Viorel Simion

Similar effects of resistin and high glucose on P-selectin and fractalkine expression and monocyte adhesion in human endothelial cells.

Resistin and high glucose (HG) are concomitantly present at elevated concentration in diabetics plasma; both are pro-inflammatory agents acting on vascular cells by mechanisms that are not fully understood. We questioned whether resistin and HG affect the expression of major adhesion molecules, P-selectin and fractalkine in human endothelial cells (HEC). The results showed that in HEC (i) resistin increased P-selectin expression; (ii) HG up-regulated Fk expression; (iii) P-selectin and fractalkine were functional increasing monocyte adhesion to activated cells. Co-stimulation with resistin and HG increased P-selectin and fractalkine mRNA and protein and induced monocyte adhesion, generated an increase in NADPH oxidase activity and of the intracellular reactive oxygen species and activated the NF-kB and AP-1 transcription factors at similar values as those of each activator. In conclusion in HEC, resistin and HG induce the up-regulation of P-selectin and fractalkine and the ensuing increased monocyte adhesion by a mechanism involving oxidative stress and NF-kB and AP-1 activation.

A novel pro-inflammatory mechanism of action of resistin in human endothelial cells: Up-regulation of SOCS3 expression through STAT3 activation

Resistin is a significant local and systemic regulatory cytokine involved in inflammation. Suppressors of cytokine signaling (SOCS) proteins are intracellular regulators of receptor signal transduction induced by several cytokines in a cytokine and cell specific manner. Resistin up-regulates SOCS3 expression in mice adipocytes but it is not known whether this is a common occurrence in other cells. We questioned whether resistin-induces SOCS3 in human endothelial cells and if signal transducer and activator of transcription (STAT) proteins are involved in the process. The Real-Time PCR and Western blot analysis showed that in resistin-activated HEC the gene and protein expression of SOCS3 were significantly increased. Furthermore, resistin induced activation of STAT3 as characterized by increased tyrosine phosphorylation. Resistin-induced SOCS3 expression was blocked by specific inhibitors of STAT3 signaling and by the transfection of siRNA specific for STAT3. Silencing of SOCS3 gene expression by transfection with SOCS3 siRNA reduced the expression of resistin induced-P-selectin and fractalkine in HEC. Together, our results demonstrate that in HEC (1) resistin up-regulates SOCS3 expression and activates STAT3 transcription factor; (2) the increase in SOCS3 mRNA and protein expression as well as STAT3 activation have a long-lasting effect (up to 18h); (3) inhibition of SOCS3 function prevents resistin-induced expression of cell adhesion molecules P-selectin and fractalkine and thus activation of endothelial cells. The data uncover a new resistin-mediated mechanism in human endothelial cells and designate SOCS3 as a novel therapeutic target to modulate resistin-dependent inflammation in vessel wall diseases.

Curcumin and a Morus alba Extract Reduce Pro‐Inflammatory Effects of Resistin in Human Endothelial Cells

Resistin is a cytokine which plays an important role in cardiovascular disease by influencing systemic inflammation and endothelial activation. In human endothelial cells (HEC) it increases the expression of P‐selectin and fractalkine, and enhances monocyte adhesion by antioxidant mechanisms. This study investigated whether the natural antioxidants curcumin (CC) and an extract of Morus alba leaves (MA) have protective effects in resistin‐activated HEC. HEC were exposed to 100 ng/mL resistin for 6 and 18 h in the absence or presence of MA or CC and the expression of fractalkine and P‐selectin was determined by RT‐PCR and western blot. Intracellular accumulation of reactive oxygen species (ROS) was monitored by fluorimetry and NADPH oxidase activity by a lucigenin‐enhanced chemiluminescence assay. In addition, adhesion assays using the monocytic U937 cells were performed. The results showed that treatment of HEC exposed to resistin with MA and CC: (1) inhibited significantly P‐selectin and fractalkine expression, (2) inhibited the increase in the intracellular ROS level, (3) reduced NADPH activation and (4) reduced monocytes adhesion to HEC. The results indicate that MA and curcumin target resistin‐induced human endothelial activation partly via antioxidant mechanisms and suggest that they may represent therapeutic agents in vascular disease mediated by resistin. Copyright

Subendothelial resistin enhances monocyte transmigration in a co-culture of human endothelial and smooth muscle cells by mechanisms involving fractalkine, MCP-1 and activation of TLR4 and Gi/o proteins signaling

The cytokine resistin and the chemokine fractalkine (FKN) were found at increased levels in human atherosclerotic plaque, in the subendothelium, but their role in this location still needs to be characterized. Recently, high local resistin in the arterial vessel wall was shown to contribute to an enhanced accumulation of macrophages by mechanisms that need to be clarified. Our recent data showed that resistin activated smooth muscle cells (SMC) by up-regulating FKN and MCP-1 expression and monocyte chemotaxis by activating toll-like receptor 4 (TLR4) and Gi/o proteins. Since in the vessel wall both endothelial cells (EC) and SMC respond to cytokines and promote atherosclerosis, we questioned whether subendothelial resistin (sR) has a role in vascular cells cross-talk leading to enhanced monocyte transmigration and we investigated the mechanisms involved. To this purpose we used an in vitro system of co-cultured SMC and EC activated by sR and we analyzed monocyte transmigration. Our results indicated that: (1) sR enhanced monocyte transmigration in EC/SMC system compared to EC cultured alone; (2) sR activated TLR4 and Gi/o signaling in EC/SMC system and induced the secretion of more FKN and MCP-1 compared to EC cultured alone and used both chemokines to specifically recruit monocytes by CX3CR1 and CCR2 receptors. Moreover, FKN produced by resistin in EC/SMC system, by acting on CX3CR1 on EC/SMC specifically contributes to MCP-1 secretion in the system and to the enhanced monocyte transmigration. Our study indicates new possible targets for therapy to reduce resistin-dependent enhanced macrophage infiltration in the atherosclerotic arterial wall.

Monocytes and smooth muscle cells cross-talk activates STAT3 and induces resistin and reactive oxygen species and production

During the early phase of atherosclerosis, monocytes attach to and migrate through the vessel wall where they activate and communicate with smooth muscle cells (SMC) affecting plaque progression by largely unknown mechanisms. Activation of STAT3 transcription factor is suggested to be critically involved in dedifferentiation, migration, and proliferation of SMC in the neointima formation after vascular injury. Monocytes‐SMC cross‐talk induces an inflammatory phenotype of the resident SMC, but the involvement of STAT3 in phenotype switching is not known. Resistin is a cytokine found in human atheroma associated to monocytes/macrophages with role in inflammation associated with cardiovascular disease. The aim of this study was to follow the effect of activated monocytes‐SMC cross‐talk on STAT3 activation and subsequent resistin and reactive oxygen species (ROS) production. Our results showed that the interaction of activated monocytes with SMC determines: (i) phosphorylation of STAT3 and reduction of SOCS3 expression in both cell types; (ii) intracellular ROS production dependent on NADPH oxidase (by increased Nox1 expression) and STAT3 activation in SMC; (iii) up‐regulation of resistin expression in monocytes dependent on STAT3 activation. Furthermore, exposure of SMC to resistin induces ROS by increasing NADPH oxidase activity and the p22phox and Nox1 expression. In conclusion, the cross‐talk between SMC and monocytes activates STAT3 transcription factor and lead to resistin up‐regulation in monocytes and ROS production in SMC. Moreover, resistin increases the ROS levels in SMC. These data indicate that monocyte‐SMC communication may represent an important factor for progression of the atherosclerotic lesion. J. Cell. Biochem. 114: 2273–2283, 2013.

Hybrid fullerene conjugates as vectors for DNA cell-delivery

The present study reports fullerene conjugates that act as efficient binders of double stranded DNA (dsDNA) into cytofriendly polyplexes. The conjugates are designed to generate dendrimeric structures, having C60 as the core and bearing linear or branched PEI and polyethyleneglycol (PEG) arms (∼2 kDa). Simple and reproducible synthesis pathways provided C60-PEI and C60-PEG-PEI conjugates. They were able to bind linear and plasmidic dsDNA and they form particulate polyplexes of 50 to 200 nm in diameter. The resulted polyplexes toggle between the anionic and cationic state at nitrogen to phosphorous ratios (N/P) of about 5, as revealed by their zeta potential and became colloidally stable at N/P ratios above 10, as determined by atomic force microscopy (AFM). They are electrophoretically unbreakable starting with N/P ratios of 3 and of 5 when salmon sperm DNA and pEYFP-C1 plasmid, respectively are loaded. Both C60-PEI·pEYFP and C60-PEG-PEI·pEYFP polyplexes are non-cytotoxic against HEK 293T cells in culture and exhibit transfection efficiency better than 25% (N/P ratios above 20) and 6% (N/P ratios above 60) respectively, measured by flow cytometry. For comparison, the commercial SuperFect® from Qiagen (positive control) was able to provide an efficiency of 15-20%, under similar conditions. Moreover, the C60-PEG-PEI conjugate is as performant as the positive control in terms of expression of EYFP reporter gene in cultured cells and exhibited high cytocompatibility, determining cell proliferation up to 200%. Our study proved that C60-PEG-PEI is effective vector for DNA delivery being, in addition, easily synthesizable, practically non-cytotoxic and as efficient the commercially available transfection tools.

Long noncoding Rnas in cardiovascular disease, diagnosis, and therapy

Purpose of review Long noncoding RNAs (lncRNAs) have emerged as powerful regulators of nearly all biological processes. Their cell-type and tissue-specific expression in health and disease provides new avenues for diagnosis and therapy. This review highlights the role of lncRNAs that are involved in cardiovascular disease (CVD) with a special focus on cell types involved in cardiac injury and remodeling, vascular injury, angiogenesis, inflammation, and lipid metabolism. Recent findings Almost 98% of the genome does not encode for proteins. LncRNAs are among the most abundant type of RNA in the noncoding genome. Accumulating studies have uncovered novel lncRNA-mediated regulation of CVD-associated genes, signaling pathways, and pathophysiological responses. Targeting lncRNAs in vivo using short antisense oligonucleotides or by gene editing has provided important insights into disease pathogenesis through epigenetic, transcriptional, or translational mechanisms. Although cross-species conservation still remains a major obstacle, there is increasing appreciation that altered expression of lncRNAs associates with stage-specific CVD and in human patient cohorts, providing new opportunities for diagnosis and therapy. Summary A better understanding of lncRNAs will not only fundamentally improve our understanding of key signaling pathways in CVD, but also aid in the development of effective new therapies and RNA-based biomarkers.

VCAM-1 directed target-sensitive liposomes carrying CCR2 antagonists bind to activated endothelium and reduce adhesion and transmigration of monocytes

Chemokines are critically involved in the development of chronic inflammatory-associated diseases such as atherosclerosis. We hypothesized that targeted delivery of compounds to the surface of activated endothelial cells (EC) interferes with chemokine/receptor interaction and thereby efficiently blocks inflammation. We developed PEGylated target-sensitive liposomes (TSL) encapsulating a CCR2 antagonist (Teijin compound 1) coupled with a specific peptide recognized by endothelial VCAM-1 (Vp-TSL-Tj). TSL were characterized for size (by dynamic light scattering), the amount of peptide coupled at the liposomal surface and Teijin release (by HPLC). We report that Vp-TSL-Tj binds specifically to activated EC in vitro and in situ, release the entrapped Teijin and prevent the transmigration of monocytes through activated EC. This is the first evidence that nanocarriers which transport and release chemokine inhibitors at specific pathological sites can reduce chemokine-dependent inflammatory processes.

Conjugation of curcumin-loaded lipid nanoemulsions with cell-penetrating peptides increases their cellular uptake and enhances the anti-inflammatory effects in endothelial cells.

To prepare and characterize in vitro and in vivo lipid nanoemulsions (LN) loaded with curcumin (Cm) and functionalized with a cell‐penetrating peptide.

Stem Cell Regenerative Potential Combined with Nanotechnology and Tissue Engineering for Myocardial Regeneration

The stem cell-based therapy for post-infarction myocardial regeneration has been introduced more than a decade ago, but the functional improvement obtained is limited due to the poor retention and short survival rate of transplanted cells into the damaged myocardium. More recently, the emerging nanotechnology concepts for advanced diagnostics and therapy provide promising opportunities of using stem cells for myocardial regeneration. In this paper will be provided an overview of the use of nanotechnology approaches in stem cell research for: 1) cell labeling to track the distribution of stem cells after transplantation, 2) nanoparticle-mediated gene delivery to stem cells to promote their homing, engraftment, survival and differentiation in the ischemic myocardium and 3) obtaining of bio-inspired materials to provide suitable myocardial scaffolds for delivery of stem cells or stem cell-derived factors.