Hippo signaling instructs ectopic but not normal organ growth.

The Hippo signaling pathway is widely considered a master regulator of organ growth because of the prominent overgrowth phenotypes caused by experimental manipulation of its activity. Contrary to this model, we show here that removing Hippo transcriptional output did not impair the ability of the mouse liver and Drosophila eyes to grow to their normal size. Moreover, the transcriptional activity of the Hippo pathway effectors Yap/Taz/Yki did not correlate with cell proliferation, and hyperactivation of these effectors induced gene expression programs that did not recapitulate normal development. Concordantly, a functional screen in Drosophila identified several Hippo pathway target genes that were required for ectopic overgrowth but not normal growth. Thus, Hippo signaling does not instruct normal growth, and the Hippo-induced overgrowth phenotypes are caused by the activation of abnormal genetic programs.

Cross-sectional association of soluble thrombomodulin with mild peripheral artery disease; the ARIC study. Atherosclerosis Risk in Communities.

Thrombomodulin, an endothelial membrane glycoprotein, is an essential part of the protein C anti-coagulant pathway. It may also have a role in the regulation of fibrinolysis. We carried out a cross-sectional study to assess the association of soluble thrombomodulin (sTM) with peripheral artery disease (PAD) in a stratified random sample (n=863) of otherwise healthy black and white participants of the Atherosclerosis Risk in Communities (ARIC) Study. PAD was more common in black than in white participants and associated with classical risk factors in an expected manner; positively with age, smoking, hypertension, diabetes (P=0.05), and LDL-cholesterol, and inversely with HDL-cholesterol. Significant positive associations were observed also with fibrinogen and white blood cell count. Overall, the sTM concentration was not a significant predictor of PAD. The association was, however, modified by the level of factor VIII:C in whites (P=0.002 for the interaction), but not in blacks. Protein C was inversely associated with PAD prevalence (odds ratio 0.33, 95% CI 0.18--0.61, P=0.0004). sTM was inversely associated with plasminogen, but no associations with t-PA, PAI-1, or D-dimer were seen. In conclusion, the present results provide some additional evidence on the role of thrombomodulin-protein C pathway in atherosclerotic disease and support our earlier observation on interaction between sTM and factor VIII:C.

Selective suppression of CCAAT/enhancer-binding protein beta binding and cyclooxygenase-2 promoter activity by sodium salicylate in quiescent human fibroblasts.

The anti-inflammatory actions of salicylates cannot be explained by inhibition of cyclooxygenase (COX) activity. This study demonstrates that sodium salicylate at a therapeutic concentration suppressed COX-2 gene transcription induced by phorbol 12-myristate 13-acetate and interleukin 1beta by inhibiting the binding of CCAAT/enhancer-binding protein beta to its promoter region of COX-2. By contrast, salicylate did not inhibit nuclear factor kappaB-dependent COX-2 induction by tumor necrosis factor alpha. The inhibitory effect of sodium salicylate was restricted to serum-deprived quiescent cells. These findings indicate that contrary to the current view that salicylate acts via inhibition of nuclear factor kappaB the pharmacological actions of aspirin and salicylates are mediated by inhibiting CCAAT/enhancer-binding protein beta binding and transactivation. These findings have a major impact on the conceptual understanding of the mechanism of action of salicylates and on new drug discovery and design.

Cell cycle-dependent expression of cyclooxygenase-2 in human fibroblasts.

The purpose of this investigation is to determine whether the levels of cyclooxygenase-2 (COX-2) expression are cell cycle dependent. We used a serum-starved human foreskin fibroblast model to determine changes in COX-2 mRNA, protein, and promoter activity in response to stimulation with interleukin-1b (IL-1b) and phorbol 12-myristate 13-acetate (PMA) at G0, G1, S and G2/M phases of the cell cycle. IL-1b (1 ng/ml) and PMA (100 nM) induced robust COX-2 expression in the G0 cells, and the level of COX-2 expression declined progressively after the cells had entered the cell cycle. The COX-2 mRNA level at G1, S and G2/M phases of the cell cycle was 76%, 46%, and 30% of that at G0, respectively. A 5-flanking promoter fragment of COX-2 constructed into a luciferase expression vector was transfected into cells. The promoter activity in response to PMA stimulation was significantly higher in G0 than in S phase cells. These results imply that G0 cells are the key players in inflammation and other COX-2-dependent pathophysiological processes. When the cells are in the proliferative phase, COX-2 inducibility becomes restrained probably by an endogenous control mechanism to avoid COX-2 mediated oxidative DNA damage.

Soluble thrombomodulin as a predictor of incident coronary heart disease and symptomless carotid artery atherosclerosis in the Atherosclerosis Risk in Communities (ARIC) Study: a case-cohort study.

BACKGROUND: Small amounts of soluble thrombomodulin in plasma are thought to reflect endothelial damage. In a case-cohort study, we examined whether soluble thrombomodulin is a predictor of incident coronary heart disease and carotid artery atherosclerosis. METHODS: The study population consisted of 14,170 black and white participants from the Atherosclerosis Risk in Communities (ARIC) study, who did not have cardiovascular disease at the start of the study and who we followed up for 6 years. After appropriate exclusions, we analysed 258 cases of incident coronary heart disease and 449 cases of carotid atherosclerosis. A stratified random sample of 753 individuals from the ARIC cohort was used as the comparison group. We used EIA to measure soluble thrombomodulin in plasma samples from both groups. For the analysis, we used quintiles of soluble thrombomodulin concentrations (< 24.7, 24.8-30.6, 30.7-40.2, 40.3-55.2, and > or = 55.3 ng/mL). FINDINGS: Soluble thrombomodulin showed a strong, graded, inverse association with incident coronary heart disease (p=0.005). The adjusted rate ratio of the highest quintile of soluble thrombomodulin compared with the lowest quintile was 0.29 (95% CI 0.15-0.57). The association with carotid atherosclerosis, however, tended to be positive, especially among white participants (odds ratio 2.94 [1.15-7.51] for highest vs lowest quintile). The relation of soluble thrombomodulin to incident coronary heart disease and carotid atherosclerosis was dependent on factor VIII coagulant activity (p=0.06 and 0.003, respectively). INTERPRETATION: The prospective association of soluble thrombomodulin with incident coronary heart disease differs from its cross-sectional association with carotid atherosclerosis. In healthy people, plasma concentrations of soluble thrombomodulin may reflect endothelial expression of thrombomodulin. High concentration of soluble thrombomodulin may be associated with decreased risk of coronary heart disease.

Suppression of inducible cyclooxygenase 2 gene transcription by aspirin and sodium salicylate.

The pharmacological action of salicylate cannot be explained by its inhibition of cyclooxygenase (COX) activity. In this report, the effects of aspirin and sodium salicylate on COX-2 expressions in human umbilical vein endothelial cells and foreskin fibroblasts were evaluated. Aspirin and sodium salicylate at therapeutic concentrations equipotently blocked COX-2 mRNA and protein levels induced by interleukin-1beta and phorbol 12-myristate 13-acetate. The suppressing effect was more pronounced in cultured cells deprived of fetal bovine serum for 24 h, suggesting that it may be cell cycle related. Salicylate inhibited nascent COX-2 transcript synthesis but had no effect on COX-2 mRNA stability. It inhibited COX-2 promoter activity in a concentration-dependent manner. In mice pretreated with aspirin (10 and 30 mg/kg), followed by challenge with lipopolysaccharide, COX-2 mRNA expression in peritoneal macrophages was markedly suppressed. These findings suggest that salicylate exerts its antiinflammatory action in part by suppressing COX-2 induction, thereby reducing the synthesis of proinflammatory prostaglandins.

Cloning and expression analysis of a novel salicylate suppressible gene, Hs-CUL-3, a member of cullin/Cdc53 family.

By using a mRNA differential display technique to search for salicylate suppressible genes, we identified a cDNA in human foreskin fibroblasts, which by GenBankTM DNA data base search shows sequence homology to the recently reported cullin/Cdc53 (CUL) family genes, especially CUL-3. We have cloned the full-length human CUL-3 (Hs-CUL-3) cDNA. It encodes a 768-amino acid polypeptide and has a predicted molecular weight of 88,939. The amino acid sequence of Hs-CUL-3 shows 46% homology to that of its Caenorhabditis elegans ortholog, Ce-CUL-3, and 27 and 23% to that of Hs-CUL-1 and Hs-CUL-2, respectively. Northern blot analysis showed that phorbol 12-myristate 13-acetate increased the expression of Hs-CUL-3 mRNA in a concentration- and time-dependent manner, and this increase was inhibited by sodium salicylate. Hs-CUL-3 widely expressed in human tissues and its expression in cultured COLO205 colon cancer cells was increased when compared with that in normal colon cells. It is likely that Hs-CUL-3 is involved in cell proliferation control.

The incidence of protein C deficiency in thrombosis-related portal hypertension.

OBJECTIVE: To know the incidence of protein C deficiency associated with noncirrhotic, thrombosis-related portal hypertension. METHODS: Thirty-six patients were studied who had thrombosis-related portal hypertension diagnosed by means of hepatic venography or abdominal echocardiography or during abdominal surgery. Liver disease was excluded in 20 patients based on normal liver function tests and normal histology on liver biopsy. At the time of protein C assays, these patients were not receiving oral anticoagulation, and, in those recently diagnosed, the assays were performed more than 14 days after the last thrombotic event. Antigenic and functional assays for protein C were performed by ELISA and chromogenic assay, respectively. RESULTS: We found 11 patients with protein C deficiency who had a median age of 28 yr (range 19-55 yr) at time of diagnosis. Five patients had a history of systemic thromboembolism, and upper GI bleeding was the most frequent symptom related to portal hypertension (six cases). Antigenic protein C levels were measured in nine of the 11 patients (mean 31.88%, range 10-49%). Functional protein C level was assayed for all 11 patients (mean 40.90%, range 15-58%). After diagnosis, all patients received oral anticoagulants (ideally International Normalized Ratio: 2-3). CONCLUSION: We suggest that protein C screening should be performed in patients with thrombosis-related portal hypertension.

[Reference values for protein C activity in healthy adults in Mexico]. / Valores de referencia de la activadad de proteína C en adultos sanos en México.

Protein C (pC) is a natural-occurring anticoagulant and its acquired or hereditary deficiency has been associated with thrombosis. For its screening, technics that appraise both its plasmatic concentration and biological activity are used. The quantitative deficiency is important, but some characteristics of pC activity (pCA) suggest an essential role of the functional deficiency. Because reference levels have not been previously described in Mexico, we report here the results of a pCA assessed by a chromogenic assay in 88 adult healthy mexican people between 15 and 69 years of age. The pCA values at the 2.5 and 97.5% percentiles in our population were 75 and 137% in normal plasma. Functional disorders of this protein have been described in the presence of normal pC quantitative levels and, therefore, there are individuals with low pC concentrations and a normal pCA without thrombosis. These data suggest that the pCA could be a more important screening test than the quantitative determination as the first step in the study of a possible deficiency state of protein C.

[The international normalization ratio in the monitoring of oral anticoagulant therapy]. / El cociente de normalización internacional en la vigilancia de la terapia anticoagulante oral.

Oral anticoagulants are employed very frequently in the prophylaxis and treatment of several diseases. For their optimal effectiveness, many vigilance schedules have been proposed but none has proved to be 100% effective. The international normalization ratio (INR) can be a safer way to monitor oral anticoagulation, and our objective was to determine its clinical usefulness. A prothrombin time test (PT) was carried out by means of either a chromogenic or a coagulometric method, and an INR was obtained using the ratio of the PT patient/PT control elevated to an exponential given by the international sensitivity index (ISI) of our thromboplastin. Our objective was to maintain our patients in a therapeutical INR range between two and three. We present our experience with 77 patients and 810 results during an 18 months period. We observed 26 cases of hemorrhage and three of thrombosis. In all these cases, the INR was out of the desired therapeutical range. No deaths occurred in our patients. Our analysis showed a significative disagreement between the INR and the prothrombin time ratio (PTR) but a better correlation with hemorrhage and thrombosis was seen with the INR than with the PTR. Our experience supports the use of INR in the clinical vigilance of oral anticoagulation as a useful method.