George F. Schreiner

B-Type Natriuretic Peptide Exerts Broad Functional Opposition to Transforming Growth Factor-β in Primary Human Cardiac Fibroblasts. Fibrosis, Myofibroblast Conversion, Proliferation, and Inflammation

Abstract— The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-&bgr; (TGF-&bgr;) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-&bgr;–induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-&bgr;–induced cell proliferation as well as the production of collagen 1 and fibronectin proteins as measured by Western blot analysis. cDNA microarray analysis was performed on RNA from cardiac fibroblasts incubated in the presence or absence of TGF-&bgr; and BNP for 24 and 48 hours. TGF-&bgr;, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-&bgr; effects; 88% and 85% of all TGF-&bgr;–regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-&bgr;–regulated genes related to fibrosis (collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (&agr;-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNF &agr;-induced protein 6, and TNF superfamily, member 4). Lastly, BNP stimulated the extracellular signal-related kinase pathway via cyclic guanosine monophosphate–dependent protein kinase signaling, and two mitogen-activated protein kinase kinase inhibitors, U0126 and PD98059, reversed BNP inhibition of TGF-&bgr;–induced collagen-1 expression. These findings demonstrate that BNP has a direct effect on cardiac fibroblasts to inhibit fibrotic responses via extracellular signal-related kinase signaling, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.

Uric Acid Induces Hepatic Steatosis by Generation of Mitochondrial Oxidative Stress POTENTIAL ROLE IN FRUCTOSE-DEPENDENT AND -INDEPENDENT FATTY LIVER

Background: Uric acid is an independent risk factor in fructose-induced fatty liver, but whether it is a marker or a cause remains unknown. Results: Hepatocytes exposed to uric acid developed mitochondrial dysfunction and increased de novo lipogenesis, and its blockade prevented fructose-induced lipogenesis. Conclusion: Rather than a consequence, uric acid induces fatty liver Significance: Hyperuricemic people are more prone to develop fructose-induced fatty liver. Metabolic syndrome represents a collection of abnormalities that includes fatty liver, and it currently affects one-third of the United States population and has become a major health concern worldwide. Fructose intake, primarily from added sugars in soft drinks, can induce fatty liver in animals and is epidemiologically associated with nonalcoholic fatty liver disease in humans. Fructose is considered lipogenic due to its ability to generate triglycerides as a direct consequence of the metabolism of the fructose molecule. Here, we show that fructose also stimulates triglyceride synthesis via a purine-degrading pathway that is triggered from the rapid phosphorylation of fructose by fructokinase. Generated AMP enters into the purine degradation pathway through the activation of AMP deaminase resulting in uric acid production and the generation of mitochondrial oxidants. Mitochondrial oxidative stress results in the inhibition of aconitase in the Krebs cycle, resulting in the accumulation of citrate and the stimulation of ATP citrate lyase and fatty-acid synthase leading to de novo lipogeneis. These studies provide new insights into the pathogenesis of hepatic fat accumulation under normal and diseased states.

The Role of p38α Mitogen-Activated Protein Kinase Activation in Renal Fibrosis

The p38 mitogen-activated protein kinase (MAPK) pathway transduces external stress stimuli and is important in extracellular matrix synthesis in cell types in vitro; however, its role in renal fibrosis is not known. Explored was the role the p38 MAPK pathway in rat unilateral ureteric obstruction (UUO), a model of renal fibrosis induced by a noninflammatory surgical insult. In a time-course study, a marked increase in phosphorylation (activation) of p38 in both interstitial myofibroblasts and tubules was shown. Rats were then treated daily with a specific inhibitor of p38alpha, NPC 31169, from the time of UUO surgery until being killed 7 d later. Compared with vehicle, NPC 31169-treated rats had a significant reduction in renal fibrosis assessed by interstitial volume, collagen IV deposition, and mRNA levels. This was primarily due to a reduction in the accumulation of interstitial myofibroblasts, as shown by a reduction in the area of immunostaining for alpha-smooth muscle actin and heat shock protein 47. The increase in renal TGF-beta1 mRNA and protein levels in UUO was unaltered with NPC 31169 treatment; however, connective tissue growth factor mRNA was reduced. These results demonstrate that p38alpha MAPK plays an important role in renal fibrosis, acting downstream of TGF-beta1. Blockade of p38 MAPK reduces extracellular matrix production and may be considered a potential therapeutic option in the treatment of renal fibrosis.

Inhibition of Growth and Metastasis of Mouse Mammary Carcinoma by Selective Inhibitor of Transforming Growth Factor-β Type I Receptor Kinase In vivo

Purpose: Transforming growth factor-β (TGF-β) suppresses tumor development by inhibiting cellular proliferation, inducing differentiation and apoptosis, and maintaining genomic integrity. However, once tumor cells escape from the tumor-suppressive effects of TGF-β, they often constitutively overexpress and activate TGF-β, which may promote tumor progression by enhancing invasion, metastasis, and angiogenesis and by suppressing antitumor immunity. The purpose of this study was to test this hypothesis using TGF-β pathway antagonists. Experimental Design: We examined the effects of selective TGF-β type I receptor kinase inhibitors, SD-093 and SD-208, on two murine mammary carcinoma cell lines (R3T and 4T1) in vitro and in vivo. Results: Both agents blocked TGF-β-induced phosphorylation of the receptor-associated Smads, Smad2 and Smad3, in a dose-dependent manner, with IC50 between 20 and 80 nmol/L. TGF-β failed to inhibit growth of these cell lines but stimulated epithelial-to-mesenchymal transdifferentiation, migration, and invasiveness into Matrigel in vitro. These effects were inhibited by SD-093, indicating that these processes are partly driven by TGF-β. Treatment of syngeneic R3T or 4T1 tumor-bearing mice with orally given SD-208 inhibited primary tumor growth as well as the number and size of metastases. In contrast, SD-208 failed to inhibit R3T tumor growth or metastasis in athymic nude mice. Moreover, in vitro anti-4T1 cell cytotoxic T-cell responses of splenocytes from drug-treated animals were enhanced compared with cells from control animals. In addition, SD-208 treatment resulted in a decrease in tumor angiogenesis. Conclusion: TGF-β type I receptor kinase inhibitors hold promise as novel therapeutic agents for metastatic breast cancer.

Uric acid stimulates fructokinase and accelerates fructose metabolism in the development of fatty liver.

Excessive dietary fructose intake may have an important role in the current epidemics of fatty liver, obesity and diabetes as its intake parallels the development of these syndromes and because it can induce features of metabolic syndrome. The effects of fructose to induce fatty liver, hypertriglyceridemia and insulin resistance, however, vary dramatically among individuals. The first step in fructose metabolism is mediated by fructokinase (KHK), which phosphorylates fructose to fructose-1-phosphate; intracellular uric acid is also generated as a consequence of the transient ATP depletion that occurs during this reaction. Here we show in human hepatocytes that uric acid up-regulates KHK expression thus leading to the amplification of the lipogenic effects of fructose. Inhibition of uric acid production markedly blocked fructose-induced triglyceride accumulation in hepatocytes in vitro and in vivo. The mechanism whereby uric acid stimulates KHK expression involves the activation of the transcription factor ChREBP, which, in turn, results in the transcriptional activation of KHK by binding to a specific sequence within its promoter. Since subjects sensitive to fructose often develop phenotypes associated with hyperuricemia, uric acid may be an underlying factor in sensitizing hepatocytes to fructose metabolism during the development of fatty liver.

Increased Oxidative Stress Following Acute and Chronic High Altitude Exposure

The generation of reactive oxygen species is typically associated with hyperoxia and ischemia reperfusion. Recent evidence has suggested that increased oxidative stress may occur with hypoxia. We hypothesized that oxidative stress would be increased in subjects exposed to high altitude hypoxia. We studied 28 control subjects living in Lima, Peru (sea level), at baseline and following 48 h exposure to high altitude (4300 m). To assess the effects of chronic altitude exposure, we studied 25 adult males resident in Cerro de Pasco, Peru (altitude 4300 m). We also studied 27 subjects living in Cerro de Pasco who develop excessive erythrocytosis (hematocrit > 65%) and chronic mountain sickness. Acute high altitude exposure led to increased urinary F(2)-isoprostane, 8-iso PGF(2 alpha) (1.31 +/- 0.8 microg/g creatinine versus 2.15 +/- 1.1, p = 0.001) and plasma total glutathione (1.29 +/- 0.10 micromol versus 1.37 +/- 0.09, p = 0.002), with a trend to increased plasma thiobarbituric acid reactive substance (TBARS) (59.7 +/- 36 pmol/mg protein versus 63.8 +/- 27, p = NS). High altitude residents had significantly elevated levels of urinary 8-iso PGF(2 alpha) (1.3 +/- 0.8 microg/g creatinine versus 4.1 +/- 3.4, p = 0.007), plasma TBARS (59.7 +/- 36 pmol/mg protein versus 85 +/- 28, p = 0.008), and plasma total glutathione (1.29 +/- 0.10 micromol versus 1.55 +/- 0.19, p < 0.0001) compared to sea level. High altitude residents with excessive erythrocytosis had higher levels of oxidative stress compared to high altitude residents with normal hematological adaptation. In conclusion, oxidative stress is increased following both acute exposure to high altitude without exercise and with chronic residence at high altitude.

Blockade of p38α MAPK Ameliorates Acute Inflammatory Renal Injury in Rat Anti-GBM Glomerulonephritis

The p38 mitogen-activated protein kinase (MAPK) pathway is a pro-inflammatory signal transduction pathway. The aim of this study was to examine the role of this pathway in acute renal inflammation. Immunostaining localized components of the p38 MAPK pathway (p38alpha, p-p38, p-ATF-2) in normal glomeruli, to podocytes, and occasional endothelial cells. This study identified an eightfold increase in glomerular activation of p38 MAPK (phosphorylated p38, p-p38) within 3 h of the induction of rat anti-glomerular basement membrane (GBM) glomerulonephritis and localized p-p38 and p-ATF-2 to infiltrating neutrophils, with increased staining of podocytes and endothelial cells. The relevance of these findings to human acute inflammatory renal disease was determined by examination of biopsy specimens. In patients with post-infectious glomerulonephritis, there was an increased number of positive p-p38 glomerular cells, including p-p38 staining of infiltrating neutrophils, compared with normal human kidney. In rats, administration of a specific p38 MAPK inhibitor, NPC 31145, before induction of anti-GBM disease prevented a loss of renal function and substantially reduced proteinuria. The reduction in renal injury was attributed to a 55% reduction in glomerular neutrophil infiltration and a 68% reduction in platelet accumulation. This was associated with an abrogation of glomerular P-selectin immunostaining and inhibition of glomerular P-selectin gene expression. In summary, this study has localized the components of the p38 MAPK pathway to cells in normal and diseased rat and human kidney and identified a number of important mechanisms by which signaling through the p38 MAPK pathway induces inflammatory renal disease. Blockade of the p38 pathway may be a novel therapeutic strategy for the treatment of acute renal inflammation.

Role of ERK1/2 and p38 Mitogen-Activated Protein Kinases in the Regulation of Thrombospondin-1 by TGF-β1 in Rat Proximal Tubular Cells and Mouse Fibroblasts

Thrombospondin-1 (TSP-1) inhibits angiogenesis and activates latent TGF-beta1, both of which are strongly associated with progression of renal disease. Recently, it was reported that Smad2 but not Smad3 regulates TSP-1 expression in response to TGF-beta1 in rat tubular epithelial cells as well as in mouse fibroblasts. This study investigated the role of ERK1/2 and p38 mitogen-activated protein kinases (MAPK). TGF-beta1 activated both ERK1/2 and p38 in the rat proximal tubular cell line NRK52E. Blocking ERK1/2 and p38 inhibited TGF-beta1-induced TSP-1 mRNA and protein expression. Next, the cross-talk between Smad2 and ERK1/2 or p38 was examined. Whereas blocking of ERK1/2 or p38 failed to inhibit TGF-beta1-induced Smad2 activation, inhibition of Smad2 by Smad7 overexpression inhibited the phosphorylation of ERK1/2 but not p38 in response to TGF-beta1. Similar results were observed using mouse fibroblasts from Smad2 knockout embryos, in that TGF-beta1 was able to activate p38 but not ERK1/2 in this cell line. In conclusion, TSP-1 expression is regulated by both ERK1/2 and p38 MAPK in rat proximal tubular cells and mouse fibroblasts in response to TGF-beta1. The ERK1/2 activation is dependent on Smad2 activation, whereas the p38 activation occurs independent of Smad2. Because TSP-1 is a major antiangiogenic molecule and an activator of TGF-beta1, this provides an important insight to the mechanism by which TGF-beta1 may mediate interstitial fibrosis and progressive renal disease.

p38α Mitogen-activated protein kinase inhibition improves cardiac function and reduces myocardial damage in isoproterenol-induced acute myocardial injury in rats

p38 mitogen-activated protein (MAP) kinase is activated during ischemic/hypoxic myocardial injury. However, the role of activated p38 MAP kinase on cardiac function after myocardial injury is not well understood. In the present study, we investigated the cardioprotective effects of p38 MAP kinase inhibition in a rat model of acute myocardial injury, induced by subcutaneous injection of isoproterenol (ISO, 20 mg/kg/d for 3 days). A synthetic p38α MAP kinase inhibitor, SD-282 (40 mg/kg) or vehicle (0.25% Tween 80 in saline) was given intraperitoneally twice a day for 3 days, concomitant with ISO treatment. Cardiac function, systolic blood pressure, gene expression including collagen I and III, fibronectin and COX-2, and the myocardial injury were analyzed. Results showed that administration of SD-282 remarkably improved ISO-induced reduction of cardiac function with increases in ejection fraction (P < 0.001), cardiac output (P < 0.05), stroke volume (P < 0.001), and cardiac index (P < 0.01). SD-282 abolished ISO-induced reduction of systolic blood pressure (106.7 ± 2.2 versus 123.1 ± 5.3 mm Hg, P < 0.05). The ISO-induced expression of COX-2, collagen I and III, and fibronectin genes was reduced significantly (P < 0.05 in all cases) by administration of SD-282. The myocardial injury induced by ISO was significantly reduced by the treatment of SD-282 as judged by the reduction of myocardial necrosis. Data suggest that p38α MAP kinase may be involved in the pathogenesis of cardiac dysfunction in ischemic myocardial injury. Inhibition of this enzyme may improve cardiac function and protect myocardium from ischemic/hypoxic injury that occurs during ischemic heart disease.

Inhibition of p38 mitogen-activated protein kinase augments progression of remnant kidney model by activating the ERK pathway.

p38, a mitogen-activated protein kinase, is a major intracellular signaling molecule involved in inflammation. To test the hypothesis that p38 mediates renal disease progression, we administered a novel p38 alpha inhibitor, NPC31169, to rats with remnant kidneys (RKs). RK rats showed increased p38 activation at 9 weeks (by p38 kinase assay), which was blocked by the inhibitor. In contrast to our expectation, treatment with the NPC31169 resulted in worse renal function, more proteinuria, and more severe glomerulosclerosis and tubulointerstitial injury. p38 inhibition resulted in marked cell proliferation in RK rats, with more proliferating tubular cells, myofibroblasts, and macrophages. In contrast, p38 suppression resulted in less tubular cell apoptosis. Interestingly, Western blot demonstrated increased ERK1/2 phosphorylation in p38-treated rats. No histological changes were observed in p38 inhibited sham-operated rats. Our findings indicate that, whereas blocking p38 usually shows benefit in inflammatory disease, in this model p38 inhibition resulted in accelerated renal progression. We conclude that blocking p38-dependent inflammation may have resulted in enhanced proliferation and increased ERK1/2 activation, and thereby explains the worse renal lesions observed.