Monica Pirvulescu

Cross talk between smooth muscle cells and monocytes/activated monocytes via CX3CL1/CX3CR1 axis augments expression of pro-atherogenic molecules.

OBJECTIVE In atherosclerotic lesions, fractalkine (CX3CL1) and its receptor (CX3CR1) expressed by smooth muscle cells (SMC) and monocytes/macrophages, mediate the heterotypic anchorage and chemotaxis of these cells. We questioned whether, during the close interaction of monocytes with SMC, the CX3CL1/CX3CR1 pair modulates the expression of pro-atherogenic molecules in these cells. METHODS AND RESULTS SMC were co-cultured with monocytes or LPS-activated monocytes (18h) and then the cells were separated and individually investigated for the gene and protein expression of TNFα, IL-1β, IL-6, CX3CR1 and metalloproteinases (MMP-2, MMP-9). We found that SMC-monocyte interaction induced, in each cell type, an increased mRNA and protein expression of TNFα, IL-1β, IL-6, CX3CR1, MMP-2 and MMP-9. Blocking the binding of fractalkine to CX3CR1 (by pre-incubation of monocytes with anti-CX3CR1 or by CX3CR1 siRNA transfection) before cell co-culture decreased the production of TNFα, CX3CR1 and MMP-9. Monocyte-SMC interaction induced the phosphorylation of p38MAPK and activation of AP-1 transcription factor. Silencing the p65 (NF-kB subunit) inhibited the IL-1β and IL-6 and silencing c-jun inhibited the TNFα, CX3CR1 and MMP-9 induced by SMC-monocyte interaction. CONCLUSIONS The cross-talk between SMC and monocytes augments the inflammatory response in both cell types as revealed by the increased expression of TNFα, IL-1β, IL-6, CX3CR1 and MMPs. Up-regulation of TNFα, CX3CR1 and MMP-9 is further increased upon interaction of SMC with activated monocytes and is dependent on fractalkine/CXRCR1 pair. These data imply that the fractalkine/CX3RCR1 axis may represent a therapeutic target to impede the inflammatory process associated with atherosclerosis.

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.

Resistin up-regulates fractalkine expression in human endothelial cells : Lack of additive effect with TNF-α

Resistin is a cytokine and fractalkine (Fk) a cell adhesion molecule and chemokine that contribute to human vascular inflammation by mechanisms not clearly defined. We questioned whether resistin induces Fk expression in human endothelial cells (HEC), compared the effect with that of the pro-inflammatory cytokine, TNF-alpha, and evaluated the consequences of co-stimulating HEC with both activators on Fk induction and on the signalling molecules involved. We found that resistin up-regulated Fk expression at comparable level to that of TNF-alpha by a mechanism involving P38 and JNK MAPK and NF-kappaB. Co-stimulation of cells with resistin and TNF-alpha did not increase Fk expression induced by every single inducer. Moreover resistin reduced the expression induced by TNF-alpha in HEC. The new data uncover Fk as a novel molecular link between resistin and inflammation and show that resistin and TNF-alpha have no additive effect in Fk up-regulation or on the signalling molecules implicated.

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.

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.

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

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.

Functional analysis of the fractalkine gene promoter in human aortic smooth muscle cells exposed to proinflammatory conditions.

Fractalkine (Fk) and its receptor CX3C receptor 1 contribute effectively to the atherosclerotic process, mediating the recruitment of leukocytes and promoting the interactions between monocytes/macrophages and smooth muscle cells (SMCs). As Fk expression is significantly increased in SMCs during atherogenesis, we aimed to uncover the detailed molecular mechanisms of transcriptional regulation of the Fk gene. For this purpose, we cloned and characterized the human Fk promoter, and studied the specific roles of different transcription factors in its regulation in human aortic SMCs activated by interferon‐γ. In silico analysis of the Fk promoter indicated the presence of binding sites for various inflammatory modulators, such as nuclear factor‐κB (NF‐κB), signal transducer and activator of transcription (STAT)1/STAT3, and activator protein‐1. Using a luciferase reporter plasmid, we identified a 2046‐bp region spanning the transcriptional start point of the Fk gene, which has strong constitutive promoter activity in SMCs. The effects of interferon‐γ on both Fk reporter activity and endogenous transcription were abolished by silencing NF‐κB, STAT1, and STAT3. Transient overexpression of p65/NF‐κB and STAT1/STAT3 increased Fk promoter activity, whereas c‐Jun/activator protein‐1 overexpression had no effect. The results obtained with chromatin immunoprecipitation assays revealed the existence of physical interactions of p65 and STAT1/STAT3 with the predicted elements of the Fk promoter. Moreover, Fk‐promoted monocyte chemotaxis was dependent on the janus kinase–STAT pathway. Investigation of the detailed molecular mechanisms by cloning and characterizing potential transcriptional response elements has identified the Fk regulatory mechanism in activated human SMCs.