Downregulation of ERRα inhibits angiogenesis in human umbilical vein endothelial cells through regulating VEGF production and PI3K/Akt/STAT3 signaling pathway.

The human estrogen related receptor α (ERRα) is a pivotal regulator involved in energy homeostasis and mitochondrial biogenesis. It has been demonstrated that activation of ERRα in various breast cancer cells results in a significant increase of vascular endothelial growth factor (VEGF) mRNA and protein secretion. However, little is known about the relationship between ERRα and angiogenesis. Thus, the present study is aimed to investigate the effects and mechanism of ERRα suppression on the angiogenesis in human umbilical vein endothelial cells (HUVECs). Here we show that ERRα suppression powerfully inhibits proliferation, migration and capillary-like structures formation of HUVECs. Importantly, we demonstrate that these inhibitory effects are associated with the significantly reduced expression and production of VEGF. Results from further experiments using western blot and luciferase reporter assay exhibit that ERRα suppression inhibits hypoxia-inducible factor 1α (HIF-1α) expression, and phosphorylation of protein kinase B (Akt) and signal transducer and activator of transcription (STAT3) which up-regulated VEGF expression. In summary, we show that ERRα suppression inhibits angiogenesis in HUVECs and deserves further studies for application of rationale therapeutic target for patient with diseases related with aberrant angiogenesis.

Tectorigenin regulates adipogenic differentiation and adipocytokines secretion via PPARγ and IKK/NF-κB signaling.

CONTEXT: Obesity is associated with a number of diseases with metabolic abnormalities such as type 2 diabetes (T2D). OBJECTIVE: We investigate the effects of tectorigenin on 3T3-L1 preadipocyte differentiation and adipocytokines secretion. MATERIALS AND METHODS: The effects of tectorigenin on adipocyte differentiation were studied using Oil Red O staining. Effects of tectorigenin on adipogenesis-related genes expression and adipocytokines secretion were measured by the real-time quantitative RT-PCR and ELISA method, respectively. Reporter gene assays were performed to determine the PPARγ and NF-κB transactivation. We also used [(3)H]-2-deoxy-d-glucose to study the glucose uptake, and the IKK/NF-κB signaling pathway was assessed by western blot analysis. HFD/STZ rats were used to evaluate the therapeutic efficacies of tectorigenin. RESULTS: Tectorigenin 10, 25, 50, and 75 µM inhibited 3T3-L1 adipogenesis and related genes transcription. TNF-α-induced changes of IL-6, MCP-1, as well as adiponectin in 3T3-L1 were markedly reversed by tectorigenin at 75 µM. Further investigation using reporter gene revealed that tectorigenin was a partial PPARγ agonist with an IC50 value of 13.3 µM. Tectorigenin improved basal and insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes. Moreover, tectorigenin antagonized TNF-α-induced NF-κB transactivation and p65 nuclear translocation. Although tectorigenin (50 and 100 mg/kg) displayed the ability to promote insulin sensitivity and improve glucose metabolism in HFD/STZ rats, it did not cause significant side effects such as body weight gain, fluid retention, or cardiac hypertrophy. DISCUSSION AND CONCLUSION: These results suggest that tectorigenin may ameliorate hyperglycemia by blocking preadipocyte differentiation and adipocytokines secretion in which PPARγ and NF-κB signaling pathways were involved.

Chlorogenic acid inhibits hypoxia-induced pulmonary artery smooth muscle cells proliferation via c-Src and Shc/Grb2/ERK2 signaling pathway.

Chlorogenic acid (CGA), abundant in coffee and particular fruits, can modulate hypertension and vascular dysfunction. Hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) proliferation has been tightly linked to vascular remodeling in pulmonary arterial hypertension (PAH). Thus, the present study was designed to investigate the effect of CGA on hypoxia-induced proliferation in cultured rat PASMCs. The data showed that CGA potently inhibited PASMCs proliferation and DNA synthesis induced by hypoxia. These inhibitory effects were associated with G1 cell cycle arrest and down-regulation of cell cycle proteins. Treatment with CGA reduced hypoxia-induced hypoxia inducible factor 1α (HIF-1α) expression and trans-activation. Furthermore, hypoxia-evoked c-Src phosphorylation was inhibited by CGA. In vitro ELISA-based tyrosine kinase assay indicated that CGA was a direct inhibitor of c-Src. Moreover, CGA attenuated physical co-association of c-Src/Shc/Grb2 and ERK2 phosphorylation in PASMCs. These results suggest that CGA inhibits hypoxia-induced proliferation in PASMCs via regulating c-Src-mediated signaling pathway. In vivo investigation showed that chronic CGA treatment inhibits monocrotaline-induced PAH in rats. These findings presented here highlight the possible therapeutic use of CGA in hypoxia-related PAH.

[XCT790 inhibits rat vascular smooth muscle cells proliferation through down-regulating the expression of estrogen-related receptor alpha].

Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in several pathological processes of cardiovascular diseases. In this study, the effects of XCT790, a potent and selective inverse agonist of estrogen-related receptor alpha (ERRalpha), on rat VSMCs proliferation and related signal pathways were investigated. The proliferative activity of VSMCs was determined by CCK-8 assay. The mRNA levels of ERRalpha, PGC-1alpha, OPN and MCAD were assayed by RT-PCR. The protein levels of ERRalpha, ERK2 and p-ERK1/2 were evaluated by Western blotting. ELISA was used to assess the protein expression of VEGF. The results showed that XCT790 (5-20 micromol x L(-1)) inhibited rat VSMCs proliferation, and the expression of ERRalpha and its target genes, as well as p-ERK1/2, were also inhibited. XCT790 inhibited VSMCs proliferation in a dose-dependent manner at the dose range from 5 to 20 micromol x L(-1) and in a time-dependent manner at the dose range from 10 to 20 micromol x L(-1). These findings demonstrate that XCT790 inhibits rat VSMCs proliferation by down-regulating the gene level of ERRalpha and thus inhibiting the ERK signal pathway, suggesting that ERRalpha may be a novel potential target for therapeutic approaches to inhibit VSMCs proliferation, which plays an important role in several cardiovascular diseases.

6-Hydroxydaidzein enhances adipocyte differentiation and glucose uptake in 3T3-L1 cells.

Fermented soybean foods have been shown to reduce incidence of diabetes and improve insulin sensitivity. 6-Hydroxydaidzein (6-HD) is a bioactive ingredient isolated from fermented soybean. In this study, we examined the effects of 6-HD on adipocyte differentiation and insulin-stimulated glucose uptake, as well as the mechanisms involved. In our experiments, 6-HD enhanced 3T3-L1 adipocyte differentiation and insulin-stimulated glucose uptake in a dosage-dependent manner. In addition, 6-HD increased peroxisome proliferator-activated receptor gamma (PPARγ) gene expression and PPARγ transcriptional activity. 6-HD increased CCAAT/enhanced binding protein alpha (C/EBPα) expression as well. Although having no effects on glucose transporter type 4 (GLUT4) gene expression, 6-HD facilitated GLUT4 protein translocation to the cell membranes. Our results indicate that 6-HD exhibited the actions of promoting adipocyte differentiation and improving insulin sensitivity by increasing the expression of C/EBPα and facilitating the translocation of GLUT4 via the activation of PPARγ, suggesting that 6-HD can be promising in diabetes management.

Discovery of novel PDE10 inhibitors by a robust homogeneous screening assay.

AIM: To develop a homogeneous assay for high-throughput screening (HTS) of inhibitors of phosphodiesterase 10 (PDE10). METHODS: Purified human PDE10 enzyme derived from E coli, [(3)H]-cAMP and yttrium silicate microbeads were used to develop an HTS assay based on the scintillation proximity assay (SPA) technology. This method was applied to a large-scale screening campaign against a diverse compound library and subsequent confirmation studies. Preliminary structure-activity relationship (SAR) studies were initiated through limited structural modifications of the hits. RESULTS: The IC50 value of the control compound (papaverine) assessed with the SPA approach was comparable and consistent with that reported in the literature. Signal to background (S/B) ratio and Z' factor of the assay system were evaluated to be 5.24 and 0.71, respectively. In an HTS campaign of 71 360 synthetic and natural compounds, 67 hits displayed reproducible PDE10 inhibition, of which, 8 were chosen as the scaffold for structural modifications and subsequent SAR analysis. CONCLUSION: The homogeneous PDE10 SPA assay is an efficient and robust tool to screen potential PDE10 inhibitors. Preliminary SAR studies suggest that potent PDE10 inhibitors could be identified and developed through this strategy.

Ginsenoside Rb1 inhibits proliferation and inflammatory responses in rat aortic smooth muscle cells.

Ginsenoside Rb1, a known phytoestrogen, is a major pharmacologically active component in ginseng. The present study was designed to investigate the effect of ginsenoside Rb1 on fetal bovine serum (FBS)-induced proliferation and tumor necrosis factor-α (TNF-α)-evoked inflammatory responses in cultured rat aortic vascular smooth muscle cells (VSMCs). The data showed that Rb1 potently inhibited VSMC proliferation and cell growth induced by 5% FBS. These inhibitory effects were associated with G(1) cell cycle arrest and down-regulation of cell cycle proteins. Treatment with Rb1 reduced FBS-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, TNF-α-evoked inflammatory responses were inhibited by Rb1. Reporter gene assay indicated that Rb1 could transactivate ERß especially. Moreover, Rb1-mediated inhibition of VSMCs proliferation was greatly blocked by transfection of ERß siRNA. These results suggest that Rb1 inhibits FBS-induced proliferation and TNF-α-evoked inflammatory responses in VSMCs. The findings presented here highlight the possible therapeutic use of Rb1 in cardiovascular disease.

Involvement of estrogen receptor-ß in farrerol inhibition of rat thoracic aorta vascular smooth muscle cell proliferation.

AIM: To investigate the effect of farrerol, a major active component isolated from a traditional Chinese herb "man-shan-hong" (the dried leaves of Rhododendron dauricum L) on fetal bovine serum (FBS)-induced proliferation of cultured vascular smooth muscle cells (VSMCs) of rat thoracic aorta. METHODS: VSMCs proliferation, DNA synthesis and cell cycle progression were studied using the MTT assay, bromodeoxyuridine (BrdU) incorporation and flow cytometry, respectively. The mRNA levels of cell cycle proteins were quantified using real-time RT-PCR, and the phosphorylation of ERK1/2 was determined using Western blotting. Reporter gene and receptor binding assays were employed to study the interaction between farrerol and estrogen receptors (ERs). RESULTS: Farrerol (0.3-10 µmol/L) inhibited VSMC proliferation and DNA synthesis induced by 5% FBS in a concentration-dependent manner. The effects were associated with G(1) cell cycle arrest, down-regulation of cell cycle proteins and reduction in FBS-induced ERK1/2 phosphorylation. Using a reporter gene, it was found that farrerol (3 µmol/L) induced 2.1-fold transcription of ER. In receptor binding assays, farrerol inhibited the binding of [(3)H]estradiol for ERα and ERß with IC(50) values of 57 µmol/L and 2.7 µmol/L, respectively, implying that farrerol had a higher affinity for ERß. Finally, the inhibition of VSMC proliferation by farrerol (3 µmol/L) was abolished by the specific ERß antagonist PHTPP (5 µmol/L). CONCLUSION: Farrerol acts as a functional phytoestrogen to inhibit FBS-induced VSMC proliferation, mainly via interaction with ERß, which may be helpful in the treatment of cardiovascular diseases related to abnormal VSMCs proliferation.

Characterization of a novel non-steroidal glucocorticoid receptor antagonist.

Selective antagonists of the glucocorticoid receptor (GR) are desirable for the treatment of hypercortisolemia associated with Cushing's syndrome, psychic depression, obesity, diabetes, neurodegenerative diseases, and glaucoma. NC3327, a non-steroidal small molecule with potent binding affinity to GR (K(i)=13.2nM), was identified in a high-throughput screening effort. As a full GR antagonist, NC3327 greatly inhibits the dexamethasone (Dex) induction of marker genes involved in hepatic gluconeogenesis, but has a minimal effect on matrix metalloproteinase 9 (MMP-9), a GR responsive pro-inflammatory gene. Interestingly, the compound recruits neither coactivators nor corepressors to the GR complex but competes with glucocorticoids for the interaction between GR and a coactivator peptide. Moreover, NC3327 does not trigger GR nuclear translocation, but significantly blocks Dex-induced GR transportation to the nucleus, and thus appears to be a 'competitive' GR antagonist. Therefore, the non-steroidal compound, NC3327, may represent a new class of GR antagonists as potential therapeutics for a variety of cortisol-related endocrine disorders.

High-throughput screening of novel antagonists on melanin-concentrating hormone receptor-1.

AIM: To find new antagonists on human melanin-concentrating hormone receptor-1 (MCHR-1) through high-throughput screening (HTS) of a diverse compound library. METHODS: MCHR-1, [3H]SNAP7941, and FlashBlue G-protein-coupled receptor beads were used to measure the receptor-binding activities of various compounds based on scintillation proximity assay (SPA) technology. The guanosine 5'(gamma-[35S]thio) triphosphate ([35S]GTPgammaS) binding assay was subsequently applied to functionally characterize the "hits" identified by the HTS campaign. RESULTS: Of the 48,240 compounds screened with the SPA method, 12 hits were confirmed to possess MCHR-1 binding activities, 8 were functionally studied subsequently with the [35S]GTPgammaS binding assay, and only 1 compound (NC127816) displayed moderate human MCHR-1 binding affinity (Ki=115.7 nmol/L) and relatively potent antagonism (KB=23.8 nmol/L). This compound shares a novel scaffold (1-ethoxy-2H-2-aza-1-phospha-naphthalene 1-oxide) with 3 other analogs in the group. CONCLUSION: Considering the marked difference in molecular shape and electrostatic status between NC127816 and the structures reported elsewhere, we anticipate that its derivatives may represent a new class of potent MCHR-1 modulators.

Rhodanine derivatives as novel peroxisome proliferator-activated receptor gamma agonists.

AIM: To characterize the in vitro bioactivities of rhodanine derivatives as novel peroxisome proliferator-activated receptor (PPAR) gamma modulators, based on a hit (SH00012671) identified during high-throughput screening (HTS) of a diverse synthetic compound library, and to preliminarily elucidate the structure-activity relationship of this class of PPARgamma agonists. METHODS: Full-length PPARgamma and retinoid X receptor alpha (RXRalpha), biotinylated PPAR response element (PPRE), [3H]BRL49653 (rosiglitazone), and streptavidin-coated FlashPlate or microbeads were used to measure the receptor-binding properties of various compounds based on the scintillation proximity assay (SPA) technology. A recombinant PPRE vector was transiently cotransfected with PPARgamma and RXRalpha plasmids into the African green monkey kidney (CV-1) cells, and the effects of BRL49653 and test compounds on transcription mediated by PPARgamma were determined by examining luciferase (reporter) responses. 3T3-L1 cells were employed to determine whether the compounds facilitated adipogenesis upon PPARgamma activation. RESULTS: Of the 16,000 samples screened with the SPA method, only 1 compound (SH00012671) displayed a similar binding affinity (Ki=186.7 nmol/L) to PPARgamma as BRL49653, but it was inactive in the cell-based assays. A series of rhodanine derivatives were synthesized based on the core structure of SH00012671 and 8 of them showed agonist activities in both cotransfection and pre-adipocyte differentiation assays. To reduce intrinsic cytotoxicities, the sulphur on the rhodanine was changed to oxygen. This alteration led to a decrease in receptor-binding affinities while modified analogues generally maintained agonist efficacies in the cell-based assays. Of the analogues studied, compound 31 exhibited about 70% the efficacy exerted by BRL49653 in both cotransfection and pre-adipocyte differentiation assays. CONCLUSION: Through minor chemical modifications on the core structure of the initial HTS hit, SH00012671 was transformed to possess both molecular (PPARgamma binding) and cellular (adipogenesis) activities. The rhodanine derivatives reported here may represent a new scaffold in further understanding the molecular mechanism of agonism at PPARgamma.