Maria Luisa Biondi

Matrix Metalloproteinase-1 and Matrix Metalloproteinase-3 Gene Promoter Polymorphisms Are Associated With Carotid Artery Stenosis

Background and Purpose— The matrix metalloproteinases (MMPs) are a family of enzymes that are important in the resorption of extracellular matrix and are involved in atherogenesis. Recently, 2 common polymorphisms on MMP-1 (1G/2G) and MMP-3 (5A/6A) gene promoters have been described. The aim of this study was to investigate a possible association between MMP polymorphisms and increased risk of internal carotid artery (ICA) stenosis. Methods— We studied 91 patients consecutively recruited for ICA stenosis who had undergone carotid endarterectomy and 133 subjects without ICA stenosis (controls). Polymorphic genotypes were determined by polymerase chain reaction and sequencing analysis. Results— The frequency of the 6A allele was significantly different between cases and controls: 0.62 and 0.50, respectively (odds ratio [OR], 1.58; 95% CI, 1.08 to 2.33;P =0.017). The frequency of 6A/6A genotype was significantly higher in cases with involvement of both carotids (OR, 3.13; 95% CI, 1.14 to 8.5;P =0.026) and in patients with stenosis >70% (OR, 2.55; 95% CI, 1.07 to 6.07;P =0.033). No significant differences were observed in MMP-1 distribution. Patients who were homozygous for both the 6A and 2G alleles had an elevated relative risk of ICA stenosis (OR, 2.66; 95% CI, 1.23 to 5.72;P =0.016). Multiple logistic regression analysis using the common risk factors and the 6A and 2G allele variants revealed that the 6A allele was an independent risk factor for ICA stenosis (P =0.049). When 6A/6A and 2G/2G were combined, the risk factor for ICA stenosis was 3-fold higher (OR, 3.31; 95% CI, 1.48 to 7.42;P =0.004). Conclusions— Homozygosity for the 6A allele of the MMP-3 promoter is associated with carotid stenosis and, in association with MMP-1 2G homozygosity, predicts an increased risk of ICA stenosis. Even if obtained from a relatively limited patient series, these results might have relevant implications for treatment of ICA stenosis and possibly prevention of carotid-related stroke.

Progression of Coronary Artery Calcification in Predialysis Patients

Background: In patients on dialysis coronary artery calcification (CAC) rapidly proceeds due to impaired mineral metabolism and/or exogenous calcium load. Progression has not been assessed in patients with chronic kidney disease not yet requiring dialysis (CKD patients). In this study, rate and determinants of CAC progression have been evaluated in CKD patients who are exposed to minor derangement of mineral metabolism and calcium load. Methods: Consecutive patients were enrolled. Exclusion criteria were: symptomatic coronary disease, arrhythmia, myocardial infarction, and diabetes. Serum calcium, phosphorus, parathyroid hormone, homocysteine, C-reactive protein, triglycerides, total cholesterol, high- and low-density lipoprotein cholesterol were serially measured. Fetuin-A was assessed at entry into the study. CAC progression was detected by measuring total calcium score (TCS) with computed tomography. Initial and final scans were obtained. Predictive factors of progression were investigated. Results: Fifty-three patients had CKD (stage 3–5 CKD; K-DOQI classification) not yet requiring dialysis, and 60 patients had normal renal function (NRF patients). Follow-up lasted 24 ± 4.2 months (mean ± SE). Patients with CAC were older with lower serum fetuin-A. TCS increased from 73 ± 17 to 80 ± 20 (mean ± SE; p = NS) in NRF patients, and from 384 ± 116 to 602 ± 140 (mean ± SE; p < 0.01) in CKD patients. Serum phosphorus [OR = 1.97 (1.14–3.41, 95% CI); p = 0.015] was the only variable that was associated with CAC progression. Cardiovascular events occurred in CKD patients with CAC. Conclusion: CAC progression was prominent in CKD patients and correlated with serum phosphorus. Fatal and nonfatal cardiovascular events were more frequent in CKD patients. Studies are required to ascertain whether the attainment of serum phosphorus concentration lower than that suggested by current guidelines may reduce CAC progression and ultimately mortality.

Matrix GLA Protein Gene Polymorphisms: Clinical Correlates and Cardiovascular Mortality in Chronic Kidney Disease Patients

Background: Increased vascular calcification plays an important role in the pathogenesis of cardiovascular events in chronic kidney disease (CKD) patients. It is the result of an active ossification process counteracted by ‘protective’ proteins, such as matrix GLA protein (MGP). Polymorphisms of MGP have been identified. Methods: The aim of this study was to define the distribution of two MGP polymorphisms (–7, –138) in 99 hemodialysis (HD) patients, in 26 patients with CKD stage 3 and in 135 age- and sex-matched healthy controls. Patients were followed up for 12 months to record any cardiovascular deaths. The cause of death was determined by medical doctors, considering the medical history of each patient. The primers were designed with Primer Express software. Results: MGP –138TT homozygotes were more frequent in the HD group versus controls (p = 0.0004). Additionally, the frequency of the T allele was significantly higher in the HD group (p = 0.0006). The frequency of the A allele of MGP-7 was significantly higher both in the HD group (p = 0.033) and in the CKD group (p = 0.0017) versus controls. MGP-7 GG homozygotes were significantly less common in the CKD group than in controls (p = 0.037). Combination –138TT –7AA was significantly more frequent in both CKD patients (p = 0.001) and in HD patients (p = 0.029) than in controls. Seventeen out of 99 HD patients experienced fatal cardiovascular events. Sixteen (94.1%) were –138TT homozygotes and either –7AA homozygotes or –7GA heterozygotes. Conclusion: This study suggests that CKD and HD patients have a different distribution of MGP gene polymorphism as compared with the normal population. Altered MGP gene polymorphism may be a negative prognostic factor for the progression to end-stage renal disease and for cardiovascular events in CKD patients.

Plasma free and intraplatelet serotonin in patients with Raynaud's phenomenon☆

Plasma free and intraplatelet serotonin concentrations were measured by high-performance liquid chromatography coupled with electrochemical detection, in 30 patients with Raynauds phenomenon of various etiologies. Serotonin was significantly higher in plasma (P less than 0.005) and in platelets (P less than 0.005) from Raynauds patients than from normal controls. Moreover, plasma circulating serotonin could differentiate primary from secondary Raynauds phenomenon, with significantly higher levels (P less than 0.05) for patients with an underlying connective tissue disease. Our data indicate a role for serotonin in Raynauds phenomenon.

Serum fetuin-A levels link inflammation and cardiovascular calcification in hemodialysis patients

Background: Cardiovascular disease (CVD) is the leading cause of mortality in hemodialysis (HD). An elevated incidence of cardiovascular calcifications (CVC) is observed in HD. Fetuin-A is an important inhibitor of CVC. Reduced fetuin-A levels associate with inflammation and increased cardiovascular (CV) mortality in HD. In this study we investigated the association of fetuin-A levels and CVC. Method: We evaluated a cohort of 115 patients (67 males), aged 63 ± 16 years with a HD vintage ≧9 months. Presence of CVC was assessed by ultrasound imaging of the abdominal aorta, common carotid arteries, bilateral ilio-femoral axis, aortic and mitral cardiac valves. The presence of CVC was analyzed as a CVC score (CVCS) (0–7) according to the number of CVC sites. Patients were arbitrary stratified in three groups: group I (CVCS = 0), group II (0 < CVCS < 6) and group III (CVCS ≧ 6). Patients without CVC were younger, non-diabetic and with a negative history for CV events. Results: Patients with evidence of CVC in more than 5 sites had lower serum fetuin-A levels (0.41 ± 0.22 g/l) compared to patients with CVCS = 0 (0.51 ± 0.17 g/l, p = 0.048). In addition a worse CVCS was associated with higher serum levels of C-reactive protein (p = 0.002) and fibrinogen (p < 0.001). Serum fetuin-A levels lower than 0.290 g/l were associated with higher risk of a worse CVCS, independently from traditional risk factors. Conclusion: Chronic inflammation in HD patients leads to lower serum fetuin-A levels. The present study confirms the independent and significant association between reduced serum fetuin-A levels and multi-site CVC in HD.

Internal carotid artery occlusive disease and polymorphisms of fractalkine receptor CX3CR1: A genetic risk factor

Background and Purpose— Fractalkine (FKN), a chemokine expressed by inflamed endothelium, induces leukocyte adhesion and migration via the receptor CX3CR1. The polymorphisms V249I and T280M affect receptor expression and function. The role of FKN in atherosclerosis has been recently demonstrated. The aim of this study was to investigate a possible association between CX3CR1 polymorphisms and increased risk of internal carotid artery (ICA) occlusive disease. Methods— We studied 108 patients consecutively recruited for ICA occlusive disease, 84 of whom underwent operation for carotid endarterectomy, and 204 subjects without ICA occlusive disease (controls). Polymorphic genotypes were determined by polymerase chain reaction and sequencing analysis. Results— The adjusted odds ratio (OR) associated with the presence of the M280 (TM+MM versus TT genotype) was 0.55 (95% CI: 0.29 to 0.99; P = 0.037). Therefore, this allele is associated with a reduced risk of ICA occlusive disease. No significant differences were observed in I249 distribution. The frequency of I249 allele was significantly higher in cases of hard plaques, which are considered more stable than soft ones (OR: 0.38; 95% CI: 0.13 to 1.05; P = 0.037). Multiple logistic regression analysis using the common risk factors and the I249 and M280 allele variants revealed that the M280 allele was an independent risk factor for ICA stenosis (P = 0.047). Conclusion— The results show that the CX3CR1 M280 is an independent genetic risk factor for ICA occlusive disease and that I249 is involved in the stability of carotid plaques. Even if obtained from a relatively limited patient series, these results might have relevant implications for treatment of ICA stenosis and possibly prevention of carotid related stroke. Further prospective cross-sectional studies are needed to confirm these results.

PPAR-γ2 Pro12Ala Variant, Insulin Resistance and Plasma Long-chain Polyunsaturated Fatty Acids in Childhood Obesity

Pro12Ala variant of peroxisome-proliferator-activated receptor-gamma2 (PPAR-γ2) may be linked to insulin sensitivity. This study examined whether an association of PPAR-γ2 Pro12Ala with insulin resistance and plasma LCPUFAs may exist in obese children. One hundred and forty Italian normolipidemic obese children (58 girls and 82 boys, mean age [SD], 10.2 [2.7] y) entered the study. Obesity was defined according to International Obesity Task Force. BMI Z-scores were calculated. Fasting blood glucose, insulin, lipids and plasma fatty acids were measured. Insulin resistance was estimated by the homeostatic model assessment (HOMA-IR). The frequency of Ala allele was 9%. Mean [SD] values of fasting insulin and HOMA-IR in Pro/Pro versus Pro12Ala groups were: 19.3 [10.6] versus 14.1 [10.4] μU/mL (p = 0.017) and 4.2 [2.3] versus 3.0 [2.3] (p = 0.022). Mean [SD] values of plasma C20:3n-9 and of C20:4n-6, C20:5n-3, C22:6n-3 and n-6/n-3 LCPUFA in phospholipds in Pro/Pro versus Pro12Ala groups were: 0.15 [0.07] versus 0.12 [0.08] % (p = 0.014), 8.9 [1.9] versus 10.2 [2.6] % (p = 0.023), 0.34 [0.15] versus 0.42 [0.11] % (p = 0.005), 2.1 [0.9] versus 2.6 [0.9] % (p = 0.032) and 4.8 [1.2] versus 4.2 [0.7] (p = 0.017). Pro12Ala may be associated with higher insulin sensitivity and higher LCPUFAs, particularly n-3, levels in plasma phosholipids of obese children.

Cigarette smoking and platelet function

To assess the influence of cigarette smoking on platelet activation, we studied the changes in intraplatelet and platelet-released serotonin (5-HT) and plasma levels and platelet-associated production of thromboxane B2 (TXB2), in 6 non smokers and 6 habitual smokers, before and after acute exposure to smoke. Before smoking, habitual smokers showed slightly higher, albeit not significantly, 5-HT platelet concentrations and TXB2 plasma levels, as well as lower TXB2 platelet production after collagen and even more after ADP stimulation (0.59 +/- 0.27 vs 1.35 +/- 0.46 and 0.99 +/- 0.47 vs 2.08 +/- 0.51 ng/10(8) platelets for habitual smokers vs controls, 4 and 10 min after ADP, p less than 0.02). No significant differences in platelet 5-HT release were observed. Acute smoking did not induce any significant change from baseline in either 5-HT or TXB2 for controls, while significantly reduced TXB2 production from ADP-challenged platelets from habitual smokers (0.30 +/- 0.15 vs 0.59 +/- 0.27 ng/10(8) platelets, immediately after smoking vs baseline, p less than 0.01). Ninety min after the completion of the smoking, the values had returned to baseline. Immediately after smoking, significant differences were found between habitual smokers and controls for TXB2 platelet production (2.76 +/- 1.78 vs 6.42 +/- 1.60, p less than 0.025 and 3.01 +/- 1.90 vs 6.44 +/- 2.26 ng/10(8) platelets, p less than 0.05, for habitual smokers vs controls, 4 and 10 min after the addition of collagen; 0.30 +/- 0.15 vs 1.20 +/- 0.84 and 0.79 +/- 0.50 vs 1.70 +/- 0.74 ng/10(8) platelets, p less than 0.05, after ADP stimulation). Differences were no longer significant 90 min after smoking. Our data indicate that cigarette smoking is associated with platelet dysfunction, which seems due to impairment of metabolic platelet capacity rather than increased platelet activation in vivo.

Polymorphism of the fractalkine receptor CX3CR1 and systemic sclerosis-associated pulmonary arterial hypertension.

Fractalkine (FKN) and its receptor CX3CR1 are critical mediators in the vascular and tissue damage of several chronic diseases, including systemic sclerosis (SSc) and pulmonary arterial hypertension (PAH). Interestingly, the V249I and T280M genetic polymorphisms influence CX3CR1 expression and function. We investigated whether these polymorphisms are associated with PAH secondary to SSc. CX3CR1 genotypes were analyzed by PCR and sequencing in 76 patients with limited SSc and 204 healthy controls. PAH was defined by colorDoppler echocardiography. Homozygosity for 249II as well as the combined presence of 249II and 280MM were significantly more frequent in patients with SSc compared to controls (17 vs 6%, p = 0.0034 and 5 vs 1%, p = 0.0027, respectively). The 249I and 280M alleles were associated with PAH (odd ratio [OR] 2.2, 95% confidence interval [CI] 1.01-4.75, p = 0.028 and OR 7.37, 95%CI: 2.45-24.60, p = 0.0001, respectively). In conclusion, the increased frequencies of 249I and 280M CX3CR1 alleles in a subgroup of patients with SSc-associated PAH suggest a role for the fractalkine system in the pathogenesis of this condition. Further, the 249I allele might be associated with susceptibility to SSc.

Genetic variants of endothelial nitric oxide synthase in patients with primary biliary cirrhosis: Association with disease severity

Background and Aims:  Primary biliary cirrhosis (PBC) presents a wide spectrum of clinical manifestations. In experimental models of liver cirrhosis and cholestasis it has been suggested that altered nitric oxide production is involved in liver injury and portal hypertension development. The present study investigated endothelial nitric oxide synthase (eNOS) genetic polymorphisms (894G/T, −786T/C) in patients with PBC and in controls to verify whether disease susceptibility and progression are associated with a particular genotype.