Biglycan expression in current cigarette smokers: a possible link between active smoking and atherogenesis.

OBJECTIVE: Cigarette smokers present early signs of vascular damage and systemic inflammation. Biglycan (BGN), an ubiquitous component of extracellular matrix orchestrating several physiological functions, has recently been indicated as a major source of low-density lipoprotein retention in the normal arterial intima-media layer. We evaluated whether BGN-mRNA expression was enhanced in peripheral monocytes of smokers with no additional cardiovascular risk factors (CVRFs), and if it was associated with altered carotid arterial stiffness (AS) or intima media thickness (cIMT). We also evaluated plasma markers of systemic and vascular inflammation, and correlation with BGN-mRNA. METHODS: Two-hundred-fifty-one young smokers were enrolled, with no additional CVRFs, and 60 controls. Plasma lipids, fibrinogen, C-reactive protein (CRP), interleukin-6 (IL-6), AS and cIMT were assessed. A smoke exposure index (SEIx) was calculated. RESULTS: Fibrinogen, CRP, AS indices, cIMT, and BGN-mRNA were higher in smokers compared to controls; HDL-C levels were lower, no difference was detected in IL-6 levels. After stratification of smokers in quartiles based on SEIx values, smokers in the highest quartiles presented highest fibrinogen, CRP, AS, cIMT, BGN, and also IL-6 values, and lowest HDL-C. CONCLUSION: BGN-mRNA was enhanced in young smokers, compared to controls, and appears associated to a proatherogenic profile, characterized by increased fibrinogen, CRP, and IL-6, lower HDL-C, altered AS and cIMT values, particularly in those with higher SEIx: the more cigarettes smoked over years, the more marked the alterations. Although we cannot state whether BGN have a direct causal role in inducing, maintaining and developing vascular damage, including intima-media wall thickening and arterial stiffening, our data could suggest that it may represent a link between proatherogenic status induced by cigarette smoking, and the development and progression of vascular damage.

Circulating progenitor cells in hypertensive patients with different degrees of cardiovascular involvement.

We investigated whether different degrees of hypertension-related cardiovascular involvement are associated with changes in circulating proangiogenic hematopoietic cell (PHC) numbers and/or phenotypes and/or in the PHC redox system in hypertensive individuals with isolated arterial stiffening (AS) hypertensives or with both carotid intima-media thickening and left ventricular hypertrophy (LVH) hypertensives. We also evaluated microRNA (miRs) 221 and 222 (miRs221/222) expression in CD34+ cells, the relationship between these miRs and cell number and reactive oxygen species (ROS) levels, and the expression of manganese superoxide dismutase (MnSOD), catalase (CAT) glutathione peroxidase type-1 (GPx-1) and gp91phox-containing nicotinamide-adenine-dinucleotide-phosphate-oxidase (NOX2). Proangiogenic hematopoietic cells (PHCs) from hypertensive patients and controls were isolated by flow cytometry. PHCs were higher in hypertensives than in controls but were lower in LVH than in AS hypertensives. In CD34+ cells from AS hypertensives, NOX2, MnSOD, CAT and GPx-1 were overexpressed; ROS, miRs and NOX2 were also increased and were associated with cell number. In LVH, we found an imbalance in the cell redox system; MnSOD showed the highest values, whereas CAT and GPx-1 were lower than in AS hypertensives. Intracellular ROS, miRs and NOX2 were higher and inversely associated with cell number. In AS hypertensives, the redox balance may sustain the increase in PHCs; by contrast, in hypertensives with more advanced lesions, redox imbalance may result in increased oxidative stress and cell reduction.

Circulating progenitor cells in rheumatoid arthritis: association with inflammation and oxidative stress.

OBJECTIVES: To evaluate the association between inflammation, oxidative stress, and circulating progenitor cell (CPC) number and redox equilibrium, vascular lesions and accelerated atherosclerosis in rheumatoid arthritis (RA). METHOD: Circulating CD34+ cells were isolated from 33 RA patients and 33 controls. Reactive oxygen species (ROS) levels and mRNA expression of manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase type 1 (GPx-1) antioxidant enzymes, and the gp91phox-containing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase NOX2 were measured in CD34+ cells. C-reactive protein (CRP), fibrinogen, erythrocyte sedimentation rate (ESR), carotid intima-media thickness (cIMT), and arterial stiffness (AS) were also evaluated. We investigated the relationships between inflammatory markers, vascular parameters, cell number, and antioxidant enzymes. RESULTS: CD34+ cell number was lower in RA patients than in controls. In CD34+ cells from RA patients, ROS levels, MnSOD mRNA, and NOX2 mRNA were higher, while mRNA expression of GPx-1 and CAT was significantly lower. The AS, pulse wave velocity (PWV), and augmentation index (AIx) were higher, as was cIMT. CD34+ cell number was inversely correlated with CRP, ROS, PWV, and AIx, and with the CAT/MnSOD and GPx-1/MnSOD ratios. CRP was correlated with MnSOD mRNA, PWV, and AIx but not with CAT and GPx-1 mRNA. CONCLUSIONS: Our data show a link between inflammation, oxidative stress, and the impairment of the antioxidant system of CPCs and their number, and with arterial stiffness in RA subjects. This could suggest a perspective on the accelerated development of vascular damage and atherosclerosis in RA.

4-mer hyaluronan oligosaccharides stimulate inflammation response in synovial fibroblasts in part via TAK-1 and in part via p38-MAPK.

4-mer hyaluronan (HA) oligosaccharides stimulate pro-inflammatory effects in different cell types by interacting with both the toll-like receptor-4 (TLR-4) and -2 (TLR-2). This interaction induces the activation of the transforming growth factor activated kinase-1 (TAK-1) that activates the nuclear factor kappaB (NF-kB) either directly and/or through the activation of p38-mitogen-activated protein kinase (p38-MAPK). This in turn induces the transcription of proinflammatory mediators that prime inflammation. Our aim was to investigate the involvement of TAK-1 and p38-MAPK in 4-mer HA oligosaccharide-induced inflammatory response in mouse synovial fibroblasts obtained from normal DBA/J1 mice (NSF) and from mice subjected to collagen-induced arthritis (CIA). Treatment of NSF and rheumatoid arthritis synovial fibroblasts (RASF) with 4-mer HA showed a marked up-regulation of TLR-4, TLR-2, TAK-1 and p38-MAPK mRNA expression and of the related proteins, as well as NF-kB activation. High levels were also detected of TNF-α, IL- 1ß, MMP-13 and iNOS. Treatment of NSF and RASF, previously stimulated with 4-mer HA oligosaccharides, with TAK- 1 and/or p38-MAPK specific inhibitors significantly reduced all the parameters, although the inhibitory effect of p38- MAPK was less effective than that of TAK-1. The addition of CD44 antibody to both NSF and RASF showed that CD44 was not involved in 4-mer HA-induced inflammation.

Effect of cytokines on hyaluronan synthase activity and response to oxidative stress by fibroblasts.

Cytokines such as tumour necrosis factor-alpha (TNFalpha), interferon-gamma (IFNgamma), and transforming growth factor-beta (TGF1beta) modulate hyaluronan synthase (HAS) gene expression and protein activity. The aim of this research is to evaluate the response of HAS gene expression and the related protein synthesis in fibroblasts after treatment with TNFalpha, IFNgamma and TGF1beta and to assess the potential protective effect of increased hyaluronan (HA) synthesis during oxidative stress. In this study, gene expression, protein synthesis, hyaluronan content, cell death, lactate dehydrogenase (LDH) activity, membrane lipid peroxidation and endogenous antioxidant depletion are determined for HAS1, HAS2 and HAS3. Messenger RNA (mRNA) expression and protein formation of the three HAS genes is modulated using different cytokines and various doses and correlated with increased HA synthesis. Protection of fibroblasts from injury induced by exposure to reactive oxygen species was significantly increased by TGF1beta and was associated with increased gene expression and protein formation of HAS1 and HAS2 enzymes synthesising high-molecular-weight HA. It is proposed that specific HAS enzyme activity and HA molecular weight specificity is involved in the protective mechanism.

The antioxidant activity of chondroitin-4-sulphate, in carbon tetrachloride-induced acute hepatitis in mice, involves NF-kappaB and caspase activation.

BACKGROUND AND PURPOSE: Reactive oxygen species (ROC) are the main causes of carbon tetrachloride (CCl4)-induced acute liver injury. Chondroitin-4-sulphate (C4S) is known to inhibit lipid peroxidation through antioxidant mechanisms. Activation of nuclear factor (NF)-kappaB and caspases may strongly intensify inflammation and cell damage, in addition to that directly exerted by ROS. We investigated whether treatment with C4S, besides exerting antioxidant activity, was able to modulate NF-kappaB and apoptosis activation in CCl4-induced liver injury in mice. EXPERIMENTAL APPROACH: Acute hepatitis was induced in mice by an i.p. injection of CCl(4). Varying doses of C4S were administered i.p. 1 h before, 6 and 12 h after CCl4 injection. 24 h after CCl4 injection, the mice were killed for biochemical and histological analysis. KEY RESULTS: CCl4 injection produced: marked elevation of alanine aminotransferase and aspartate aminotransferase; hepatic membrane lipid peroxidation, assayed by 8-isoprostane levels; and depletion of reduced glutathione and superoxide dismutase. CCl4 also decreased NF-kappaB translocation and IkBalpha, and increased gene expression of mRNA and protein of metalloproteases (MMP)-2 and -9, and of pro- and cleaved forms of caspases-3 and -7. There was also increased liver polymorphonuclear infiltration, evaluated by elastase assay, and hepatic cell disruption.C4S treatment inhibited lipid peroxidation; blocked NF-kappaB activation and IkBalpha protein loss; decreased mRNA and proteins for MMPs and caspases; restored endogenous antioxidants; limited hepatic polymorphonuclear accumulation and tissue damage. CONCLUSIONS AND IMPLICATIONS: As antioxidants may inhibit NF-kappaB and caspase activation, we hypothesize that treatment with C4S was able to inhibit NF-kappaB and apoptosis activation in hepatic injury.

Chondroitin-4-sulphate inhibits NF-kB translocation and caspase activation in collagen-induced arthritis in mice.

OBJECTIVE: Free radical damage, inflammation, and apoptosis play a critical role in the onset and progression of cartilage erosion in arthritis. Many studies have demonstrated that glycosaminoglycans (GAGs), and chondroitin-4-sulphate (C4S) in particular, possess antioxidant activity that is able to inhibit lipid peroxidation which is the main mechanism of free radical-mediated biological injury. In addition to the effect directly exerted by reactive oxygen species (ROS), the activation of nuclear factor kB (NF-kB) and caspases may contribute substantially to increase inflammation and cell damage. We studied whether the antioxidant action of chronic C4S treatment to reduce ROS injury involves NF-kB and caspases modulation using an experimental model of collagen-induced arthritis in mice. METHODS: Arthritis was induced in mice via an intradermal injection at the base of the tail of 100 microl of emulsion containing bovine type II collagen in complete Freund's adjuvant. RESULTS: Arthritis provoked the following: severe oedema and inflammation in the hind paws; lipid peroxidation in the joints [measured by 8-isoprostane (8-IPE) levels]; reduction of the endogenous antioxidants catalase (CAT) activity and reduced glutathione (GSH) levels; induction of NF-kB translocation; a loss of cytoplasmic NF-kB inhibitor alpha (IkBalpha); an increase in metalloproteinase-13 (MMP-13), caspase-3 and caspase-7 gene expression and their related protein; the induction of cartilage polymorphonuclear (PMN) activation and infiltration [evaluated by elastase (ELA) assay] and cartilage alterations evaluated by histological analysis. Intraperitoneal administration of different doses of C4S (for 25 days), ameliorated all the symptoms of inflammation in the articular knee and paw joints, limited lipid peroxidation, inhibited NF-kB activation and IkBalpha protein loss, decreased mRNA MMP-13 and caspases expression and their related protein, restored endogenous antioxidants, and reduced PMN accumulation in the damaged cartilage. CONCLUSION: The evidence that C4S was able to inhibit NF-kB and apoptosis activation supports the hypothesis that the C4S effect depends on reduction of ROS production, although other direct effects cannot be excluded.

Differential effect of growth factors on hyaluronan synthase gene expression in fibroblasts exposed to oxidative stress.

The aim of this study was to evaluate how growth factors (PDGF-BB, EGF, and TGF-1beta) modulate hyaluronan synthase (HAS) activities in normal or stressed cultured human skin fibroblasts. The effects of concomitant treatment with cytokines and FeSO4 plus ascorbate on HAS mRNA expression, protein synthesis, and hyaluronic acid (HA) concentrations were also studied. Treatment of fibroblasts with growth factors up-regulated HAS gene expression and increased HAS enzymes and HA production. PDGF-BB induced HAS mRNA expression, protein synthesis, and HA production more efficiently than EGF and TGF-1beta. EGF was less effective than TGF-1beta. In addition, TGF-1beta reduced the expression and synthesis of HAS3, while PDGF-BB and EGF had the opposite effect. Concomitant treatment with growth factors and the oxidant was able to further increase HAS mRNA expression, once again with the exception of HAS3 with TGF-1beta. HAS protein synthesis was reduced, while HA levels were unaffected in comparison to those obtained from exposure to FeSO4 plus ascorbate alone. In conclusion, although growth factors plus the oxidant synergistically induced HAS mRNA expression in part, enzyme production was not correlated with this increase. Moreover, the increase in HAS mRNA levels was not translated into a consequent rise in HA concentration.