reflect compensatory responses in vascular inflammation. Our data suggest the transfer of genetic information via shed MP as a putative mechanism of intercellular communication—with implications in organ regeneration.     

Proteomic profiling of endorepellin angiostatic activity on human endothelial cells

Background

Endorepellin, the C-terminal domain V of the heparan sulfate proteoglycan perlecan, exhibits powerful and targeted anti-angiogenic activity on endothelial cells. To identify proteins involved with endorepellin anti-angiogenic action, we performed an extensive comparative proteomic analysis between vehicle- and endorepellin-treated human endothelial cells.

Results

Proteomic analysis of endorepellin influence on human umbilical vein endothelial cells identified five differentially expressed proteins, three of which (β-actin, calreticulin, and chaperonin/Hsp60) were down-regulated and two of which (vimentin and the β subunit of prolyl 4-hydroxylase also known as protein disulfide isomerase) were up-regulated in response to endorepellin treatment—and associated with a fold change (endorepellin/control) ≤ 0.75 and ≥ 2.00, and a statistically significant p-value as determined by Student's t test.

Conclusion

The proteins identified represent potential target areas involved with endorepellin anti-angiogenic mechanism of action. Further elucidation as such will ultimately provide useful in utilizing endorepellin as an anti-angiogenic therapy in humans.     

Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke

Purpose

Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke.

Methods

Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3–7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls.

Results

Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions.

Conclusions

Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions associate with deeper vessel injury affecting vascular smooth muscle cells.     

Acute ST-segment elevation myocardial infarction after amoxycillin-induced anaphylactic shock in a young adult with normal coronary arteries: a case report

Background

Sirolimus-eluting stents (CYPHER stents) demonstrated remarkable efficacy in reducing restenosis rates in patients with coronary artery disease. There is a concern of sub-acute and late stent thrombosis. Tissue factor (TF) is critical in thrombosis. This study investigated the effect of sirolimus on TF expression and activity in cultured human vascular smooth muscle cells (SMCs).

Methods

SMCs were cultured from human saphenous veins and aortas. Quiescent cells were stimulated with sirolimus (0.1 – 20 ng/ml) over 24 hours. Cellular TF expression and activity released into culture medium were measured. The effect of sirolimus on activation of mammalian target of rapamycin (mTOR) was measured by phosphorylation of the substrate p70s6k at T389, and activation of RhoA was measured by pull-down assay.

Results

Sirolimus increased TF protein level in cultured human SMCs in a concentration and time-dependent manner (about 2-fold, p < 0.01) reaching maximal effect at 5 ng/ml. The stimulation of TF expression by sirolimus was associated with inhibition of basal activity of mTOR. No effects of sirolimus on RhoA or p38mapk activation that are positive regulators of TF in vascular wall cells were observed. The stimulation of TF expression by sirolimus (20 ng/ml) was prevented by the HMG-CoA reductase inhibitor fluvastatin (1 μmol/L). However, no increase in TF activity released from SMC into culture medium was observed after sirolimus treatment.

Conclusion

Although sirolimus stimulates TF protein expression in human SMC associated with inhibition of mTOR, it does not enhance TF activity released from the cells, suggesting a relatively safe profile of CYPHER stents. The inhibition of TF expression by fluvastatin favors clinical use of statins in patients undergoing coronary stenting.     

Treatment with TNF-α or bacterial lipopolysaccharide attenuates endocardial endothelial cell-mediated stimulation of cardiac fibroblasts

Background

The endocardial endothelium that lines the inner cavity of the heart is distinct from the microvascular endothelial cells and modulates cardiac muscle performance in a manner similar to the vascular endothelial modulation of vascular structure and vasomotor tone. Although the modulatory effects of endocardial endothelium (EE) on cardiomyocytes are firmly established, the regulatory effects of endocardial endothelium on the cardiac interstitium and its cellular components remain ill defined.

Methods and Results

We investigated whether the stimulatory effect of EE on cardiac fibroblasts would be altered when EECs are activated by the cytokine tumor necrosis factor-α (TNF-α) or the endotoxin bacterial lipopolysaccharide (LPS). Both TNF-α and LPS were found to independently attenuate the stimulatory effect of EE on cardiac fibroblasts. These agents lowered the synthesis or release of ET-1 and increased the secretion of TGF-β and NO.

Conclusion

The findings of this study using endocardial endothelial cells (EECs) and neonatal cardiac fibroblasts demonstrate that pro-inflammatory cytokines cause altered secretion of paracrine factors by EECs and inhibit proliferation and lower collagen synthesis in fibroblasts. These changes may influence fibroblast response and extra cellular matrix remodeling in pathological conditions of the heart.