Hyperuricemia and Risk of Cardiovascular Outcomes: The Experience of the URRAH (Uric Acid Right for Heart Health) Project.

The latest European Guidelines of Arterial Hypertension have officially introduced uric acid evaluation among the cardiovascular risk factors that should be evaluated in order to stratify patient's risk. In fact, it has been extensively evaluated and demonstrated to be an independent predictor not only of all-cause and cardiovascular mortality, but also of myocardial infraction, stroke and heart failure. Despite the large number of studies on this topic, an important open question that still need to be answered is the identification of a cardiovascular uric acid cut-off value. The actual hyperuricemia cut-off (> 6 mg/dL in women and 7 mg/dL in men) is principally based on the saturation point of uric acid but previous evidence suggests that the negative impact of cardiovascular system could occur also at lower levels. In this context, the Working Group on uric acid and CV risk of the Italian Society of Hypertension has designed the Uric acid Right for heArt Health project. The primary objective of this project is to define the level of uricemia above which the independent risk of CV disease may increase in a significantly manner. In this review we will summarize the first results obtained and describe the further planned analysis.

GM2 ganglioside lysosomal storage disease in cats with beta-hexosaminidase deficiency.

Two kitteens with progressive neurologic disease had increased concentrations of GM2 ganglioside in their cerebral cortex. Examination under the light microscope revealed cytoplasmic vacuolation of neurons and hepatocytes. Transmission and scanning electron microscopy demosntrated cytoplasmic inclusions encompassed by membranes in various central nervous system cell types and in hepatocytes. Beta-D-N-acetyl-hexosaminidase activity was reduced to about 1.0 percent of normal in brain, liver, and cultured skin fibroblasts of the diseased kittens; both major electrophoretic forms, A and B, of the enzyme were deficient. In fibroblasts from the parents of the diseased kittens, this enzyme activity was intermediate between that of affected and normal cats, suggesting an autosomal recessive mode of inheritance of the enzyme defect. Histopahtological and ultrastructural lesions, glycolipid storage, enzyme defect, and pattern of inheritance are similar to those of human GM2 gangliosidosis type 2.

Genetic polymorphisms of human mitochondrial glutamic oxaloacetic transaminase.

In a survey of 860 unselected human placental extracts, three variants of mitochondrial glutamic oxaloacetic transaminase were found, all of which were common enough to be considered polymorphisms. Family studies showed that this enzyme is under the control of nuclear rather than mitochondrial DNA.

Excretion-reuptake route of beta-hexosaminidase in normal and I-cell disease cultured fibroblasts.

It has been proposed that in cultured fibroblasts the final packaging of enzymes in lysosomes requires excretion followed by pinocytosis by neighboring cells via a carbohydrate-specific receptor mechanism. It has also been proposed that the abnormally high activity of lysosomal enzymes in the medium of cultured fibroblasts from patients with I-cell disease (mucolipidosis II) results from an altered carbohydrate recognition residue on the enzymes which prevents reuptake into the cells. With beta-hexosaminidase as a marker, and competitive inhibition of uptake by 2 mM mannose-6-phosphate, we have determined that only 12% of the total (intra- and extracellular) beta-hexosaminidase in normal fibroblasts is channeled through the excretion-reuptake route. After 9 d of exposure to mannose-6-phosphate, normal fibroblast cultures accumulated in the medium only a fraction of the enzyme excreted by I-cell disease fibroblasts in the same period. Furthermore, this minimal loss of enzyme to the medium did not result in a decrease of intracellular enzyme activity. Finally, if the defect in I-cell disease were only because of an impairment of a reuptake mechanism that involves only 12% of the total enzyme, then 88% of the newly synthesized enzyme should be retained by I-cell fibroblasts, resulting in intracellular activity three to nine times higher than that which is observed. These data are consistent with our previous proposal that excessive lysosomal enzyme activity in the medium of I-cell disease fibroblasts results from preferential exocytosis.

Mitogenic effect of lysosomal hydrolases on bovine tracheal myocytes in culture.

Studies were conducted to assess the mitogenic effect of lysosomal hydrolases, enzymes known to have an association with allergen- or ozone-induced airway hyperreactivity, on bovine tracheal myocytes in culture. Addition of purified human placental beta-hexosaminidase and partially purified bovine liver beta-glucuronidase resulted in the doubling of cell count after 4 d of incubation in medium M199 with 0.4% FBS. Unstimulated cells remained quiescent without a significant increase of cell count. Lysosomal hydrolases also selectively enhanced 3H-thymidine incorporation four to seven times more than that in vehicle-treated cells or cells treated with endotoxin, a common contaminant of purified enzymes. Ovalbumin (glycoprotein control), pronase, and lysozyme caused a modest but statistically insignificant increase (up to twofold) in 3H-thymidine incorporation. Elastase, collagenase and dialyzed E. coli beta-glucuronidase had no effect. The mitogenic effect of hydrolases was equally seen in quiescent, serum-depleted cells as well as in those maintained in medium with 10% FBS, suggesting that it was independent of serum factors. The effect of lysosomal hydrolases was inhibited by exposure to yeast mannan, and mannosylated human serum albumin had a mitogenic effect, suggesting the involvement of a mannose receptor. We conclude that lysosomal hydrolases may play a role in the development of the hyperplasia/hypertrophy of respiratory smooth muscle.

A mannose receptor mediates mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle cells.

The putative mannose receptor (MR), previously implicated in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle (ASM) cells, was studied to determine its properties. Specific binding of the mitogenic neoglycoprotein, mannosylated bovine serum albumin (Man-BSA) to ASM cells was saturable, with an apparent Kd = 5.0 x 10(-8) M. Cell-bound ManBSA-colloidal gold conjugate was localized by electron microscopy to clathrin-coated pits on the cell surface, and was found to undergo internalization to endosomes; this was inhibitable by weak bases and swainsonine, that also inhibited ligand-induced mitogenesis. The ASM-MR, isolated by mannose-affinity chromatography, had the same apparent molecular mass as the macrophage (Mø) MR (M(r) = 175 kD), and was immunoprecipitated by an anti-MøMR immune serum. This antiserum blocked 125I-labeled-ManBSA binding to intact ASM cells, stimulated mitogenesis, and immunolocalized the ASM-MR in cytoplasmic vesicles compatible with endosomes. A monoclonal antibody directed against the MøMR also reacted with the ASM-MR; like the polyclonal antibodies, it stimulated mitogenesis as effectively as beta-hexosaminidases. These data indicate that the ASM-MR shares a number of functional and structural properties with the MøMR and suggest that similar receptors may have different main functions in different cells.

Cell disease: desialylation of beta-hexosaminidase and its effect on uptake by fibroblasts.

The pinocytosis by fibroblasts of beta-hexosaminidase (EC 3.2.1.30) excreted by cultured skin fibroblasts from a patient with I-cell disease was not enhanced by neuraminidase treatment of the enzyme. The uptake of sialic acid-rich normal plasma beta-hexosaminidase was minimal and neuraminidase treatment did not appreciably enhance uptake. In contrast, sialic acid-rich normal seminal fluid beta-hexosaminidase was readily pinocytosed regardless of neuraminidase treatment. Thus the presence of sialic acid on beta-hexosaminidase does not influence uptake and a neuraminidase deficiency in I-cell disease may not be directly responsible for excessive extracellular enzyme.

Antigenic homology of feline and human beta-hexosaminidase.

The immunological characteristics of feline beta-hexosaminidase (beta-D-N-acetylglucosaminidase, EC 3.2.1.30) isoenzymes, Hex A and Hex B, were studied. Immunization of rabbits and goats with either cat Hex A or Hex B produced antibodies which reacted with a common antigenic marker shared by both Hex A and Hex B. With properly absorbed antisera, a unique antigenic marker was demonstrated on cat Hex A, but not on Hex B. This antigenic profile is comparable to that of the human beta-hexosaminidase isozymes, in which both Hex A and Hex B share the antigenic determinant, beta, while only Hex A possesses the antigenic determinant, alpha. Ho cross-reactivity between the two species could be demonstrated using goat or rabbit antisera to either feline of human beta-hexosaminidase. These immunological data validate feline Gm2 gangliosidosis as a model for human Gm2 gangliosidosis type II, and facilitate the investigation of enzyme replacement therapy.

Visceral obesity, but not metabolic syndrome, is associated with the presence of post-thrombotic syndrome.

INTRODUCTION: The relationship between metabolic syndrome (MetS), and the development of post-thrombotic syndrome (PTS) is currently unknown. MATERIALS AND METHODS: We enrolled 120 patients with a previous episode of deep venous thrombosis (DVT) diagnosed more than 2years apart from the enrollment. Presence of MetS was identified according to NCEP ATP III criteria and Villalta Score (VS) was used to establish the presence of PTS (VS≥5). RESULTS: We identified 49 (40.8%) subjects with clinical diagnosed of PTS. Patients with or without PTS showed comparable age and temporal distance from DVT event. We observed higher BMI (p=0.005) and waist circumference (p=0.006) among subjects with VS≥5 as compared to patients without PTS. No differences between the two groups were found in terms of lipid profile, blood pressure, diabetes, hs-CRP level and ongoing medications. The prevalence of MetS was equally distributed among patients with or without PTS (20% vs 26% respectively, p=0.64). Among the individual components of MetS only the prevalence of visceral adiposity was significantly increased in subjects affected by PTS (OR 2.81, p=0.008). Moreover, a significant linear correlation was found between VS and waist circumference in the entire cohort (r=0,354, p<0.0001). CONCLUSION: There is no evidence of association between MetS and PTS. However, the degree of visceral adiposity is strongly correlated with the presence and severity of post-thrombotic disease.

Predictors of vascular remodelling in hypertensive subjects with well-controlled blood pressure levels.

We evaluated the structural/functional characteristics of the arterial wall in a cohort of hypertensives with well-controlled blood pressure (BP) levels. We studied 40 hypertensives with well-controlled BP. We assessed by B-mode ultrasound the mean intima-media thickness (mean-IMT) and maximum-IMT (M-MAX) of carotid artery (common, bulb, internal) bilaterally. Endothelial function was evaluated by post-occlusion flow-mediated dilation (FMD) of the brachial artery. Along with traditional risk factors, we studied the impact of serum high-sensitivity C-reactive protein (hs-CRP) and osteoprotegerin (OPG). Forty normotensive subjects served as controls. In the hypertensives, the BP levels were well controlled (office BP: 129/79 mm Hg, ambulatory BP monitoring: 121/75 mm Hg). Compared with controls, higher BP levels and body mass index were present in hypertensives, whereas age and metabolic parameters were similar. In hypertensives, the IMT (mean-IMT 0.68 mm, M-MAX 0.81 mm) was significantly higher than in controls (mean-IMT 0.60 mm, M-MAX 0.71 mm). FMD was impaired in hypertensives (5.9%) compared with controls (9.2%). In multivariate analyses, it turned out that in hypertensives IMT parameters were related to age, hs-CRP and OPG. Low-density lipoprotein (LDL) cholesterol was the only factor related to FMD. IMT and FMD had no relationship with BP levels. In conclusion, in hypertensives with well-controlled BP, the pro-atherogenic remodelling (IMT) is mainly dependent on age and the inflammatory cytokines, OPG in particular. The functional impairment of the arterial wall (FMD) was related to the levels of LDL cholesterol. Under these conditions, when the impact of BP is minimized, the role of inflammatory cytokines and lipids on structural/functional remodelling becomes predominant.

Conversion of radiolabeled human growth hormone into higher molecular weight moieties in human plasma in vivo and in vitro.

Purified human growth hormone (hGH) was iodinated with 131I ([131I]iodo-hGH) and purified on Sephadex G-100. The monomeric [131I]iodo-hGH (mol wt about 20,000 daltons) was injected as a bolus iv in healthy volunteers and plasma obtained at 10, 20, and 40 min. Then continuous infusion with [131I]-iodo-hGH was started and further plasma samples obtained after 120 min when a plateau of immunoreactive hGH (IR-hGH) had been attained. In one individual the bolus injection was followed further to 60 and 120 min. Fresh plasma was immediately chromatographed on Sephadex G-100 and the IR-hGH profiles evaluated. In all instances following iv injection, larger and occasionally smaller immunoreactive moieties than the injected [131I]iodo-hGH appeared. When [131I]iodo-hGH was incubated at 37 C with fresh human plasma from 4 different individuals from 10 to 120 min, the larger molecular weight forms, noted in the in vivo studies, were again demonstrable. Only the monomeric [131I]iodo-hGH was found when [131I]iodo-hGH was incubated at 37 C for 120 min with human serum albumin or gamma globulin.

Distribution of ectopic neurite growth and other geometrical distortions of CNS neurons in feline GM2 gangliosidosis.

Golgi and combined Golgi-electron microscopic (EM) studies were carried out on cats in the terminal stages of GM2 ganglioside storage disease and the resulting data were compared with those from similar studies of other neuronal storage diseases in cats, including GM1 gangliosidosis. The results support the view that only limited types of neurons affected by the lysosomal hydrolase deficiency and subsequent intracellular storage have the capacity to sprout new dendritic-like growth processes from their axon hillocks, and that these neurons are essentially the same in all of these diseases studied to date. Golgi studies of CNS tissues from GM2 gangliosidosis cats revealed ectopic neurite growth on pyramidal neurons of cerebral cortex and multipolar cells of amygdala and claustrum, whereas other types of neurons responded to the metabolic defect with aspiny meganeurite formation or somatic enlargement, or appeared normal in terms of soma-dendritic morphology. Combined Golgi-EM studies of cortical pyramidal neurons revealed that ectopic, axon hillock neurites commonly possessed asymmetrical synapses which were similar to those observed in other storage disorders.

Initiation and growth of ectopic neurites and meganeurites during postnatal cortical development in ganglioside storage disease.

The incidence of cortical pyramidal neurons displaying meganeurites or enlarged axon hillocks with ectopic spines and neurites was evaluated developmentally using feline models of GM1 and GM2 gangliosidosis. Results of these studies demonstrated that the onset of ectopic neurite growth occurred after the elaboration of dendrites on cortical pyramidal neurons, and that the time of onset of this renewed dendritogenesis was similar in the two diseases. Initiation and growth of ectopic neurites also correlated in a general way with onset and progression of clinical deterioration in both diseases. In GM1 gangliosidosis there was a greater tendency toward formation of meganeurites, whereas in cats with GM2 gangliosidosis the growth of ectopic axon hillock neurites without meganeurites predominated. At end-stage disease in GM2 gangliosidosis, nearly 90% of pyramidal cells displayed some degree of axon hillock neurite growth as opposed to less than half this number for GM1 gangliosidosis cats at the same age. These data are consistent with the hypothesis that there are two separate driving forces behind these somadendritic abnormalities of pyramidal neurons in the gangliosidoses. Excessive intraneuronal accumulation of storage vacuoles accounts for the formation of meganeurites, whereas some type of intrinsic metabolic defect results in axon hillock neurite growth which in turn offers new surface area for synaptic input. Currently available data indicate that GM2 or GM3 ganglioside, or a closely related metabolic product other than GM1 ganglioside, may be primarily associated with the growth of ectopic dendritic processes on morphologically mature neurons in storage diseases.

Neuroaxonal dystrophy in neuronal storage disorders: evidence for major GABAergic neuron involvement.

The formation of focal granular enlargements within axons (axonal spheroids or "torpedoes"; neuroaxonal dystrophy) is a well known phenomenon occurring in a variety of neurological diseases. The relative susceptibility of different types of neurons to this kind of axonal pathology, however, is largely unknown. An immunocytochemical study directed at localizing glutamic acid decarboxylase (GAD), the synthetic enzyme for the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), in various CNS regions in feline models of lysosomal storage disorders has revealed vast numbers of axonal spheroids containing this enzyme. In some storage diseases (GM1 and GM2 gangliosidosis), GAD-immunoreactive spheroids were a common occurrence in many brain regions, whereas in other disorders these structures were more limited in distribution (alpha-mannosidosis), or were absent (mucopolysaccharidosis type I). Axonal spheroids unreactive for GAD were encountered in large numbers in subcortical white matter in GM2 gangliosidosis, but were infrequently observed in the other diseases. The incidence and distribution of GAD-immunoreactive spheroids in the various diseases under study were found to correlate closely with the type and degree of neurological deficits exhibited by affected animals. This study indicates that the neuroaxonal dystrophy occurring in some types of storage disorders commonly involves axons of GABAergic neurons and suggests that a resulting defect in neurotransmission in inhibitory circuits may be an important factor underlying brain dysfunction in this family of diseases.

Impact of hypertension on vascular remodeling in patients with psoriatic arthritis.

We studied the impact of hypertension along with traditional and new cardiovascular risk factors on the structural and functional properties of arteries in psoriatic arthritis (PsA) patients. We examined 42 PsA subjects (aged 51±9 years) stratified according to hypertensive status (19 normotensive, PsA-NT and 23 hypertensives, PsA-HT). Thirty-eight normotensive subjects (C-NT) and 23 hypertensives (C-HT) comparable by age and sex served as controls. Mean carotid intima-media thickness (mean-IMT) and mean of the maximum IMT (M-Max) were evaluated by ultrasound in carotid artery segment bilaterally. Post-occlusion flow-mediated dilation (FMD) of the brachial artery was evaluated by ultrasonography. These parameters were correlated with risk factors, markers of inflammation and disease activity. Values of mean-IMT were higher in both groups of PsA patients compared with C-NT (0.68 mm in PsA-NT and 0.75 mm in PsA-HT versus 0.61 mm in C-NT). PsA-HT displayed higher M-Max (0.95 mm) versus both C-HT (0.71 mm) and PsA-NT (0.79 mm). FMD was impaired in PsA subjects compared with C-NT (5.7% in PsA-NT and 6.0% PsA-HT versus 9.3% in C-NT), whereas there was no difference among PsA-HT, PsA-NT, and C-HT groups. Values of carotid IMT were directly related to tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), blood pressure and lipid profile levels. FMD showed an inverse relationship with TNF-α and blood pressure, but no correlation with lipids. In conclusion, PsA per se implies a pro-atherogenic remodeling, which is enhanced by the hypertensive status. TNF-α and OPG may have an independent role in the development of such vascular damage.