Abstract: Skeletal muscle mitochondrial dysfunction and insulin resistance occur in chronic kidney disease. Ghrelin is a gastric hormone previously shown to enhance muscle mitochondrial enzyme activities and AKT-mediated insulin signaling independent of food intake in healthy rats. Here we determined the impact of ghrelin treatment on anorexia, skeletal muscle mitochondrial oxidative capacity, AKT phosphorylation as a measure of insulin signaling, and lean body mass in a rat model of chronic kidney disease. Ghrelin infusion promoted higher food intake and lean body mass. Further, although muscle mitochondrial enzyme activities were low in the rats with CKD (chronic kidney disease), they normalized with ghrelin treatment, a change that was consistent with the increase in the transcript levels of regulators of mitochondrial biogenesis and lipid metabolism. This was associated with a lower muscle triglyceride content and higher AKT phosphorylation. Pair-feeding showed that mitochondrial effects of ghrelin are independent of changes in food intake, whereas combined ghrelin treatment and higher food intake were needed to enhance AKT phosphorylation. Thus, ghrelin-induced muscle mitochondrial changes and lower tissue triglycerides could favor insulin action and muscle anabolism in the presence of improvement in food intake. Our study shows that combined effects of ghrelin on appetite and muscle mitochondria improve muscle metabolic and nutritional alterations in chronic kidney disease. This could have potential beneficial impact on patient morbidity and survival.
Abstract: Insulin resistance often characterizes chronic uremia, and is associated with enhanced morbidity and mortality, because it may contribute to protein-energy wasting (in turn, an independent predictor of reduced survival), atherosclerosis, and cardiovascular death. Causes of insulin resistance in chronic uremia are complex and multifactorial. Obesity is emerging as an independent risk factor for chronic kidney disease, and an expected rise in number of obese uremic patients because of the ongoing worldwide obesity epidemic is likely to increase the prevalence of insulin resistance in chronic uremia in the near future. Similar to the general population, reported associations between obesity and insulin resistance in chronic uremia support a role of adipose tissue and altered adipokine profiles in insulin resistance in obese chronic kidney disease patients. Hormonal imbalances, chronic acidosis, and systemic inflammation and oxidative stress are uremia-associated relevant causes of insulin resistance in nonobese individuals. A further understanding of the causes of insulin resistance in chronic uremia represents a potential important tool in the design of more effective therapeutic strategies to reduce uremia-associated morbidity and mortality.
Abstract: Despite the broad anti-tumour potential of the proteasome inhibitor bortezomib, partial information is available with regard to its effects on hepatocellular carcinoma (HCC) cells. Here we studied the effects of bortezomib on two human HCC cell lines displaying a different phenotype, hepatocyte-like for HepG2 and undifferentiated for JHH6. Bortezomib induced a dose- and time-dependent increase in cell toxicity and decrease of cell viability, with JHH6 being less sensitive than HepG2. Moreover, a differential influence on major cell cycle regulatory genes was responsible for the observed decrease of S and increase of G(2)-M phase cells. In HepG2, bortezomib induced a post-transcriptional increase of cyclin E1 together with a transcriptional-mediated decrease of the transcription factor E2F1. This in turn resulted in the reduction of the hyper-phosphorylated form of pRB and in the transcriptional down-regulation of the E2F1 targets cyclin D1, cyclin A2 and CdK2 but not cyclin E1. Up-regulation of LRH1, a liver specific cyclin E1 transcription factor, accounted for the unvaried cyclin E1 mRNA levels. Additionally, bortezomib induced both transcriptional and post-translational increase of p21(waf1/cip1) and p27(kip1). In JHH6, an overall more contained variation in cell cycle mediators was observed with the reduction of E2F1, cyclin A2, LRH1 and the increase of p21(waf1/cip1) being the most evident. In conclusion, the presented data show the mechanisms regulating cell proliferation inhibition by bortezomib in two different HCC cell lines. Despite a certain phenotype-dependent effect, the potent action exerted by bortezomib makes this drug attractive for future experimentation in animal models, possibly leading to novel treatments for HCC.
Abstract: Metabolic syndrome is characterized by increased cardiovascular risk. Pentraxin 3 (PTX3), an acute phase protein, is involved in atherosclerosis. No information is available on PTX3 plasma concentrations in metabolic syndrome and on its associations with metabolic alterations and subclinical atherosclerosis. The aim of this study was to assess PTX3 plasma levels in metabolic syndrome patients compared to control subjects and their potential associations with anthropometric and clinical components of the syndrome as well as with carotid artery intima-media thickness (cIMT), a marker of subclinical atherosclerosis. Plasma was obtained from metabolic syndrome patients (NCEP-ATP III criteria n = 41, 20 M/21F) and by age-matched control subjects (n = 32, 16 M/16F). PTX3 was measured using sandwich ELISA and cIMT with ultrasound. Compared to those of the control subjects, plasma levels of PTX3 were higher (? * 100%, P = 0.0009) in metabolic syndrome patients. In univariate analysis, plasma PTX3 was negatively (P = 0.005) associated with high-density lipoprotein (HDL) cholesterol and positively (P = 0.046) with plasma triglycerides and with cIMT (P = 0.045) in the patients (n = 41). In multivariate analysis the direct association between PTX3 and cIMT was no longer significant after correction for HDL. None of these associations were detected in the control patients. These data demonstrate that PTX3, a novel marker of vascular disease, is higher in patients with metabolic syndrome associated with subclinical atherosclerosis. In addition, PTX3 is significantly independently correlated with low HDL cholesterol, but not with cIMT, suggesting a novel association between PTX3 and atherogenic lipid profile.
Abstract: Low-grade systemic inflammation is an important potential factor in the pathogenesis of insulin resistance in end-stage renal disease (ESRD). Insulin resistance and diabetes, characterized by impaired skeletal muscle glucose uptake or excess hepatic glucose production, are in turn relevant contributors to morbidity and mortality in ESRD patients. Oxidative stress is increased in ESRD, in conservative therapy as well as hemodialysis treatment. Recent evidence suggests that oxidative stress contributes, at least in part, to both inflammation and insulin resistance by modulating the production of proinflammatory cytokines and adipokines in monocytes and in adipose tissue. This review focuses on the pathogenesis of inflammation and oxidative stress, and the effects of their interplay on insulin resistance in ESRD.
Abstract: BACKGROUND: Impaired protein anabolism and insulin resistance are characteristic features of maintenance haemodialysis patients. We have used a randomised, matched-paired, double-blind, placebo-controlled experimental design to determine the capability of intravenous L-carnitine supplementation to modify insulin resistance and protein catabolism in non-diabetic patients with end-stage renal disease (ESRD) undergoing chronic haemodialysis treatment. METHODS: L-carnitine (20 mg x kg(-1)) (n = 9) or placebo (n = 10) were given intravenously at the end of seven consecutive dialysis sessions. Whole-body protein and glucose metabolism were assessed on interdialytic days by the L[1-(13)C]leucine and the [2,2-(2)H(2)]glucose kinetic models in the postabsorptive state and during euglicemic hyperinsulinemic clamp studies at baseline and at the end of the treatment period. RESULTS: L-carnitine supplementation was associated with lower (P < 0.05) rates of leucine oxidation (-11 +/- 12%) and appearance from proteolysis (-6 +/- 2%) during the clamp studies than after placebo supplementation. The rates of glucose appearance in the postabsorptive state did not change significantly in the patients receiving L-carnitine treatment. Insulin-mediated glucose disappearance was improved by L-carnitine only in those patients (n = 5) (+18 +/- 3%, P < 0.05 vs placebo group, n = 5) with greater baseline insulin resistance, selected according to the median value of insulin sensitivity before treatment. CONCLUSIONS: L-carnitine supplementation was associated with protein-sparing effects in maintenance haemodialysis patients during hyperinsulinemia.
Abstract: Fenofibrate has beneficial effects on the progression and clinical emergence of atherosclerosis in normoglycemic and in diabetic patients. Given the involvement of endothelium in these processes, we speculated that fenofibrate may influence endothelial cell apoptosis and proliferation, regulators of endothelium integrity. Fenofibrate effects on apoptosis and proliferation were studied in human umbilical vein endothelial cells under normal (5.5 mmol/l, NG) and high (22 mmol/l, HG) glucose with or without fenofibrate (50 micromol/l). Apoptosis was evaluated by annexin V, by poly(ADP-ribose) polymerase protein cleavage, and cyclooxygenase-2 (COX-2), Bax/Bcl-2, and p53 protein levels; proliferation was assessed by determining cell cycle phase distribution and the amounts of the cell cycle regulators E2F1, cyclin D1, E1, and A and the levels of the hyper-phosphorylated form of the retinoblastoma protein (ppRb). HG resulted in increased (p<0.05) apoptosis rate associated with COX-2 protein overexpression, without modification of Bax/Bcl2 ratio and p53 levels. Fenofibrate decreased apoptosis and normalized increased COX-2 expression in HG (p<0.05). Both in HG and NG, fenofibrate dramatically reduced cell proliferation (p<0.05) through a G1/G0 block mediated by the reduction in ppRb and the decrease in E2F1, cyclin E1, A, and D1 protein expression, with a mechanism that, for cyclin E1, occurred at the posttranscriptional level. In conclusion, our data show that fenofibrate reduces apoptosis caused by HG but severely interferes with endothelial cell proliferation both in NG and HG. The resulting effect may influence endothelium integrity in vivo and may impact the outcome of acute complications of atherosclerosis in diabetes.
Abstract: Alcoholic beverages are known to exert a protective effect on atherosclerotic disease. This study aimed to assess the in vivo and in vitro effects of alcohol on matrix metalloproteinase (MMP) -2 and -9, known to determine atherosclerosis progression. Eighteen healthy volunteers, regular drinkers (two standard alcohol servings/day, on average) at first examination (baseline) were asked to abstain from any alcoholic beverage for one week (abstention), and then to assume two standard alcohol servings of beer daily for 1 week (re-exposure). Activity of MMP-2 and -9, total antioxidant activity (AOA), glutathione (GSH) plasma levels were carried out at baseline, at the end of abstention, and after 1 week of re-exposure. To validate the in vivo results, MMP-2 activity and expression, AOA, and GSH, were determined in human smooth muscle cells treated for 96 h with increasing concentrations (12.5-100 mM) of ethanol. MMP-2, but not MMP-9 plasma activity was higher at abstention than at baseline or re-exposure (P<.001 and P< or =.005, respectively). Changes in AOA and GSH throughout the study were not significant. No correlation was found between MMPs and antioxidant activity. In vitro, ethanol at 25 mM reduced by around 10% MMP-2 activity (P=.003) in smooth muscle cells, whereas MMP-2 expression, AOA, and GSH were unaffected. Alcohol reduces MMP-2 plasma activity in healthy humans and in isolated vascular smooth muscle cells. This in vitro reduction is unrelated to MMP-2 expression in vascular cells or to antioxidant levels changes.
Abstract: INTRODUCTION: Experimental models and ex-vivo studies suggest a crucial role of some matrix metalloproteinases (MMPs) in the development of acute coronary syndromes, but expression levels of MMP-2, MMP-9 and TIMP-1 in human coronary plaques causing stable angina or an acute coronary syndrome have not been reported, yet. METHODS: MMP-2, -9 and TIMP-1 expressions were assessed by real-time PCR from the debris collected into distal protective vascular guards from patients with stable angina (SA-Group, n=16), acute coronary syndrome (ACS-Group, n=16) undergoing percutaneous coronary interventions (PCI). MMP-2 and -9 activities were also evaluated by gelatin-substrate zymography on plasma samples collected immediately before PCI, and compared to those of healthy subjects (Control-Group). RESULTS: The expression of MMP-2 was similar in ACS and SA-Groups. MMP-9 (P=0.011), but not TIMP-1, expression was higher in debris samples from patients in the ACS-Group than in SA-Group. In both groups, the expression of MMP-2 and MMP-9 were inversely correlated (rho=-0.7; P<0.004). Zymography data indicated that pro and active MMP-9 were higher in ACS than in SA-Group, while no difference in MMP-2 was found. CONCLUSIONS: MMP-9, but not TIMP-1 or MMP-2 expression is increased in plaques causing acute coronary syndrome.
Abstract: We tested the relative ability of rapidly digested whey and slowly digested casein to stimulate net whole-body protein synthesis during prolonged physical inactivity. We studied 8 young male volunteers after they consumed isonitrogenous casein or whey mixed meals on d 12 or d 14 of experimental bed rest. Rates of phenylalanine hydroxylation were measured by primed-constant oral administration of L[2-(2)H(2)]tyrosine and L[ring-(2)H(5)]phenylalanine for 3 h in the postabsorptive state and 6 h after an isonitrogenous bolus meal containing sucrose (0.27 g/kg) and casein or whey (0.40 g/kg). Net protein synthesis in the fed state was calculated during the first 6 h postmeal as the difference between phenylalanine hydroxylation and phenylalanine content in the ingested casein or whey. In the fed state, the integrated changes in phenylalanine hydroxylation were lower (P < 0.05) after whey (-2 +/- 8 micromol x kg(-1) x 6 h(-1)) than after casein ingestion (34 +/- 7 micromol x kg(-1) x 6 h(-1)). During bed rest, net postprandial protein synthesis was greater (P < 0.05) after whey (96 +/- 8 micromol phenylalanine x kg(-1) x 6 h(-1)) than after casein ingestion (82 +/- 7 micromol phenylalanine x kg(-1) x 6 h(-1)). The rapidly digested whey protein was more efficient than the slowly digested casein in increasing postprandial net protein synthesis during short-term bed rest.
Abstract: Glutamine synthesis and utilization are strictly linked to energy metabolism and physical activity. To investigate the interaction between bed rest and moderate energy restriction on whole body glutamine kinetics in healthy volunteers, we performed a four-period study in which each subject randomly underwent a bed rest or ambulatory 14 day period both in eucaloric or hypocaloric (-20% of energy requirement) conditions. Glutamine kinetics were measured by l-[5-(15)N]glutamine and l-[1-(13)C]leucine primed continuous infusions in the postabsorptive state and during a 3 h infusion of a glutamine-free amino acid mixture (0.13 g amino acids (kg lean body mass (LBM))(-1) h(-1)). Bed rest decreased glutamine de novo synthesis in the postabsorptive state both in eucaloric (from 4.17 +/- 0.14 to 3.56 +/- 0.13 micromol (kg LBM)(-1) min(-1); P < 0.001) and in hypocaloric (from 3.79 +/- 0.19 to 3.49 +/- 0.14 micromol (kg LBM)(-1) min(-1); P < 0.001) conditions, independently of changes in whole body proteolysis. Bed rest did not affect glutamine clearance. We failed to detect either significant effects of energy intake or energy x activity interactions on glutamine kinetics. Bed rest significantly decreased postabsorptive plasma glutamine concentrations (P < 0.05). Amino acid infusion increased glutamine de novo synthesis rate and plasma glutamine concentrations in all conditions, without significant effects of bed rest or energy levels. We conclude that inactivity is associated with decreased whole body glutamine availability due to down-regulated de novo synthesis.
Abstract: CONTEXT: Energy balance and physical activity potentially influence systemic inflammation. OBJECTIVE: Our objective was to test the hypothesis that moderate energy restriction may prevent activation of inactivity-induced inflammatory response. DESIGN: Participants were studied four times at the end of 14-d periods of experimental bed rest or controlled ambulation, after receiving eucaloric or hypocaloric diets. SETTING: The study was conducted at the clinical research center of the German Space Agency. SUBJECTS: Nine healthy young volunteers participated. INTERVENTIONS: Energy intake was calibrated to physical activity and decreased by about 20% in hypocaloric conditions. MAIN OUTCOME MEASURES: Changes in body fat by dual-energy x-ray absorptiometry as well as plasma inflammatory markers and cytokine mRNA levels in blood cells were measured. Results: Fat mass did not change significantly in eucaloric conditions and decreased in hypocaloric periods (-1.0 +/- 0.3 and -1.0 +/- 0.3 kg in ambulatory and bed rest, respectively). Bed rest in eucaloric conditions increased plasma C-reactive protein (CRP) (+143 +/- 53%) and both the ratios between plasma IL-6 and IL-10 (4+/-1 times) and white blood cell IL-6 and IL-10 mRNAs (5 +/- 1 times). Energy restriction prevented bed-rest-mediated increases in CRP and the IL-6 to IL-10 ratio. Bed rest increased (P = 0.03) long pentraxin-3 (PTX3) plasma concentration, without significant activity-by-diet interaction. In all conditions (n = 36), CRP and PTX3 were inversely correlated (r = -0.61; P < 0.001). Changes in fat mass, leptin, and IL-6 directly correlated with CRP and inversely correlated with PTX3. IL-10 inversely correlated with CRP and directly correlated with PTX3 (r = 0.52; P < 0.01). CONCLUSIONS: Calorie restriction prevents the inflammatory response induced by 14 d of bed rest. We suggest an inverse regulation of CRP and PTX3 in response to changes in energy balance.
Abstract: OBJECTIVE: Cancer and surgical stress interact to aggravate insulin resistance, protein catabolism, and glutamine depletion in skeletal muscle. We compared the effects of insulin-mediated euglycemia and moderate hyperglycemia on kinetics of protein and selected amino acids in skeletal muscle of female cancer patients after major surgery. DESIGN: In each patient, a 24-hr period of insulin-mediated tight euglycemia (mean blood glucose, 5.8 +/- 0.4 mmol/L) preceded or followed a 24-hr control period of moderate hyperglycemia (mean blood glucose, 9.6 +/- 0.6 mmol/L) on the first and second day after surgery, in randomized order, according to a crossover experimental design. SETTING: Intensive care unit, cancer hospital. PATIENTS: Cancer patients after abdominal radical surgery combined with intraoperative radiation therapy. INTERVENTIONS: Intensive (57 +/- 11 units/24 hrs) and conventional (25 +/- 5 units/24 hrs) insulin treatment during total parenteral nutrition. MEASUREMENTS AND MAIN RESULTS: Muscle metabolism was assessed at the end of each 24-hr period of euglycemia and of hyperglycemia by leg arteriovenous catheterization with stable isotopic tracers. We found that euglycemia as compared with hyperglycemia was associated with higher (p < .05) fractional glucose uptake (16% +/- 4% vs. 9% +/- 3%); higher (p < .05) muscle protein synthesis and neutral net protein balance (-3 +/- 3 vs. -11 +/- 3 nmol phenylalanine x 100 mL(-1) x min(-1), respectively); lower (-52% +/- 12%, p < .01) muscle nonprotein leucine disposal (an index of leucine oxidation) and higher (p < .05) plasma leucine concentrations; and higher (3.6 +/- 1.7 times, p < .01) net de novo muscle glutamine synthesis and plasma glutamine concentrations (p < .05). Euglycemia was associated with higher (23% +/- 7%, p < .05) plasma concentrations of arginine but did not affect either arginine release from muscle or plasma concentration and muscle flux of asymmetrical dimethylarginine. Rate of muscle proteolysis correlated (p < .05) with muscle release of asymmetrical dimethylarginine. CONCLUSIONS: Treating hyperglycemia improves skeletal muscle protein and amino acid metabolism in cancer patients after major surgery.
Abstract: Carnitine is a conditionally essential metabolite that plays a critical role in cell physiology. Carnitine is necessary for fatty acid transport to sites of beta-oxidation in the mitochondria, where it helps to prevent organic acid accumulation. Because of these key regulatory functions, carnitine represents a crucial determinant of mitochondrial energy metabolism, whose deficiency may lead to metabolic and clinical disturbances. Loss of carnitine through dialytic membranes occurs in maintenance hemodialysis, resulting in potential carnitine depletion and relative increments of esterified carnitine forms. Carnitine supplementation has been shown to counteract such alterations and may be associated with clinical benefit. In particular, carnitine supplementation in patients on hemodialysis may enhance response to erythropoietin, resulting in improved hematologic status. Carnitine was also reported to improve exercise tolerance and intradialytic symptoms. Carnitine supplementation may enhance insulin resistance, inflammatory and antioxidant status, protein balance, lipid profile, and cardiac function. Carnitine administration can be useful for selected patients on dialysis who do not adequately respond to standard therapy.
Abstract: CONTEXT: Metabolic syndrome shows clustered metabolic abnormalities with major roles for insulin resistance and obesity. Ghrelin is a gastric hormone whose total plasma concentration (T-Ghr) is associated positively with insulin sensitivity and is reduced in obesity. Ghrelin circulates in acylated (A-Ghr) and desacylated (D-Ghr) forms, but their potential differential associations with insulin resistance and whether they are differentially altered in obesity remain undefined. OBJECTIVE: Our objective was to determine potential differential associations of ghrelin forms with insulin resistance [homeostasis model assessment of insulin resistance (HOMA-IR)] and the impact of obesity on their plasma concentrations in metabolic syndrome. DESIGN: This is a cross-sectional study. SETTING: The study was performed in a metabolic outpatient unit. PATIENTS: Patients with metabolic syndrome (National Cholesterol Education Program-Adult Treatment Panel III; n = 45, 23 males/22 females) were included in the study. MAIN OUTCOMES: The main study outcomes were metabolic syndrome criteria, HOMA-IR, and ghrelin forms. RESULTS: Plasma insulin and HOMA-IR were associated negatively with T-Ghr and D-Ghr but positively with A-Ghr and acylated to desacylated ghrelin (A/D-Ghr) ratio (n = 45; P < 0.05). Compared with nonobese [body mass index (BMI) < 27.5 kg/m(2); n = 12, six males/six females], obese metabolic syndrome patients (BMI > 27.5 kg/m(2); n = 33) had lower T-Ghr and D-Ghr but comparable A-Ghr and higher A/D-Ghr ratio (P < 0.05). BMI and waist circumference (WC) were positively related with HOMA-IR (n = 45; P < 0.05). However, opposite associations between A/D-Ghr ratio and HOMA-IR remained significant after adjustment for sex and BMI (or WC). Additional obese individuals without metabolic syndrome (n = 10: age-, sex-, BMI-, and WC-matched to obese metabolic syndrome patients) had lower T-Ghr but higher A-Ghr (P < 0.05) compared with age-, sex-matched healthy nonobese counterparts (n = 15). T-Ghr and A-Ghr were comparable in obese with or without metabolic syndrome. CONCLUSION: Obesity could alter circulating ghrelin profile, and relative A-Ghr excess could contribute to obesity-associated insulin resistance in metabolic syndrome.
Abstract: Our goal was to set up a pilot study to explore the possible relation between the expression of p66((ShcA)) and PTX3, two emerging regulators of stress response and inflammation processes, respectively, and the circulating levels of LDL-cholesterol (LDL), a factor implicated in the development of inflammation and oxidative-stress associated diseases such as atherosclerosis. p66((ShcA)) and PTX3 mRNA contents were determined locally, in subcutaneous adipose specimens of non-diabetic pacemaker-implanted patients, and systemically in the circulating white blood cells (WBC) obtained from the same patients. The mean of the circulating LDL levels (125 mg/dl) was chosen as a threshold to identify two groups here considered to have high (>125 mg/dl) and low (<125 mg/dl) LDL plasma levels. Our data show that PTX3 and p66((ShcA)) mRNA levels are significantly more elevated in WBCs and in adipose tissue samples of patients with high levels of LDL compared to those with low levels. Additionally, a multiple regression analysis indicates that among LDL, TG, HDL, total cholesterol, CRP, creatinine and glucose levels, the only variable significantly affecting p66((ShcA)) and PTX3 mRNA expressions either in adipose tissue or in WBCs is represented by the circulating amount of LDL. In conclusion, our results suggest a potential link between the level of LDL and the expression of two genes involved in inflammation/oxidative stress pathways, i.e., p66((ShcA)) and PTX3, thus contributing to further understand the mechanism through which LDL may mediate the pathogenesis of inflammation and oxidative-stress associated diseases such as atherosclerosis.
Abstract: Chronic oxidative stress that characterizes uremia has potentially devastating effects on the vasculature and has been advocated in the pathogenesis of accelerated atherosclerosis in this disease. Recent advances have been made in our understanding of the molecular mechanisms that regulate expression and activity of key enzymes of vascular oxidative stress (eg, nicotinamide adenine dinucleotide phosphate [NAD{P}H] oxidase) and that dissect their interactions with signalling pathways of inflammation. The finding that NAD(P)H oxidase is upregulated in experimental uremia has important consequences from a physiologic and a therapeutic standpoint. In addition, identification of novel proteins involved in systemic oxidative stress has shed some new light on the pathogenesis of vascular disease. p66(shc) is a cytoplasmic protein that is expressed in a wide range of cell types. Initially believed to be involved in signalling pathways that regulate cell growth and oxidative stress, it has now been shown to play a pivotal role in promoting endothelial dysfunction and atherosclerosis. Although a specific role in uremia-related vascular disease has not yet been shown, available data in humans suggest involvement of p66(shc) in clinical conditions associated with increased oxidative stress.
Abstract: OBJECTIVES AND METHODS: The present study was designed to evaluate the role of some inflammation [interleukin (IL)-1beta, soluble IL-1 receptor, IL-1 receptor antagonist (IL-1RA), high-sensitivity C-reactive protein (hsCRP) and fibrinogen], and remodeling markers [matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2] in patients with acute coronary syndrome (ACS; 40 patients), or chronic stable angina (CSA; 40 patients) compared to age- and sex-matched healthy controls (20 subjects). RESULTS: IL-1RA, hsCRP, fibrinogen, MMP-9, and TIMP-1 plasma levels were significantly higher in patients than in controls, whereas soluble IL-1 receptor had an opposite pattern. Among patients with ACS, hsCRP plasma levels were higher in patients with non-ST segment elevation myocardial infarction (NSTEMI) than in those with unstable angina (UA). TIMP-1 plasma levels were higher in those patients with ACS who did not respond to medical therapy (non-responsive unstable angina; NR-UA). A CRP plasma level higher than 0.86 mg/dl had a 91% positive predictive value (PPV) and 63% negative predictive value for NSTEMI (odds ratio = 6.4, 95% confidence interval = 1.5-27.4). TIMP-1 plasma level higher than 21.5 ng/ml had a 100% PPV for patients with NR-UA or NSTEMI. Binary logistic analysis confirmed TIMP-1 levels as being able to predict responsiveness to therapy. CONCLUSIONS: In conclusion, a different biochemical pattern characterizes ACS patients: those with NR-UA show only an increase of remodeling markers, whereas ACS patients with NSTEMI have an increase of both remodeling and inflammation markers.
Abstract: In spite of association between high plasma adiponectin and high metabolic and cardiovascular (CV) risk, highest adiponectin increments retain CV and metabolic protective effects in advanced chronic kidney disease (CKD). Passive accumulation can favor CKD-associated hyperadiponectinemia but potential additional regulation by adipose tissue remains undefined. Oxidative stress (OS) is associated with metabolic and CV disease and with CKD [increasing from conservative treatment (CT) to maintenance hemodialysis (MHD)], and OS can reduce adiponectin expression in experimental models. OS (in the form of plasma thiobarbituric acid-reactive substances: TBARS), subcutaneous adipose adiponectin mRNA, and plasma adiponectin were studied in CKD patients (stages 4 and 5) on CT (n = 7) or MHD (n = 11). Compared with CT and controls (C: n = 6) MHD had highest TBARS and lowest adiponectin mRNA (P < 0.05) with lower adipose adiponectin protein (P < 0.05 vs. CT). MHD also had lower plasma adiponectin than CT, although both had higher adiponectin than C (P < 0.05). In renal transplant recipients (RT: CKD stage 3; n = 5) normal TBARS were, in turn, associated with normal adiponectin mRNA (P < 0.05 vs. MHD). In all CKD (n = 23), adiponectin mRNA was associated positively with adiponectin plasma concentration (P < 0.01). In all subjects (n = 29), adiponectin mRNA was related (P < 0.05) negatively with TBARS after adjusting for plasma C-reactive protein (CRP) or CRP and creatinine. Thus altered OS, adiponectin expression, and plasma concentration represent a novel cluster of metabolic and CV risk factors in MHD that are normalized in RT. The data suggest novel roles of 1) MHD-associated OS in modulating adiponectin expression and 2) adipose tissue in contributing to circulating adiponectin in advanced CKD.
Abstract: OBJECTIVE: Ghrelin administration can induce fat weight gain and hyperglycemia (potentially through ghrelin-induced hepatic glucose production), but plasma ghrelin is positively associated with whole-body insulin sensitivity (mainly reflecting muscle insulin action) being increased in lean individuals or after diet-induced weight loss and reduced in obesity or after diet-induced weight gain. To investigate potential mechanisms, we measured in vivo effects of sustained ghrelin administration at a non-orexigenic dose on skeletal muscle and liver insulin signaling at the AKT level and adipokine expression changes. RESEARCH METHODS AND PROCEDURES: Young-adult male rats received 4-day, twice daily subcutaneous ghrelin (200 mug/injection) or saline. We measured skeletal muscle (mixed, gastrocnemius; oxidative, soleus) and liver protein levels of activated [phosphorylated (P)] and total (T) AKT and glycogen synthase kinase (GSK; reflecting AKT-dependent GSK inactivation) and epididymal adipose tissue adipokine mRNA. RESULTS: Ghrelin increased body weight (+1.4%) and blood glucose (both p < 0.05 vs. saline) but not food intake, plasma insulin, or free fatty acids. Ghrelin, however, enhanced P/T/AKT and P/T/GSK ratios and glucose transporter-4 mRNA in soleus (p < 0.05), but not in gastrocnemius, muscle. In contrast, ghrelin reduced hepatic P/T-AKT and P/T-GSK. No alterations occurred in adiponectin, leptin, or resistin transcripts or plasma adiponectin. DISCUSSION: Despite moderate weight gain and in the absence of insulin-free fatty acid changes, sustained ghrelin administration enhanced oxidative muscle AKT activation. Reduced liver AKT signaling could potentially contribute to concomitant blood glucose increments. These findings support ghrelin as a novel tissue-specific modulator of lean tissue AKT signaling with insulin-sensitizing effects in skeletal muscle but not in liver in vivo.
Abstract: BACKGROUND: Muscle inactivity and low energy intake commonly occur in persons with acute or chronic disease, in astronauts during space flight, and during aging. OBJECTIVE: We used a crossover design to investigate the effects of the interactions of inactivity and calorie restriction on whole-body composition and protein kinetic regulation in 9 healthy volunteers. DESIGN: Lean body mass (LBM) was measured by using dual-energy X-ray absorptionmetry before and at the end of 14-d periods of bed rest (B) and controlled ambulation (A) in patients receiving eucaloric (E) or hypocaloric (H) (approximately 80% of total energy expenditure) diets. Whole-body leucine kinetics were determined at the end of the 4 study periods by using a standard stable-isotope technique in the postabsorptive state and during a 3-h infusion of a 0.13 g x kg LBM(-1) x h(-1) amino acid mixture. RESULTS: In the postabsorptive state, we found a significant (P = 0.04) bed rest x hypocaloric diet interaction for the rate of leucine oxidation, an index of net protein catabolism (A+E: 0.23 +/- 0.01; B+E: 25 +/- 0.01; A+H: 0.23 +/- 0.01; B+H: 0.28 +/- 0.01 micromol x min(-1) x kg LBM(-1)). Bed rest significantly (P < 0.01) decreased amino acid-mediated stimulation of nonoxidative leucine disappearance, an index of protein synthesis (A+E: 35 +/- 2%; B+E: 30 +/- 2%; A+H: 41 +/- 3%; B+H: 32 +/- 2%). B+H decreased LBM by 1.10 +/- 0.1 kg, which is significantly (P < 0.01) greater than the decrease seen with A+E, A+H, or B+E. CONCLUSION: Calorie restriction enhanced the catabolic response to inactivity by combining greater protein catabolism in the postabsorptive state with an impaired postprandial anabolic utilization of free amino acids.
Abstract: The eukaryotic elongation factors (eEF1A2 and eEF1A1) play a key role in translation of messenger RNA (mRNA) to protein. In skeletal muscle of healthy humans, EEF1A2 is overexpressed and selected over EEF1A1. In cellular stress models, muscle EEF1A1 expression increased and was associated with apoptosis and catabolism. We have determined mRNA levels of EEF1A1 and EEF1A2, as well as those of other proapoptotic genes, such as p66(ShcA) and c-MYC, in skeletal muscle of severely traumatized patients and healthy volunteers. Muscle protein kinetic was determined by stable isotopes and the arteriovenous technique. The patients were in a hypercatabolic condition because the rate of muscle proteolysis exceeded that of synthesis. Mean mRNA levels of EEF1A1 and EEF1A2 were 165- and 29-fold greater (P < .01) in patients than in the control group, respectively. Mean p66(ShcA) mRNA levels were 3-fold greater (P < .05) in patients than in the controls. In contrast, c-MYC mRNA levels were not significantly different in patients and healthy controls. In patients, muscle mRNA levels of EEF1A1 and p66(ShcA) directly correlated (P < .05) with the rate of proteolysis (R = 0.901 and R = 0.826, respectively). This is in agreement with a reduction in actin and tubulin protein content, both markers of cytoskeletal and sarcomeric disorganization, and with an increased poly(adenosine diphosphate-ribose) polymerase cleavage, a marker of apoptosis. In conclusion, in hypercatabolic traumatized patients, an up-regulation of muscle EEF1A1 and p66(ShcA) relates to proteolysis rate, suggesting an involvement of these genes in muscle catabolic response.
Abstract: Chronic renal failure (CRF) is characterized by persistent systemic inflammatory response. We tested the hypothesis that the balance between synthetic capacity of pro-inflammatory, as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and anti-inflammatory cytokines, as IL-10, may become progressively impaired during decline of renal function. Cytokine mRNA transcript levels (fraction of GAPDH mRNA) were detected by real time RT-PCR technique in whole blood cells of patients with far-advanced or less-advanced CRF (glomerular filtration rate lower or greater than 15ml/min per 1.73m(2), respectively) undergoing conservative treatment and in healthy controls. TNF-alpha mRNA levels were greater (p<0.05) in the patients with far-advanced CRF than in those with less-advanced CRF. IL-6 mRNA levels were not significantly different in the two groups. Both groups of patients exhibited greater (p<0.05) TNF-alpha and IL-6 mRNA levels than the healthy controls. IL-10 mRNA levels were greater (p<0.05) in the patients with less-advanced CRF (65+/-18) than in the healthy controls (12+/-2). Nonetheless, in the patients with far-advanced CRF, IL-10 mRNA levels (20+/-10) were lower (p<0.05) than in the patients with less-advanced CRF and not significantly different than in the healthy controls. In conclusion, advanced renal failure is characterized by unbalanced synthetic capacity of pro- and anti-inflammatory cytokines. A progressive decrease in IL-10 synthetic capacity during the course of CRF could contribute to increasing cardiovascular risk.
Abstract: BACKGROUND: We have studied the effects of interferon (IFN)-gamma allelic variations on expression levels of pro- and anti-inflammatory cytokines and on long-term inflammatory status in haemodialysis patients. METHODS: Genotyping was performed in 123 patients for single nucleotide polymorphisms in the first intron of the IFN-gamma gene (+874 T/A). They were prospectively followed for 2 years. Cytokine mRNA levels in whole blood cells (detected by real time (RT)-PCR technique) and serum C-reactive protein (CRP) concentrations were compared in patient groups with different IFN-gamma genotypes. Serum CRP was evaluated every month and inflammatory state was defined as percent of abnormal values (above 5 mg/l) over total determinations. Of the total, 102 patients survived and completed 24+/-1 monthly CRP determinations. The IFN-gamma +/-874 A/A, 'low-producer' genotype was associated with decreased (P<0.05) mRNA levels of IFN-gamma and of interleukin-6 and with a lower (P<0.05) frequency of CRP elevation (37+/-6%) than the +/-874 A/T and T/T, 'intermediate and high-producer' genotypes (59+/-6%, and 60+/-5%, respectively). The mRNA levels of tumor necrosis factor-alpha, IL-10 and of transforming growth factor-beta1 were not different in the three groups of patients. Pooled analysis in deceased (10+/-3 monthly CRP determinations) and survived patients confirmed the results obtained in the patients who completed the follow-up period. CONCLUSIONS: The 'low-producer' IFN-gamma +874 A/A genotype was associated with a preventive effect on long-term CRP elevation in haemodialysis patients possibly mediated by decreased gene expression of IFN-gamma and IL-6.
Abstract: OBJECTIVE: Patients with cancer are characterized by decreased muscle protein synthesis and glutamine availability that contribute to an impaired immune response. These abnormalities worsen after surgical stress. We tested the hypothesis that pharmacologic doses of branched-chain amino acids would improve the early metabolic response after major cancer surgery. METHODS: By using a crossover experimental design, we compared the metabolic effects of isonitrogenous solutions of balanced and branched-chain-enriched amino acid mixtures infused at the rate of 82 mg x h(-1) x kg(-1) for 3 h in patients with colorectal or cervical cancer on the first and second days after radical surgery combined with intraoperative radiation therapy. The ratios of leucine to total amino acid (grams) in the two mixtures were 0.09 and 0.22, respectively. Muscle protein and glutamine kinetics were determined by using stable isotope of amino acids and the leg arteriovenous balance technique. Glucose and insulin were continuously infused throughout the 2-d study to maintain near euglycemia. RESULTS: Rates of muscle protein synthesis and degradation were not significantly affected by the balanced amino acid infusion. In contrast, the isonitrogenous, branched-chain-enriched amino acid mixture accelerated muscle protein turnover by stimulating (P <or= 0.05) protein synthesis. The rate of muscle glutamine de novo synthesis did not significantly change after infusion of the balanced amino acid mixture but increased (P <or= 0.05) by 263 +/- 69% during infusion of the branched-chain-enriched amino acid mixture. CONCLUSIONS: An excess of branched-chain amino acids in the presence of an optimal profile of other essential amino acids acutely increased muscle protein synthesis and glutamine flux from skeletal muscle in cancer patients after surgery.
Abstract: Rheological and erosion studies regarding a liposome-containing polymeric blend that is propaedeutic to its use in paving techniques in tubular organs, such as blood vessels, are reported. Attention is focused on an aqueous polymeric blend composed of Pluronic (PF127) and alginate (Protanal LF 10/60) because both polymers, when dissolved in water at a sufficiently high concentration, are subjected to different structural mechanisms, which are driven by temperature increase and addition of bivalent cations, respectively, and both result in marked viscoelastic and plastic properties. After proving the compatibility between PF127 and alginate, we show that the structural transition temperature of the blend, T(ST), can be properly modulated. In particular, we found that T(ST) for an aqueous solution of pure Pluronic 20% w/w is about 21 degrees C and that even slight reductions in polymer concentration result in considerable T(ST) decrease. The addition of salts or alginate (provided as Na-alginate) provokes a substantial decrease of T(ST) and thus the alginate concentration in the blend should not exceed 1% w/w. In addition, liposomes slow down the structural transition but do not substantially affect the rheological properties of the system in the final state at higher temperatures, thus showing that they can be added to the polymeric blend without significant effects. Finally, erosion tests show that after contact with a source of bivalent cations, the polymeric blend containing PF127 and alginate shows an erosion resistance neatly improved with respect to the simple structured Pluronic system having the same polymer concentration. As a whole, all these results constitute the basis for future potential applications of the considered polymeric blend in tubular organs such as blood vessels.
Abstract: OBJECTIVE: The aims of this study were to compare a microsatellite polymorphism (PM) of matrix metalloproteinase (MMP)-9 in patients with carotid atherosclerosis and control population, and to assess the relationship between this PM and plaque structure. METHODS AND RESULTS: One hundred fifty patients referring to vascular diagnostic centers for suspected carotid atherosclerosis (at ultrasound examination: 110 positive, 40 negative) and controls (n=110) have been genotyped for MMP-9 PM. After controlling for risk factors, allelic and genotype frequencies were significantly different among the groups, with significant prevalence of long microsatellites in patients with carotid atherosclerosis. Long microsatellites (settled as 22 to 27 repeats) were associated with carotid atherosclerosis (odds ratio [OR], 5.2; 95% confidence interval [CI], 2.9 to 9.2), compared with controls; an independent case control study on patients with coronary atherosclerosis confirmed such result. Binary logistic regression showed that hypertension, long microsatellites in MMP-9 PM and smoking habits were variables accounting for the difference between ultrasound-positive patients and controls. Long microsatellites were also associated to plaques with thin fibrous cap and echolucent core (OR, 13.1; 95% CI, 1.6 to 100). These alleles were slightly more represented in female patients (chi2 test=0.019; OR, 2.7; 95% CI, 1.2 to 6) but not associated with other risk factors. Plasma MMP-9 levels were related neither to MMP-9 PM nor to plaque type, and were related to gender and extension of atherosclerosis in carotid arteries. CONCLUSIONS: The number of repeats (> or =22 CA) in the microsatellite of MMP-9 promoter, but not MMP-9 plasma levels, is associated to carotid atherosclerosis and particularly to plaques with a thin fibrous cap.
Abstract: Enhanced chronic systemic inflammation and reduced insulin sensitivity are often associated in patients with chronic renal failure, contributing to cardiovascular morbidity and mortality in these patients. Adipose tissue produces several hormones (adipocytokines including leptin, resistin, tumor necrosis factor-alpha, and adiponectin) that modulate both systemic inflammatory response and insulin action. High leptin, resistin, and tumor necrosis factor-alpha and low adiponectin are associated with proinflammatory conditions, whereas opposite patterns are commonly observed in the presence of increased insulin sensitivity, low inflammation, and reduced cardiovascular risk. Oxidative stress has also been shown recently to modulate adipocytokine production, resulting in a proinflammatory profile. Increments of plasma concentrations of both proinflammatory and anti-inflammatory adipocytokines have been reported in chronic renal failure, possibly caused by both passive accumulation from reduced renal excretion and metabolic abnormalities induced by uremia. The potential role of altered adipose tissue adipocytokine production in the onset of renal failure-associated inflammatory and metabolic derangements remains largely to be elucidated and is discussed in the current report.
Abstract: We studied the mechanism governing the delivery of nucleic acid-based drugs (NABD) from microparticles and nanoparticles in zero shear conditions, a situation occurring in applications such as in situ delivery to organ parenchyma. The delivery of a NABD molecule from poly(DL-lactide-co-glycolide) (PLGA) microparticles and stearic acid (SA) nanoparticles was studied using an experimental apparatus comprising a donor chamber separated from the receiver chamber by a synthetic membrane. A possible toxic effect on cell biology, as evaluated by studying cell proliferation, was also conducted forjust PLGA microparticles. A mathematical model based on the hypothesis that NABD release from particles is due to particle erosion was used to interpret experimental release data. Despite zero shear conditions imposed in the donor chamber, particle erosion was the leading mechanism for NABD release from both PLGA microparticles and SA nanoparticles. PLGA microparticle erosion speed is one order of magnitude higher than that of competing SA nanoparticles. Finally, no deleterious effects of PLGA microparticles on cell proliferation were detected. Thus, the data here reported can help optimize the delivery systems aimed at release of NABD from micro- and nanoparticles.
Abstract: Ghrelin is a recently described hormone secreted by the stomach. Ghrelin administration in ad-libitum-fed rodents was shown to increase appetite as well as body weight and body fat content, showing metabolic effects of ghrelin in vivo and suggesting its involvement in the pathogenesis of obesity. However, plasma ghrelin concentration was shown to be inversely correlated with body weight and body fat in people and rodents. Increased plasma ghrelin concentration was also reported during diet-induced weight loss and in malnourished states. These findings suggest that circulating ghrelin is regulated by nutritional state and body fat with a feedback mechanism opposing changes in body composition, with a potential key adaptive role during calorie restriction. Plasma ghrelin concentration was shown to be increased in advanced renal failure and hemodialysis patients. The known metabolic effects of ghrelin and the potential implications of hyperghrelinemia in kidney disease will be discussed in this article.
Abstract: Physical inactivity is associated with alteration of normal physiologic processes leading to muscle atrophy, reduced exercise capacity, insulin resistance, and altered energy balance. Bed rest studies in human beings using stable isotopes of amino acids indicate that muscle unloading decreases the turnover rates of muscle and whole-body proteins, with a prevailing inhibition of protein synthesis. In the fasting state, muscle and whole-body nitrogen loss was not accelerated during bed rest. In experimental postprandial states, the amino acid-mediated stimulation of protein synthesis was impaired, whereas the ability of combined insulin and glucose infusion to decrease whole-body proteolysis was not affected by muscle inactivity. Thus, an impaired ability of protein/amino acid feeding to stimulate body protein synthesis is the major catabolic mechanism for the effect of bed rest on protein metabolism. This suggests that a protein intake level greater than normal could be required to achieve the same postprandial anabolic effect during muscle inactivity. Metabolic adaptation to muscle inactivity also involves development of resistance to the glucoregulatory action of insulin, decreased energy requirements, and increased insulin and leptin secretion. These alterations may lead to the development of the metabolic syndrome that is defined as the association of hyperinsulinemia, dyslipidemia, hypertension, hyperglycemia, and abdominal obesity. This cluster of metabolic abnormalities is a risk factor for coronary artery disease and stroke. Evidence indicates that exercise training programs may counteract all of these abnormalities both in healthy sedentary subjects and in patients affected by a variety of chronic disease states.
Abstract: Chronically uremic patients are characterized by a low-grade systemic inflammation that reflects the consequences of an unbalanced production of proinflammatory and anti-inflammatory cytokines and contributes to the progression of atherosclerotic vascular disease and malnutrition. The causes of inflammation in end-stage kidney disease have been studied in details. Nonetheless, the degree of activation of the systemic inflammatory response shows great interindividual variability that cannot be explained by renal disease or dialysis. The amount of cytokine that is produced on a definite stimulus varies among individuals. Single nucleotide polymorphisms in the promoter or coding regions of cytokine genes lead to high or low productions of these mediators and may genetically explain this heterogeneity. The "low-producer" genotypes for the anti-inflammatory cytokine interleukin-10 are more permissive for a greater level of systemic inflammation and for increased cardiovascular morbidity and mortality in patients on hemodialysis. Potential pharmacologic and nutritional approaches for treatment of systemic inflammation have been identified in recent years. In addition, physical exercise training may reduce the systemic inflammatory response. Definition of the relationships between different cytokine gene polymorphisms and systemic inflammation in chronically uremic patients will improve efficacy of targeted anti-inflammatory treatments.
Abstract: Antiatherogenic effects of nitric oxide (NO) are mediated by activation of soluble guanylate cyclase (sGC) and are impaired by diabetes in animals and humans. We investigated whether uncontrolled diabetes and insulin therapy effect expression and function of the main enzymes of the endothelial nitric oxide (eNOS)-sGC signaling pathway in vivo. Expression and function of eNOS, sGC and protein kinase G (PKG) were studied by Western blot analysis and vasorelaxation to NO-donor in thoracic aortas from control (CON) and streptozotocin (SZT)-induced diabetic rats during uncontrolled diabetes (DM) and insulin treatment (INS) for 8 weeks. Protein level of eNOS was increased (+300%, P < 0.05), while sGC (-50%) and PKG (-65%) proteins were reduced (P < 0.03) in aortas of DM. Insulin treatment normalized these defects resulting in eNOS, sGC and PKG aortic protein content comparable to control. In aortic rings, diethylamine NONOate (DEA-NONOate)-induced vasorelaxation was attenuated (P< or =0.05) in DM compared to control and returned to normal in INS. Thus, experimental diabetes decreases sGC and PKG expression and their NO-dependent activation in aorta despite overexpression of eNOS. These abnormalities are normalized by insulin treatment and improved metabolic control.
Abstract: Intima-media thickening (IMT) of carotid arteries and constrictive remodeling (CR) of atherosclerotic plaques are vascular pathologic characteristics that precede the onset of vascular events. SMC migration and proliferation are linked both to IMT and CR and are matrix metalloproteinase 9 (MMP-9) dependent. A genetic polymorphism (PM) of MMP-9, a CA (13-27) microsatellite in the promoter region, which accounts for differential expression of MMP-9, could be linked to progression of IMT and CR. Progression of IMT and CR of plaques in carotid arteries were studied in 55 consecutive patients with a 12-18 months follow-up. All patients were genotyped for MMP-9 PM. A positive linear relationship between the number of repeats and the progression of IMT (P=0.028) as well as of CR (P=0.018) was found. The analogous relationship was obtained when only the allele with longer microsatellite was considered. Carriers of more than 20 repeats in one allele showed faster both IMT growth (P=0.045) and stenosis progressions of plaques (P=0.019). In multivariate analysis, age, dyslipidemia, and MMP-9 PM were determinants of IMT progression, while MMP-9 PM was the only one of CR. In conclusion, the high number of CA repeats in MMP-9 promoter is positively correlated with faster IMT and CR progression.
Abstract: PURPOSE: To assess if a polymorphism (PM) of the microsatellite (CA(13-27)) in the promoter region of Matrix Metalloproteinase 9 (MMP-9) was associated with the exudative form of age-related macular degeneration (AMD) and to its risk factors. METHODS: In 107 patients with AMD (AMD Group) and 223 age- and gender-matched controls (Control Group) with cataract, demographic, clinical data, and MMP-9 PM have been compared. RESULTS: The comparison of allelic frequencies showed a different pattern of CA repeats between AMD and Control Group (P < 0.00005), in particular the prevalence of longer microsatellites (> or = 22 CA repeats) was higher in AMD than in Control Group (O.R. 2.49, 95% CI 1.71-3.37, P < 0.001). Analyses of genetic frequencies gave similar results. Logistic regression confirmed that 22 or more CA repeats are associated to AMD. The only association between MMP-9 PM and other risk factors for AMD was with BMI (Spearman's R = 0.298, P < 0.00005): all patients with both microsatellites > or = 22 CA repeats were overweight or obese (chi2 test P < 0.0005, compared to other genotypes). CONCLUSIONS: Longer microsatellites in the promoter of MMP-9 are associated to the exudative form of AMD and to body mass index, a well-known risk factor for the disease.
