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Background: Endocan is a soluble dermatan sulfate PG (50kDa), expressed and secreted by endothelial cells of the dermal microvasculature, the coronary, the pulmonary arteries and the capillaries from adipose tissue. It plays an important role in the pathogenesis of vascular disorders, inflammation, endothelium dysfunction and neoangiogenesis. The biosynthesis of endocan is upregulated by inflammatory cytokines like TNF-α, IL-1, TGF-β1 and by proangiogenic and growth factors like VEGF, EGF and FGF-2; conversely, it is downregulated by INF-γ and insulin level, but this event in obese patients is attenuated by insulin resistance. Methods: The study included 19 patients with obesity (10 males and 9 females), aged between 2-18 years, screened at the pediatric Endocrinology Clinic of our Department in Messina (Italy). Results: The results, compared with the healthy controlled group, have shown upraised endocan serum levels in children with BMI higher than normal (30,044 ±0,984), demonstrating a positive correlation trend between endocan and BMI (rho 0.356, p = 0.13). Furthermore endocan serum levels were significantly correlated to the insulin levels (rho 0.474, p = 0.047) and HOMA-IR (rho 0.434, p = 0.072). These findings confirm the correlation between endothelial damage and insulin resistance in obese children. Instead we did not found any association between Endocan and lipid serum levels, nor with the fasting blood glucose value (rho 0.024, p = 0.926). An elevated correlation was also revealed with steatosis, and with the ultrasound grade of liver steatosis (rho 0.514, p = 0.087 and rho 0.512, p = 0.088, respectively), despite not raising statistically significant results, given the small sample size, indeed only 12 children of the study underwent ultrasound. Conclusions: We can assume that endocan could be used also in children with obesity as a biomarker and that could be a valid predictor in order to stratify the future cardiovascular risk in adulthood. Retrospectively registered

Endocan is a circulating proteoglycan, involved in immunity, inflammation, and endothelial function. It has been recently suggested as a biomarker of inflammation, increased angiogenesis, and cancer. In vitro studies have shown that endocan expression could be upregulated by inflammatory cytokines and proangiogenic molecules. High endocan levels were also shown in arthritic joint tissues and particularly in sites characterized by severe inflammation. This study was performed to evaluate endocan expression in chondrocytes stimulated with IL-ß. mRNA and related protein production were measured for endocan, TNF-α, and IL-6. NF-kB activity was also evaluated. IL-1ß treatment induced a significant upregulation of both endocan and the inflammatory parameters as well as NF-kB activity. The treatment of chondrocytes with the specific NF-kB inhibitor before IL-1ß stimulation was able to reduce endocan and the inflammatory markers over-expression. The results of our study indicated that endocan is also expressed in human chondrocytes; furthermore, consistent with previous results in other cell types and tissues, IL-1ß-induced inflammatory response involves the expression of endocan through NF-kB activation. In this context, endocan seems to be an important inflammatory marker associated with the activation of NF-kB pathway.

miR profile could be associated to CV risk, and also to prognosis/outcome in response to therapeutic approach. We aimed to evaluate if anti-hypertensive drugs enalapril, losartan or olmesartan have effects on monocyte miR profile in essential hypertensives without target organ involvement. For this purpose, 82 hypertensives and 49 controls were included, we evaluated SBP/DBP, lipid profile, glucose, CRP, fibrinogen, arterial stiffness indices (PWV, AIx), and cIMT at baseline (T0) and after 24 weeks of treatment (T1). Subjects with LDL-C ≥ 160 mg/dL, TG ≥ 200 mg/dL, BMI ≥ 30, and other additional CV risk factors were excluded. Patients who were prescribed to receive once-a-day enalapril 20 mg, losartan 100 mg or olmesartan 20 mg were eligible for the study. At T1, we found a significant improvement of SBP (−18.5%), DBP (−18%), HDL-C and LDL-C (+3% and −5.42%), glucose (−2.15%), BMI (−3.23%), fibrinogen (−11%), CRP (−17.5%,), AIx (−49.1%) PWV (−32.2%), and monocyte miR expression (miR-221: −28.4%, miR-222: −36%, miR-145: +41.7%) with respect to baseline. miR profile was compared to control subjects at baseline and at T1. We found some little difference in the behaviour of the three treatments on some variables: olmesartan was the most effective in reducing fibrinogen, DBP, CRP, and AIx (−13.1%, −19.3%, −21.4%, and −56.8%, respectively). Enalapril was the drug more significantly increasing the expression of miR-145. In conclusion, enalapril, losartan and olmesartan are effective in improving mechanical and humoral factors associated to AS and atherogenesis. These drugs appear to be able to modify miRs 221/222 and miR-145 expression in drug-naïve hypertensives, making it closer to that of control subjects, additionally, this provides a good blood pressure compensation, contributing to slow the progression of vascular damage.

The present study aimed to investigate the expression of miR9 and its correlation with cytokines, proteolytic enzymes and apoptosis in an experimental model of 6-mer HA induced inflammation in human chondrocytes. Human articular chondrocytes, transfected with a miR-9 mimic and miR-9 inhibitor, were stimulated with 6-mer HA in presence/absence of a specific NF-kB inhibitor. 6-mer HA induced a significant increase of TLR-4, CD44, IL-8, IL-18, MMP-9, ADAMTS-5, BAX and BCL-2 mRNAs expression and the related proteins, as well as NF-kB activation, associated with a significant up regulation of miR-9. In chondrocytes transfected with the miR-9 mimic before 6-mer HA treatment we found a decrease of such inflammatory cytokines, metalloproteases and pro-apoptotic molecules, while we found them increased in chondrocytes transfected with the miR9 inhibitor before 6-mer HA stimulation. The activities of TLR-4 and CD44, up regulated by 6-mer HA, were not modified by miR9 mimic/inhibitor, while the NF-kB activation was significantly affected. We suggested that the up regulation of miR9, induced by 6-mer HA, could be a cellular attempt to limit cell damage during inflammation.

BACKGROUND AND AIMS Neutrophil-to-lymphocyte ratio (NLR) is a novel inflammatory biomarker strongly associated with atherosclerotic cardiovascular disease (ASCVD). Our aim was to evaluate the role of NLR on pulse wave velocity (PWV) after adding-on proprotein convertase subtilisin/kexin type 9 inhibitor (PCSK9-i) in familial hypercholesterolemia (FH) subjects with ASCVD. METHODS AND RESULTS In this prospective observational study, we evaluated 45 FH subjects with ASCVD on high-intensity statins plus ezetimibe and with an off-target LDL-C. Study population was divided into two groups according to the mean value of NLR. All patients received PCSK9-i therapy and obtained biochemical analysis as well as PWV evaluation at baseline and after six months of PCSK9-i. After six months of add-on PCSK9-i therapy, a significant reduction of TC, LDL-C, Non-HDL-C, Lp(a) and ApoB plasma levels was observed in the two groups; while low-NLR group exhibited a significant PWV reduction after six-month therapy with PCSK9-i (Δ -16.2%, p

Autoimmune thyroid diseases, such as Hashimoto's thyroiditis, are characterized by lymphocytic infiltration and altered function of the thyroid. During inflammation, it has been reported a decreased expression in Tg and NIS, accompanied by an increase in HA production that accumulates in the gland. HA fragments produced in different pathological states can modulate gene expression in a variety of cell types and may prime inflammatory response by interacting with the TLR-2, TLR-4 and CD44 that, in turn, induce NF-kB activation finally responsible of inflammatory mediator transcription, such as IL-1β, TNF-α and IL-6. The aim of this study was to investigate the potential inflammatory effect and the biochemical pathways activated by 6-mer HA oligosaccharides in cultured human thyrocytes. 6-mer HA treatment induced up-regulation of TLR-2, TLR-4, CD44 mRNA and related protein levels, increased HA production and NF-kB activation, that in turn increased IL-1β and IL-6 concentrations. Instead, we found evidence of an opposite effect on thyroid specific-gene Tg and NIS, that were decreased after 6-mer HA addition. Thyrocytes exposition to specific blocking antibodies for TLR-2, TLR-4 and CD44 abolished up-regulation of NF-κB activation and the consequent pro-inflammatory cytokine production, while restored Tg and NIS levels. A further goal of this study was demonstrate that also other LMW HA have pro inflammatory proprieties. These data suggest that HA fragments, through the involvement of TLR-2, TLR-4 and CD44 signaling cascade, contribute to prime the inflammatory response in thyrocytes and, by reducing the expression of thyroid-specific genes, could promote the loss of function of gland such as in Hashimoto's thyroiditis.

The mechanisms that modulate the response to tissue injury are not fully understood. Abnormalities in the repair response are associated with a variety of chronic disease states characterized by inflammation, followed subsequently by excessive ECM deposition. As cell-matrix interactions are able to regulate cellular homeostasis, modification of ECM integrity appears to be an unspecific factor in promoting the onset and progression of inflammatory diseases. Evidence is emerging to show that endogenous ECM molecules supply signals to damage tissues and cells in order to promote further ECM degradation and inflammation progression. Several investigations have been confirmed that HA fragments of different molecular sizes exhibit different biological effects and responses. In fact, the increased deposition of HA into the ECM is a strong hallmark of inflammation processes. In the context of inflammatory pathologies, highly polymerized HA is broken down into small components, which are able to exacerbate the inflammatory response by inducing the release of various detrimental mediators such as reactive oxygen species, cytokines, chemokines and destructive enzymes and by facilitating the recruitment of leukocytes. However, strategies involving the modulation of the HA fragment with specific receptors on cell surface could represent different promising effects for therapeutic scope. : This review will focus on the inflammation action of small HA fragments in recent years obtained by in vivo reports.

Atherosclerosis (ATH) is a chronic, dynamic, evolutive process involving morphological and structural subversion of artery walls, leading to the formation of atherosclerotic plaques. ATH generally initiates during the childhood, occurring as a result of a number of changes in the intima tunica and in the media of arteries. A key event occurring during the pathobiology of ATH is the accumulation of lipoproteins in the sub-intimal spaces mediated by extracellular matrix (ECM) molecules, especially by the chondroitin sulfate/dermatan sulfate (CS/DS) -containing proteoglycans (CS/DSPGs). Among them, the proteoglycan biglycan (BGN) is critically involved in the onset and progression of ATH and evidences show that BGN represents the missing link between the pro-atherogenic status induced by both traditional and non-traditional cardiovascular risk factors and the development and progression of vascular damage. In the light of these findings, the role of BGN in dyslipidemia, hypertension, cigarette smoking, diabetes, chronic kidney disease and inflammatory status is briefly analyzed and discussed in order to shed new light on the underlying mechanisms governing the association between BGN and ATH.

Background Familial hypercholesterolemia (FH) is characterized by increased cardiovascular risk; despite-high intensity statins, only few patients with FH achieve the recommended low-density lipoprotein cholesterol (LDL-C) targets. Objective We aimed to evaluate the effectiveness of six-month add-on therapy with proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9-i) or ezetimibe on lipid profile and pulse wave velocity (PWV) in patients with FH. Methods In this observational study, we evaluated 98 genetically confirmed patients with FH with an LDL-C off-target despite high-intensity statins with or without ezetimibe; of these, 53 patients (statin plus ezetimibe) added PCSK9-i (PCSK9-i group) and 45 (statin only) added ezetimibe (EZE group) per applicable guidelines and reimbursement rules. All patients obtained biochemical analysis and PWV evaluation at baseline and after six months of optimized treatment. Results After 6 months of add-on therapy, most patients achieving LDL-C targets were in the PCSK9-i group (77.3% PCSK9-i group vs 37.8% EZE group, P Conclusions Lipid and PWV profiles in patients with FH significantly improved after addition of PCSK9-i or ezetimibe to high-intensity statin therapy; moreover, ΔPWV was associated with ΔLDL. Our results are consistent with a beneficial role of these novel therapies in FH subjects.

Serglycin is expressed by a variety of cell types and mediates different functions in both normal and pathological conditions by interacting with different biological molecules, such as the CD44 receptor. Many studies suggest that serglycin has a crucial role in inflammatory response, but there are limited data on the functions of this proteoglycan in chondrocytes. In this study we investigated the effect of serglycin knockdown induced by a specific serglycin small interfering RNA (SRGN siRNA) in normal mouse chondrocytes stimulated with lipopolysaccharide (LPS). LPS administration in normal chondrocytes increased the expression of serglycin mRNA and related protein and the production of the pro-inflammatory mediators TNF-alpha, IL-1beta, IL-6, iNOS and MMP-9, through NF-kB activation. In addition, a marked increased expression of CD44 after LPS stimulation was observed. Notably, the CD44 expression and the inflammatory response were significantly reduced by SRGN siRNA treatment in LPS treated chondrocytes. Similar results were obtained in normal mouse chondrocytes exposed to LPS, using a specific blocking antibody against CD44. These results indicate that serglycin produced in LPS-induced inflammation in normal mouse chondrocytes is able to modulate inflammation by interacting with CD44 receptor, suggesting a possible key role in the cartilage inflammation.

Background and aims Young cigarette smokers may already present with early signs of vascular inflammation and damage; biglycan (BGN) has been shown to play a critical role in the initiation and progression of vascular lesions, also in young smokers. We investigated whether after smoke cessation, monocyte BGN expression is reduced; moreover, we evaluated any improvement of pro-atherogenic profile and arterial stiffness (AS), and their relationship with BGN in abstinent smokers. Methods Two-hundred-fifty-one young people who had decided to quit smoking were enrolled; of these, 71 had completed the 12-month observation period maintaining smoking abstinence. At enrollment and 12 months later, we evaluated anthropometrics, laboratory profile, carotid-femoral pulse wave velocity (cf-PWV), carotid intima-media thickness (cIMT), BGN expression. Results After 12-month smoke abstinence, we found a significant decrease in inflammatory markers (Hs-CRP: −23.3%; fibrinogen: −11.8%; IL-6: −9.2%), and increased HDL-C levels (+9.3%); blood pressure values were also slightly reduced. cf-PWV (−8.9%) appeared to be improved; cIMT remained unchanged. BGN expression appeared to be reduced (−42.8% relative reduction). BGN reduction appeared to be associated with fibrinogen reduction, and smoking burden. Reduced cf-PWV appeared to be dependent on change in fibrinogen, SBP, IL-6, and BGN by multiple regression analysis. Conclusions After the first year of smoke abstinence, the levels of IL-6, CRP, fibrinogen, HDL-C, and BGN expression, as well cf-PWV, are significantly improved as compared to baseline. This is the first evidence that removing exposure to a well-known cardiovascular risk factor, such as cigarette smoking, leads to significant reduction of BGN expression.

Serglycin (SRGN) is an intracellular proteoglycan produced and secreted by several cell types. The increased expression of SRGN was associated with greater aggressiveness in cancer and inflammation. In this study, we demonstrated that SRGN is increased in human chondrocytes after IL-β stimulation. Furthermore, we found that secreted SRGN was able to bind the CD44 receptor thus participating in the extension of the inflammatory response. Using SRGN knockdown cells we observed a significantly decrease in specific inflammatory markers and NF-kB activation. Similar results were observed by blocking the CD44 receptor. These data provide further evidences for a direct involvement of SRGN in the mechanisms regulating the non-infectious chondrocytes damage, and the consequent joint inflammation and cartilage destruction in arthritis.

Teleost haemoglobins vary in polymorphisms and primary structure, although display similar functional properties. Key amino acids for Root effect (a reduction in oxygen-carrying capacity and loss of cooperativity with declining pH) are conserved throughout fish evolution. For the first time, we cloned and characterised Sparus aurata L. embryonic globin chains (eα1, eα2, eβ). We also studied haemoglobins (eHbI, eHbII) behaviour in normal and low-oxygen conditions. Several amino acids in fry globins are different in chemical type (e.g. polar → non-polar and vice versa), compared to adult globins. His55α1, crucial for Root effect, is substituted by Ala in fry, presumably enhancing oxygen capture, transport and reducing the dependence of Root effect from pH. Phylogenetic trees demonstrate that eα1 globin diversified more recently than eα2; moreover, eα1, eα2 and eβ globins evolved earlier than adult α and β globins. In low-oxygen conditions, fry haemoglobins display the same behaviour of the adult haemoglobins (probably, embryonic and adult-type I Hbs display a higher oxygen affinity than type II Hbs, operating through a rapid cycle of heme-Fe auto-oxidation/reduction). Therefore, based on our results and on the comparison with adult haemoglobins, we hypothesise that embryonic haemoglobins have evolved to better adapt fry to variable habitats. We studied Sparus aurata for its economical relevance in Mediterranean aquaculture. The information we provide can help understand Sparus aurata behaviour in the wild and in rearing conditions. Further studies with functional assays will deepen the knowledge on the molecular mechanisms of fry haemoglobin physiology.

Background: Systemic sclerosis (SSc) is characterized by early vasculopathy and fibrosis in the skin, lungs, and other tissues. Vascular manifestations of SSc include Raynaud’s phenomenon, digital ulcers, and pulmonary artery hypertension (PAH). PAH is the second most common cause of mortality in SSc. Circulating CD34+ cells associated with cardiovascular health status in several conditions, including chronic immune-inflammatory disease. CD34+ cell numbers have been found inconstantly reduced in SSc. Endocan, a proteoglycan expressed by endothelial cells, was recently suggested as a marker of vascular stress. We tested the relationships among CD34+ cells, endocan, inflammatory markers, vitamin D levels, and clinical parameters in SSc patients with PAH. METHODS: Standard echocardiography was performed. Vitamin D levels, CD34+ cells, inflammatory markers, endocan plasma levels were determined in 36 female SSc patients (24 diffuse/12 limited) and 36 matched controls (HC). RESULTS: We found no difference in CD34+ and vitamin D levels in SSc as compared to controls, ESR, CRP, fibrinogen, endocan, sPAP were higher in SSc with respect to controls. We found a correlation between endocan and: CD34+ cells (r: −0.540, p = 0.002), pulmonary arterial pressure (sPAP) (r: 0.565, p &lt, 0.001), tricuspid annular plane excursion (TAPSE) (r: −0.311, p &lt, 0.01), and E/A ratio (r: −0.487, p &lt, 0.001), but not with ejection fraction (r: −0.057, p = 0.785) in SSc. CD34+ cells correlate with fibrinogen (r: −0.619, p &lt, 0.001), sPAP (r: −0.404, p = 0.011), E/A (r: 0.470, p &lt, 0.005 in SSc. CONCLUSION: CD34+ cell number was significantly correlated with endocan levels and with sPAP in SSc, endocan and CD34+ progenitor cells might be suggested as a potential marker of disease status.

Monoclonal gammopathy of undetermined significance (MGUS) is a pre-malignant abnormality of plasma cells, with increased serum levels of immunoglobulins. Patients with MGUS may evolve to multiple myeloma through a multistep process including deregulated gene expression. microRNAs are small non-coding RNA molecules involved in post-transcriptional regulation of crucial biological processes, such as morphogenesis, cell differentiation, apoptosis, and cancer. This study aimed to evaluate microRNA expression on peripheral lymph-monocytes from MGUS subjects compared with healthy controls using qPCR arrays. Blood samples were collected by venipuncture from fifteen, newly diagnosed MGUS patients and fifteen healthy subjects. A further group (validation group) of six newly diagnosed MGUS patients and five healthy control were enrolled for the validation of miRNAs and their mRNAs target. The study was conducted performing miProfile miRNA qPCR arrays, followed by validation of miRNAs and related mRNA targets through RT-qPCR. The functional interaction between microRNAs and target gene were obtained by Ingenuity Pathways Analysis (IPA). IPA network analysis identified only molecules and relationships experimentally observed in peripheral lymphomonocytes. The following miRNAs :133a-3p, 16−5p, 291−3p, 23a-3p, 205−5p, 17−5p, 7a-5p, 221−3p, 30c-5p, 126a-3p,155−5p, let-7a-5p and 26a-5p, involved in the regulation of genes with a role in lymphocyte homeostasis, cell proliferation, apoptosis, and multiple myeloma (MM) progression, were differently expressed in MGUS with respect to healthy subjects. This miRNA signature and its relative targets could be considered for the formulation of new therapeutic strategies in the prophylaxis or treatment of monoclonal gammopathies.

Background Small HA fragments are produced during cartilage degradation and their role seems to be preponderant during pathologies in which cartilage injury contribute to trigger and perpetuate the inflammatory mechanism. Several reports have increasingly shown that MicroRNAs (miRs), a small non-coding mRNAs are involved in the regulation of multiple biological processes, including cell proliferation and inflammation response in different pathologies, among them miR146a seems to be involved in inflammatory processes. Methods Starting by these evidences we investigated the levels of miR146a and its correlation with inflammatory mediators in an experimental model of 6-mer HA-induced inflammatory response in human cultured chondrocytes. Results Treatment of chondrocytes with 6-mer HA showed up-regulation in inflammation parameters such as TLR-4, and CD44 receptors activation, IL-6, IL-1β and MMP-13 mRNA expression and proteins production, as well as NF-kB activation. We also revealed an up-regulation of miR146a. Transfection with a miR146a mimic or miR146a inhibitor produced the following effects: chondrocytes receiving miR146a mimic and then 6-mer HA significantly reduced inflammatory cytokines and MMP-13, while exposition of chondrocytes with miR146a inhibitor and then the 6-mer HA incremented the activity of damaging cytokines and MMP13. Expression of CD44 receptor was not affected by miR-146a treatments, while TLR-4 expression and NF-kB activation were modified. Conclusions We concluded that up-regulation of miR146a occurred in 6-mer HA-induced inflammation response may reduce the inflammatory cascade by modulating TLR-4 and NF-kB activation. General significance These results could be useful in develop new therapeutic strategies with the aim to reduce OA and RA incidence.

Several studies have shown the degradation of the extracellular matrix at the site of neuroinflammation and increased release of degradation products of glycosaminoglycans. Among these, low molecular weight fragments of hyaluronan (HA) may play a key role in the events leading to neuroinflammation and/or neuronal degeneration. Small HA fragments are able to induce inflammation by stimulating both TLR-2 and TLR-4 as well as CD44 receptors. This stimulation culminates in the nuclear translocation of NF-kB that in turn induces the production of pro-inflammatory intermediates such as TNF-α and IL-1β. The potential of HA fragments, as mediators of inflammation, it has been poorly investigated in neuron-like SH-SY5Y cells so the aim of this study was to investigate the neuroinflammatory effects of very small HA oligosaccharides, the involvement of TLR-2, TLR-4, and CD44 and the production of α-synuclein in such cells. The addition of HA fragments to cell cultures up-regulated TLR-2, TLR-4, and CD44 levels, induced NF-kB activity and increased both TNF-α and IL-β as well as α-synuclein production. On blocking the activity of TLR-2, TLR-4, and CD44 the levels of inflammatory parameters and of α-synuclein were significantly reduced. Since several data have shown as α-synuclein, produced from neurons, is able to initiate ex novo or to maintain an existing neuroinflammatory response, which has been suggested as one of the principal components involved in neurodegenerative pathologies, as PD, we suggest that HA pathways should be given careful consideration when devising future anti-neuroinflammatory strategies to defend against the onset of neurodegenerative disorders. J. Cell. Biochem. 117: 2835-2843, 2016. © 2016 Wiley Periodicals, Inc.

Inflammation is a complex mechanism that plays a key role during diseases. Dynamic features of the extracellular matrix (ECM), in particular, during phases of tissue inflammation, have long been appreciated, and a great deal of several investigations has focused on the effects of ECM derivatives on cell function. It has been well defined that during inflammatory and tissue injury, ECM components were degraded. ECM degradation direct consequence is the loss of cell homeostasis, while a further consequence is the generation of fragments from larger precursor molecules. These bio-functional ECM shred defined matrikines as capable of playing different actions, especially when they function as powerful initiators, able to prime the inflammatory mechanism. Non-sulphated glycosaminoglycan hyaluronan (HA) is the major component of the ECM that undergoes specific modulation during tissue damage and inflammation. HA fragments at very low molecular weight are produced as a result of HA depolymerization. Several evidence has considered the plausibility that HA breakdown products play a modulatory action in the sequential stages of inflammation, although the effective mechanism of these HA derivative compounds act is not completely defined. This review will focus on the pro-inflammatory effects of HA fragments in recent years obtained by in vitro investigations.

Rheumatic diseases are associated with accelerated atherosclerosis and with increased risk of cardiovascular morbidity and mortality. The mechanisms underlying the higher prevalence of cardiovascular disease are not completely clarified, but it is likely that a pivotal role is played by vascular inflammation and consequently to altered vascular endothelium homeostasis. Also, high prevalence of traditional risk factors, proatherogenic activation and endothelial dysfunction further contribute to vascular damage. Circulating endothelial progenitor cells (EPCs) can restore dysfunctional endothelium and protect against atherosclerotic vascular disease. However, abnormalities in number and function of these cells in patients with rheumatic condition have been extensively reported. During the last years, growing interest in the mechanisms of endothelial renewal and its potential as a therapy for CVD has been shown; in addition, pioneering studies show that EPC dysfunction might be improved with pharmacological strategies. However, how to restore EPC function, and whether achieving this aim may be effective in preventing cardiovascular complications in rheumatic disease, remain to be established. In this review we report an overview on the current stand of knowledge on the effect of pharmaceutical and lifestyle intervention in improving EPCs number and function in rheumatic disease.

Our knowledge of extracellular matrix (ECM) structure and function has increased enormously over the last decade or so. There is evidence demonstrating that ECM provides signals affecting cell adhesion, shape, migration, proliferation, survival, and differentiation. ECM presents many domains that become active after proteolytic cleavage. These active ECM fragments are called matrikines which play different roles; in particular, they may act as potent inflammatory mediators during cartilage injury. A major component of the ECM that undergoes dynamic regulation during cartilage damage and inflammation is the non-sulphated glycosaminoglycan (GAG) hyaluronan (HA). In this contest, HA is the most studied because of its different activity due to the different polymerization state. In vivo evidences have shown that low molecular weight HA exerts pro-inflammatory action, while high molecular weight HA possesses anti-inflammatory properties. Therefore, the beneficial HA effects on arthritis are not only limited to its viscosity and lubricant action on the joints, but it is especially due to a specific and effective anti-inflammatory activity. Several in vitro experimental investigations demonstrated that HA treatment may regulate different biochemical pathways involved during the cartilage damage. Emerging reports are suggesting that the ability to recognize receptors both for the HA degraded fragments, whether for the high-polymerized native HA involve interaction with integrins, toll-like receptors (TLRs), and the cluster determinant (CD44). The activation of these receptors induced by small HA fragments, via the nuclear factor kappa-light-chain enhancer of activated B cell (NF-kB) mediation, directly or other different pathways, produces the transcription of a large number of damaging intermediates that lead to cartilage erosion. This review briefly summarizes a number of findings of the recent studies focused on the protective effects of HA, at the different polymerization states, on experimental arthritis in vitro both in animal and human cultured chondrocytes.

Cartilage degeneration are hallmarks of wear, tear, mechanical and inflammatory damage of the joint cartilage. Tissue degradation as well as compromising the integrity and function of the organ, produces different intermediates, directly able to stimulate further inflammatory effect, therefore, amplifying the inflammation response. Biglycan is a soluble component of the extracellular matrix that is released during tissue injury. It has been reported that released biglycan is an endogenous ligand for TLR-2/4 in some cell type. We studied the role of biglycan in an experimental model of biglycan-induced inflammatory response in human chondrocytes and the effect of high polymerized HA on reducing its activity. Exposition of chondrocytes to LPS generated cell injury, including high levels of biglycan. Chondrocyte treatment with biglycan produces a high mRNA expression of several detrimental inflammation mediators such as IL-1β, IL-6, MMP-13, and IL-17, as well as NF-kB and TLR-4 activation. Administration of high polymerized HA to chondrocytes exposed to biglycan was able to attenuate the inflammatory response by decreasing the expression of the inflammatory mediators. Involvement of the TLR-4 in the mediation of the biglycan action was confirmed using a specific silent agent (siRNA). Taken together, these data could be used to develop new anti-inflammatory approaches.

Recent studies have found that the inactivation of small hyaluronan (HA) fragments originating from native HA during inflammation reduced the inflammatory response in models of experimental arthritis. The stimulation of adenosine receptors A2A reduced inflammation by inhibiting NF-kB activation. The combination of both treatments was significantly more effective than either of the individual treatments. The aim of this study was to further investigate the effects of a combined treatment using the HA inhibitor Pep-1 and a selective A2AR agonist (CV-1808) on the structure and ultrastructure of the articular cartilage and on apoptosis in a model of collagen-induced arthritis (CIA) in mice. Arthritic mice were treated with Pep-1 and/or CV-1808 intraperitoneally daily for 20 days. At day 35, the hind limbs were processed for light microscopy (hematoxylin/eosin and Safranin-O-Fast Green) and for transmission and scanning electron microscopy. CIA increased IL-6, caspase-3 and caspase-7 mRNA expression and the related protein levels in arthritic articular cartilage, and significantly increased concentrations of Bcl-2-associated X protein (Bax), while B cell-lymphoma-2 protein (Bcl-2) was markedly reduced. The combined Pep-1/CV-1808 treatment significantly reduced CIA injury, particularly at the highest doses, demonstrated by the presence of Safranin-O-positive cartilage, with a smooth surface and normal chondrocytes in the superficial, intermediate and deep zones. Morphological data and histological scoring were strongly supported by the reduction in inflammation and apoptotic markers. The results further support the role of HA degradation and A2A receptors in arthritis.

CD34+ circulating progenitor cells (CD34+CPCs) are a population of multipotent cells which can delay the development of atherosclerosis and cardiovascular disease (CVD) in conditions of increased CV risk. MicroRNAs (miRs) 221 and 222 modulate different genes regulating angiogenesis and inflammation; moreover, miR221/22 have beenshown to participate in differentiation and proliferation of CD34+CPCs, inhibiting cell migration and homing. miR221/222 in CD34+CPCs from hypertensive subjects are also increased and associated with CD34+cell number and reactive oxygen species (ROS). We evaluated CD34+CPC number, intracellular miR221/222 and ROS levels, arterial stiffness (AS)and echocardiography indices at baseline (T0).Then, after a six-month treatment with olmesartan, 20 mg/day (T1), in 57 hypertensive patients with left ventricular hypertrophy (LVH) and with no additional risk factor for CVD, and in 29 healthy controls (baseline),fibrinogen, C-reactive protein (CRP), glucose and lipid profiles were also evaluated.At T1, blood pressure values, CRP and fibrinogen levels, ROS and miR221/222 were significantly decreased (all p

Angiotensin I converting enzyme (ACE) insertion/deletion (I/D) polymorphism is thought to affect renin–angiotensin system (RAS) activity and development of cardiovascular disease; significant associations between I/D polymorphism and atherosclerosis, stroke, nephropathy, and early mortality were already found. We investigated whether Southern Italy resistant hypertensives presented an association between the presence of I and/or D alleles and early vascular damage, inflammation, and insulin resistance. One-hundred-fifty resistant hypertensives were enrolled, studied, and genotyped; carotid intima-media thickness (cIMT), arterial stiffness (AS), and HOMA indices were also evaluated. D allele was more prevalent, and 74 patients presented DD homozygosis. Sixty-eight patients had metabolic syndrome (MetS), without significant differences between DD and I allele carriers. DD genotype appeared strongly associated with higher HOMA values (p

The aim of this study was to investigate the involvement of exchange proteins directly activated by cyclic adenosine (ADO) monophosphate (EPAC) in 4-mer hyaluronan (HA) oligosaccharide-induced inflammatory response in mouse normal synovial fibroblasts (NSF). Treatment of NSF with 4-mer HA increased Toll-like receptor-4, TNF-alpha and IL-1beta mRNA expression and of the related proteins, as well as nuclear factor kappaB (NF-kB) activation. Addition to NSF, previously stimulated with 4-mer HA oligosaccharides, of ADO significantly reduced NF-kB activation, TNF-alpha and IL-1beta expression. The pre-treatment of NSF with cyclic ADO monophosphate and/or PKA and/or EPAC-specific inhibitors significantly inhibited the anti-inflammatory effect exerted by ADO. In particular, the EPAC inhibitor reduced the ADO effect to a major extent than the PKA inhibitor. These results mean that both PKA and EPAC pathways are involved in ADO-induced NF-kB inhibition although EPAC seems to be more involved than PKA.

In pathological conditions, oxidative burst generates hyaluronan (HA) fragmentation with a consequent increase in the number of small HA oligosaccharides. These fragments are able to stimulate an inflammatory response in different cell types by activating the CD44 and the toll-like receptors 4 (TLR-4) and 2 (TLR-2). The stimulation of CD44 and TLRs in turn activates the NF-kB which induces the production of several pro-inflammatory mediators that amplify and perpetuate inflammation. We aimed to study the antioxidant effect of the SOD mimic, synthetic manganese porphyrin, Mn(III) 5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin (MnTM-2-PyP(5+)) on preventing HA degradation in mouse articular chondrocytes stimulated with Fe (II) plus ascorbate. Fe (II) plus ascorbate stimulation induced oxidative burst confirmed by high levels of hydroxyl radical/peroxynitrite production, increased lipid peroxidation and HA degradation. HA fragments highly induced mRNA expression and the related protein production of CD44, TLR-4 and TLR-2, NF-kB activation and significantly up-regulated the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and other pro-inflammatory mediators, i.e. matrix metalloprotease 13 (MMP-13) and inducible nitric oxide synthase (iNOS). Treatment of cells with MnTM-2-PyP(5+)was able to attenuate oxidative burst, HA degradation and NF-kB activation, and markedly decreased mRNA expression of CD44, and TLRs and the related protein synthesis, as well as the levels of up-regulated inflammatory mediators. Adding a specific HA-blocking peptide (PEP-1) to cells significantly reduced all the inflammatory parameters up-regulated by Fe (II) plus ascorbate, and increased MnTM-2-PyP(5+) activity. These findings suggest that HA degradation plays a key role in the initial inflammatory response of cartilage and antioxidants and could be a useful tool to prevent the propagation of this mechanism.

BAY 11-7082 antagonizes I-κB kinase-β preventing nuclear translocation of nuclear factor-κB (NF-κB); it also inhibits NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation. NF-κB is involved in psoriasis, whereas the role of NLRP3 is controversial. We investigated BAY 11-7082 effects in an experimental model of psoriasis-like dermatitis. Psoriasis-like lesions were induced by a topical application of imiquimod (IMQ) cream (62.5 mg/day) on the shaved back skin of C57BL/6 and NLRP3 knockout (KO) mice for 7 consecutive days. Sham psoriasis animals were challenged with Vaseline cream. Sham and IMQ animals were randomized to receive BAY 11-7082 (20 mg/kg/i.p.) or its vehicle (100 μl/i.p of 0.9% NaCl). Skin of IMQ animals developed erythema, scales, thickening and epidermal acanthosis. IMQ skin samples showed increased expression of pNF-κB and NLRP3 activation. BAY 11-7082 blunted epidermal thickness, acanthosis and inflammatory infiltrate. BAY 11-7082 reduced pNF-κB, NLRP3, tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β expression, blunted the phosphorylation of signal transducer and activators of transcription 3 (STAT3) and decreased IL-23 levels. In addition, BAY 11-7082 reawakened the apoptotic machinery. NLRP3 KO animals showed a reduced total histological score but persistent mild acanthosis, dermal thickness and expression of pNF-κB and pSTAT3, following IMQ application. Our data suggest that BAY 11-7082 might represent an interesting approach for the management of psoriasis-like dermatitis depending on the dual inhibition of NF-κB and NLRP3.

Small hyaluronan (HA) fragments produced from native HA during inflammation contribute greatly to cell injury in many pathologies. HA oligosaccharides increase proinflammatory cytokine levels by activating both CD44 and toll-like receptor (TLR)-4. Stimulation of CD44 and TLR-4 then activates nuclear factor-κB, which induces the production of proinflammatory cytokines. The adenosine 2A receptor (A(2A)R) is also involved in several inflammation pathologies, and the nucleoside adenosine acts as a potent endogenous inhibitor of inflammation in various tissues by interacting with this receptor. The aim of this study was to investigate the effects of an HA-blocking peptide that inhibits the proinflammatory action of HA oligosaccharides produced during inflammation, together with a specific A(2A)R agonist in a model of normal mouse articular chondrocytes stimulated with interleukin (IL)-1β. IL-1β stimulation significantly increased mRNA expression and the related protein production of TLR-4, TLR-2, CD44 and A(2A)R in articular chondrocytes. The induced nuclear factor-κB activation was also associated with increased levels of inflammatory cytokines, including tumor necrosis factor-α and IL-6, and other inflammatory mediators, such as matrix metalloprotease-13 and inducible nitric oxide synthase. Treatment of chondrocytes with the HA-blocking peptide Pep-1 and/or a specific A(2A)R agonist (CGS-21680) significantly reduced all of the inflammatory parameters upregulated by IL-1β. These results suggest that the inflammatory response may be reduced either by blocking oligosaccharides from HA degradation or by A(2A)R stimulation.

Interleukin-1beta (IL-1beta) elicits the expression of inflammatory mediators through a mechanism involving the CD44 receptor. Hyaluronan (HA) depolymerization also contributes to CD44 activation. This study investigated the potential of HA fragments, obtained by hyaluronidase (HYAL) treatment, as mediators of CD44 activation on IL-1beta-induced inflammation in mouse chondrocytes. mRNA and related protein levels were measured for CD44, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), matrix metalloproteinase-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in chondrocytes, treated or untreated with IL-1beta, either with or without the addition of HYAL. The level of NF-kB activation was also assayed. CD44 mRNA expression was higher than controls in chondrocytes treated with IL-1beta. IL-1beta also induced NF-kB up-regulation and increased TNF-alpha, IL-6, MMP-13 and iNOS expression. Different effects resulted from HYAL treatment. Treatment of chondrocytes exposed to IL-1beta with HYAL synergistically increased the same parameters up-regulated by IL-1beta, while the same parameters were increased by HYAL in chondrocytes not exposed to IL-1beta but to a lesser extent. Specific CD44 blocking antibody and hyaluronan binding protein (HABP), which inhibit HA activity, were used to confirm CD44 to be the target of IL-1beta action through HA mediation. HA levels and molecular size further confirm the role of degraded HA. These findings suggest that IL-1beta exerts inflammatory activity via CD44 by the mediation of HA fragments derived from HA depolymerization.

Hyaluronan (HA) oligosaccharides stimulate pro-inflammatory responses in different cell types by modulating both cluster determinant 44 (CD44) and TLR4. The activation of these receptors is also mediated by collagen-induced arthritis (CIA) that, via two different pathways, culminates in the liberation of NF-κB. This then stimulates the production of pro-inflammatory cytokines, including IL-18 and IL-33, that are greatly involved in rheumatoid arthritis. The aim of this study was to investigate the effects of 6-mer HA oligosaccharides on mouse synovial fibroblasts obtained from normal DBA/J1 mice or mice subjected to CIA. Compared with normal synovial fibroblasts (NSF), rheumatoid arthritis synovial fibroblasts (RASF) showed no up-regulation of CD44 and TLR4 mRNA expression and the related proteins, as well as no activation of NF-κB. Very low levels of both mRNA and related proteins were also detected for IL-18 and IL-33. Treatment of NSF and RASF with 6-mer HA oligosaccharides significantly increased all the parameters in both fibroblast groups, although to a greater extent in RASF. The addition of hyaluronan binding protein to both NSF and RASF inhibited HA activity and was able to reduce the effects of 6-mer HA oligosaccharides and the consequent inflammatory response.

Objective. To identify, evaluate, and synthesize evidence on the predictive power of circulating endothelial progenitor cells (EPCs) in cardiovascular disease, through a systematic review of quantitative studies.Data Sources. MEDLINE was searched using keywords related to “endothelial progenitor cells” and “endothelium” and, for the different categories, respectively, “smoking”; “blood pressure”; “diabetes mellitus” or “insulin resistance”; “dyslipidemia”; “aging” or “elderly”; “angina pectoris” or “myocardial infarction”; “stroke” or “cerebrovascular disease”; “homocysteine”; “C-reactive protein”; “vitamin D”.Study Selection. Database hits were evaluated against explicit inclusion criteria. From 927 database hits, 43 quantitative studies were included.Data Syntheses. EPC count has been suggested for cardiovascular risk estimation in the clinical practice, since it is currently accepted that EPCs can work as proangiogenic support cells, maintaining their importance as regenerative/reparative potential, and also as prognostic markers.Conclusions. EPCs showed an important role in identifying cardiovascular risk conditions, and to suggest their evaluation as predictor of outcomes appears to be reasonable in different defined clinical settings. Due to their capability of proliferation, circulation, and the development of functional progeny, great interest has been directed to therapeutic use of progenitor cells in atherosclerotic diseases. This trial is registered with registration number: ProsperoCRD42015023717.

Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factor-activated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of pro-inflammatory mediators, including tumor necrosis factor alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the anti-inflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies.