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

FT011M reduced Müller cell gliosis in the retina of Ren-2 rats diabetic for 8 weeks.

<p>Non-diab, non-diabetic. Diab, diabetic. V, vehicle. Three-μm paraffin sections. IPL, inner plexiform layer. INL, inner nuclear layer. OPL, outer plexiform layer. ONL, outer nuclear layer. Original magnification, 400X. Bar, 40 μm. (A to C) Central retina. (D to F) Mid retina. (G to I) Peripheral retina. In non-diab + V, GFAP immunolabeling is present on the retinal surface (asterisk) and in Müller cell processes (arrows) extending throughout the retinal layers and in the central, mid and peripheral retina (A, D, G). GFAP immunolabeling is increased in diab + V (B, E, H). In diabetic rats, FT011M reduced GFAP immunolabeling in the central (G), mid (F) and peripheral (I) retina to the level of non-diab + V. (J to M) *P < 0.01 and ***P < 0.001 to non-diab + V. ##P < 0.01 and ###P < 0.001 to diab + V. N = 4 to 6 rats per group. Values are Mean ± SEM.</p

FT011M reduced acellular capillaries and extracellular matrix proteins in retina from Ren-2 rats diabetic for 32 weeks.

<p>Non-diab, non-diabetic. Diab, diabetic. V, vehicle. Stain, Periodic acid-Schiff’s reagent. Bar, 40 μm. (A to C) In diab + V, acellular capillaries (asterisk) were increased in all regions of retina compared to non-diabetic + V. In diabetic rats, FT011M reduced acellular capillaries in all regions of the retina. Arrow, pericyte ghost. (D to G) *P < 0.05 to non-diab + V. #P < 0.05 to diab + V. N = 8 to 11 rats per group. (H) Collagen IV mRNA levels in retina. (I) Fibronectin mRNA levels in retina. *P < 0.05, **P < 0.01 to non-diab + V. ###P < 0.001 to diab + V. N = 5 to 9 rats per group. Values are Mean±SEM.</p

FT011M reduced VEGF immunolabeling in retina of Ren-2 rats diabetic for 8 weeks.

<p>Non-diab, non-diabetic. Diab, diabetic. V, vehicle. Three-μm paraffin sections. GCL, ganglion cell layer. IPL, inner plexiform layer. INL, inner nuclear layer. ONL, outer nuclear layer. Counterstain, haematoxylin. (A). In non-diab + V, VEGF immunolabeling is occasionally detected in ganglion cells (arrowheads) in the GCL. (B) In diab + V, VEGF immunolabeling is increased compared to non-diab + V and detected in ganglion cells and Müller cell processes (arrows) extending throughout the retina. Inset showing higher powered image of Müller cell processes. (C) In diabetic rats treated with FT011M, VEGF immunolabeling was reduced to the level of non-diab + V. (A to C) Original magnification, 400X. Bar, 40 μm. Inset: Bar, 75 μm. (D) **P < 0.01 to non-diab + V. ##P < 0.01 to diab + V. N = 4 to 6 rats per group. Values are Mean ± SEM.</p

Animal characteristics.

<p>*P < 0.05 versus control. N = 8 rats per group. Values are mean ± SEM.</p><p>Animal characteristics.</p