Abstract-Human cardiac fibroblasts are the main source of cardiac fibrosis associated with cardiac hypertrophy and heart failure. Transforming growth factor-␤1 (TGF-␤1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle ␣-actin (SM ␣-actin) de novo and produce extracellular matrix. We hypothesized that TGF-␤1-stimulated conversion of fibroblasts to myofibroblasts requires reactive oxygen species derived from NAD(P)H oxidases (Nox). We found that TGF-␤1 potently upregulates the contractile marker SM ␣-actin mRNA (7.5Ϯ0.8-fold versus control). To determine whether Nox enzymes are involved, we first performed quantitative real time polymerase chain reaction and found that Nox5 and Nox4 are abundantly expressed in cardiac fibroblasts, whereas Nox1 and Nox2 are barely detectable. On stimulation with TGF-␤1, Nox4 mRNA is dramatically upregulated by 16.2Ϯ0.8-fold (nϭ3, PϽ0.005), whereas Nox5 is downregulated. Small interference RNA against Nox4 downregulates Nox4 mRNA by 80Ϯ5%, inhibits NADPH-driven superoxide production in response to TGF-␤1 by 65Ϯ7%, and reduces TGF-␤1-induced expression of SM ␣-actin by 95Ϯ2% (nϭ6, PϽ0.05). Because activation of small mothers against decapentaplegic (Smads) 2/3 is critical for myofibroblast conversion in response to TGF-␤1, we also determined whether Nox4 affects Smad 2/3 phosphorylation. Depletion of Nox4 but not Nox5 inhibits baseline and TGF-␤1 stimulation of Smad 2/3 phosphorylation by 75Ϯ5% and 68Ϯ3%, respectively (nϭ7, PϽ0.0001). We conclude that Nox 4 mediates TGF-␤1-induced conversion of fibroblasts to myofibroblasts by regulating Smad 2/3 activation. Thus, Nox4 may play a critical role in the pathological activation of cardiac fibroblasts in cardiac fibrosis associated with human heart failure. (Circ Res. 2005;97:900-907.)Key Words: Nox4 Ⅲ human cardiac fibroblasts Ⅲ transforming growth factor Ⅲ reactive oxygen species Ⅲ Smad 2/3 H eart failure remains the leading cause of hospital admissions in the United States, with more than 550 000 new patients diagnosed each year. 1 Regardless of etiology, cardiac fibrosis is a major contributor to cardiac remodeling associated with cardiomyopathies. It is characterized by expansion of the interstitial compartment due to increased deposition of extracellular matrix by activated myofibroblasts. 2 Cardiac myofibroblasts are specialized contractile fibroblasts formed by irreversible acquisition of contractile proteins such as smooth muscle ␣-actin (SM ␣-actin) in response to potent fibrogenic cytokines. 3 The expression of SM ␣-actin is regulated by transforming growth factor-␤1 (TGF-␤1), a primary fibrogenic growth factor in heart failure that is downstream of many of the pro-fibrotic actions of other fibroblast growth factors, such as angiotensin II, aldosterone, and norepinephrine. 4 TGF-␤1 is upregulated in failing human hearts and various experimental models of cardiac hypertrophy, 4 and functional blockade of TGF-␤1 prevents cardiac interstitial fibrosis induced by pressure overload in...

Background Bronchial anastomotic complications develop in 31% of lung transplant recipients, leading to additional operative procedures and increased morbidity. Advances in surgical technique have thus far resulted in only modestly improved outcomes. We hypothesized that creating the bronchial anastomosis at the secondary carina using a combination of running and figure-of-eight sutures would minimize donor bronchial ischemia and airway complications. Methods This retrospective review of a single surgeon’s operative experience from 2000 to 2007 compares a new bronchial anastomotic technique with the conventional technique. The primary outcome was the occurrence of bronchial anastomotic complications requiring invasive intervention. The secondary outcome was distal airway complications. Patients were monitored for 1 year after transplant. Recipient and donor demographic data as well as relevant variables from their preoperative, perioperative, and postoperative courses were collected for analysis. These data were compared using t tests for normally distributed continuous variables, Mann-Whitney tests for nonnormally distributed continuous variables, and χ2 tests or Fisher exact test for categoric variables. Logistic regression was used to control for covariates while comparing the primary outcome between the new and conventional bronchial anastomotic techniques. Results The analysis included 230 patients, representing 407 anastomoses. The occurrence of anastomotic complications requiring intervention and distal airway complications decreased from 18.1% to 2.3% of anastomoses and 12.2% to 4.4% of patients, respectively. After controlling for available risk factors, the new technique significantly reduced both anastomotic (p < 0.001) and distal (p = 0.03) airway complications. Conclusions This new anastomotic technique dramatically reduces anastomotic and distal airway complications after lung transplantation.

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Objective The requirement for toll-like receptors in lung ischemia reperfusion injury (LIRI) has been demonstrated but not fully characterized. We have previously reported that toll-like receptor-4 is required by alveolar macrophages but not pulmonary endothelial or epithelial cells for the development of LIRI. Additionally, we have demonstrated differential patterns of mitogen-activated protein kinase activation and cytokine release in these cell types during LIRI. We sought to determine whether the differences in their activation responses related to cell specific toll-like receptor activation requirements. Methods Primary cultures of alveolar macrophages, pulmonary endothelial, and immortalized epithelial cells were pretreated with toll-like receptor-2 or -4 short interference (si)RNA prior to hypoxia and reoxygenation. Cell lysates and media were analyzed for receptor knockdown, mitogen-activated protein kinase activation, and cytokine production. Rats were pretreated with toll-like receptor-2 or -4 siRNA prior to lung ischemia reperfusion and changes in lung vascular permeability were assessed. Results Toll-like receptor-2 knockdown in alveolar macrophages did not affect mitogen-activated protein kinase phosphorylation or cytokine secretion. Conversely, toll-like receptor-2 knockdown in pulmonary endothelial and epithelial cells demonstrated significant reductions in ERK 1/2 activation and cytokine secretion. Toll-like receptor-4, but not toll-like receptor-2, decreased lung permeability index in LIRI. Conclusions Differential toll-like receptor signaling and mitogen-activated protein kinase activation in response to LIRI appear to be cell specific. siRNA provides an outstanding tool for examination of the underlying mechanism.