ABSTRACT
The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.
Subject(s)
Adenosine Triphosphate , Mitochondria , Mice , Animals , Adenosine Triphosphate/metabolism , Mitochondria/metabolism , Proton-Translocating ATPases/metabolism , Proteins/metabolism , Homeostasis , HydrolysisABSTRACT
INTRODUCTION: We conducted an open-label study to examine the effects of the flavonoid (-)-epicatechin in seven ambulatory adult patients with Becker muscular dystrophy (BMD). METHODS: Seven participants received (-)-epicatechin 50 mg twice per day for 8 weeks. Pre- and postprocedures included biceps brachii biopsy to assess muscle structure and growth-relevant endpoints by western blotting, mitochondria volume measurement, and cristae abundance by electron microscopy, graded exercise testing, and muscle strength and function tests. RESULTS: Western blotting showed significantly increased levels of enzymes modulating cellular bioenergetics (liver kinase B1 and 5'-adenosine monophosphate-activated protein kinase). Peroxisome proliferator-activated receptor gamma coactivator-1alpha, a transcriptional coactivator of genes involved in mitochondrial biogenesis and cristae-associated mitofilin levels, increased as did cristae abundance. Muscle and plasma follistatin increased significantly while myostatin decreased. Markers of skeletal muscle regeneration myogenin, myogenic regulatory factor-5, myoblast determination protein 1, myocyte enhancer factor-2, and structure-associated proteins, including dysferlin, utrophin, and intracellular creatine kinase, also increased. Exercise testing demonstrated decreased heart rate, maximal oxygen consumption per kilogram, and plasma lactate levels at defined workloads. Tissue saturation index improved in resting and postexercise states. DISCUSSION: (-)-Epicatechin, an exercise mimetic, appears to have short-term positive effects on tissue biomarkers indicative of mitochondrial biogenesis and muscle regeneration, and produced improvements in graded exercise testing parameters in patients with BMD.
Subject(s)
Catechin/therapeutic use , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/drug therapy , Adult , Biopsy , Blotting, Western , Creatine Kinase/metabolism , Dysferlin/metabolism , Exercise Test , Follistatin/metabolism , Heart Rate , Humans , Lactic Acid/blood , MEF2 Transcription Factors/metabolism , Male , Microscopy, Electron , Middle Aged , Mitochondria/ultrastructure , Mitochondrial Proteins/metabolism , Mitochondrial Size , Muscle Proteins/metabolism , Muscle Strength , Muscle, Skeletal/physiopathology , Muscle, Skeletal/ultrastructure , Muscular Dystrophy, Duchenne/metabolism , Muscular Dystrophy, Duchenne/pathology , Muscular Dystrophy, Duchenne/physiopathology , MyoD Protein/metabolism , Myogenic Regulatory Factor 5/metabolism , Myogenin/metabolism , Myostatin/metabolism , Organelle Biogenesis , Oxygen Consumption , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Regeneration , Utrophin/metabolismABSTRACT
BACKGROUND: (+)-Epicatechin (EPI) induces mitochondrial biogenesis and antioxidant metabolism in muscle fibers and neurons. We aimed to evaluate safety and efficacy of (+)-EPI in pediatric subjects with Friedreich's ataxia (FRDA). METHODS: This was a phase II, open-label, baseline-controlled single-center trial including 10 participants ages 10 to 22 with confirmed FA diagnosis. (+)-EPI was administered orally at 75 mg/d for 24 weeks, with escalation to 150 mg/d at 12 weeks for subjects not showing improvement of neuromuscular, neurological or cardiac endpoints. Neurological endpoints were change from baseline in Friedreich's Ataxia Rating Scale (FARS) and 8-m timed walk. Cardiac endpoints were changes from baseline in left ventricular (LV) structure and function by cardiac magnetic resonance imaging (MRI) and echocardiogram, changes in cardiac electrophysiology, and changes in biomarkers for heart failure and hypertrophy. RESULTS: Mean FARS/modified (m)FARS scores showed nonstatistically significant improvement by both group and individual analysis. FARS/mFARS scores improved in 5/9 subjects (56%), 8-m walk in 3/9 (33%), 9-peg hole test in 6/10 (60%). LV mass index by cardiac MRI was significantly reduced at 12 weeks (P = .045), and was improved in 7/10 (70%) subjects at 24 weeks. Mean LV ejection fraction was increased at 24 weeks (P = .008) compared to baseline. Mean maximal septal thickness by echocardiography was increased at 24 weeks (P = .031). There were no serious adverse events. CONCLUSION: (+)-EPI was well tolerated over 24 weeks at up to 150 mg/d. Improvement was observed in cardiac structure and function in subset of subjects with FRDA without statistically significant improvement in primary neurological outcomes. SYNOPSIS: A (+)-epicatechin showed improvement of cardiac function, nonsignificant reduction of FARS/mFARS scores, and sustained significant upregulation of muscle-regeneration biomarker follistatin.
Subject(s)
Antioxidants/administration & dosage , Catechin/administration & dosage , Friedreich Ataxia/drug therapy , Heart/diagnostic imaging , Adolescent , Child , Echocardiography , Female , Friedreich Ataxia/physiopathology , Humans , Magnetic Resonance Imaging , Male , Prospective Studies , Severity of Illness Index , Treatment Outcome , WalkingABSTRACT
Currently, there is great interest in identifying endogenous (i.e. physiological) stimulators of mitochondrial biogenesis (MB), in particular, those that may mediate the effects of exercise. The molecular size of the cacao flavanols (epicatechin and catechin) highly resembles that of sterols and epicatechin has been reported to activate cells surface receptors leading to the stimulation of MB in endothelial and skeletal muscle cells translating into enhanced exercise capacity. We therefore hypothesize, that epicatechin may be acting as a structural mimic of an as yet unknown sterol capable of stimulating MB. We developed a new synthetic process for obtaining enantiomerically pure preparations of (-)-epicatechin and (+)-epicatechin. Applying spatial analytics and molecular modeling, we found that the two isoforms of epicatechin, (-) and (+), have a structural resemblance to 11-ß-hydroxypregnenolone, a sterol with no previously described biological activity. As reported in this proof-of-concept study performed in primary cultures of endothelial and muscle cells, 11-ß-hydroxypregnenolone is one of the most potent inducers of MB as significant activity can be detected at femtomolar levels. The relative potency of (-)/(+)-epicatechin isoforms and on inducing MB correlates with their degree of spatial homology towards the 11-ß-hydroxypregnenolone. On the basis of these results, the detailed in vivo characterization of the potential for these sterols to act as endogenous modulators of MB is warranted.
Subject(s)
Catechin/chemistry , Catechin/pharmacology , Molecular Mimicry , Organelle Biogenesis , Sterols/chemistry , Sterols/pharmacology , Animals , Cattle , Cell Line , Cells, Cultured , Mice , Models, Molecular , StereoisomerismABSTRACT
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.
Subject(s)
Fatty Liver/metabolism , Mitochondria/metabolism , Oxidative Stress , Uric Acid/metabolism , Fructose/metabolism , Hep G2 Cells , Humans , Lipogenesis , Triglycerides/metabolism , Uric Acid/adverse effectsABSTRACT
Nicorandil exhibits a protective effect in the vascular system, which is thought to be due to vasodilatation from opening ATP-dependent potassium channels and donation of nitric oxide. Recently, nicorandil was shown to be renoprotective in models of acute kidney injury and glomerulonephritis. However, the specific mechanisms of renoprotection are unclear. We evaluated the effect of nicorandil on the rat remnant kidney model of chronic kidney disease. Blood pressure was unchanged by a 10-wk course of nicorandil, while albuminuria was significantly reduced. Glomerular injury and tubulointerstitial injury were also ameliorated by nicorandil. Oxidative stress, as noted by renal nitrotyrosine level and urine 8-hydroxy-2'-deoxyguanosine, were elevated in this model and was significantly reduced by nicorandil treatment. Treatment was associated with maintenance of the mitochondrial antioxidant, manganese SOD, in podocytes and with suppression of xanthine oxidase expression in infiltrating macrophages. Interestingly, these two cell types express sulfonylurea receptor 2 (SUR2), a binding site of nicorandil in the ATP-dependent K channel. Consistently, we found that stimulating SUR2 with nicorandil prevented angiotensin II-mediated upregulation of xanthine oxidase in the cultured macrophage, while xanthine oxidase expression was rather induced by blocking SUR2 with glibenclamide. In conclusion, nicorandil reduces albuminuria and ameliorates renal injury by blocking oxidative stress in chronic kidney disease.
Subject(s)
KATP Channels/agonists , Kidney Failure, Chronic/drug therapy , Kidney/metabolism , Nicorandil/pharmacology , ATP-Binding Cassette Transporters/metabolism , Albuminuria/prevention & control , Animals , Blotting, Western , Cells, Cultured , Disease Progression , Immunohistochemistry , Kidney/drug effects , Kidney/pathology , Kidney Failure, Chronic/pathology , Kidney Glomerulus/pathology , Macrophages/drug effects , Macrophages/enzymology , Macrophages/metabolism , Male , Mice , Nephritis, Interstitial/pathology , Nephritis, Interstitial/prevention & control , Nitric Oxide/metabolism , Oxidative Stress/drug effects , Paraffin Embedding , Potassium Channels, Inwardly Rectifying/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Drug/metabolism , Sulfonylurea Receptors , Xanthine Oxidase/biosynthesisABSTRACT
Nicorandil is an orally available drug that can act as a nitric oxide donor, an antioxidant, and an ATP-dependent K channel activator. We hypothesized that it may have a beneficial role in treating diabetic nephropathy. We administered nicorandil to a model of advanced diabetic nephropathy (the streptozotocin-induced diabetes in mice lacking endothelial nitric oxide synthase, eNOSKO); controls included diabetic eNOS KO mice without nicorandil and nondiabetic eNOS KO mice treated with either nicorandil or vehicle. Mice were treated for 8 wk. Histology, blood pressure, and renal function were determined. Additional studies involved examining the effects of nicorandil on cultured human podocytes. Here, we found that nicorandil did not affect blood glucose levels, blood pressure, or systemic endothelial function, but significantly reduced proteinuria and glomerular injury (mesangiolysis and glomerulosclerosis). Nicorandil protected against podocyte loss and podocyte oxidative stress. Studies in cultured podocytes showed that nicorandil likely protects against glucose-mediated oxidant stress via the ATP-dependent K channel as opposed to its NO-stimulating effects. In conclusion, nicorandil may be beneficial in diabetic nephropathy by preserving podocyte function. We recommend clinical trials to determine whether nicorandil may benefit diabetic nephropathy or other conditions associated with podocyte dysfunction.
Subject(s)
Antioxidants/therapeutic use , Diabetic Nephropathies/drug therapy , Nicorandil/therapeutic use , Nitric Oxide Donors/therapeutic use , Nitric Oxide Synthase Type III/deficiency , Severity of Illness Index , Animals , Antioxidants/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Blood Pressure/drug effects , Blood Pressure/physiology , Cells, Cultured , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/complications , Diabetic Nephropathies/etiology , Diabetic Nephropathies/physiopathology , Disease Models, Animal , Disease Progression , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nicorandil/pharmacology , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase Type III/genetics , Oxidative Stress/drug effects , Oxidative Stress/physiology , Podocytes/cytology , Podocytes/drug effects , Podocytes/metabolism , Reactive Oxygen Species/metabolism , Streptozocin/adverse effectsABSTRACT
Cisplatin nephropathy can be regarded as a mitochondrial disease. Intervention to halt such deleterious injury is under investigation. Recently, the flavanol (-)-epicatechin emerges as a novel compound to protect the cardiovascular system, owing in part to mitochondrial protection. Here, we have hypothesized that epicatechin prevents the progression of cisplatin-induced kidney injury by protecting mitochondria. Epicatechin was administered 8 h after cisplatin injury was induced in the mouse kidney. Cisplatin significantly induced renal dysfunction and tubular injury along with an increase in oxidative stress. Mitochondrial damages were also evident as a decrease in loss of mitochondrial mass with a reduction in the oxidative phosphorylation complexes and low levels of MnSOD. The renal damages and mitochondrial injuries were significantly prevented by epicatechin treatment. Consistent with these observations, an in vitro study using cultured mouse proximal tubular cells demonstrated that cisplatin-induced mitochondrial injury, as revealed by a decrease in mitochondrial succinate dehydrogenase activity, an induction of cytochrome c release, mitochondrial fragmentation, and a reduction in complex IV protein, was prevented by epicatechin. Such a protective effect of epicatechin might be attributed to decreased oxidative stress and reduced ERK activity. Finally, we confirmed that epicatechin did not perturb the anticancer effect of cisplatin in HeLa cells. In conclusion, epicatechin exhibits protective effects due in part to its ability to prevent the progression of mitochondrial injury in mouse cisplatin nephropathy. Epicatechin may be a novel option to treat renal disorders associated with mitochondrial dysfunction.
Subject(s)
Catechin/therapeutic use , Cisplatin/adverse effects , Kidney Diseases/chemically induced , Kidney Diseases/prevention & control , Mitochondria/physiology , Mitochondrial Diseases/chemically induced , Mitochondrial Diseases/prevention & control , Animals , Antioxidants/pharmacology , Antioxidants/therapeutic use , Catechin/pharmacology , Cells, Cultured , Cytochromes c/metabolism , Disease Models, Animal , HeLa Cells/drug effects , HeLa Cells/pathology , Humans , In Vitro Techniques , Kidney Diseases/physiopathology , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/pathology , Kidney Tubules, Proximal/physiopathology , Male , Mice , Mice, Inbred C57BL , Mitochondria/drug effects , Mitochondrial Diseases/physiopathology , Oxidative Stress/drug effects , Oxidative Stress/physiology , Reactive Oxygen Species/metabolism , Succinate Dehydrogenase/metabolismABSTRACT
Consumption of (-)-epicatechin (Epi), a cacao flavanol improves cognition. The aim was to compare the effects of (-)-Epi or its stereoisomer (+)-Epi on mouse frontal cortex-dependent short-term working memory and modulators of neurogenesis. Three-month-old male mice (n = 7 per group) were provided by gavage either water (vehicle; Veh), (-)-Epi, at 1 mg kg-1 or (+)-Epi at 0.1 mg per kg of body weight for 15 days. After treatment, spontaneous alternation was evaluated by Y-maze. Brain frontal cortex was isolated for nitrate/nitrite measurements, Western blotting for nerve growth factor (NGF), microtubule associated protein 2 (MAP2), endothelial and neuronal nitric oxide synthase (eNOS and nNOS) and immunohistochemistry for neuronal specific protein (NeuN), doublecortin (DCX), capillary (CD31) and neurofilaments (NF200). Results demonstrate the stimulatory capacity of (-)-Epi and (+)-Epi on markers of neuronal proliferation as per increases in immunoreactive cells for NeuN (74 and 120% respectively), DCX (70 and 124%) as well as in NGF (34.4, 63.6%) and MAP2 (41.8, 63.8%). Capillary density yielded significant increases with (-)-Epi (â¼80%) vs. (+)-Epi (â¼160%). CD31 protein levels increased with (-)-Epi (â¼70%) and (+)-Epi (â¼140%). Effects correlated with nitrate/nitrite stimulation by (-)-Epi and (+)-Epi (110.2, 246.5%) and enhanced eNOS phosphorylation (Ser1177) with (-)-Epi and (+)-Epi (21.4, 41.2%) while nNOS phosphorylation only increased with (+)-Epi (18%). Neurofilament staining was increased in (-)-Epi by 135.6 and 84% with (+)-Epi. NF200 increased with (-)-Epi (116%) vs. (+)-Epi (84.5%). Frontal cortex-dependent short-term spatial working improved with (-)-Epi and (+)-Epi (15, 13%). In conclusion, results suggest that both enantiomers, but more effectively (+)-Epi, upregulate neurogenesis markers likely through stimulation of capillary formation and NO triggering, improvements in memory.
Subject(s)
Catechin/pharmacology , Frontal Lobe/physiology , Memory, Short-Term/drug effects , Neurogenesis/drug effects , Animals , Biomarkers/analysis , Brain Chemistry , Cacao/chemistry , Catechin/analysis , Cell Proliferation/drug effects , Doublecortin Protein , Frontal Lobe/blood supply , Frontal Lobe/drug effects , Male , Maze Learning , Mice , Mice, Inbred C57BL , Neurons/physiology , Nitric Oxide/metabolism , StereoisomerismABSTRACT
We reported that (-)-epicatechin can stimulate mitochondria biogenesis and improve metabolism. However, preliminary studies indicate that the (+) stereoisomer form may be more potent. We evaluated in a preliminary manner, the pharmacokinetics (PK) and initial safety analysis of (+)-epicatechin ((+)-Epi) in healthy and pre-diabetic subjects. Using a mouse model of diet-induced obesity and insulin resistance, we also evaluated the metabolic effects of (+)-Epi vs. (+)-catechin (Cat) to determine class effects. In the Phase I PK study, subjects were provided a single incremental oral dose of (+)-Epi (10, 30 or 100 mg). For the PD study, subjects were provided a single 30 mg dose per day for 7 days. Blood samples were collected and safety measures were performed. Incremental doses of (+)-Epi increase the half-life of blood metabolites from 1.2-4.9 h. The compound was well tolerated and no adverse effects were reported. Seven day dosing of pre-diabetic subjects led to tendencies for reductions in circulating levels of tumor necrosis factor-α and monocyte chemoattractant protein-1, which returned to baseline by 7 days after treatment. In animals, 2 weeks of oral dosing (0.003, 0.01, 0.03, 0.1 and 0.3 mg kg-1 day-1) dose dependently improved metabolism-related endpoints (weight gain, glucose, cholesterol, triglyceride, with thresholds as low as 0.01 mg kg-1 day-1). Cat yielded no effects at 0.1 mg kg-1 day-1. Results indicate that (+)-Epi evidences a favorable PK and safety profile. Using a pre-clinical model, the compound positively modulates metabolism, which may link to mitochondrial effects. Effects are not due to general antioxidant actions, as Cat yielded no effects.
Subject(s)
Catechin/pharmacokinetics , Prediabetic State/drug therapy , Adult , Aged , Animals , Blood Glucose , Catechin/administration & dosage , Catechin/blood , Cholesterol , Female , Humans , Male , Mice , Middle Aged , Pilot Projects , Prediabetic State/blood , Prediabetic State/metabolism , Triglycerides , Young AdultABSTRACT
Pancreatic cancer is one of the deadliest forms of cancer and effective treatment remains a clinical challenge. Transforming growth factor-beta (TGF-beta) has important roles in primary tumor progression and in promoting metastasis, and has become an attractive target for therapy. Previously, we reported that treatment of pancreatic cancer cells in vitro with SD-208, a small molecule inhibitor of the TGF-beta receptor I kinase (TGF-betaRI), inhibited expression of genes associated with tumor progression and inhibited invasiveness in a cell-based assay. In a demonstration of efficacy of TGF-beta signaling inhibition in an in vivo model of pancreatic cancer, we showed significantly reduced primary tumor weight and decreased incidence of metastasis in the Panc-1 orthotopic xenograft model of established pancreatic cancer. In this report, we extend these in vivo findings to examine the mechanistic consequences of TGF-betaRI inhibition on Panc-1 primary tumors and their microenvironment in situ. In a longitudinal study of TGF-betaRI inhibition in the Panc-1 orthotopic model, we show that SD-208 treatment significantly reduced tumor growth measured as bioluminescence intensity throughout the study. Histological evaluation revealed that SD-208 treatment reduced proliferation and induced apoptosis in the primary tumors, and reduced fibrosis in the tumor microenvironment. An immune contribution (greater B-cell infiltration in SD-208-treated tumors) was also suggested by the histological analyses. SD-208 not only blocked direct TGF-beta signaling in Panc-1 primary tumors (reduced phospho SMAD2/3), but also down-regulated the expression of TGF-beta-regulated genes (PAI-1 and COL7A1). Taken together, our results indicate that a TGF-betaRI kinase inhibitor has a potential therapeutic benefit for pancreatic cancer patients.
Subject(s)
Adenocarcinoma/drug therapy , Pancreatic Neoplasms/drug therapy , Pteridines/pharmacology , Transforming Growth Factor beta/antagonists & inhibitors , Adenocarcinoma/immunology , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , B-Lymphocytes/immunology , Cell Growth Processes/drug effects , Cell Growth Processes/physiology , Female , Humans , Mice , Mice, Nude , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Receptors, Transforming Growth Factor beta/genetics , Signal Transduction , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Transforming growth factor-beta (TGF-beta) 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-beta, they often constitutively overexpress and activate TGF-beta, 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-beta pathway antagonists. EXPERIMENTAL DESIGN: We examined the effects of selective TGF-beta 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-beta-induced phosphorylation of the receptor-associated Smads, Smad2 and Smad3, in a dose-dependent manner, with IC50 between 20 and 80 nmol/L. TGF-beta 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-beta. 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-beta type I receptor kinase inhibitors hold promise as novel therapeutic agents for metastatic breast cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma/drug therapy , Gene Expression Regulation, Neoplastic , Mammary Neoplasms, Animal/drug therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Animals , Apoptosis , Cell Proliferation , Disease Progression , Epithelium/pathology , Humans , Inhibitory Concentration 50 , Mesoderm/pathology , Mice , Mice, Nude , Neoplasm Metastasis , Plasminogen Activator Inhibitor 1/metabolism , Pteridines/pharmacology , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor betaABSTRACT
The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-beta (TGF-beta) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-beta-induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-beta-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-beta and BNP for 24 and 48 hours. TGF-beta, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-beta effects; 88% and 85% of all TGF-beta-regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-beta-regulated genes related to fibrosis (collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (alpha-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNFalpha-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-beta-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.
Subject(s)
Fibroblasts/drug effects , Gene Expression Regulation/drug effects , Natriuretic Peptide, Brain/pharmacology , Transforming Growth Factor beta/antagonists & inhibitors , Ventricular Remodeling , Adolescent , Blotting, Western , Butadienes/pharmacology , Cell Division , Cells, Cultured/drug effects , Cyclic GMP/biosynthesis , Enzyme Inhibitors/pharmacology , Extracellular Matrix Proteins/biosynthesis , Extracellular Matrix Proteins/genetics , Fibrosis , Flavonoids/pharmacology , Gene Expression Profiling , Humans , Inflammation , MAP Kinase Signaling System/drug effects , Male , Middle Aged , Muscle Proteins/biosynthesis , Muscle Proteins/genetics , Natriuretic Peptide, Brain/physiology , Nitriles/pharmacology , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Cardiometabolic disruptions such as insulin resistance, obesity, high blood pressure, hyperglycemia, and dyslipidemias, are known to increase the risk for cardiovascular and metabolic diseases such as type 2 diabetes mellitus and atherosclerosis. Several screening tools for assessing cardiometabolic risk have been developed including the TG/HDLc ratio, which has been, demonstrated to possess a strong association with insulin resistance and coronary disease. Dietary modifications, together with regular moderate exercise have proven to be effective in attenuating cardiometabolic disruptions. However, they often exhibit poor long-term patient compliance. Nutraceutics, including (-)-epicatechin (EPI), have gained increasing interest as coadjuvant effective and safe therapies that are able to attenuate hypertension, hyperglycemia, hyperinsulinemia, hypertriglyceridemia and hypoalphalipoproteinemia. METHODS: The aims of this study were: 1) to compare the in vitro effect of EPI vs. (+)-catechin on fructose induced triglyceride accumulation and mitochondrial function in Hep2 cells in culture, 2) to evaluate the efficacy of EPI treatment in reducing fasting blood triglycerides and improving the TG/HDLc ratio in hypertriglyceridemic patients with a total daily dose of 100mg of EPI. Secondary clinical variables included total cholesterol, LDLc, fructosamine, glucose, insulin, and high sensitivity C-reactive protein blood levels. RESULTS AND CONCLUSION: Our results provide preliminary evidence as to favorable effects of EPI on glycemia homeostasis, lipid profile and systemic inflammation such bioactive actions are not class-effects (i.e. limited to their antioxidant potential) but instead, may result from the specific activation of associated downstream signaling pathways since catechin has no effects.
Subject(s)
Cardiovascular Diseases/etiology , Catechin/therapeutic use , Cholesterol, HDL/blood , Hypertriglyceridemia/drug therapy , Metabolic Syndrome/etiology , Triglycerides/blood , Adolescent , Adult , Cardiovascular Diseases/blood , Cardiovascular Diseases/drug therapy , Cells, Cultured , Double-Blind Method , Female , Humans , Hypertriglyceridemia/blood , Hypertriglyceridemia/etiology , Male , Metabolic Syndrome/blood , Metabolic Syndrome/drug therapy , Middle Aged , Risk Factors , Young AdultABSTRACT
Although PS-341 (bortezomib) is a promising agent to improve multiple myeloma (MM) patient outcome, 65% of patients with relapsed and refractory disease do not respond. We have previously shown that heat shock protein (Hsp)27 is upregulated after PS-341 treatment, that overexpression of Hsp27 confers PS-341 resistance, and that inhibition of Hsp27 overcomes PS-341 resistance. Since Hsp27 is a downstream target of p38 mitogen-activated protein kinase (MAPK)/MAPK-mitogen-activated protein kinase-2 (MAPKAPK2), we hypothesized that inhibition of p38 MAPK activity could augment PS-341 cytotoxicity by downregulating Hsp27. Although p38 MAPK inhibitor SCIO-469 (Scios Inc, CA, USA) alone did not induce significant growth inhibition, it blocked baseline and PS-341-triggered phosphorylation of p38 MAPK as well as upregulation of Hsp27, associated with enhanced cytotoxicity in MM.1S cells. Importantly, SCIO-469 enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK) and augmented cleavage of caspase-8 and poly(ADP)-ribose polymerase. Moreover, SCIO-469 downregulated PS-341-induced increases in G2/M-phase cells, associated with downregulation of p21Cip1 expression. Importantly, SCIO-469 treatment augmented cytotoxicity of PS-341 even against PS-341-resistant cell lines and patient MM cells. These studies therefore provide the framework for clinical trials of SCIO-469 to enhance sensitivity and overcome resistance to PS-341, thereby improving patient outcome in MM.
Subject(s)
Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Multiple Myeloma/pathology , Pyrazines/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Bortezomib , Cell Line, Tumor , Down-Regulation , Heat-Shock Proteins/metabolism , HumansABSTRACT
BACKGROUND: Therapeutic angiogenesis is a new approach to treating ischemic heart disease, and the optimal method for assessing its efficacy is unclear. We used myocardial contrast echocardiography (MCE) to evaluate the therapeutic response to the angiogenic agent, vascular endothelial growth factor-121 (VEGF121). METHODS AND RESULTS: After placement of an ameroid constrictor (day 0) around the left anterior descending artery (LAD), dogs were given intracoronary VEGF121 protein (108 microg, n=6) or placebo (n=6) on days 7 and 21, and subcutaneous VEGF121 (1 mg) or placebo on days 8 to 20 and 22 to 27. On day 48, MCE was performed during rest and dobutamine stress. Videointensity (y) and pulsing interval (t) were fit to an exponential model (y=A[1-e(-beta(t))]) used to derive indices of red cell velocity (beta) and capillary area (A), and parameters were compared with radiolabeled microsphere flow data. VEGF(121) treatment resulted in higher resting left anterior descending artery/left circumflex flow ratio compared with placebo (P<0.03) and improved collateral flow reserve. Beta was 0.94+/-0.37 in VEGF121 dogs versus 0.38+/-0.31 in controls (P<0.02), with the greatest difference in the endocardium. The parameter A was comparable in both groups, suggesting that microvascular changes did not alter capillary cross-sectional area, and histology indicated a trend toward higher arteriolar density in VEGF121-treated animals. CONCLUSIONS: VEGF121 protein improves collateral flow and reserve. MCE can evaluate the transmural location and structural and functional responses of the microvasculature to angiogenic interventions.
Subject(s)
Coronary Disease/diagnostic imaging , Coronary Disease/drug therapy , Echocardiography , Endothelial Growth Factors/pharmacology , Lymphokines/pharmacology , Microcirculation/drug effects , Microcirculation/diagnostic imaging , Animals , Blood Flow Velocity/drug effects , Collateral Circulation/drug effects , Coronary Circulation/drug effects , Disease Models, Animal , Dogs , Electrocardiography , Heart/drug effects , Myocardial Revascularization , Myocardium/pathology , Predictive Value of Tests , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
BACKGROUND: A method for identifying tissue experiencing hypoxic stress due to atherosclerotic vascular disease would be clinically useful. Vascular endothelial growth factor-121 (VEGF121) is an angiogenic protein secreted in response to hypoxia that binds to VEGF receptors overexpressed by ischemic microvasculature. We tested the hypothesis that VEGF receptors could serve as markers for ischemic tissue and hence provide a target for imaging such tissue with radiolabeled human VEGF121. METHODS AND RESULTS: A rabbit model of unilateral hindlimb ischemia was created by femoral artery excision (n=14). Control rabbits (n=5) underwent identical surgery without femoral excision. On postoperative day 10, rabbits were intravenously administered 100 microCi of 111In-labeled recombinant human VEGF121, and biodistribution studies and planar imaging were conducted at 3, 24, and 48 hours. On postmortem gamma counting, there was greater accumulation of 111In-labeled VEGF121 in ischemic than in control tissue (P<0.02). Differential uptake of isotope by ischemic muscle was not seen in rabbits injected with 125I-labeled human serum albumin (n=6). Radioactivity imaged in hindlimb regions of interest was significantly higher in ischemic muscle than in sham-operated and contralateral nonoperated hindlimb at 3 hours (P<0.02). Immunohistochemical staining confirmed upregulation of VEGF receptors in ischemic skeletal muscle. CONCLUSIONS: Identification of the ischemic state via targeted radiolabeling of hypoxia-induced angiogenic receptors is possible. This approach could be useful for monitoring the efficacy of revascularization strategies such as therapeutic angiogenesis.
Subject(s)
Hindlimb/blood supply , Ischemia/diagnostic imaging , Ischemia/physiopathology , Receptors, Vascular Endothelial Growth Factor/metabolism , Animals , Binding, Competitive , Disease Models, Animal , Endothelial Growth Factors/pharmacokinetics , Femoral Artery/physiopathology , Hindlimb/diagnostic imaging , Immunohistochemistry , Indium Radioisotopes , Intercellular Signaling Peptides and Proteins/pharmacokinetics , Ischemia/pathology , Lymphokines/pharmacokinetics , Metabolic Clearance Rate , Muscle, Skeletal/blood supply , Muscle, Skeletal/diagnostic imaging , Muscle, Skeletal/physiopathology , Predictive Value of Tests , Rabbits , Radionuclide Imaging , Scintillation Counting , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Since their discovery 20 years ago, the natriuretic peptides have emerged as an important endocrine response to excessive increases in intravascular volume. In a cGMP-dependent fashion, the natriuretic peptides induce a balanced vasodilation, decreasing preload and afterload in states of cardiac impairment and stimulating the excretion of salt and water by the kidneys. Recombinant B-type natriuretic peptide (nesiritide), identical to the principle natriuretic peptide produced by human cardiac ventricles, has just been approved for the treatment of acute decompensated congestive heart failure. Nesiritide has been shown in controlled trials in congestive heart failure to decrease pulmonary capillary wedge pressure, improve cardiac output, stimulate natriuresis and diuresis, and rapidly induce symptomatic relief. As a naturally occurring stress hormone of the heart, with particular rectifying effects on the pulmonary, cardiac and renal vasculatures, B-type natriuretic peptide may prove useful in a variety of cardiovascular disease states.
Subject(s)
Atrial Natriuretic Factor/therapeutic use , Cardiotonic Agents/therapeutic use , Heart Failure/drug therapy , Amino Acid Sequence , Animals , Atrial Natriuretic Factor/physiology , Heart Failure/physiopathology , Humans , Molecular Sequence Data , Natriuretic Peptide, Brain , Recombinant Proteins/therapeutic useABSTRACT
Microarrays have helped researchers gain much insight into gene expression profiles in the context of many diseases including those in the injured heart. Our genomic investigations have been focused on elucidation of host gene responses to enterovirus infection. We have gained valuable technical expertise in using Affymetrix oligonucleotide arrays, also known as GeneChips, and cDNA spotted arrays to probe differential gene expression in both cultured cells and in heart tissue. Here, we provide a technique-focused supplement to the Affymetrix GeneChip Expression Analysis Manual for sample preparation, processing, and array hybridization. We provide expanded explanations to highlight important points within the existing protocol and offer variations to standard procedures when appropriate. For investigators using myocardial tissues for microarray experiments, we further address the necessity of and methods for in situ flushing of the vasculature, tissue homogenization, and considerations for limits of expression detection in rare cells. It is our intention to provide useful technical information, based on our experience, to assist those researchers using Affymetrix GeneChips in their own genomic research.
Subject(s)
Heart Injuries , Oligonucleotide Array Sequence Analysis/methods , Animals , Cells, Cultured , Gene Expression Profiling , Genomics , Myocardium/cytologyABSTRACT
(-)-Epicatechin ((-)-EPI), a naturally occurring flavanol, has emerged as a likely candidate for cocoa-based product reported reductions in cardiometabolic risk. The present study aimed to determine the safety, tolerability, pharmacokinetics and pharmacodynamics of purified (-)-EPI administered to healthy volunteers. In this phase I, open-label, two-part single- and multiple-dose study, subjects received either a single dose (n = 9) of 50, 100 or 200 mg or multiple doses (n = 8) of 50 mg daily (q.d.) or twice daily (b.i.d) for 5 days. Blood was collected at 0, 0.5, 1, 2, 4 and 6 h after (-)-EPI administration in the single and multiple dose groups (blood collection repeated in day 5). Samples were analyzed by HPLC-HR-ESI-MS for EPI and metabolite quantification. In the q.d. and b.i.d. groups, blood samples were analyzed for NO surrogates and follistatin levels as well as, platelet mitochondrial complexes I, V and citrate synthase activity levels. (-)-EPI was well tolerated and readily absorbed with further phase 2 metabolism. On day 5, in the q.d. and b.i.d. groups, there were significant increases in plasma nitrite of 30% and 17%, respectively. In the q.d. group on day 5 vs. day 1, platelet mitochondrial complexes I, IV and citrate synthase activities demonstrated a significant increase of â¼92, 62 and 8%, respectively. Average day 5 follistatin AUC levels were â¼2.5 fold higher vs. day 1 AUC levels in the b.i.d. group. (-)-EPI was safe to use, with no observed adverse effects, and our findings suggest that increases in NO metabolites, mitochondrial enzyme function and plasma follistatin levels may underlie some of the beneficial effects of cocoa products or (-)-EPI as reported in other studies.