RNA promotes phase separation of glycolysis enzymes into yeast G bodies in hypoxia.

In hypoxic stress conditions, glycolysis enzymes assemble into singular cytoplasmic granules called glycolytic (G) bodies. G body formation in yeast correlates with increased glucose consumption and cell survival. However, the physical properties and organizing principles that define G body formation are unclear. We demonstrate that glycolysis enzymes are non-canonical RNA binding proteins, sharing many common mRNA substrates that are also integral constituents of G bodies. Targeting nonspecific endoribonucleases to G bodies reveals that RNA nucleates G body formation and maintains its structural integrity. Consistent with a phase separation mechanism of biogenesis, recruitment of glycolysis enzymes to G bodies relies on multivalent homotypic and heterotypic interactions. Furthermore, G bodies fuse in vivo and are largely insensitive to 1,6-hexanediol, consistent with a hydrogel-like composition. Taken together, our results elucidate the biophysical nature of G bodies and demonstrate that RNA nucleates phase separation of the glycolysis machinery in response to hypoxic stress

3′,4′-Dihydroxyflavonol Antioxidant Attenuates Diastolic Dysfunction and Cardiac Remodeling in Streptozotocin-Induced Diabetic m(Ren2)27 Rats

Diabetic cardiomyopathy (DCM) is an increasingly recognized cause of chronic heart failure amongst diabetic patients. Both increased reactive oxygen species (ROS) generation and impaired ROS scavenging have been implicated in the pathogenesis of hyperglycemia-induced left ventricular dysfunction, cardiac fibrosis, apoptosis and hypertrophy. We hypothesized that 3',4'-dihydroxyflavonol (DiOHF), a small highly lipid soluble synthetic flavonol, may prevent DCM by scavenging ROS, thus preventing ROS-induced cardiac damage.Six week old homozygous Ren-2 rats were randomized to receive either streptozotocin or citrate buffer, then further randomized to receive either DiOHF (1 mg/kg/day) by oral gavage or vehicle for six weeks. Cardiac function was assessed via echocardiography and left ventricular cardiac catheterization before the animals were sacrificed and hearts removed for histological and molecular analyses. Diabetic Ren-2 rats showed evidence of diastolic dysfunction with prolonged deceleration time, reduced E/A ratio, and increased slope of end-diastolic pressure volume relationship (EDPVR) in association with marked interstitial fibrosis and oxidative stress (all P<0.05 vs control Ren-2). Treatment with DiOHF prevented the development of diastolic dysfunction and was associated with reduced oxidative stress and interstitial fibrosis (all P<0.05 vs untreated diabetic Ren-2 rats). In contrast, few changes were seen in non-diabetic treated animals compared to untreated counterparts.Inhibition of ROS production and action by DiOHF improved diastolic function and reduced myocyte hypertrophy as well as collagen deposition. These findings suggest the potential clinical utility of antioxidative compounds such as flavonols in the prevention of diabetes-associated cardiac dysfunction

Novel synthetic flavonoids in acute and chronic heart and kidney diseases

© 2012 Dr. Fay Lin KhongCardiovascular disease remains one of the leading causes of death worldwide despite the dramatic reduction in mortality rates over the past two decades due to significant advancement in medical and public health interventions. Diabetes exacerbates the complex interaction of cardiovascular risk factors, resulting in the greater incidence of heart failure among individuals with diabetes when compared to those without diabetes. The relative risk of cardiovascular mortality is further increased when individuals with diabetes are diagnosed with the comorbidity of established chronic kidney disease. Therefore, there is a continuous need for the development of novel therapeutic strategies to prevent the progression of cardiac and renal dysfunction in the presence or absence of diabetes, since the protective effects from the routine use of current pharmacotherapy for the management of elevated blood glucose, high blood pressure and abnormal blood lipid profiles remain limited and controversial. The focus of this thesis is to further elucidate the pathophysiological roles of oxidative stress and inflammation in the disease progression and to explore the therapeutic potential of novel synthetic flavonoids, DiOHF and NP202, in the prevention of acute and chronic heart and kidney diseases. Cardiovascular disease remains one of the leading causes of death worldwide despite the dramatic reduction in mortality rates over the past two decades due to significant advancement in medical and public health interventions. Diabetes exacerbates the complex interaction of cardiovascular risk factors, resulting in the greater incidence of heart failure among individuals with diabetes when compared to those without diabetes. The relative risk of cardiovascular mortality is further increased when individuals with diabetes are diagnosed with the comorbidity of established chronic kidney disease. Therefore, there is a continuous need for the development of novel therapeutic strategies to prevent the progression of cardiac and renal dysfunction in the presence or absence of diabetes, since the protective effects from the routine use of current pharmacotherapy for the management of elevated blood glucose, high blood pressure and abnormal blood lipid profiles remain limited and controversial. The focus of this thesis is to further elucidate the pathophysiological roles of oxidative stress and inflammation in the disease progression and to explore the therapeutic potential of novel synthetic flavonoids, DiOHF and NP202, in the prevention of acute and chronic heart and kidney diseases. The manifestation of acute myocardial infarction remains a major contributor to the subsequent development of heart failure despite the successful implementation of coronary reperfusion strategies. There has been substantial evidence showing that the dynamic progression of LV contractile dysfunction persisted up to several weeks following the restoration of coronary blood flow. The oral administration of NP202 during the reperfusion period in an experimental model of AMI resulted in the sustained improvement of LV contractile function in association with the reduction in the accumulation of inflammatory cells in the infarct zone of the heart even after seven days following the induction of AMI. This thesis has provided a strong basis for further therapeutic advancement of synthetic flavonoids as novel pharmacological agents to prevent the progression of heart and kidney diseases in the absence and presence of diabetes. There are clear indications that the modulation of oxidative stress and inflammation in the presence of synthetic flavonoids could be responsible for the prevention of the disease progression. The comprehensive understanding of the pharmacokinetics and oral bioavailability of the synthetic flavonoids is necessary for further preclinical evaluation of these promising therapeutic interventions. The enhancement of the biological actions of the synthetic flavonoids in the oral formulation would thus enable the translation of the basic research for potential clinical utility

FT011, a novel cardiorenal protective drug, reduces inflammation, gliosis and vascular injury in rats with diabetic retinopathy

Diabetic retinopathy features inflammation as well as injury to glial cells and the microvasculature, which are influenced by hypertension and overactivity of the renin-angiotensin system. FT011 is an anti-inflammatory and anti-fibrotic agent that has been reported to attenuate organ damage in diabetic rats with cardiomyopathy and nephropathy. However, the potential therapeutic utility of FT011 for diabetic retinopathy has not been evaluated. We hypothesized that FT011 would attenuate retinopathy in diabetic Ren-2 rats, which exhibit hypertension due to an overactive extra-renal renin-angiotensin system. Diabetic rats were studied for 8 and 32 weeks and received intravitreal injections of FT011 (50 μM) or vehicle (0.9% NaCl). Comparisons were to age-matched controls. In the 8-week study, retinal inflammation was examined by quantitating vascular leukocyte adherence, microglial/macrophage density and the expression of inflammatory mediators. Macroglial Müller cells, which exhibit a pro-inflammatory and pro-angiogenic phenotype in diabetes, were evaluated in the 8-week study as well as in culture following exposure to hyperglycaemia and FT011 (10, 30, 100 μM) for 72 hours. In the 32-week study, severe retinal vasculopathy was examined by quantitating acellular capillaries and extracellular matrix proteins. In diabetic rats, FT011 reduced retinal leukostasis, microglial density and mRNA levels of intercellular adhesion molecule-1 (ICAM-1). In Müller cells, FT011 reduced diabetes-induced gliosis and vascular endothelial growth factor (VEGF) immunolabeling and the hyperglycaemic-induced increase in ICAM-1, monocyte chemoattractant protein-1, CCL20, cytokine-induced neutrophil chemoattractant-1, VEGF and IL-6. Late intervention with FT011 reduced acellular capillaries and the elevated mRNA levels of collagen IV and fibronectin in diabetic rats. In conclusion, the protective effects of FT011 in cardiorenal disease extend to key elements of diabetic retinopathy and highlight its potential as a treatment approach

Immumohistochemistry staining for collagen type I.

<p>(A) Representative images. Brown region represents positive immunostaining for collagen type I which was substantially increased in diabetic rats and reduced by DiOHF treatment. The amount of collagen type I was found to be similar in control and control-treated rats. Collagen type I interstitial accumulation as assessed by percentage proportional area showing positive immunostaining in control and diabetic rats treated with or without DiOHF (B). Data expressed as mean ± SE. *<i>P</i>&lt;0.05 vs control (non-diabetic) rats; †<i>P</i>&lt;0.05 vs diabetic rats. Original magnification ×200.</p

Immunohistochemistry staining for collagen type III.

<p>(A) Representative images. Brown region represents positive immunostaining for collagen type III which was substantially increased in diabetic rats and reduced by DiOHF treatment. The amount of collagen type III was found to be similar in control and control-treated rats. Collagen type III interstitial accumulation as assessed by proportional area on sections showing positive immunostaining in control and diabetic rats treated with or without DiOHF (B). Data expressed as mean ± SE. Data expressed as mean ± SE. *<i>P</i>&lt;0.05 vs control (non-diabetic) rats; †<i>P</i>&lt;0.05 vs diabetic rats. Original magnification ×200.</p

Measurements of oxidative stress.

<p>Representative images for the localization of 3-nitrotyrosine in LV tissues, as assessed by the proportional area of positive immunostaining in control and diabetic rats treated with or without DiOHF (A). The marked increase in positive immunostaining (brown region) for 3-nitrotyrosine in diabetic rats was reduced by DiOHF treatment. The amount of 3-nitrotyrosine was found to be comparable in control and control-treated Ren-2 rats (B). Original magnification ×200. (C) NADPH-activated superoxide production in LV tissues as measured by lucigenin-enhanced chemiluminescence. Data expressed as mean ± SE. *<i>P</i>&lt;0.05 vs control (non-diabetic) rats; †<i>P</i>&lt;0.05 vs diabetic rats.</p

Representative pressure volume (PV) loops during preload reduction.

<p>Note the steeper slope of EDPVR (green line) and rightward shift in the diabetic group (C) compared to control (A). An increase in the slope of EDPVR indicated decreased chamber compliance as seen in the diabetic animals which was reduced with DiOHF treatment (D). *<i>P</i>&lt;0.05 vs control (non-diabetic) rats; †<i>P</i>&lt;0.05 vs diabetic rats.</p

Txnip protein expression.

<p>(A) Representative western blot and (B) quantitation of Txnip normalized to loading control pan-actin. The significant increase in Txnip protein in diabetic rats was moderately reduced by DiOHF treatment. Data expressed as mean ± SE. *<i>P</i>&lt;0.05 vs control (non-diabetic) rats.</p