Abstract P303: Cilostazol Attenuates Angii-induced Cardiac Fibrosis in Mice

Background: Cilostazol, a phosphodiesterase-3 inhibitor, plays vasoprotective roles such as an improvement of endothelial function, a vasodilatation and a suppression of proliferation of vascular smooth muscle cells. The aim of study was to investigate a cardioprotective effect of cilostazol. Method: Male apolipoprotein E deficient mice (8-12 weeks old) were fed with either normal chow diet or cilostazol-containing (0.1% wt/v) diet. After 1 week of cilostazol administration, mice were infused subcutaneously with either angiotensin II (AngII, 1,000 ng/kg/min, n = 16 - 19) or saline (n = 5 - 6) by osmotic minipumps for 4 weeks. Results: AngII equivalently increased systolic blood pressure, irrespective of cilostazol administration. Cilostazol had no effect on serum cholesterol concentrations, triglycerides, high-density lipoprotein-cholesterol, body weights, heart rates, and systolic blood pressures. AngII increased heart weight but was attenuated by cilostazol administration (6.7±0.8 to 6.0±0.7 mg/gBW, p < 0.05). Cilostazol prevented both perivascular and interstitial cardiac fibrosis induced by AngII (p < 0.05, each). Quantitative real-time PCR revealed that mRNA expressions of Ctgf , Collagen I , Collagen III , Tgf- , Hgf and Spp-1 increased by AngII infusion but were attenuated by cilostazol administration (p < 0.05). Immunohistochemical analysis demonstrated that AngII administration enhanced OPN expression in heart but was suppressed by cilostazol administration. Further, to investigate the mechanism, human cardiac myocytes were cultured and stimulated with AngII (1x10 -7 M). Co-treatment of Cilostazol (1x10 -7 to 1x10 -5 M) attenuated AngII-induced increase of Spp-1 gene expression in dose-dependent manner. This effect was mimic by a treatment with forskolin, which was diminished by co-treatment with H-89. Conclusion: Cilostazol attenuated AngII-induced cardiac fibrosis in vivo. Cilostazol attenuated AngII-induced increment of Spp-1 gene expression through cAMP-PKA dependent pathway.

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Flow-dependent regulation of endothelial Tie2 by GATA3 in vivo

Intensive Care Medicine Experimental ◽

10.1186/s40635-021-00402-x ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Temitayo O. Idowu ◽

Valerie Etzrodt ◽

Thorben Pape ◽

Joerg Heineke ◽

Klaus Stahl ◽

...

Keyword(s):

Gene Expression ◽

Regulation Of Gene Expression ◽

Experimental Models ◽

Common Denominator ◽

Dependent Manner ◽

Pulmonary Endothelium ◽

Delivery Platform ◽

Transient Overexpression ◽

Plasmid Delivery

Abstract Background Reduced endothelial Tie2 expression occurs in diverse experimental models of critical illness, and experimental Tie2 suppression is sufficient to increase spontaneous vascular permeability. Looking for a common denominator among different critical illnesses that could drive the same Tie2 suppressive (thereby leak inducing) phenotype, we identified “circulatory shock” as a shared feature and postulated a flow-dependency of Tie2 gene expression in a GATA3 dependent manner. Here, we analyzed if this mechanism of flow-regulation of gene expression exists in vivo in the absence of inflammation. Results To experimentally mimic a shock-like situation, we developed a murine model of clonidine-induced hypotension by targeting a reduced mean arterial pressure (MAP) of approximately 50% over 4 h. We found that hypotension-induced reduction of flow in the absence of confounding disease factors (i.e., inflammation, injury, among others) is sufficient to suppress GATA3 and Tie2 transcription. Conditional endothelial-specific GATA3 knockdown (B6-Gata3tm1-Jfz VE-Cadherin(PAC)-cerERT2) led to baseline Tie2 suppression inducing spontaneous vascular leak. On the contrary, the transient overexpression of GATA3 in the pulmonary endothelium (jet-PEI plasmid delivery platform) was sufficient to increase Tie2 at baseline and completely block its hypotension-induced acute drop. On the functional level, the Tie2 protection by GATA3 overexpression abrogated the development of pulmonary capillary leakage. Conclusions The data suggest that the GATA3–Tie2 signaling pathway might play a pivotal role in controlling vascular barrier function and that it is affected in diverse critical illnesses with shock as a consequence of a flow-regulated gene response. Targeting this novel mechanism might offer therapeutic opportunities to treat vascular leakage of diverse etiologies.

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Hepatic HuR protects against the pathogenesis of non-alcoholic fatty liver disease by targeting PTEN

Cell Death and Disease ◽

10.1038/s41419-021-03514-0 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Mi Tian ◽

Jingjing Wang ◽

Shangming Liu ◽

Xinyun Li ◽

Jingyuan Li ◽

...

Keyword(s):

Glucose Metabolism ◽

Hepatic Steatosis ◽

Chow Diet ◽

Alcoholic Fatty Liver ◽

Phosphatase And Tensin Homolog ◽

Tensin Homolog ◽

Pten Mrna ◽

Normal Chow ◽

Mice Liver

AbstractThe liver plays an important role in lipid and glucose metabolism. Here, we show the role of human antigen R (HuR), an RNA regulator protein, in hepatocyte steatosis and glucose metabolism. We investigated the level of HuR in the liver of mice fed a normal chow diet (NCD) and a high-fat diet (HFD). HuR was downregulated in the livers of HFD-fed mice. Liver-specific HuR knockout (HuRLKO) mice showed exacerbated HFD-induced hepatic steatosis along with enhanced glucose tolerance as compared with control mice. Mechanistically, HuR could bind to the adenylate uridylate-rich elements of phosphatase and tensin homolog deleted on the chromosome 10 (PTEN) mRNA 3′ untranslated region, resulting in the increased stability of Pten mRNA; genetic knockdown of HuR decreased the expression of PTEN. Finally, lentiviral overexpression of PTEN alleviated the development of hepatic steatosis in HuRLKO mice in vivo. Overall, HuR regulates lipid and glucose metabolism by targeting PTEN.

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Modulatory Effects of Osthole on Lipopolysaccharides-Induced Inflammation in Caco-2 Cell Monolayer and Co-Cultures with THP-1 and THP-1-Derived Macrophages

Nutrients ◽

10.3390/nu13010123 ◽

2020 ◽

Vol 13 (1) ◽

pp. 123

Author(s):

Natalia K. Kordulewska ◽

Justyna Topa ◽

Małgorzata Tańska ◽

Anna Cieślińska ◽

Ewa Fiedorowicz ◽

...

Keyword(s):

Gene Expression ◽

Inflammatory Reaction ◽

Wide Spectrum ◽

Cell Monolayer ◽

In Vivo Studies ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Tnf Α

Lipopolysaccharydes (LPS) are responsible for the intestinal inflammatory reaction, as they may disrupt tight junctions and induce cytokines (CKs) secretion. Osthole has a wide spectrum of pharmacological effects, thus its anti-inflammatory potential in the LPS-treated Caco-2 cell line as well as in Caco-2/THP-1 and Caco-2/macrophages co-cultures was investigated. In brief, Caco-2 cells and co-cultures were incubated with LPS to induce an inflammatory reaction, after which osthole (150–450 ng/mL) was applied to reduce this effect. After 24 h, the level of secreted CKs and changes in gene expression were examined. LPS significantly increased the levels of IL-1β, -6, -8, and TNF-α, while osthole reduced this effect in a concentration-dependent manner, with the most significant decrease when a 450 ng/mL dose was applied (p < 0.0001). A similar trend was observed in changes in gene expression, with the significant osthole efficiency at a concentration of 450 ng/μL for IL1R1 and COX-2 (p < 0.01) and 300 ng/μL for NF-κB (p < 0.001). Osthole increased Caco-2 monolayer permeability, thus if it would ever be considered as a potential drug for minimizing intestinal inflammatory symptoms, its safety should be confirmed in extended in vitro and in vivo studies.

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Anti-Obesity and Anti-Adipogenic Effects of Chitosan Oligosaccharide (GO2KA1) in SD Rats and in 3T3-L1 Preadipocytes Models

Molecules ◽

10.3390/molecules26020331 ◽

2021 ◽

Vol 26 (2) ◽

pp. 331

Author(s):

Jung-Yun Lee ◽

Tae Yang Kim ◽

Hanna Kang ◽

Jungbae Oh ◽

Joo Woong Park ◽

...

Keyword(s):

Gene Expression ◽

Body Weight ◽

Density Lipoprotein ◽

Treated Group ◽

Control Group ◽

Chitosan Oligosaccharide ◽

Sd Rats ◽

Adipogenic Gene

Excess body weight is a major risk factor for type 2 diabetes (T2D) and associated metabolic complications, and weight loss has been shown to improve glycemic control and decrease morbidity and mortality in T2D patients. Weight-loss strategies using dietary interventions produce a significant decrease in diabetes-related metabolic disturbance. We have previously reported that the supplementation of low molecular chitosan oligosaccharide (GO2KA1) significantly inhibited blood glucose levels in both animals and humans. However, the effect of GO2KA1 on obesity still remains unclear. The aim of the study was to evaluate the anti-obesity effect of GO2KA1 on lipid accumulation and adipogenic gene expression using 3T3-L1 adipocytes in vitro and plasma lipid profiles using a Sprague-Dawley (SD) rat model. Murine 3T3-L1 preadipocytes were stimulated to differentiate under the adipogenic stimulation in the presence and absence of varying concentrations of GO2KA1. Adipocyte differentiation was confirmed by Oil Red O staining of lipids and the expression of adipogenic gene expression. Compared to control group, the cells treated with GO2KA1 significantly decreased in intracellular lipid accumulation with concomitant decreases in the expression of key transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (CEBP/α). Consistently, the mRNA expression of downstream adipogenic target genes such as fatty acid binding protein 4 (FABP4), fatty acid synthase (FAS), were significantly lower in the GO2KA1-treated group than in the control group. In vivo, male SD rats were fed a high fat diet (HFD) for 6 weeks to induced obesity, followed by oral administration of GO2KA1 at 0.1 g/kg/body weight or vehicle control in HFD. We assessed body weight, food intake, plasma lipids, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for liver function, and serum level of adiponectin, a marker for obesity-mediated metabolic syndrome. Compared to control group GO2KA1 significantly suppressed body weight gain (185.8 ± 8.8 g vs. 211.6 ± 20.1 g, p < 0.05) with no significant difference in food intake. The serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels were significantly lower in the GO2KA1-treated group than in the control group, whereas the high-density lipoprotein (HDL) level was higher in the GO2KA1 group. The GO2KA1-treated group also showed a significant reduction in ALT and AST levels compared to the control. Moreover, serum adiponectin levels were significantly 1.5-folder higher than the control group. These in vivo and in vitro findings suggest that dietary supplementation of GO2KA1 may prevent diet-induced weight gain and the anti-obesity effect is mediated in part by inhibiting adipogenesis and increasing adiponectin level.

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Platelet CD36 signaling through ERK5 promotes caspase-dependent procoagulant activity and fibrin deposition in vivo

Blood Advances ◽

10.1182/bloodadvances.2018025411 ◽

2018 ◽

Vol 2 (21) ◽

pp. 2848-2861 ◽

Cited By ~ 16

Author(s):

Moua Yang ◽

Andaleb Kholmukhamedov ◽

Marie L. Schulte ◽

Brian C. Cooley ◽

Na’il O. Scoggins ◽

...

Keyword(s):

Platelet Reactivity ◽

Low Density Lipoprotein ◽

Scavenger Receptor ◽

Density Lipoprotein ◽

Signaling Molecules ◽

Chow Diet ◽

Glycoprotein Vi ◽

Platelet Signaling ◽

Clinically Significant

Abstract Dyslipidemia is a risk factor for clinically significant thrombotic events. In this condition, scavenger receptor CD36 potentiates platelet reactivity through recognition of circulating oxidized lipids. CD36 promotes thrombosis by activating redox-sensitive signaling molecules, such as the MAPK extracellular signal-regulated kinase 5 (ERK5). However, the events downstream of platelet ERK5 are not clear. In this study, we report that oxidized low-density lipoprotein (oxLDL) promotes exposure of procoagulant phosphatidylserine (PSer) on platelet surfaces. Studies using pharmacologic inhibitors indicate that oxLDL-CD36 interaction–induced PSer exposure requires apoptotic caspases in addition to the downstream CD36-signaling molecules Src kinases, hydrogen peroxide, and ERK5. Caspases promote PSer exposure and, subsequently, recruitment of the prothrombinase complex, resulting in the generation of fibrin from the activation of thrombin. Caspase activity was observed when platelets were stimulated with oxLDL. This was prevented by inhibiting CD36 and ERK5. Furthermore, oxLDL potentiates convulxin/glycoprotein VI–mediated fibrin formation by platelets, which was prevented when CD36, ERK5, and caspases were inhibited. Using 2 in vivo arterial thrombosis models in apoE-null hyperlipidemic mice demonstrated enhanced arterial fibrin accumulation upon vessel injury. Importantly, absence of ERK5 in platelets or mice lacking CD36 displayed decreased fibrin accumulation in high-fat diet–fed conditions comparable to that seen in chow diet–fed animals. These findings suggest that platelet signaling through CD36 and ERK5 induces a procoagulant phenotype in the hyperlipidemic environment by enhancing caspase-mediated PSer exposure.

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Metabolic in Vivo Labeling Highlights Differences of Metabolically Active Microbes from the Mucosal Gastrointestinal Microbiome between High-Fat and Normal Chow Diet

Journal of Proteome Research ◽

10.1021/acs.jproteome.6b00973 ◽

2017 ◽

Vol 16 (4) ◽

pp. 1593-1604 ◽

Cited By ~ 19

Author(s):

Andreas Oberbach ◽

Sven-Bastiaan Haange ◽

Nadine Schlichting ◽

Marco Heinrich ◽

Stefanie Lehmann ◽

...

Keyword(s):

Chow Diet ◽

High Fat ◽

Gastrointestinal Microbiome ◽

In Vivo Labeling ◽

Normal Chow ◽

Normal Chow Diet

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Cardiac phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase increases glycolysis, hypertrophy, and myocyte resistance to hypoxia

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.91501.2007 ◽

2008 ◽

Vol 294 (6) ◽

pp. H2889-H2897 ◽

Cited By ~ 24

Author(s):

Qianwen Wang ◽

Rajakumar V. Donthi ◽

Jianxun Wang ◽

Alex J. Lange ◽

Lewis J. Watson ◽

...

Keyword(s):

Transgenic Mice ◽

Cardiac Fibrosis ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Weight Ratio ◽

Cardiac Metabolism ◽

Heart Weight ◽

Acid Oxidation ◽

Important Regulator

During ischemia and heart failure, there is an increase in cardiac glycolysis. To understand if this is beneficial or detrimental to the heart, we chronically elevated glycolysis by cardiac-specific overexpression of phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) in transgenic mice. PFK-2 controls the level of fructose-2,6-bisphosphate (Fru-2,6-P2), an important regulator of phosphofructokinase and glycolysis. Transgenic mice had over a threefold elevation in levels of Fru-2,6-P2. Cardiac metabolites upstream of phosphofructokinase were significantly reduced, as would be expected by the activation of phosphofructokinase. In perfused hearts, the transgene caused a significant increase in glycolysis that was less sensitive to inhibition by palmitate. Conversely, oxidation of palmitate was reduced by close to 50%. The elevation in glycolysis made isolated cardiomyocytes highly resistant to contractile inhibition by hypoxia, but in vivo the transgene had no effect on ischemia-reperfusion injury. Transgenic hearts exhibited pathology: the heart weight-to-body weight ratio was increased 17%, cardiomyocyte length was greater, and cardiac fibrosis was increased. However, the transgene did not change insulin sensitivity. These results show that the elevation in glycolysis provides acute benefits against hypoxia, but the chronic increase in glycolysis or reduction in fatty acid oxidation interferes with normal cardiac metabolism, which may be detrimental to the heart.

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Pygo1 regulates pathological cardiac hypertrophy via a β-catenin-dependent mechanism

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00538.2020 ◽

2021 ◽

Author(s):

Li Lin ◽

Wei Xu ◽

Yongqing Li ◽

Ping Zhu ◽

Wuzhou Yuan ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Cardiac Tissue ◽

Heart Weight ◽

Dependent Manner ◽

Tissue Specific ◽

Cardiac Tissues ◽

Pathological Cardiac Hypertrophy ◽

Interaction Factor ◽

Myocardial Remodelling

Wnt/β-catenin signalling plays a key role in pathological cardiac remodelling in adults. The identification of a tissue-specific Wnt/β-catenin interaction factor may realise a tissue-specific clinical targeting strategy. Drosophila Pygo codes for the core interaction factor of Wnt/β-catenin. Two Pygo homologs, Pygo1 and Pygo2, have been identified in mammals. Different from the ubiquitous expression profile of Pygo2, Pygo1is enriched in cardiac tissue. However, the role of Pygo1 in mammalian cardiac disease remains unelucidated. Here, we found that Pygo1 was upregulated in human cardiac tissues with pathological hypertrophy. Cardiac-specific overexpression of Pygo1 in mice spontaneously led to cardiac hypertrophy accompanied by declined cardiac function, increased heart weight/body weight and heart weight/tibial length ratios and increased cell size. The canonical β-catenin/T-cell transcription factor 4 complex was abundant in Pygo1-overexpressingtransgenic(Pygo1-TG) cardiac tissue,and the downstream genes of Wnt signaling, i.e., Axin2, Ephb3, and C-myc, were upregulated. A tail vein injection of β-catenin inhibitor effectively rescued the phenotype of cardiac failure and pathological myocardial remodelling in Pygo1-TG mice. Furthermore, in vivo downregulated pygo1 during cardiac hypertrophic condition antagonized agonist-induced cardiac hypertrophy. Therefore, our study is the first to present in vivo evidence demonstrating that Pygo1 regulates pathological cardiac hypertrophy in a canonical Wnt/β-catenin-dependent manner, which may provide new clues for a tissue-specific clinical treatment targeting this pathway.

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Abstract 268: Strain-specific Cardiac Fibroblast Response to Isoproterenol-induced Cardiac Fibrosis

Circulation Research ◽

10.1161/res.121.suppl_1.268 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Shuin Park ◽

Sara Ranjbarvaziri ◽

Fides Lay ◽

Peng Zhao ◽

Aldons J Lusis ◽

...

Keyword(s):

Gene Expression ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Expression Profiles ◽

Inbred Mouse ◽

Gene Expression Profiles ◽

Mouse Strains ◽

Sequencing Analysis ◽

Different Strains

Fibroblasts are a heterogeneous population of cells that function within the injury response mechanisms across various tissues. Despite their importance in pathophysiology, the effects of different genetic backgrounds on fibroblast contribution to the development of disease has yet to be addressed. It has previously been shown that mice in the Hybrid Mouse Diversity Panel, which consists of 110 inbred mouse strains, display a spectrum in severity of cardiac fibrosis in response to chronic treatment of isoproterenol (ISO). Here, we characterized cardiac fibroblasts (CFbs) from three different mouse strains (C57BL/6J, C3H/HeJ, and KK/HIJ) which exhibited varying degrees of fibrosis after ISO treatment. The select strains of mice underwent sham or ISO treatment via intraperitoneally-implanted osmotic pumps for 21 days. Masson’s Trichrome staining showed significant differences in fibrosis in response to ISO, with KK/HIJ mice demonstrating the highest levels, C3H/HeJ exhibiting milder levels, and C57BL/6J demonstrating little to no fibrosis. When CFbs were isolated and cultured from each strain, the cells demonstrated similar traits at the basal level but responded to ISO stimuli in a strain-specific manner. Likewise, CFbs demonstrated differential behavior and gene expression in vivo in response to ISO. ISO treatment caused CFbs to proliferate similarly across all strains, however, immunofluorescence staining showed differential levels of CFb activation. Additionally, RNA-sequencing analysis revealed unique gene expression profiles of all three strains upon ISO treatment. Our study depicts the phenotypic heterogeneity of CFbs across different strains of mice and our results suggest that ISO-induced cardiac fibrosis is a complex process that is independent of fibroblast proliferation and is mainly driven by the activation/inhibition of genes involved in pro-fibrotic pathways.

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Abstract 28: In Vivo Knockdown of Mirna-15b Results in Increased Cardiac Hypertrophy and Fibrosis in Response to Pressure Overload of the Mouse Heart

Circulation Research ◽

10.1161/res.111.suppl_1.a28 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Anke J Tijsen ◽

Ingeborg van der Made ◽

Elza D van Deel ◽

Monika Hiller ◽

Yolan J Reckman ◽

...

Keyword(s):

Transgenic Mice ◽

Cardiac Function ◽

Cardiac Fibrosis ◽

Negative Control ◽

Heart Weight ◽

Mouse Heart ◽

Hypertrophic Response ◽

Wide Range ◽

Tgfβ Pathway

MiRNAs play an important role in the control of diverse aspects of cardiac function. MiR-15b is highly expressed in the heart and is found consistently upregulated in hypertrophic and failing hearts. To investigate the function of miR-15b in the heart we set out two experiments. In the first experiment we generated two independent transgenic mouse lines that drive miR-15b expression under the αMHC-promotor and show a three and four fold overexpression of miR-15b. Strikingly, both lines show a decrease in heart weight/tibia length of 20% in adult and aged mice when compared to littermate controls. We investigated the response of these transgenic mice to thoracic aorta constriction (TAC) and found no differences in the hypertrophic response or in cardiac function measured by echocardiography between wild-type and transgenic mice. In a second experiment, we inhibited miR-15b using LNA-based antimiRs. In these mice, TAC resulted in an increased hypertrophic response and increased cardiac fibrosis when compared to a negative control antimiR. A wide range of predicted targets of miR-15 belong to the pathways of the TGFβ-superfamily and using a smad-dependent reporter we show that miR-15b inhibits TGFβ-induced Smad activity in HepG2 cells. One of the predicted targets in the TGFβ pathway is TGFβ receptor 1 (TGFβR1), of which the 3’UTR contains six predicted miR-15 binding sites. This suggests that the phenotype in the transgenic mice and after knockdown of miR-15b may be (partly) mediated by repression of TGFβR1. Indeed, in the adult miR-15b transgenic hearts we found a downregulation of TGFβR1 mRNA and protein and we confirmed binding of miR-15 to the TGFβR1 3’UTR by luciferase assays. In conclusion, miR-15b causes a cardiac hypotrophic phenotype at baseline in transgenic mice and inhibition of miR-15b leads to a stronger hypertrophic and fibrotic response after TAC. Furthermore miR-15b inhibits the TGFβ pathway by targeting the TGFβR1 and possibly other targets in this pathway. This research is funded by the Dutch Heart Foundation (NHF grant #2007B077).

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