Beta-lapachone inhibits pathological retinal neovascularization in oxygen-induced retinopathy via regulation of HIF-1α.

Retinal neovascularization in retinopathy of prematurity (ROP) is the most common cause of blindness for children. Despite evidence that hypoxia inducible factor (HIF)-1α -VEGF axis is associated with the pathogenesis of ROP, the inhibitors of HIF-1α have not been established as a therapeutic target in the control of ROP pathophysiology. We investigated the hypothesis that degradation of HIF-1α as a master regulator of angiogenesis in hypoxic condition, using ß-lapachone, would confer protection against hypoxia-induced retinopathy without affecting physiological vascular development in mice with oxygen-induced retinopathy (OIR), an animal model of ROP. The effects of ß-lapachone were examined after intraocular injection in mice with OIR. Intraocular administration of ß-lapachone resulted in significant reduction in hypoxia-induced retinal neovascularization without retinal toxicity or perturbation of developmental retinal angiogenesis. Our results demonstrate that HIF-1α-mediated VEGF expression in OIR is associated with pathological neovascularization, not physiological angiogenesis. Thus, strategies blocking HIF-1α in the developing eye in the pathological hypoxia could serve as a novel therapeutic target for ROP.

ß-Lapachone attenuates mitochondrial dysfunction in MELAS cybrid cells.

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disease caused by mutations in the mitochondrial genome. This study investigated the efficacy of ß-lapachone (ß-lap), a natural quinone compound, in rescuing mitochondrial dysfunction in MELAS cybrid cells. ß-Lap significantly restored energy production and mitochondrial membrane potential as well as normalized the elevated ROS level in MELAS cybrid cells. Additionally, ß-lap reduced lactic acidosis and restored glucose uptake in the MELAS cybrid cells. Finally, ß-lap activated Sirt1 by increasing the intracellular NAD(+)/NADH ratio, which was accompanied by increased mtDNA content. Two other quinone compounds (idebenone and CoQ10) that have rescued mitochondrial dysfunction in previous studies of MELAS cybrid cells had a minimal effect in the current study. Taken together, these results demonstrated that ß-lap may provide a novel therapeutic modality for the treatment of MELAS.

Identification of novel microRNAs negatively regulating cardiac hypertrophy.

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules that suppress gene expression via degradation or translational inhibition of their target genes. Many miRNAs are associated with cardiac hypertrophy and heart failure. In this study, we pursued to identify miRNAs that negatively regulate cardiac hypertrophy by utilizing a surgical model for regression of cardiac hypertrophy. Microarray analysis revealed that 15 miRNAs out of the 696 miRNAs tested were specifically up-regulated during the regression period. Among these regression-specific miRNAs, nine microRNAs, which have not been previously reported, were further tested for their effects on phenylephrine (PE)-treated neonatal cardiomyocytes. Consequently, five miRNAs (miR-101b, 142-3p, 181d, 24-2(∗), and 450a) completely abrogated PE-induced hypertrophy as determined by measurements of cell size and fetal gene expression. Conversely, antagomers of these miRNAs exacerbated the PE-induced hypertrophy. Collectively, these findings suggest that the five miRNAs newly identified by using our cardiac hypertrophy-regression surgical model negatively regulate cardiac hypertrophy and could be used as potential therapeutic targets for the treatment of heart diseases.

The opposing effects of CCN2 and CCN5 on the development of cardiac hypertrophy and fibrosis.

CCN family members are matricellular proteins with diverse roles in cell function. The differential expression of CCN2 and CCN5 during cardiac remodeling suggests that these two members of the CCN family play opposing roles during the development of cardiac hypertrophy and fibrosis. We aimed to evaluate the role of CCN2 and CCN5 in the development of cardiac hypertrophy and fibrosis. In isolated cardiomyocytes, overexpression of CCN2 induced hypertrophic growth, whereas the overexpression of CCN5 inhibited both phenylephrine (PE)- and CCN2-induced hypertrophic responses. Deletion of the C-terminal (CT) domain of CCN2 transformed CCN2 into a CCN5-like dominant negative molecule. Fusion of the CT domain to the Carboxy-terminus of CCN5 transformed CCN5 into a CCN2-like pro-hypertrophic molecule. CCN2 transgenic (TG) mice did not develop cardiac hypertrophy at baseline but showed significantly increased fibrosis in response to pressure overload. In contrast, hypertrophy and fibrosis were both significantly inhibited in CCN5 TG mice. CCN2 TG mice showed an accelerated deterioration of cardiac function in response to pressure overload, whereas CCN5 TG mice showed conserved cardiac function. TGF-beta-SMAD signaling was elevated in CCN2 TG mice, but was inhibited in CCN5 TG mice. CCN2 is pro-hypertrophic and -fibrotic, whereas CCN5 is anti-hypertrophic and -fibrotic. CCN5 lacking the CT domain acts as a dominant negative molecule. CCN5 may provide a novel therapeutic target for the treatment of cardiac hypertrophy and heart failure.

A Pilot Study of Chronological Microbiota Changes in a Rat Apical Periodontitis Model.

Apical periodontitis caused by microbial infection in the dental pulp is characterized by inflammation, destruction of the pulpal and periradicular tissues, and alveolar bone resorption. We analyzed the chronological changes in microbiota using a pyrosequencing-based approach combined with radiologic and histopathologic changes in a rat apical periodontitis model. During the three-week observation, the pulp and periapical area showed a typical progress of apical periodontitis. A total of 27 phyla, 645 genera, and 1276 species were identified. The root apex had a lower bacterial species diversity than the pulp chamber. Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were dominant phyla in both the pulp chamber and root apex. Remarkably, bacterial communities showed a tendency to change in the root apex based on the disease progression. At the genus level, Escherichia, Streptococcus, Lactobacillus, Rodentibacter, and Bacteroidetes were dominant genera in the pulp chamber. The most abundant genera in the root apex were Bradyrhizobium, Halomonas, and Escherichia. The species Azospirillum oryzae increased in the pulp chamber, whereas the species Bradyrhizobium japonicum and Halomonas stevensii were highly observed in the root apex as the disease progressed. The experimental rat model of apical periodontitis demonstrated a relationship between the microbiota and the apical periodontitis progression.

PICOT is a critical regulator of cardiac hypertrophy and cardiomyocyte contractility.

PICOT (PKC-interacting cousin of thioredoxin) was previously shown to inhibit the development of cardiac hypertrophy, concomitant with an increase in cardiomyocyte contractility. To explore the physiological function of PICOT in the hearts, we generated a PICOT-deficient mouse line by using a gene trap approach. PICOT(-/-) mice were embryonic lethal indicating that PICOT plays an essential role during embryogenesis, whereas PICOT(+/-) mice were viable with no apparent morphological defects. The PICOT protein levels were reduced by about 50% in the hearts of PICOT(+/-) mice. Significantly exacerbated cardiac hypertrophy was induced by pressure overload in PICOT(+/-) mice relative to that seen in wild type littermates. In line with this observation, calcineurin-NFAT signaling was greatly enhanced by pressure overload in the hearts of PICOT(+/-) mice. Cardiomyocytes from PICOT(+/-) mice exhibited significantly reduced contractility, which may be due in part to hypophosphorylation of phospholamban and reduced SERCA activity. These data indicate that the precise PICOT protein level significantly affects the process of cardiac hypertrophy and cardiomyocyte contractility. We suggest that PICOT plays as a critical negative regulator of cardiac hypertrophy and a positive inotropic regulator.

Cucurbitacin I Attenuates Cardiomyocyte Hypertrophy via Inhibition of Connective Tissue Growth Factor (CCN2) and TGF- ß/Smads Signalings.

Cucurbitacin I is a naturally occurring triterpenoid derived from Cucurbitaceae family plants that exhibits a number of potentially useful pharmacological and biological activities. However, the therapeutic impact of cucurbitacin I on the heart has not heretofore been reported. To evaluate the functional role of cucurbitacin I in an in vitro model of cardiac hypertrophy, phenylephrine (PE)-stimulated cardiomyocytes were treated with a sub-cytotoxic concentration of the compound, and the effects on cell size and mRNA expression levels of ANF and ß-MHC were investigated. Consequently, PE-induced cell enlargement and upregulation of ANF and ß-MHC were significantly suppressed by pretreatment of the cardiomyocytes with cucurbitacin I. Notably, cucurbitacin I also impaired connective tissue growth factor (CTGF) and MAPK signaling, pro-hypertrophic factors, as well as TGF-ß/Smad signaling, the important contributing factors to fibrosis. The protective impact of cucurbitacin I was significantly blunted in CTGF-silenced or TGF-ß1-silenced hypertrophic cardiomyocytes, indicating that the compound exerts its beneficial actions through CTGF. Taken together, these findings signify that cucurbitacin I protects the heart against cardiac hypertrophy via inhibition of CTGF/MAPK, and TGF- ß/Smad-facilitated events. Accordingly, the present study provides new insights into the defensive capacity of cucurbitacin I against cardiac hypertrophy, and further suggesting cucurbitacin I's utility as a novel therapeutic agent for the management of heart diseases.

ß-Lapachone ameliorates lipotoxic cardiomyopathy in acyl CoA synthase transgenic mice.

Lipotoxic cardiomyopathy is caused by myocardial lipid accumulation and often occurs in patients with diabetes and obesity. This study investigated the effects of ß-lapachone (ß-lap), a natural compound that activates Sirt1 through elevation of the intracellular NAD+ level, on acyl CoA synthase (ACS) transgenic (Tg) mice, which have lipotoxic cardiomyopathy. Oral administration of ß-lap to ACS Tg mice significantly attenuated heart failure and inhibited myocardial accumulation of triacylglycerol. Electron microscopy and measurement of mitochondrial complex II protein and mitochondrial DNA revealed that administration of ß-lap restored mitochondrial integrity and biogenesis in ACS Tg hearts. Accordingly, ß-lap administration significantly increased the expression of genes associated with mitochondrial biogenesis and fatty acid metabolism that were down-regulated in ACS Tg hearts. ß-lap also restored the activities of Sirt1 and AMP-activated protein kinase (AMPK), the two key regulators of metabolism, which were suppressed in ACS Tg hearts. In H9C2 cells, ß-lap-mediated elevation of AMPK activity was retarded when the level of Sirt1 was reduced by transfection of siRNA against Sirt1. Taken together, these results indicate that ß-lap exerts cardioprotective effects against cardiac lipotoxicity through the activation of Sirt1 and AMPK. ß-lap may be a novel therapeutic agent for the treatment of lipotoxic cardiomyopathy.