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.

Pulse wave velocity and augmentation index, but not intima-media thickness, are early indicators of vascular damage in hypercholesterolemic children.

BACKGROUND: Arterial stiffness is an important determinant of cardiovascular risk. It is associated with several cardiovascular risk factors, including hypertension, diabetes and cigarette smoking. However, there are conflicting data about the relationship between arterial stiffness and hypercholesterolemia. Furthermore, augmentation index (AIx), a measure of systemic arterial stiffness, has not been previously investigated in hypercholesterolemic (HCh) children. Aim of our study was to evaluate local and systemic arterial stiffness as well as carotid intima-media thickness (IMT) in HCh children and also to investigate the relation between serum cholesterol levels and arterial stiffness. MATERIALS AND METHODS: We determined lipid profile, body mass index, blood pressure, heart rate, carotid IMT and several arterial stiffness parameters, as beta-index, elastic modulus (E(p)), arterial compliance (AC), pulse wave velocity (PWV) and AIx, in 44 untreated HCh children (mean age 10.7 +/- 2.8 years; 18 with familial hypercholesterolemia, FH, and 26 with primary hypercholesterolemia, PHC) and 18 age- and sex-matched controls. HCh children never received any medication, including antihypertensive and lipid lowering drugs. RESULTS: Respect to controls and to PHC, FH had significantly higher (P < 0.001) beta-index (5.22 +/- 1.13 vs. 3.13 +/- 0.74 and 3.60 +/- 1.02), PWV (4.72 +/- 0.72 m s(-1) vs. 3.66 +/- 0.55 m s(-1) and 4.10 +/- 0.67 m s(-1)), AIx (3.55 +/- 3.97% vs. -4.43 +/- 4.09% and 0.61 +/- 2.39%) and E(p) (64.4 +/- 19.6 kPa vs. 36.2 +/- 11.3 kPa and 42.9 +/- 13.1), whereas AC (1.25 +/- 0.48 mm(2) kPa(-1) vs. 1.9 +/- 0.43 mm(2) kPa(-1) and 1.62 +/- 0.43 mm(2) kPa(-1)) was lower (P < 0.001). There was no significant difference in carotid IMT and blood pressure values between the groups. The multiple regression analysis showed a significant association of arterial stiffness values with plasma cholesterol levels (P < 0.0001). CONCLUSION: Our findings show that local and systemic arterial stiffness are increased in asymptomatic, normotensive HCh children, suggesting that HCh plays a key role in arterial mechanical impairment since the paediatric age.

Effects of simvastatin treatment on sICAM-1 and sE-selectin levels in hypercholesterolemic subjects.

This study was performed to determine whether the levels of soluble intercellular adhesion molecule-1 (sICAM-l) and soluble endothelial molecule-1 (sE-selectin) were elevated in subjects with hypercholesterolemia who presented with no other risk factors or evidence of atherosclerosis. The effects of administration of an HMG-CoA reductase inhibitor on the serum levels of these molecules were also examined. Forty hypercholesterolemic subjects (HCh) (19 males and 21 females), without hypertension or cardiovascular disease, received placebo for 4 weeks. The patients were then randomized in two groups; 20 of them (simvastatin group) were treated with simvastatin (20 mg/day) and the other 20 (placebo group) continued placebo administration. After 12 and 24 weeks of either simvastatin or placebo treatment, sICAM-1 and sE-selectin levels were measured. The same parameters were measured in 20 control subjects (C) with normal cholesterol levels, matched for sex and age. HCh had sICAM-1 basal values higher than C (352.4+/-57.9 ng/ml versus 114.9+/-89.6 ng/ml; P<0.001); however, sE-selectin basal values were not different in the two groups. No correlation was observed between HCh sICAM-1 levels and cholesterol levels (total and low-density lipoprotein). Furthermore, cholesterol-lowering treatment with simvastatin did not significantly diminish sICAM-1 levels. Our findings would support the hypothesis that patients with isolated hypercholesterolemia and without clinical atherosclerosis may be silent carriers of arterial subendothelial inflammation, expressed as an increase of sICAM-1.

Inapparent "wild-type" and "e-minus variant" HBV infection in patients with HCV-related chronic hepatitis.

We analysed DNA extracted from liver biopsy specimens and serum samples from 42 HCV-RNA-positive/HBsAg-negative subjects with chronic hepatitis. Twenty-eight of them were anti-HBs/anti-HBc-positive (group A), while 14 were negative for all HBV markers (group B). HBV sequences were found in hepatic DNA of 12 cases (11 of group A, one of group B), but in the serum of only two cases of group A. Sequencing analysis of pre-core region of HBV-DNA showed the presence of wild-type HBV in three cases, HBeAg-defective HBV in three cases, and the coexistence of both viral populations in six cases. These results indicate that HBV and HCV infection may coexist in HBsAg-negative chronic hepatitis, particularly in anti-HBs/anti-HBc-positive patients. However, HBV replication appears suppressed in these cases, and this state of latency may involve both wild and HBeAg-defective HBV types.

Persistence of "wild-type" and "e-minus" hepatitis B virus infection in chronic healthy HBsAg/anti-HBe positive carriers.

We examined nine chronic healthy hepatitis B surface antigen/antibody to hepatitis Be carriers with consistently normal liver chemistries and negative serum hepatitis B virus-DNA. Liver biopsy, performed twice, 10-11 years apart in all patients, showed normal histology and negative hepatitis B core antigen. DNA extracted from the second liver biopsy specimen, from 1 ml of serum from each patient and from an additional serum sample of 6 ml from two patients, was tested for pre-C/C and pre-S regions of hepatitis B virus-DNA by polymerase chain reaction amplification. Viral sequences were found in six of nine liver DNA extracts. In four cases both pre-C/C and pre-S regions were amplified, while the pre-C/C alone and the pre-S alone were detected in one case each. Direct sequencing of the amplified DNAs revealed no significant genomic changes in the pre-S and Core regions, while analysis of the pre-Core demonstrated the presence of a double viral population (wild-type and "e-defective") in four cases, and only "e-defective" hepatitis B virus in one case. No hepatitis B virus genomes were revealed in the serum sample when DNA was extracted from 1 ml of serum, while viral sequences were detected in both extracts of 6 ml of serum, indicating the presence of very low levels of viremia. These data suggest that episomal hepatitis B virus-DNA may persist for years in the liver of chronic healthy carriers in a latent state which may involve both wild-type and HBeAg-defective hepatitis B virus.

Is the course of perinatal hepatitis B virus infection influenced by genetic heterogeneity of the virus?

We studied the relations between genetic heterogeneity of pre-C region of hepatitis B virus (HBV) DNA and outcome of HBV infection in 5 infants with perinatal infection, 3 born to anti-hepatitis B e antigen (HBeAg), and 2 to HBeAg positive mothers. HBV infection developed in the babies at 3-4 months of age, but it resolved with seroconversion to anti-HBs in infants born to anti-HBe positive mothers, while the infection became chronic in the 2 babies born to HBeAg positive mothers. HBV-DNA extracted from the first hepatitis B surface antigen (HBsAg) positive serum sample of each baby was amplified and directly sequenced for the pre-core region. HBV-DNA sequences from 3 babies born to anti-HBe positive mothers showed at position 1896 the contemporary presence of 2 nucleotides (G+A), indicating a mixture of wild-type and "e minus" variant HBV. These findings suggest a possible co-transmission of the 2 viruses from anti-HBe positive mothers to newborns. HBV-DNA from babies born to HBeAg positive mothers showed wild-type sequences only. The results of this study suggest that the outcome of HBV infection in newborns depends not only on the host's immunocompetence and on viremia level in maternal blood, but also on heterogeneity of HBV. Transmission of mixed HBV populations appears associated with an early immunoelimination of the virus, while infection with wild-type HBV alone contributes to induction of chronicity.

HBe antibody unrelated to 'e minus' hepatitis B virus variant infection in patients with chronic type D hepatitis.

The presence of HBV-DNA sequences was evaluated in DNA extracted from serum samples, peripheral blood lymphocytes and liver biopsy specimens of five HBsAg/anti-HBe-positive carriers with chronic HDV infection. DNAs were tested by polymerase chain reaction (PCR) amplification technique using two pairs of oligonucleotide primers specific for the preC/C and S regions of the HBV; viral sequences were found exclusively in liver extracts and only in three out of the five cases. The direct sequencing of the amplified preC/C regions showed wild-type sequences in two cases, while in the third case a combination of 'wild' and 'e minus variant' viral populations was observed. Moreover, liver DNA of one positive case was electrophoresed through a low melting agarose gel and the following amplification, performed on DNA re-extracted from three different fragments of the gel, showed the presence of free HBV genomes but the absence of replicative intermediate forms. These results show that anti-HBe positivity is not constantly related to precore mutant HBV infection and suggest that HDV inhibits HBeAg production. Moreover, as it was observed in 'e minus' HBV variants, also during a chronic HBV wild-type infection, the viral replication might be suppressed to undetectable levels.

Hepatitis B virus variant, with a deletion in the preS2 and two translational stop codons in the precore regions, in a patient with hepatocellular carcinoma.

We analyzed hepatitis B virus (HBV) genomes obtained from serum samples and liver biopsy specimen of a chronic HBsAg/anti-HBe carrier with hepatocellular carcinoma (HCC). Before the liver biopsy, performed at the time of HCC diagnosis, the patient had been followed for 2 years; the serum samples collected in that period resulted negative for HBV-DNA dot blot hybridization. The hepatic DNA was at first examined by Southern blot, but no HBV sequence was detected. Polymerase chain reaction (PCR) amplification revealed the presence of HBV genomes in DNA extracted from the liver tissue and from two serum samples collected, respectively, 1 and 2 years before the biopsy. Direct sequence of the amplified preC/C and preS regions showed that the viral populations present in serum and liver were identical and that they had a 34 nucleotide deletion in the preS2 region, while the preC region presented two mutations each introducing a translational stop codon, one at the carboxy terminal end and the other at the second codon of the region, both able to prevent HBeAg expression. These results identify a new HBV variant which was selected during a chronic infection, and had very low levels of replication as shown by its detection only after PCR amplification.