Biomimetic Nanocarriers of Pro-Resolving Lipid Mediators for Resolution of Inflammation in Atherosclerosis.

Atherosclerosis (ATH) is a systemic disease characterized by a chronic inflammatory process and lipid deposition in the arterial walls. The chronic inflammation within ATH lesions results, at least in part, from the failed resolution of inflammation. This process is controlled actively by specialized pro-resolving lipid mediators (SPMs), namely lipoxins, resolvins, protectins, and maresins. Herein, biomimetic nanocarriers are produced comprising a cocktail of SPMs-loaded lipid nanoemulsions (LN) covered with macrophage membranes (Bio-LN/SPMs). Bio-LN/SPMs retain on their surface the macrophage receptors involved in cellular interactions and the "marker of self" CD47, which impede their recognition and uptake by other macrophages. The binding of Bio-LN/SPMs to the surface of endothelial cells (EC) and smooth muscle cells (SMC) is facilitated by the receptors on the macrophage membranes and partly by SPMs receptors. In addition, Bio-LN/SPMs prove functional by reducing monocyte adhesion and transmigration to/through activated EC and by stimulating macrophage phagocytic activity. After intravenous administration, Bio-LN/SPMs accumulate in the aorta of ApoE-deficient mice at the level of atherosclerotic lesions. Also, the safety assessment testing reveals no side effects or immunotoxicity of Bio-LN/SPMs. Thus, the newly developed Bio-LN/SPMs represent a reliable targeted nanomedicine for the resolution of inflammation in atherosclerosis.

Synthetic lipoproteins based on apolipoprotein E coupled to fullerenol have anti-atherosclerotic properties.

BACKGROUND: Apolipoprotein E (apoE) is an anti-atherosclerotic protein associated with almost all plasma lipoproteins. Fullerenol (Full-OH) contains the fullerene hydrophobic cage and several hydroxyl groups that could be derivatized to covalently bind various molecules. Herein, we aimed to produce fullerenol-based nanoparticles carrying apoE3 (Full-apoE) and test their anti-atherosclerotic effects. METHODS: Full-apoE nanoparticles were obtained from Full-OH activated to reactive cyanide ester fullerenol derivative that was further reacted with apoE protein. To test their effect, the nanoparticles were administered to apoE-deficient mice for 24 h or 3 weeks. ApoE part of the nanoparticles was determined by Western Blot and quantified by ELISA. Atherosclerotic plaque size was evaluated after Oil Red O staining and the gene expression was determined by Real-Time PCR. RESULTS: Full-apoE nanoparticles were detected mainly in the liver, and to a lesser extent in the kidney, lung, and brain. In the plasma of the Full-apoE-treated mice, apoE was found associated with very-low-density lipoproteins and high-density lipoproteins. Treatment for 3 weeks with Full-apoE nanoparticles decreased plasma cholesterol levels, increased the expression of apolipoprotein A-I, ABCA1 transporter, scavenger receptor-B1, and sortilin, and reduced the evolution of the atheromatous plaques in the atherosclerotic mice. CONCLUSIONS: In experimental atherosclerosis, the administration of Full-apoE nanoparticles limits the evolution of the atheromatous plaques by decreasing the plasma cholesterol level and increasing the expression of major proteins involved in lipid metabolism. Thus, they represent a novel promising strategy for atherosclerosis therapy.

Apolipoprotein A-II, a Player in Multiple Processes and Diseases.

Apolipoprotein A-II (apoA-II) is the second most abundant apolipoprotein in high-density lipoprotein (HDL) particles, playing an important role in lipid metabolism. Human and murine apoA-II proteins have dissimilar properties, partially because human apoA-II is dimeric whereas the murine homolog is a monomer, suggesting that the role of apoA-II may be quite different in humans and mice. As a component of HDL, apoA-II influences lipid metabolism, being directly or indirectly involved in vascular diseases. Clinical and epidemiological studies resulted in conflicting findings regarding the proatherogenic or atheroprotective role of apoA-II. Human apoA-II deficiency has little influence on lipoprotein levels with no obvious clinical consequences, while murine apoA-II deficiency causes HDL deficit in mice. In humans, an increased plasma apoA-II concentration causes hypertriglyceridemia and lowers HDL levels. This dyslipidemia leads to glucose intolerance, and the ensuing high blood glucose enhances apoA-II transcription, generating a vicious circle that may cause type 2 diabetes (T2D). ApoA-II is also used as a biomarker in various diseases, such as pancreatic cancer. Herein, we provide a review of the most recent findings regarding the roles of apoA-II and its functions in various physiological processes and disease states, such as cardiovascular disease, cancer, amyloidosis, hepatitis, insulin resistance, obesity, and T2D.

VLA4-Enhanced Allogeneic Endothelial Progenitor Cell-Based Therapy Preserves the Aortic Valve Function in a Mouse Model of Dyslipidemia and Diabetes.

The number and function of endothelial progenitor cells (EPCs) are reduced in diabetes, contributing to deteriorated vascular repair and the occurrence of cardiovascular complications. Here, we present the results of treating early diabetic dyslipidemic mice or dyslipidemic with disease-matched EPCs modified to overexpress VLA4 (VLA4-EPCs) as compared with the treatment of EPCs transfected with GFP (GFP-EPCs) as well as EPCs from healthy animals. Organ imaging of injected PKH26-stained cells showed little pulmonary first-pass effects and distribution in highly vascularized organs, with splenic removal from circulation, mostly in non-diabetic animals. Plasma measurements showed pronounced dyslipidemia in all animals and glycaemia indicative of diabetes in streptozotocin-injected animals. Echocardiographic measurements performed 3 days after the treatment showed significantly improved aortic valve function in animals treated with VLA4-overexpressing EPCs compared with GFP-EPCs, and similar results in the groups treated with healthy EPCs and VLA4-EPCs. Immunohistochemical analyses revealed active inflammation and remodelling in all groups but different profiles, with higher MMP9 and lower P-selectin levels in GFP-EPCs, treated animals. In conclusion, our experiments show that genetically modified allogeneic EPCs might be a safe treatment option, with bioavailability in the desired target compartments and the ability to preserve aortic valve function in dyslipidemia and diabetes.

Treatment with Mesenchymal Stromal Cells Overexpressing Fas-Ligand Ameliorates Acute Graft-versus-Host Disease in Mice.

Allogeneic hematopoietic cell transplantation (allo-HCT) has the potential to cure malignant and non-malignant hematological disorders, but because of the serious side effects of this intervention its applications are limited to a restricted number of diseases. Graft-versus-host disease (GvHD) is the most frequent complication and the leading cause of mortality and morbidity following allo-HCT. It results from the attack of the transplanted T cells from the graft against the cells of the recipient. There is no clear treatment for this severe complication. Due to their immunomodulatory properties, mesenchymal stromal cells (MSC) have been proposed to treat GvHD, but the results did not meet expectations. We have previously showed that the immunomodulatory effect of the MSC was significantly enhanced through adenoviral-mediated overexpression of FasL. In this study, we have tested the properties of FasL-overexpressing MSC in vivo, in a mouse model for acute GvHD. We found that treatment with FasL-overexpressing MSC delayed the onset of the disease and increased survival of the mice.

Inflammatory status of the pancreas in NOD mice that do not develop overt diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which immune cells target the pancreatic islets and destroy the ß-cells, resulting in hyperglycemia and decreased plasmatic insulin levels. The non-obese diabetic (NOD) mouse is the most used animal model for studying diabetes because it spontaneously develops T1D and shares similarities with the human disease. A hallmark feature of this model is the appearance of insulitis, defined as an inflammatory cell infiltration of the pancreatic islets. However, a small percentage of NOD mice do not develop overt diabetes even after 28-35 weeks of age. Thus, we questioned the status of the pancreatic islets in these non-diabetic NOD mice, with particular focus on islet inflammation and plasmatic insulin levels, in comparison to pre-diabetic (11 weeks old) and new-onset diabetic mice. Diabetes progression was evaluated by assessing blood glucose and pancreas histology. The inflammatory score was determined on Hematoxylin-Eosin (HE)-stained sections of pancreas. Plasma insulin was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that inflammation increased in an age-dependent manner in all mice, irrespective of their diabetic status. Mostly affected within the analyzed groups were the 28 weeks old non-diabetic NOD mice, in which insulin production was reduced and inversely correlated with the inflammatory status. We conclude that in NOD mice, pancreatic inflammation progresses independently of diabetes onset and clinical signs of disease. Most likely, the NOD females that do not develop overt diabetes preserve a small mass of functional ß-cells, which is able to provide the physiological insulin levels and avoid diabetes onset.

P-selectin targeted RAGE-shRNA lipoplexes alleviate atherosclerosis-associated inflammation.

The receptor for advanced glycation end products (RAGE) plays a central role in the chronic inflammatory process associated with atherosclerosis development. We aimed to develop lipoplexes carrying RAGE-short hairpin (sh) RNA, targeted to the adhesion molecule P-selectin, selectively expressed on the surface of activated endothelium (Psel-lipo/shRAGE) to down-regulate RAGE expression as a therapeutic strategy for atherosclerosis. In vitro, Psel-lipo/shRAGE lipoplexes were efficiently taken up by activated endothelial cells (EC), decreased the expression of RAGE protein, and proved to be functional by reducing the monocyte adhesion to activated EC. In ApoE-deficient mice, the targeted lipoplexes accumulated specifically and efficiently transfected the aorta. The repeated administration of Psel-lipo/shRAGE lipoplexes, twice per week for one month: i) reduced the expression of RAGE protein in the aorta by decreasing the expression of NF-kB and TNF-α; ii) diminished the plasma levels of TNF-α, IL6, IL-1ß, and MCP-1; iii) inhibited the atherosclerotic plaque development and iv) had no significant adverse effects. In conclusion, the newly developed Psel-lipo/shRAGE lipoplexes reduce the inflammatory processes associated with RAGE signaling and the progression of atherosclerosis in ApoE-deficient mice. Downregulation of RAGE employing these lipoplexes may represent a promising new targeted therapy to block atherosclerosis progression.

An Efficient Method for Adenovirus Production.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by "negative" and "positive" PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Short lifespan of syngeneic transplanted MSC is a consequence of in vivo apoptosis and immune cell recruitment in mice.

Mesenchymal stromal cells (MSC) are attractive tools for cell-based therapy, yet the mechanisms underlying their migration and survival post-transplantation are unclear. Accumulating evidence indicates that MSC apoptosis modulates both innate and adaptive immune responses which impact on MSC therapeutic effects. Using a dual tracking system, namely the Luciferase expression and VivoTrack680 labelling, and in vivo optical imaging, we investigated the survival and migration of MSC transplanted by various routes (intravenous, subcutaneous, intrapancreatic and intrasplenic) in order to identify the best delivery approach that provides an accumulation of therapeutic cells to the injured pancreas in the non-obese diabetic (NOD) mouse. The results showed that transplanted MSC had limited migration capacity, irrespective of the administration route, and were short-lived with almost total disappearance at 7 days after transplantation. Within one day after transplantation, cells activated hypoxia signalling pathways, followed by Caspase 3-mediated apoptosis. These were subsequently followed by local recruitment of immune cells at the transplantation site, and the engulfment of apoptotic MSC by macrophages. Our results argue for a "hit and die" mechanism of transplanted MSC. Further investigations will elucidate the molecular crosstalk between the inoculated and the host-immune cells.

Detection of Vascular Reactive Oxygen Species in Experimental Atherosclerosis by High-Resolution Near-Infrared Fluorescence Imaging Using VCAM-1-Targeted Liposomes Entrapping a Fluorogenic Redox-Sensitive Probe.

Excessive production of reactive oxygen species (ROS) and the ensuing oxidative stress are instrumental in all phases of atherosclerosis. Despite the major achievements in understanding the regulatory pathways and molecular sources of ROS in the vasculature, the specific detection and quantification of ROS in experimental models of disease remain a challenge. We aimed to develop a reliable and straightforward imaging procedure to interrogate the ROS overproduction in the vasculature and in various organs/tissues in atherosclerosis. To this purpose, the cell-impermeant ROS Brite™ 700 (RB700) probe that produces bright near-infrared fluorescence upon ROS oxidation was encapsulated into VCAM-1-targeted, sterically stabilized liposomes (VLp). Cultured human endothelial cells (EC) and macrophages (Mac) were used for in vitro experiments. C57BL6/J and ApoE-/- mice were randomized to receive normal or high-fat, cholesterol-rich diet for 10 or 32 weeks. The mice received a retroorbital injection with fluorescent tagged VLp incorporating RB700 (VLp-RB700). After two hours, the specific signals of the oxidized RB700 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-DSPE), inserted into liposome bilayers, were measured ex vivo in the mouse aorta and various organs by high-resolution fluorescent imaging. VLp-RB700 was efficiently taken up by cultured human EC and Mac, as confirmed by fluorescence microscopy and spectrofluorimetry. After systemic administration in atherosclerotic ApoE-/- mice, VLp-RB700 were efficiently concentrated at the sites of aortic lesions, as indicated by the augmented NBD fluorescence. Significant increases in oxidized RB700 signal were detected in the aorta and in the liver and kidney of atherosclerotic ApoE-/- mice. RB700 encapsulation into sterically stabilized VCAM-1-sensitive Lp could be a novel strategy for the qualitative and quantitative detection of ROS in the vasculature and various organs and tissues in animal models of disease. The accurate and precise detection of ROS in experimental models of disease could ease the translation of the results to human pathologies.

K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells.

Adenoviral vectors are important vehicles for delivering therapeutic genes into mammalian cells. However, the yield of the adenoviral transduction of murine mesenchymal stromal cells (MSC) is low. Here, we aimed to improve the adenoviral transduction efficiency of bone marrow-derived MSC. Our data showed that among all the potential transduction boosters that we tested, the K2 Transfection System (K2TS) greatly increased the transduction efficiency. After optimization of both K2TS components, the yield of the adenoviral transduction increased from 18% to 96% for non-obese diabetic (NOD)-derived MSC, from 30% to 86% for C57BL/6-derived MSC, and from 0.6% to 63% for BALB/c-derived MSC, when 250 transduction units/cell were used. We found that MSC derived from these mouse strains expressed different levels of the coxsackievirus and adenovirus receptors (MSC from C57BL/6≥NOD>>>BALB/c). K2TS did not increase the level of the receptor expression, but desensitized the cells to foreign DNA and facilitated the virus entry into the cell. The expression of Stem cells antigen-1 (Sca-1) and 5'-nucleotidase (CD73) MSC markers, the adipogenic and osteogenic differentiation potential, and the immunosuppressive capacity were preserved after the adenoviral transduction of MSC in the presence of the K2TS. In conclusion, K2TS significantly enhanced the adenoviral transduction of MSC, without interfering with their main characteristics and properties.

Evidence of mesenchymal stromal cell adaptation to local microenvironment following subcutaneous transplantation.

Subcutaneous transplantation of mesenchymal stromal cells (MSC) emerged as an alternative to intravenous administration because it avoids the pulmonary embolism and prolongs post-transplantation lifetime. The goal of this study was to investigate the mechanisms by which these cells could affect remote organs. To this aim, murine bone marrow-derived MSC were subcutaneously transplanted in different anatomical regions and the survival and behaviour have been followed. The results showed that upon subcutaneous transplantation in mice, MSC formed multicellular aggregates and did not migrate significantly from the site of injection. Our data suggest an important role of hypoxia-inducible signalling pathways in stimulating local angiogenesis and the ensuing modulation of the kinetics of circulating cytokines with putative protective effects at distant sites. These data expand the current understanding of cell behaviour after subcutaneous transplantation and contribute to the development of a non-invasive cell-based therapy for distant organ protection.

Enhanced Suppression of Immune Cells In Vitro by MSC Overexpressing FasL.

Mesenchymal stromal cells (MSC) display several mechanisms of action that may be harnessed for therapeutic purposes. One of their most attractive features is their immunomodulatory activity that has been extensively characterized both in vitro and in vivo. While this activity has proven to be very efficient, it is transient. We aimed to enhance it by transforming MSC to overexpress a first apoptosis signal (Fas) ligand (FasL). In this study, our goal was to induce FasL overexpression through adenoviral transduction in MSC to improve their immunomodulatory activity. We characterized the impact of FasL overexpression on the morphology, proliferation, viability, phenotype, multilineage differentiation potential and immunomodulation of MSC. Moreover, we determined their suppressive properties in mixed reactions with A20 cells, as well as with stimulated splenocytes. Our findings demonstrate that FasL-overexpressing MSC exhibit improved immunosuppressive properties, while maintaining their MSC-characteristic features. In conclusion, we establish, in a proof-of-concept set-up, that FasL-overexpressing MSC represent good candidates for therapeutic intervention targeted at autoimmune disorders.

Pharmacological inhibition of histone deacetylase reduces NADPH oxidase expression, oxidative stress and the progression of atherosclerotic lesions in hypercholesterolemic apolipoprotein E-deficient mice; potential implications for human atherosclerosis.

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are instrumental in all inflammatory phases of atherosclerosis. Dysregulated histone deacetylase (HDAC)-related epigenetic pathways have been mechanistically linked to alterations in gene expression in experimental models of cardiovascular disorders. Hitherto, the relation between HDAC and Nox in atherosclerosis is not known. We aimed at uncovering whether HDAC plays a role in mediating Nox up-regulation, oxidative stress, inflammation, and atherosclerotic lesion progression. Human non-atherosclerotic and atherosclerotic arterial samples, ApoE-/- mice, and in vitro polarized monocyte-derived M1/M2-macrophages (Mac) were examined. Male ApoE-/- mice, maintained on normal or high-fat, cholesterol-rich diet, were randomized to receive 10 mg/kg suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor, or its vehicle, for 4 weeks. In the human/animal studies, real-time PCR, Western blot, lipid staining, lucigenin-enhanced chemiluminescence assay, and enzyme-linked immunosorbent assay were employed. The protein levels of class I, class IIa, class IIb, and class IV HDAC isoenzymes were significantly elevated both in human atherosclerotic tissue samples and in atherosclerotic aorta of ApoE-/- mice. Treatment of ApoE-/- mice with SAHA reduced significantly the extent of atherosclerotic lesions, and the aortic expression of Nox subtypes, NADPH-stimulated ROS production, oxidative stress and pro-inflammatory markers. Significantly up-regulated HDAC and Nox subtypes were detected in inflammatory M1-Mac. In these cells, SAHA reduced the Nox1/2/4 transcript levels. Collectively, HDAC inhibition reduced atherosclerotic lesion progression in ApoE-/- mice, possibly by intertwined mechanisms involving negative regulation of Nox expression and inflammation. The data propose that HDAC-oriented pharmacological interventions could represent an effective therapeutic strategy in atherosclerosis.

Epigenetic regulation of vascular NADPH oxidase expression and reactive oxygen species production by histone deacetylase-dependent mechanisms in experimental diabetes.

Reactive oxygen species (ROS) generated by up-regulated NADPH oxidase (Nox) contribute to structural-functional alterations of the vascular wall in diabetes. Epigenetic mechanisms, such as histone acetylation, emerged as important regulators of gene expression in cardiovascular disorders. Since their role in diabetes is still elusive we hypothesized that histone deacetylase (HDAC)-dependent mechanisms could mediate vascular Nox overexpression in diabetic conditions. Non-diabetic and streptozotocin-induced diabetic C57BL/6J mice were randomized to receive vehicle or suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor. In vitro studies were performed on a human aortic smooth muscle cell (SMC) line. Aortic SMCs typically express Nox1, Nox4, and Nox5 subtypes. HDAC1 and HDAC2 proteins along with Nox1, Nox2, and Nox4 levels were found significantly elevated in the aortas of diabetic mice compared to non-diabetic animals. Treatment of diabetic mice with SAHA mitigated the aortic expression of Nox1, Nox2, and Nox4 subtypes and NADPH-stimulated ROS production. High concentrations of glucose increased HDAC1 and HDAC2 protein levels in cultured SMCs. SAHA significantly reduced the high glucose-induced Nox1/4/5 expression, ROS production, and the formation malondialdehyde-protein adducts in SMCs. Overexpression of HDAC2 up-regulated the Nox1/4/5 gene promoter activities in SMCs. Physical interactions of HDAC1/2 and p300 proteins with Nox1/4/5 promoters were detected at the sites of active transcription. High glucose induced histone H3K27 acetylation enrichment at the promoters of Nox1/4/5 genes in SMCs. The novel data of this study indicate that HDACs mediate vascular Nox up-regulation in diabetes. HDAC inhibition reduces vascular ROS production in experimental diabetes, possibly by a mechanism involving negative regulation of Nox expression.

Differential action of glucocorticoids on apolipoprotein E gene expression in macrophages and hepatocytes.

Apolipoprotein E (apoE) has anti-atherosclerotic properties, being involved in the transport and clearance of cholesterol-rich lipoproteins as well as in cholesterol efflux from cells. We hypothesized that glucocorticoids may exert anti-inflammatory properties by increasing the level of macrophage-derived apoE. Our data showed that glucocorticoids increased apoE expression in macrophages in vitro as well as in vivo. Dexamethasone increased ~6 fold apoE mRNA levels in cultured peritoneal macrophages and RAW 264.7 cells. Administered to C57BL/6J mice, dexamethasone induced a two-fold increase in apoE expression in peritoneal macrophages. By contrast, glucocorticoids did not influence apoE expression in hepatocytes, in vitro and in vivo. Moreover, dexamethasone enhanced apoE promoter transcriptional activity in RAW 264.7 macrophages, but not in HepG2 cells, as tested by transient transfections. Analysis of apoE proximal promoter deletion mutants, complemented by protein-DNA interaction assays demonstrated the functionality of a putative glucocorticoid receptors (GR) binding site predicted by in silico analysis in the -111/-104 region of the human apoE promoter. In hepatocytes, GR can bind to their specific site within apoE promoter but are not able to modulate the gene expression. The modulatory blockade in hepatocytes is a consequence of partial involvement of transcription factors and other signaling molecules activated through MEK1/2 and PLA2/PLC pathways. In conclusion, our study indicates that glucocorticoids (1) differentially target apoE gene expression; (2) induce a significant increase in apoE level specifically in macrophages. The local increase of apoE gene expression in macrophages at the level of the atheromatous plaque may have therapeutic implications in atherosclerosis.

c-Src tyrosine kinase mediates high glucose-induced endothelin-1 expression.

Endothelin-1 (ET-1) plays an important role in the pathophysiology of diabetes-associated cardiovascular disorders. The molecular mechanisms leading to ET-1 upregulation in diabetes are not entirely defined. c-Src tyrosine kinase regulates important pathophysiological aspects of vascular response to insults. In this study, we aimed to elucidate whether high glucose-activated c-Src signaling plays a role in the regulation of ET-1 expression. Human endothelial cells EAhy926 (ECs) were exposed to normal or high levels of glucose for 24h. Male C57BL/6J mice were rendered diabetic with streptozotocin and then treated with a specific c-Src inhibitor (Src I1) or c-Src siRNA. Real-time PCR, Western blot, and ELISA, were used to investigate ET-1 regulation. The c-Src activity and expression were selectively downregulated by pharmacological inhibition and siRNA-mediated gene silencing, respectively. High glucose dose-dependently up-regulated c-Src phosphorylation and ET-1 gene and protein expression levels in human ECs. Chemical inhibition or silencing of c-Src significantly decreased the high-glucose augmented ET-1 expression in cultured ECs. In vivo studies showed significant elevations in the aortic ET-1 mRNA expression and plasma ET-1 concentration in diabetic mice compared to non-diabetic animals. Treatment with Src I1, as well as in vivo silencing of c-Src, significantly reduced the upregulated ET-1 expression in diabetic mice. These data provide new insights into the regulation of ET-1 expression in endothelial cells in diabetes. Pharmacological targeting of c-Src activity and/or expression may represent a potential therapeutic strategy to reduce ET-1 level and to counteract diabetes-induced deleterious vascular effects.

Human monocytes and macrophages express NADPH oxidase 5; a potential source of reactive oxygen species in atherosclerosis.

Monocytes (Mon) and Mon-derived macrophages (Mac) orchestrate important oxidative and inflammatory reactions in atherosclerosis by secreting reactive oxygen species (ROS) due, in large part, to the upregulated NADPH oxidases (Nox). The Nox enzymes have been extensively investigated in human Mon and Mac. However, the expression and functional significance of the Nox5 subtypes is not known. We aimed at elucidating whether Nox5 is expressed in human Mon and Mac, and examine its potential role in atherosclerosis. Human monocytic THP-1 cell line and CD14(+) Mon were employed to search for Nox5 expression. RT-PCR, Western blot, lucigenin-enhanced chemiluminescence and dihydroethidium assays were utilized to examine Nox5 in these cells. We found that Nox5 transcription variants and proteins are constitutively expressed in THP-1 cells and primary CD14(+) Mon. Silencing of Nox5 protein expression by siRNA reduced the Ca(2+)-dependent Nox activity and the formation of ROS in Mac induced by A23187, a selective Ca(2+) ionophore. Exposure of Mac to increasing concentrations of IFNγ (5-100 ng/ml) or oxidized LDL (5-100 µg/ml) resulted in a dose-dependent increase in Nox5 protein expression and elevation in intracellular Ca(2+) concentration. Immunohistochemical staining revealed that Nox5 is present in CD68(+) Mac-rich area within human carotid artery atherosclerotic plaques. To the best of our knowledge, this is the first evidence that Nox5 is constitutively expressed in human Mon. Induction of Nox5 expression in IFNγ- and oxidized LDL-exposed Mac and the presence of Nox5 in Mac-rich atheroma are indicative of the implication of Nox5 in atherogenesis.

The involvement of the monocytes/macrophages in chronic inflammation associated with atherosclerosis.

Atherosclerosis is a progressive chronic disease of large and medium arteries, characterized by the formation of atherosclerotic plaques. Monocytes and macrophages are key factors in lesion development, participating to the processes that mediate the progression of the atherosclerotic plaque (lipid accumulation, secretion of pro-inflammatory and cytotoxic factors, extracellular matrix remodeling). The recruitment of the monocytes in the vascular wall represents a hallmark in the pathology of the atherosclerotic lesion. Monocyte adhesion and transmigration are dependent on the complementary adhesion molecules expressed on the endothelial surface, whose expression is modulated by chemical mediators. The atherosclerotic plaque is characterized by a heterogeneous population of macrophages reflecting the complexity and diversity of the micro-environment to which cells are exposed after entering the arterial wall. Within the atherosclerotic lesions, macrophages differentiate, proliferate and undergo apoptosis. Taking into account that their behavior has a direct and critical influence on all lesional stages, the development of therapeutic approaches to target monocytes/macrophages in the atherosclerotic plaque became a focal interest point for researchers in the field.