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Valve disease and particularly calcific aortic valve disease (CAVD) and diabetes (DM) are progressive diseases constituting a global health burden for all aging societies (Progress in Cardiovascular Diseases. 2014;56(6):565: Circulation Research. 2021;128(9):1344). Compared to non‐diabetic individuals (The Lancet. 2008;371(9626):1800: The American Journal of Cardiology. 1983;51(3):403: Journal of the American College of Cardiology. 2017;69(12):1523), the diabetic patients have a significantly greater propensity for cardiovascular disorders and faster degeneration of implanted bioprosthetic aortic valves. Previously, using an original experimental model, the diabetic‐hyperlipemic hamsters, we have shown that the earliest alterations induced by these conditions occur at the level of the aortic valves and, with time these changes lead to calcifications and CAVD. However, there are no pharmacological treatments available to reverse or retard the progression of aortic valve disease in diabetes, despite the significant advances in the field. Therefore, it is critical to uncover the mechanisms of valve disease progression, find biomarkers for diagnosis and new targets for therapies. This review aims at presenting an update on the basic research in CAVD in the context of diabetes. We provide an insight into the accumulated data including our results on diabetes‐induced progressive cell and molecular alterations in the aortic valve, new potential biomarkers to assess the evolution and therapy of the disease, advancement in targeted nanotherapies, tissue engineering and the potential use of circulating endothelial progenitor cells in CAVD.

Hepatic cancer is one of the most widespread maladies worldwide that requires urgent therapies and thus reliable means for testing anti-cancer drugs. The switch from two-dimensional (2D) to three-dimensional (3D) cell cultures produced an improvement in the in vitro outcomes for testing anti-cancer drugs. We aimed to develop a novel hyaluronic acid (HA)-based 3D cell model of human hepatocellular carcinoma (HepG2 cells) for drug testing and to assess comparatively in 3D vs. 2D, the cytotoxicity and the apoptotic response to the anti-tumor agent, cisplatin. The 3D model was developed by seeding HepG2 cells in a HA/poly(methylvinylether-alt-maleic acid) (HA3P50)-based scaffold. Compared to 2D, the cells grown in the HA3P50 scaffold proliferate into larger-cellular aggregates that exhibit liver-like functions by controlling the release of hepatocyte-specific biomarkers (albumin, urea, bile acids, transaminases) and the synthesis of cytochrome-P450 (CYP)7A1 enzyme. Also, growing the cells in the scaffold sensitize the hepatocytes to the anti-tumor effect of cisplatin, by a mechanism involving the activation of ERK/p38α-MAPK and dysregulation of NF-kB/STAT3/Bcl-2 pathways. In conclusion, the newly developed HA-based 3D model is suitable for chemotherapeutic drug testing on hepatocellular carcinoma. Moreover, the system can be adapted and employed as experimental platform functioning as a proper tissue/tumor surrogate.

Calcific aortic valve disease (CAVD)—the most common valvular heart disease—is accelerated in diabetes and has no pharmacotherapy. Although it is known that early CAVD is associated with inflammation and osteogenesis, the molecular mechanisms involved in diabetes‐associated CAVD still need to be uncovered. In this context, we have developed a 3D construct based on gelatin populated with human valvular endothelial cells (VEC) and valvular interstitial cells (VIC) and evaluated the effect of high glucose (HG) concentration on osteogenic molecules expression and on calcification mechanisms. First, we characterized the 3D model and assessed VIC remodelling properties at different time‐points. Then, we exposed it to normal glucose (NG) or high glucose (HG) for 7, 14 and 21 days after which the cells were isolated, separated and investigated individually. Our results showed that encapsulated VIC actively remodel the hydrogel, as demonstrated by an increased expression of extracellular matrix (ECM) proteins and matrix metalloproteinases (MMPs). Moreover, exposure of the construct to HG triggered bone morphogenetic protein (BMP) and TGF‐β signalling pathways, up‐regulating expression of osteogenic molecules—BMP‐2/‐4, osteocalcin, osteopontin, SMADs and Runt‐related transcription factor (Runx‐2)—and increased calcium deposits in an osteogenic environment. These findings underline the potential of the developed 3D model as a suitable system to investigate the mechanisms of human CAVD and may help to better understand the calcification mechanisms in CAVD associated to diabetes.

Calcific aortic valve disease (CAVD) is a progressive inflammatory disorder characterized by extracellular matrix remodeling and valvular interstitial cells (VIC) osteodifferentiation leading to valve leaflets calcification and impairment movement. Runx2, the master transcription factor involved in VIC osteodifferentiation, modulates the expression of other osteogenic molecules. Previously, we have demonstrated that the osteoblastic phenotypic shift of cultured VIC is impeded by Runx2 silencing using fullerene (C60)-polyethyleneimine (PEI)/short hairpin (sh)RNA-Runx2 (shRunx2) polyplexes. Since the use of polyplexes for in vivo delivery is limited by their instability in the plasma and the non-specific tissue interactions, we designed and obtained targeted, lipid-enveloped polyplexes (lipopolyplexes) suitable for (1) systemic administration and (2) targeted delivery of shRunx2 to osteoblast-differentiated VIC (oVIC). Vascular cell adhesion molecule (VCAM)-1 expressed on the surface of oVIC was used as a target, and a peptide with high affinity for VCAM-1 was coupled to the surface of lipopolyplexes encapsulating C60-PEI/shRunx2 (V-LPP/shRunx2). We report here that V-LPP/shRunx2 lipopolyplexes are cyto- and hemo-compatible and specifically taken up by oVIC. These lipopolyplexes are functional as they downregulate the Runx2 gene and protein expression, and their uptake leads to a significant decrease in the expression of osteogenic molecules (OSP, BSP, BMP-2). These results identify V-LPP/shRunx2 as a new, appropriately directed vehicle that could be instrumental in developing novel strategies for blocking the progression of CAVD using a targeted nanomedicine approach.

Parathyroid hormone (PTH) is a key regulator of calcium, phosphate and vitamin D metabolism. Although it has been reported that aortic valve calcification was positively associated with PTH, the pathophysiological mechanisms and the direct effects of PTH on human valvular cells remain unclear. Here we investigated if PTH induces human valvular endothelial cells (VEC) dysfunction that in turn could impact the switch of valvular interstitial cells (VIC) to an osteoblastic phenotype. Human VEC exposed to PTH were analyzed by qPCR, western blot, Seahorse, ELISA and immunofluorescence. Our results showed that exposure of VEC to PTH affects VEC metabolism and functions, modifications that were accompanied by the activation of p38MAPK and ERK1/2 signaling pathways and by an increased expression of osteogenic molecules (BMP-2, BSP, osteocalcin and Runx2). The impact of dysfunctional VEC on VIC was investigated by exposure of VIC to VEC secretome, and the results showed that VIC upregulate molecules associated with osteogenesis (BMP-2/4, osteocalcin and TGF-β1) and downregulate collagen I and III. In summary, our data show that PTH induces VEC dysfunction, which further stimulates VIC to differentiate into a pro-osteogenic pathological phenotype related to the calcification process. These findings shed light on the mechanisms by which PTH participates in valve calcification pathology and suggests that PTH and the treatment of hyperparathyroidism represent a therapeutic strategy to reduce valvular calcification.

Patients with diabetes mellitus have an increased risk of myocardial infarction and coronary artery disease‐related death, exhibiting highly vulnerable plaques. Many studies have highlighted the major role of macrophages (MAC) and smooth muscle cells (SMC) and the essential part of metalloproteases (MMPs) in atherosclerotic plaque vulnerability. We hypothesize that in diabetes, the interplay between MAC and SMC in high glucose conditions may modify the expression of MMPs involved in plaque vulnerability. The SMC‐MAC cross‐talk was achieved using trans‐well chambers, where human SMC were grown at the bottom and human MAC in the upper chamber in normal (NG) or high (HG) glucose concentration. After cross‐talk, the conditioned media and cells were isolated and investigated for the expression of MMPs, MCP‐1 and signalling molecules. We found that upon cross‐talk with MAC in HG, SMC exhibit: (i) augmented expression of MMP‐1 and MMP‐9; (ii) significant increase in the enzymatic activity of MMP‐9; (iii) higher levels of soluble MCP‐1 chemokine which is functionally active and involved in MMPs up‐regulation; (iv) activated PKCα signalling pathway which, together with NF‐kB are responsible for MMP‐1 and MMP‐9 up‐regulation, and (v) impaired function of collagen assembly. Taken together, our data indicate that MCP‐1 released by cell cross‐talk in diabetic conditions binds to CCR2 and triggers MMP‐1 and MMP‐9 over‐expression and activity, features that could explain the high vulnerability of atherosclerotic plaque found at diabetic patients.

Exploitation of nanotechnology advances in the cardiovascular medicine has led to new approaches for the diagnosis and therapy of vascular inflammation associated to cardiovascular diseases. Innovative nanoparticles targeted to the main players in the arterial wall inflammation in atherosclerosis (i.e. inflammatory molecules, immune or resident cells) have been already developed to diagnose and/or reduce excessive vascular inflammation and is eagerly awaited that promising preclinical and early clinical results to boost the approval of the first nanocarrier in this field. This review discusses the challenges and the steps undertaken so far to produce powerful multifunctional nanoparticles capable of raising new scenarios for future precision medicine in atherosclerosis.

Coronary atherosclerosis complicated by plaque disruption and thrombosis is a critical event in myocardial infarction and stroke, the major causes of cardiovascular death. In atherogenesis, macrophages (MAC) and smooth muscle cells (SMC) are key actors; they synthesize matrix components and numerous factors involved in the process. Here, we design experiments to investigate whether SMC-MAC communication induces changes in ECM protein composition and/or neo-angiogenesis. Cell to cell communication was achieved using trans-well chambers, where SMCs were grown in the upper chamber and differentiated MAC in the bottom chamber for 24 or 72h. We found that cross-talk between MAC and SMC during co-culture: (i) significantly decreased the expression of ECM proteins (collagen I, III, elastin) in SMC; (ii) increased the expression and activity of metalloprotease MMP-9 and expression of collagenase MMP-1, in both MAC and SMC; (iii) augmented the secretion of soluble VEGF in the conditioned media of cell co-culture and VEGF gene expression in both cell types, compared with control cells. Moreover, the conditioned media collected from MAC-SMC co-culture promoted endothelial cell tube formation in Matrigel, signifying an increased angiogenic effect. In addition, the MAC-SMC communication led to an increase in inflammatory IL-1β and TLR-2, which could be responsible for cellular signaling. In conclusion, MAC-SMC communication affects factors and molecules that could alter ECM composition and neo-angiogenesis, features that could directly dictate the progression of atheroma towards the vulnerable plaque. Targeting the MAC-SMC cross-talk may represent a novel therapeutic strategy to slow-down or retard the plaque progression.

Objectives To prepare and characterize in vitro and in vivo lipid nanoemulsions (LN) loaded with curcumin (Cm) and functionalized with a cell-penetrating peptide. Methods Curcumin-loaded lipid nanoemulsions (CmLN) functionalized with a nona-arginine peptide (R9-CmLN) have been obtained, characterized and optimized for size, entrapment efficiency and in vitro Cm release. The interaction of R9-CmLN with human endothelial cells (HEC) was investigated using cultured EA.hy926 cells, and in vivo biodistribution studies were performed using C57BL6 mice. Key findings When used in therapeutically relevant concentration, R9-CmLN have low haemolytic activity, low cytotoxicity on HEC, and show anti-inflammatory effects by reducing the monocytes adhesion to TNF-α activated HEC. Moreover, HEC uptake and internalization of R9-CmLN was significantly higher compared to the non-functionalized CmLN. In vivo biodistribution studies in mice revealed a higher accumulation of R9-CmLN in the liver and the lungs compared to CmLN and the body clearance of the both nanoformulations after 72 h. Conclusions Cell-penetrating peptides-functionalized CmLN have superior characteristics compared to their non-functionalized counterparts: are more efficiently internalized by the cells, produces anti-inflammatory effects in HEC and when administrated intravenously in mice exhibit increased accumulation in the liver and the lungs, suggesting their potential therapeutic applications in different inflammatory pathologies localized in the liver or the lungs.

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 has emerged as a significant local and systemic regulatory cytokine involved in inflammation. In diabetic patients, the serum resistin level is increased, monocytes/macrophages being an important source of resistin production. We therefore hypothesize that high glucose concentrations (HG) regulate resistin expression in human monocytes. Our aim has been to uncover the potential signalling pathways involved in this process. We have also questioned whether insulin has an effect on the regulation of resistin expression induced by HG. Human monocytes (U937 cell line) were exposed to 25 mM glucose for 24 h and then resistin gene expression and protein levels were determined by reverse transcription with the polymerase chain reaction and Western blot assays. We found that (1) the gene expression and protein level of resistin were up-regulated by HG; (2) the inhibitors of the mitogen-activated protein kinases (MAPKs) p38 (SB203580), extracellular signal-regulated kinases 1/2 (ERK1/2; PD98059) and c-Jun N-terminal kinase (SP600125) and of the transcription factor nuclear factor kappa-B (PDTC) inhibited HG-induced resistin protein production and (3) insulin reduced HG-induced resistin expression via a mechanism independent of phosphatidylinositol 3-kinase (PI3K) or p38 and ERK1/2. Therefore, HG significantly increases resistin gene expression and protein production in the U937 cell line by mechanisms involving MAPKs and the transcription factor NF-kB, whereas insulin reduces its expression. This study adds new data concerning the molecular mechanisms involved in the pro-inflammatory effects of HG on human monocytes.

Resistin and high glucose (HG) are concomitantly present at elevated concentration in diabetic's 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.

In atherosclerotic plaques, macrophages (MAC) and smooth muscle cells (SMC) frequently reside in close proximity and resistin (Rs) and fractalkine (Fk) are present at increased levels, resistin being associated with CD68 macrophages and fractalkine predominantly associated with intimal SMC; however, their role in this location is not clear, yet. The objective of this study was to determine whether the cross-talk between MAC-SMC induces changes in MAC cytokine phenotype and if Fk and Rs have a role in the process. To this purpose, macrophages (THP-1 monocytes differentiated with phorbol myristate acetate) were interacted with SMC cultured on the membrane inserts in the presence or absence of Rs or Fk. After 24h, MAC were removed from the co-culture and the gene and protein expression of 57 cytokines was assessed by QPCR and Proteome Profiler™ Array. Fk secreted in the culture medium following MAC-SMC interaction was determined (ELISA assay) and the role of Fk in MAC cytokine gene expression was assessed by silencing the Fk receptor in both cell types. The results showed that subsequent to the interaction with SMC, MAC exhibit: (1) a general increased expression of chemokines (the highest fold increase: VCC-1 and GRO-α) and of some interleukins, such as interleukins IL-5 (∼8-fold) and IL-6; (2) an increased Fk expression that in turn induces expression of: CXCL17, CCL19, CCL2, CXCL10, CXCL12, CXCL4, CXCL7, CCL4, CCL18, CXCL16, CXCL1 and IL-27; (3) in the presence of Rs, a predominant increased expression of interleukins (the highest fold increase: IL-6, IL-27, IL-23 and IL-5) and an augmented expression of some chemokines such as MIP-1β, GRO-α and CCL1. In addition, the secretome collected from the SMC-MAC co-culture increased human monocytes chemotaxis. DAVID analysis of the data revealed that the switch of MAC to a pro-inflammatory phenotype, prime the cells to intervene in the immune response, chemotaxis and inflammatory response. In conclusion, MAC cytokines expression is considerable augmented upon their interaction with SMC and Fk and Rs have distinct immunomodulatory roles: Fk predominantly increases the pro-angiogenic and inflammatory chemokines expression and Rs mostly the pro-inflammatory interleukins with consequences on monocyte chemotaxis. The novel data could help to develop targeted nanotherapies to reduce leukocyte chemotaxis and the ensuing inflammatory process associated with atherosclerosis.

Activated endothelial cells express monocyte chemoattractant protein-1 (MCP-1), a chemokine which is reportedly involved in the recruitment of plasma monocytes in the early stages of atherosclerosis. Since accelerated atherosclerosis is the main complication of diabetes and both diseases encompass an inflammatory reaction, we hypothesized that the anti-inflammatory drugs, aspirin and peroxisome proliferator-activated receptor (PPAR-alpha) activators (fenofibrate and clofibrate), could have an effect on the high glucose-induced MCP-1 expression in endothelial cells. To test this assumption, as well as the possible mechanisms involved, the MCP-1 expression and secretion, the reactive oxygen species levels, nuclear factor-kB (NF-kB) and activator protein-1 (AP-1) expression were determined in human endothelial cells exposed to high glucose concentrations in the presence of aspirin, fenofibrate and clofibrate. Human endothelial cells kept in normal glucose concentration in the absence of drugs were used as control. The results showed that (i) aspirin, fenofibrate and clofibrate decrease significantly the MCP-1 expression and secretion in human endothelial cells; (ii) the high glucose up-regulated expression of MCP-1 in endothelial cells was significantly reduced by inhibitors of NF-kB and reactive oxygen species; (iii) all drugs notably decrease the level of the reactive oxygen species and activation of NF-kB and AP-1. Together, the findings indicate that in endothelial cells aspirin and PPAR-alpha activators reduce the high glucose-increased expression of MCP-1 by a mechanism that includes the inhibition of reactive oxygen species, and decrease of AP-1 and NF-kB activation.

Aspirin's pharmacological action is mainly related to its property to inhibit prostaglandin synthesis; apart from this, aspirin has some beneficial side effects that are not completely understood, yet. Since aspirin possesses antioxidant properties and antioxidants prevent high d-glucose enhanced endothelial [Ca(2+)](i), we questioned whether aspirin also has an effect on this process as well as on high-glucose-impaired nitric oxide (NO) production. For these purposes, human endothelial cells (HECs) were cultured in normal concentration (5 mM) glucose (NG) or high concentration (33 mM) glucose (HG) and after confluence, exposed for 48 h to HG in the absence or presence of 1 mM aspirin. Then, the [Ca(2+)](i) was measured fluorimetrically using fura-2, NO production was determined by Griess reaction, superoxide anions (O(2)) was evaluated by ferricytochrome c reduction, the intracellular reactive oxygen species (ROS) were evaluated by fluorimetry, and the levels of protein kinase C (PKC) by Western blot. The results showed that HECs exposed to HG displayed: (i) increased [Ca(2+)](i); (ii) enhanced O(2) release; (iii) augmented level of intracellular ROS; and (iv) PKC translocation to the membrane fraction. By comparison, exposure to cells grown in HG to 1 mM aspirin resulted in: (i) a reduction of histamine stimulated [Ca(2+)](i) release to control level and of [Ca(2+)](i) entry by 30%; (ii) a twofold increase in NO production; (iii) a decrease of O(2)(-) accumulation in both culture medium and cell homogenate (by 60.4% and 70%, respectively); (iv) a decline of ROS to the control levels; and (v) a reduction of PKC translocation to the control levels. These data indicate that aspirin corrects the high-glucose-induced changes in cellular Ca(2+) homeostasis and NO production, via a mechanism involving the reduction of the O(2)(-) levels possible by acting on PKC-induced NADPH activity.

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.

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.

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.

In the atherosclerotic plaque, smooth muscle cells (SMC) acquire an inflammatory phenotype. Resistin and fractalkine (CX3CL1) are found in human atheroma and not in normal arteries. CX3CL1 and CX3CR1 are predominately associated with SMC. We have questioned whether resistin has a role in the expression of CX3CL1 and CX3CR1 in SMC thus contributing to the pro-inflammatory status of these cells. Cultured human aortic SMC were stimulated with 100 ng/ml resistin for 4, 6, 12, and 24 h, and then CX3CL1 and CX3CR1 expression was assessed by quantitative reverse transcription with the polymerase chain reaction and Western blot. We found that resistin up-regulated CX3CL1 and CX3CR1 in SMC and induced the phosphorylation of p38MAPK and STAT3. Inhibitors of p38MAPK, JAK-STAT, NF-kB, and AP-1 significantly reduced CX3CL1 and CX3CR1 expression. Knockdown of STAT1 and STAT3 with decoy oligodeoxinucleotides and the silencing of p65 and cjun with short interfering RNA decreased CX3CL1 and CX3CR1 expression. Anti-TLR4 antibody and pertussis toxin also reduced CX3CL1 and CX3CR1 protein expression. xCELLigence experiments revealed that resistin probably uses Gi-proteins for its effect on SMC. The CX3CL1 induced by resistin exhibited a chemotactic effect on monocyte transmigration. Thus, (1) resistin contributes to the pro-inflammatory state of SMC by the up-regulation of CX3CL1 and CX3CR1 expression via a mechanism involving NF-kB, AP-1, and STAT1/3 transcription factors, (2) resistin employs TLR4 and Gi-protein signaling for its effect on SMC, (3) CX3CL1 induced by resistin is functional in monocyte chemotaxis. The data reveal new mechanisms by which resistin promotes the inflammatory phenotype of SMC.

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.

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.

SummaryThe major complication of diabetes mellitus is accelerated atherosclerosis that entails an inflammatory process, in which fractalkine and monocyte chemotactic protein-1 (MCP-1) play a key role.We investigated the effect of diabetes-associated high glucose (HG) on these chemokines and signalling mechanisms involved in human aortic smooth muscle cells (SMC). Exposure of SMC to HG resulted in an increase of fractalkine and MCP-1 expression and the activated mitogen-activated protein kinase (MAPK) signalling pathway, a process associated with elevated oxidative stress.Transfection with decoy oligodeoxynucleotides identified the involvement of transcription factors activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB) in the observed up-regulation of chemokines. The MAPK inhibitors blocked the phosphorylation of IkBα and c-jun, indicating the role of MAPK in NF-κB and AP-1 activation in SMC under HG conditions.The up-regulation of MCP-1 and fractalkine was associated with increased adhesive interactions between HG-exposed SMC and monocytes. Treatment of HG-exposed SMC with peroxisome proliferator-activated receptors α (PPAR α ) activators (fenofibrate and clofibrate) resulted in a reduction of mRNA and protein expression of MCP-1 and fractalkine.In conclusion, HG upregulates the expression of fractalkine and MCP-1 in SMC leading to increased monocyte-SMC adhesive interactions by a mechanism involving activation of MAPK, activator protein-1 (AP-1) and NF-κB. The increased expression of these two pro-inflammatory chemokines and the ensuing increased adhesion between SMC and monocytes may trigger the inflammatory process associated with further vascular complications of diabetes.

There are data that document the anti-inflammatory effect of enoxaparin (EP) and its possible antioxidant potential. This study was designed to search for the antioxidant mechanism(s) of EP directly on endothelial cells exposed to an oxidant stimulus. For this purpose cultured human endothelial cells were exposed to nontoxic concentrations of hydrogen peroxide in the presence or absence of EP, and the adhesion of monocytes, the expression of cell adhesion molecules and transcription factors possibly involved in the process were tested. Adhesion assays, ELISA and Western blot analysis revealed that EP reduced monocyte adhesion, ICAM-1 and P-selectin expression, decreased the nuclear levels of c-Jun and p65 proteins, and diminished the phosphorylation of c-Jun protein, MAPK p38 and JNK. Together, the data demonstrate the antioxidant effect of EP and the involvement of ICAM-1, P-selectin, MAPK p38, JNK and the transcription factors NF-kappaB and AP-1 in the mechanism of action of this drug.

Immunoliposomes can be potentially used as carriers for drug delivery to specific cells. The aim of this paper was to exploit the overexpression of vascular cell adhesion molecule-1 (VCAM-1) on activated human endothelial cells (HEC) for targeting of anti-VCAM-1 coupled liposomes with the intent for further use as drug carriers.TNF-alpha-activated HEC were exposed to liposomes, either plain or coupled with antibodies to VCAM-1 (L-VCAM-1) or to irrelevant IgG (L-IgG); nonactivated HEC subjected to the same conditions were used as control. For binding studies, the cells were incubated with fluorescently labeled liposomes at 4 degrees C, and after 2 h, fluorescence intensity was assessed by flow cytometry; specificity of binding was determined by performing the experiments in the presence of excess anti-VCAM-1. Cellular internalization of liposomes was studied employing radioactively or fluorescently labelled liposomes; to detect the mechanisms of uptake, experiments were performed in the presence of agents that interfere in the endocytotic pathway. Transmigration of liposomes was monitored in a two-chamber culture model. The effect of L-VCAM-1 binding to HEC on intracellular calcium ([Ca(2+)](i)) and distribution of actin was determined by fluorimetry and fluorescence microscopy.(1) L-VCAM-1 binds selectively and specifically to TNF-alpha activated HEC. (2) Approximately 50% of L-VCAM-1 is taken up by receptor-mediated endocytosis via clathrin-coated vesicles. (3) Binding of L-VCAM-1 to HEC surface induces a rise in [Ca(2+)](i) and reorganization of actin filaments. (4) A small percentage of liposomes migrates across HEC.The data indicate that VCAM-1 may be an appropriate target for specific drug delivery to activated HEC using immunoliposomes.

Clinical and experimental studies indicate that low-molecular-weight heparins reduce inflammation. To uncover the possible mechanisms involved, we investigated the effect of a low-molecular-weight heparin, enoxaparin, on high glucose-induced activation of endothelial cells. Bovine valvular endothelial cells and human endothelial cell line, EA hy926 were cultured in medium containing 5 mM (normal glucose) or 33 mM (high glucose) glucose. Postconfluency, the cells were exposed for 48 h to high glucose in the absence or presence of 16 microg/ml enoxaparin and tested for monocyte adhesion, expression of cell adhesion molecules, and translocation of nuclear transcription factor-kappaB (NF-kappaB), using adhesion assays, enzyme-linked immunosorbent assay (ELISA), and Western blotting. Statistical data revealed that treatment with enoxaparin resulted in a significant decrease in monocyte adhesion, expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, translocation of NF-kappaB, as well as in the level of intracellular reactive oxygen species. These results suggest that enoxaparin reduces the high glucose-induced activation of endothelial cells by inhibiting monocyte adhesion through a mechanism that involves cell adhesion molecules and NF-kappaB.

Enoxaparin is a low molecular weight heparin, widely accepted as anticoagulant or antithrombotic drug, and is likely to have a role in acute inflammation. To evaluate the anti-inflammatory potential of enoxaparin, we investigated the direct effect of the drug on the activation of endothelial cells. For this purpose we set up an in vitro system in which cultured valvular endothelial cells (VEC) activated by tumor necrosis factor alpha or lipopolysaccharide were exposed to a monocytic cell line; these conditions induced a significant adhesion of monocytes to VEC. Adhesion assays, ELISA, and flow cytometric analysis revealed that pretreatment with enoxaparin, at a relevant plasma concentration (16 µg/ml), acts upon activation of VEC by inhibition of lipopolysaccharide-induced E-selectin expression and tumor necrosis factor stimulated ICAM-1 expression, thus reducing monocyte adhesion to VEC. These results suggest a novel function of enoxaparin, namely to protect VEC from activation and inhibiting the expression of cell adhesion molecules.

Upon induction of experimental hyperglycemia (i.e. diabetes) pathological modifications are early detected (approximately 7 days) at the level of the cardiac valves leading rapidly to the development of valvular atheroma. Monocyte adhesion to the vascular endothelium is one of the initial event at the onset of atherosclerosis. We questioned whether high glucose enhances monocyte adhesion to the valvular endothelial cells (VEC) so as to explain, in part, the accelerated atheroma formation that occur in diabetic conditions. To this purpose we compared the adhesion of monocytes to VEC cultured in 5.5 mM (normal) glucose (NG) or in 33 mM (high) glucose (HG) or in high mannitol (HM) (27.5 mM mannitol plus 5.5 mM glucose), a concentration known to simulate the hyperosmolar effect of high glucose. After incubation for 30 min at 37 degrees C, the adhesion of monocyte cell line (U937 cells) to VEC was quantitated by a fluorimetric assay or by direct counting. Statistical data showed a significant increased adhesion of monocytes to VEC grown in HG (up to 4 fold) or in HM (up to 2.7) when compared to normal conditions. Using a battery of specific monoclonal antibodies molecules it was found that the increased adhesion of monocytes to VEC grown in high glucose was specifically inhibited (p < 0.05) by anti-ICAM-1, anti-VCAM-1 and anti-CD18 monoclonal antibodies. Together, the results indicate that high glucose induces enhanced monocyte adhesion to VEC via a mechanism involving in part an osmotic effect and mainly the cell adhesion molecules: ICAM-1, VCAM-1 and CD18.