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...

Human cardiac fibroblasts are protected from oxidative stress triggered by inflammation after myocardial injury (Li, P. F., Dietz, R., and von Harsdorf, R. (1999) FEBS Lett. 448, 206 -210) by expressing potent antioxidant defenses such as superoxide dismutases, catalases, glutathione-peroxidases, and peroxiredoxins. Recently the transcription factor FOXO3A has been shown to increase resistance to oxidative stress by up-regulation of mitochondrial superoxide dismutase and peroxisomal catalase (Kops, G.

Transgenic mice (TG) were used to define mitochondrial oxidative stress and cardiomyopathy (CM) induced by zidovudine (AZT), an antiretroviral used to treat HIV/AIDS. Genetically engineered mice either depleted or overexpressed mitochondrial superoxide dismutase (SOD2 þ /À KOs and SOD2-OX, respectively) or expressed mitochondrially targeted catalase (mCAT). TGs and wild-type (WT) littermates were treated (oral AZT, 35 days). Cardiac mitochondrial H 2 O 2 , aconitase activity, histology and ultrastructure were analyzed. Left ventricle (LV) mass and LV end-diastolic dimension were determined echocardiographically. AZT induced cardiac oxidative stress and LV dysfunction in WTs. Cardiac mitochondrial H 2 O 2 increased and aconitase was inactivated in SOD2 þ /À KOs, and cardiac dysfunction was worsened by AZT. Conversely, the cardiac function in SOD2-OX and mCAT hearts was protected. In SOD2-OX and mCAT TG hearts, mitochondrial H 2 O 2 , LV mass and LV cavity volume resembled corresponding values from vehicle-treated WTs. AZT worsens cardiac dysfunction and increases mitochondrial H 2 O 2 in SOD2 þ /À KO. Conversely, both SOD2-OX and mCAT TGs prevent or attenuate AZT-induced cardiac oxidative stress and LV dysfunction. As dysfunctional changes are ameliorated by decreasing and worsened by increasing H 2 O 2 abundance, oxidative stress from H 2 O 2 is crucial pathogenetically in AZT-induced mitochondrial CM.

Mitochondrial (mt) DNA biogenesis is critical to cardiac contractility. DNA polymerase gamma (Pol gamma) replicates mtDNA, whereas thymidine kinase 2 (TK2) monophosphorylates pyrimidines intramitochondrially. Point mutations in POLG and TK2 result in clinical diseases associated with mtDNA depletion and organ dysfunction. Pyrimidine analogs (NRTIs) inhibit Pol gamma and mtDNA replication. Cardiac "dominant negative" murine transgenes (TGs; Pol gamma Y955C, and TK2 H121N or I212N) defined the role of each in the heart. mtDNA abundance, histopathological features, histochemistry, mitochondrial protein abundance, morphometry, and echocardiography were determined for TGs in "2 x 2" studies with or without pyrimidine analogs. Cardiac mtDNA abundance decreased in Y955C TGs ( approximately 50%) but increased in H121N and I212N TGs (20-70%). Succinate dehydrogenase (SDH) increased in hearts of all mutants. Ultrastructural changes occurred in Y955C and H121N TGs. Histopathology demonstrated hypertrophy in H121N, LV dilation in I212N, and both hypertrophy and dilation in Y955C TGs. Antiretrovirals increased LV mass ( approximately 50%) for all three TGs which combined with dilation indicates cardiomyopathy. Taken together, these studies demonstrate three manifestations of cardiac dysfunction that depend on the nature of the specific mutation and antiretroviral treatment. Mutations in genes for mtDNA biogenesis increase risk for defective mtDNA replication, leading to LV hypertrophy.

Background:Photomicrographs in Anatomic Pathology provide a means of quickly sharing information from a glass slide for consultation, education, documentation and publication. While static image acquisition historically involved the use of a permanently mounted camera unit on a microscope, such cameras may be expensive, need to be connected to a computer, and often require proprietary software to acquire and process images. Another novel approach for capturing digital microscopic images is to use smartphones coupled with the eyepiece of a microscope. Recently, several smartphone adapters have emerged that allow users to attach mobile phones to the microscope. The aim of this study was to test the utility of these various smartphone adapters.Materials and Methods:We surveyed the market for adapters to attach smartphones to the ocular lens of a conventional light microscope. Three adapters (Magnifi, Skylight and Snapzoom) were tested. We assessed the designs of these adapters and their effectiveness at acquiring static microscopic digital images.Results:All adapters facilitated the acquisition of digital microscopic images with a smartphone. The optimal adapter was dependent on the type of phone used. The Magnifi adapters for iPhone were incompatible when using a protective case. The Snapzoom adapter was easiest to use with iPhones and other smartphones even with protective cases.Conclusions:Smartphone adapters are inexpensive and easy to use for acquiring digital microscopic images. However, they require some adjustment by the user in order to optimize focus and obtain good quality images. Smartphone microscope adapters provide an economically feasible method of acquiring and sharing digital pathology photomicrographs.