Smad proteins are intracellular signaling effectors of the TGF beta superfamily. We show that endogenous Smad2, 3, and 4 bind microtubules (MTs) in several cell lines. Binding of Smads to MTs does not require TGF beta stimulation. TGF beta triggers dissociation from MTs, phosphorylation, and nuclear translocation of Smad2 and 3, with consequent activation of transcription in CCL64 cells. Destabilization of the MT network by nocodazole, colchicine, or a tubulin mutant disrupts the complex between Smads and MTs and increases TGF beta-induced Smad2 phosphorylation and transcriptional response in CCL64 cells. These data demonstrate that MTs may serve as a cytoplasmic sequestering network for Smads, controlling Smad2 association with and phosphorylation by activated TGF beta receptor I, and suggest a novel mechanism for the MT network to negatively regulate TGF beta function.

Scleroderma is a chronic systemic disease that leads to fibrosis of affected organs. Transforming growth factor (TGF) β has been implicated in the pathogenesis of scleroderma. Smad proteins are signaling transducers downstream from TGF-β receptors. Three families of Smads have been identified: ( i ) receptor-regulated Smad2 and -3 (R-Smads); ( ii ) common partner Smad4 (Co-Smad); and ( iii ) inhibitory Smad6 and -7 (I-Smads, part of a negative feedback loop). We have investigated the signaling components for the TGF-β pathway and TGF-β activity in scleroderma lesions in vivo and in scleroderma fibroblasts in vitro . Basal level and TGF-β-inducible expression of Smad7 are selectively decreased, whereas Smad3 expression is increased both in scleroderma skin and in explanted scleroderma fibroblasts in culture. TGF-β signaling events, including phosphorylation of Smad2 and -3, and transcription of the PAI-1 gene are increased in scleroderma fibroblasts, relative to normal fibroblasts. In vitro adenoviral gene transfer with Smad7 restores normal TGF-β signaling in scleroderma fibroblasts. These results suggest that alterations in the Smad pathway, including marked Smad7 deficiency and Smad3 up-regulation, may be responsible for TGF-β hyperresponsiveness observed in scleroderma.

Background Therapeutic hypothermia (TH) improves outcomes in comatose survivors of cardiac arrest. Few hospitals have protocol-driven plans that include TH. We implemented a series of process interventions designed to increase TH use and improve outcomes in patients successfully resuscitated from out-of-hospital cardiac arrest (OHCA) or in-hospital cardiac arrest (IHCA). Methods and Results Linked interventions including a TH order sheet, verbal and written feedback to individual providers, an educational program, TH “kit” and on-call consultants to assist with patient care and hypothermia induction were implemented between January 1, 2005 and December 31, 2007 in a large, university-affiliated, tertiary care center. We then completed a retrospective review of all patients treated for cardiac arrest during the study period. Descriptive statistics, chi-squared analyses, or Fisher’s exact test were used as appropriate. A p value <0.05 was considered significant. 135 OHCA patients and 106 IHCA patients were eligible for post-arrest care. TH use increased each year in the OHCA group (from 6% to 65% to 76%; p<0.001) and IHCA group (from 0% to 36% to 53%; p=.02). A good outcome was achieved in 21% and 8% of comatose patients with OHCA and IHCA, respectively. Patients with OHCA and ventricular dysrhythmia were more likely to have a good outcome with TH treatment than without it (good outcome in 57% vs. 8%; p=.005). Conclusion Implementing a series of aggressive interventions increased appropriate TH use and was associated with improved outcomes in our facility.

Tubulointerstitial changes in the diabetic kidney correlate closely with the decline in glomerular filtration. In this study, we used a cell culture system of mouse proximal tubule epithelial cells to test the effects of glucose on cell growth, size, and matrix biosynthesis. [3H]thymidine incorporation was significantly inhibited in cells grown in 450 mg/dl glucose, compared with cells grown in 100 mg/dl glucose. The cells grown in the higher glucose concentration were slightly larger, their protein content and the total protein synthetic rate were significantly increased, and they secreted approximately twice as much procollagens type IV and type I. Concordantly, steady-state procollagen mRNA levels were also increased: 2.6-fold for the alpha 1(IV) and 2.2-fold for the alpha 2(I) procollagens. Additionally, nuclear run-off studies demonstrated that procollagen gene transcription rate was stimulated approximately 50%; beta-actin transcription rate was not altered. We used chloramphenicol acetyltransferase (CAT) reporter gene constructs to determine whether the increased transcription rate of alpha 2(I) gene was associated with activation of its enhancer sequence. Cells transfected with the enhancer demonstrated more than fivefold increase in CAT activity when cultured in the high-glucose medium. These studies demonstrate a multitude of effects of high ambient glucose concentrations on proximal tubule cell growth and collagen biosynthesis; cell proliferation is decreased although cell hypertrophy occurs. Procollagen gene transcription rate is stimulated and this response contributes to the observed increase in procollagen mRNA content. Activation of an enhancer sequence may be one possible mode through which high glucose levels increase the transcription of procollagen type I, presumably involving trans-acting factor(s).

Introduction-Coronary angiography (CATH) is associated with survival in patients suffering out-of-hospital cardiac arrest (OHCA) from ventricular fibrillation or ventricular tachycardia (VF/ VT). The effect of CATH on outcome in other cohorts is unknown. We hypothesized clinical parameters of resuscitated patients predict CATH performance and receiving CATH was associated with outcome.

In this small retrospective study of women with peripartum cardiomyopathy, patients treated with immune globulin had a greater improvement in ejection fraction during early follow-up than patients treated conventionally. Given the poor prognosis of women with peripartum cardiomyopathy who do not improve, this therapy merits further study.

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