RGS2 (regulators of G-protein signaling) is a negative regulator of Galphaq protein signaling, which mediates the action of several vasoconstrictors. RGS2-deficient mouse line exhibits a hypertensive phenotype and a prolonged response to vasoconstrictors. To compare RGS2 expression in peripheral blood mononuclear cells (PBMs) and cultured fibroblasts from normotensive subjects and hypertensive patients. PBMs were isolated from 100 controls and 150 essential hypertensives. Additionally, fibroblasts were isolated from skin biopsy of 11 normotensives and 12 hypertensives and cultured up to the third passage. Quantitative mRNA and protein RGS2 expression were performed by real-time quantitative reverse transcriptase-polymerase chain reaction and by immunoblotting, respectively. Free Ca measurement was performed in monolayers of 24-h serum-deprived cells, using FURA-2 AM. Phosphorylation of the extracellular signal-regulated kinases ERK1/2 was measured by immunoblotting. Polymorphism (C1114G) in the 3' untranslated region of the RGS2 gene was investigated by direct sequencing and real-time polymerase chain reaction (PCR). RGS2 mRNA expression was significantly lower in PBM and in fibroblasts from hypertensives, in comparison to normotensives. C1114G polymorphism was associated with RGS2 expression, with the lowest values in GG hypertensives. The 1114G allele frequency was increased in hypertensives compared with normotensives. Angiotensin II-stimulated intracellular Ca increase and ERK1/2 phosphorylation were higher in fibroblasts from hypertensive patients compared with control subjects, and in those with the G allele, independently of the blood pressure status. The angiotensin II-stimulated Ca mobilization and ERK1/2 phosphorylation were negatively correlated with RGS2 mRNA expression. Low expression of RGS2 contributes to increased G-protein-coupled signaling in hypertensive patients. The allele G is associated with low RGS2 expression and blood pressure increase in humans.

Summary: The natural history of calf deep-vein thrombosis (DVT) is still uncertain and it is debated whether it warrants to be diagnosed and treated. We aimed to investigate the complication rate of untreated isolated calf DVT (ICDVT). Symptomatic outpatients were prospectively managed with serial compression ultrasonography (SCUS). Those without proximal DVT and with likely pre-test clinical probability (PCP) or altered D-dimer received immediate subsequent complete examination of calf deep veins (CCUS) by a different operator. The result of CCUS was kept blind both to the managing doctor and the patient and disclosed after three months. Primary outcome was the rate of venous thromboembolism at three months. We examined 431 subjects (196 males; median age 68.0 years) in whom five outcomes were recorded (1.2%; 95% confidence intervals [CI]: 0.4–2.7). If CCUS results had been available, outcomes would have been recorded in 3/424 patients (0.7%; 95% CI: 0.2–2.1) with two events in subjects negative at both serial and complete CUS. ICDVT was diagnosed in 65 subjects (15.3%; 95% CI: 12–19); of whom 59 remained uneventful (one was lost to follow-up). A significant higher rate of outcomes was recorded in subjects with than without ICDVT (5/64; 7.8%; 95% CI: 3–17 vs. 3/351; 0.8%; 95% CI: 0–2; p=0.003). However, after excluding two events picked at serial CUS in subjects with ICDVT, the difference became barely significant (3/64; 4.7%; 95% CI: 1–13; p=0.049). Thrombotic evolution of untreated ICDVT in high-risk subjects may be relevant. Larger studies are needed to address this issue.

STROKE IS the third most common cause of death in western populations, after coronary heart disease and cancer. Furthermore, stroke is the most common life-threatening neurological disorder and the most important single cause of disability among these populations.

Regulator of G protein signaling-2 (RGS-2) plays a key role in the G protein-coupled receptor (GPCR) angiotensin II (Ang II) signaling. NO and cGMP exert a vasodilating action also through activation and binding to RGS-2 of cGMP dependent protein kinase 1-α, which phosphorylates RGS-2 and dephosphorylates myosin light chain. In Bartter’s/Gitelman’s patients (BS/GS) Ang II related signaling and vasomotor tone are blunted. Experiments were planned to explore whether RGS-2 may play a role in BS/GS vascular hyporeactivity. NO metabolites and cGMP urinary excretion were also measured. Mononuclear cells (PBM) from six BS/GS patients and six healthy controls were used. PBM RGS-2 mRNA and RGS-2 protein were increased in BS/GS: 0.47 ± 0.06 d.u. vs 0.32 ± 0.04, (p < 0.006) (RGS-2 mRNA), and 0.692 ± 0.02 vs 0.363 ± 0.06 (p < 0.0001) (RGS2 protein). Incubation of PBM with Ang II increased RGS-2 protein in controls (from 0.363 ± 0.06 d.u. to 0.602 ± 0.05; p < 0.0001) but not in BS/GS (from 0.692 ± 0.02 to 0.711 ± 0.02). NO2-/NO3- and cGMP urinary excretion were increased in BS/GS (0.46 ± 0.13 vs 0.26 ± 0.05 μmol/μmol of urinary creatinine, p < 0.005, and 0.060 ± 0.030 vs 0.020 ± 0.01 p < 0.009, respectively). These results demonstrate that RGS-2 is increased and maximally stimulated in BS/GS and human RGS-2 system reacts as predicted by knockout mice experiments. This is the first report of RGS-2 level in a human clinical condition characterized by altered vascular tone, underlines the importance of RGS-2 as a key regulator element for Ang II signaling and provides insight into the links between BS/GS genetic abnormalities and abnormal vascular tone regulation.

Summary. Background: Pretest clinical probability with the Wells rule and D‐dimer have been widely investigated for the diagnosis of symptomatic proximal deep vein thrombosis (DVT) of the lower limbs, but they have not been formally tested for symptomatic isolated distal DVT diagnosis. Objective: To evaluate the diagnostic accuracy of the Wells rule and D‐dimer for isolated distal DVT. Design, Setting, and Patients: This was a single‐center, cross‐sectional study including 873 consecutive outpatients with suspected DVT, in whom pretest clinical probability determination, D‐dimer determination (STA Liatest; cut‐off of < 500 ng mL⁻¹) and complete compression ultrasonography of both lower limbs were performed. Results: The isolated distal DVT prevalence was 12.4% (90/725). The sensitivity of the Wells rule for isolated distal DVT was 47% (95% confidence interval [CI] 36–57%), the specificity was 74% (95% CI 70–77%), and the negative and positive predictive values were 91% (95% CI 88–93%) and 20% (95% CI 15–26%), respectively. Patients with isolated distal DVT had higher D‐dimer levels than patients without DVT (1759 ± 1576 vs. 862 ± 1079 ng mL⁻¹, P = 0.0001). D‐dimer was negative in 13 patients with isolated distal DVT. D‐dimer sensitivity and specificity for isolated distal DVT were 84% (95% CI 75–91%) and 50% (95% CI 46–54%), respectively, with a negative predictive value of 96% (95% CI 93–98%). In patients with low pretest clinical probability, the D‐dimer negative predictive value was 99% (95% CI 95–100%). Conclusion: In clinically suspected DVT with negative proximal compression ultrasonography, pretest clinical probability with the Wells rule has a low diagnostic accuracy for isolated distal DVT. D‐dimer has a better negative predictive value, but alone it does not exclude isolated distal DVT. In patients with low pretest clinical probability, D‐dimer had a negative predictive value of > 95% for isolated distal DVT.

Activation of protein kinase C (PKC) by hyperglycemia is implicated in the pathogenesis of long-term diabetic complications. Monocyte activation and transformation into macrophages is a key step in the atherosclerotic process. Therefore, in this study, we sought to determine 1) the effect of hyperglycemia on monocyte PKC activity and on the distribution of Ca2+-dependent and diacylglycerol-sensitive PKC isoforms; and 2) whether the effects on these parameters are determined by hyperglycemia per se, independent of the diabetic state. The studies were performed in 19 type 2 diabetic patients and 14 control subjects. Plasma glucose concentration was higher and insulin sensitivity lower (both P < 0.01) in diabetic patients than in control subjects. Monocytes from diabetic patients showed similar cytosol PKC activity to those from control subjects but higher membrane PKC activity (78+/-6 vs. 50+/-5 pmol x min(-1) x mg(-1) protein; P < 0.01). A direct correlation was observed between fasting plasma glucose and membrane PKC activity (r2 = 0.4008, P = 0.0001). In contrast, a reciprocal correlation was observed between membrane PKC activity and insulin sensitivity index (r2 = 0.28, P < 0.05). Using immunoblotting analysis, we found that membrane beta2, but not alpha, isoform of PKC was more abundant in monocytes from diabetic patients. In diabetic patients, when euglycemia was acutely induced, membrane PKC activity decreased by approximately 42% and beta2 isoform by approximately 15%. In two normal subjects in whom hyperglycemia was induced, membrane PKC increased from 63 and 57 to 92 and 128.6 pmol x min(-1) x mg(-1) protein, respectively. This increase was associated with an increase in the membrane isoform beta2; alpha isoform was unchanged. We conclude that 1) monocytes express the glucose-sensitive beta2 isoform of PKC; 2) the prevailing plasma glucose acutely regulates the activity of the membrane PKC and the content of membrane PKC beta2 isoform; and 3) this effect appears to be a direct effect of glucose per se, since the phenomenon was observed in normal control subjects when hyperglycemia was induced. Monocyte PKC activation may account for the accelerated atherosclerosis of patients with type 2 diabetes.