Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 in Pulmonary Arterial Hypertension.

BACKGROUND: Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. METHODS: We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH. RESULTS: Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses. CONCLUSIONS: Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.

Restoration of impaired endothelial myocyte enhancer factor 2 function rescues pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary arterioles, characterized by increased pulmonary arterial pressure and right ventricular failure. The cause of PAH is complex, but aberrant proliferation of the pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells is thought to play an important role in its pathogenesis. Understanding the mechanisms of transcriptional gene regulation involved in pulmonary vascular homeostasis can provide key insights into potential therapeutic strategies. METHODS AND RESULTS: We demonstrate that the activity of the transcription factor myocyte enhancer factor 2 (MEF2) is significantly impaired in the PAECs derived from subjects with PAH. We identified MEF2 as the key cis-acting factor that regulates expression of a number of transcriptional targets involved in pulmonary vascular homeostasis, including microRNAs 424 and 503, connexins 37, and 40, and Kruppel Like Factors 2 and 4, which were found to be significantly decreased in PAH PAECs. The impaired MEF2 activity in PAH PAECs was mediated by excess nuclear accumulation of 2 class IIa histone deacetylases (HDACs) that inhibit its function, namely HDAC4 and HDAC5. Selective, pharmacological inhibition of class IIa HDACs led to restoration of MEF2 activity in PAECs, as demonstrated by increased expression of its transcriptional targets, decreased cell migration and proliferation, and rescue of experimental pulmonary hypertension models. CONCLUSIONS: Our results demonstrate that strategies to augment MEF2 activity hold potential therapeutic value in PAH. Moreover, we identify selective HDAC IIa inhibition as a viable alternative approach to avoid the potential adverse effects of broad spectrum HDAC inhibition in PAH.

Virtual screening and synthesis of quinazolines as novel JAK2 inhibitors.

JAK2 is an important target in multiple processes associated with tumor growth. In this study, virtual screening was employed for hit compound identification with chemical libraries using SurflexDock. Subsequently, hit optimization for potent and selective candidate JAK2 inhibitors was performed through synthesis of diverse C-1 substituted quinazoline derivatives. A novel compound 5p, (6,7-dimethoxyquinazolin-4-yl)naphthalen-1-ylamine, was thus obtained. JAK2 inhibitory activity of 5p was 43% at 20µM and this was comparable to AG490, a representative JAK2 inhibitor. Moreover, 5p showed a positive correlation between JAK2 inhibition and cytotoxicity; 5p treatment in HT-29 cells strongly inhibited JAK2 activation and subsequent STAT3 phosphorylation, reduced anti-apoptotic protein levels, and finally induced apoptosis. This suggests that compound 5p is a candidate inhibitor of JAK2 and its downstream STAT3 signaling pathway for antitumor therapy. In the docking model, the quinazoline template of 5k, the lead compound, occupied a hydrophobic region such as Leu856, Leu855, Ala880, Leu932 and Gly935, and the highly conserved hydrogen bond was created by 6-OMe of the ring template, which binds to the NH of Arg980. Moreover, hydrophobic interactions were identified between morpholine moiety and the hydrophobic region formed by Leu855, Ala880, Tyr931, Val911 and Met929. Also, compound 5k more strongly inhibited JAK2 phosphorylation in mouse embryonic stem cells than AG490. Our study shows the successful application of virtual screening for lead discovery and we propose that the novel compound 5p can be an effective JAK2 inhibitor candidate for further antitumor agent research.

Endothelial APLNR regulates tissue fatty acid uptake and is essential for apelin's glucose-lowering effects.

Treatment of type 2 diabetes mellitus continues to pose an important clinical challenge, with most existing therapies lacking demonstrable ability to improve cardiovascular outcomes. The atheroprotective peptide apelin (APLN) enhances glucose utilization and improves insulin sensitivity. However, the mechanism of these effects remains poorly defined. We demonstrate that the expression of APLNR (APJ/AGTRL1), the only known receptor for apelin, is predominantly restricted to the endothelial cells (ECs) of multiple adult metabolic organs, including skeletal muscle and adipose tissue. Conditional endothelial-specific deletion of Aplnr (AplnrECKO ) resulted in markedly impaired glucose utilization and abrogation of apelin-induced glucose lowering. Furthermore, we identified inactivation of Forkhead box protein O1 (FOXO1) and inhibition of endothelial expression of fatty acid (FA) binding protein 4 (FABP4) as key downstream signaling targets of apelin/APLNR signaling. Both the Apln-/- and AplnrECKO mice demonstrated increased endothelial FABP4 expression and excess tissue FA accumulation, whereas concurrent endothelial Foxo1 deletion or pharmacologic FABP4 inhibition rescued the excess FA accumulation phenotype of the Apln-/- mice. The impaired glucose utilization in the AplnrECKO mice was associated with excess FA accumulation in the skeletal muscle. Treatment of these mice with an FABP4 inhibitor abrogated these metabolic phenotypes. These findings provide mechanistic insights that could greatly expand the therapeutic repertoire for type 2 diabetes and related metabolic disorders.

MicroRNA 139-5p coordinates APLNR-CXCR4 crosstalk during vascular maturation.

G protein-coupled receptor (GPCR) signalling, including that involving apelin (APLN) and its receptor APLNR, is known to be important in vascular development. How this ligand-receptor pair regulates the downstream signalling cascades in this context remains poorly understood. Here, we show that mice with Apln, Aplnr or endothelial-specific Aplnr deletion develop profound retinal vascular defects, which are at least in part due to dysregulated increase in endothelial CXCR4 expression. Endothelial CXCR4 is negatively regulated by miR-139-5p, whose transcription is in turn induced by laminar flow and APLN/APLNR signalling. Inhibition of miR-139-5p in vivo partially phenocopies the retinal vascular defects of APLN/APLNR deficiency. Pharmacological inhibition of CXCR4 signalling or augmentation of the miR-139-5p-CXCR4 axis can ameliorate the vascular phenotype of APLN/APLNR deficient state. Overall, we identify an important microRNA-mediated GPCR crosstalk, which plays a key role in vascular development.

Standardized flavonoid-rich fraction of Artemisia princeps Pampanini cv. Sajabal induces apoptosis via mitochondrial pathway in human cervical cancer HeLa cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia princeps Pampanini is widely used in Eastern traditional medicine for the treatment of circulatory disorders, such as, dysmenorrhea, hematuria, hemorrhoids, and inflammation, and is also used to treat chronic conditions, such as, cancers, ulcers, and digestive disorders. AIM OF THE STUDY: The purpose of this study is to investigate the effect of a standardized flavonoid-rich fraction of Artemisia princeps Pampanini cv. Sajabal (FRAP) on the induction of apoptosis and the molecular mechanism involved in human cervical cancer HeLa cells. MATERIALS AND METHODS: Human cervical cancer HeLa cells were treated with FRAP and apoptosis was detected by cell morphologic observation, annexin-V-PI staning and western blot analysis on the expression of protein associated with cell death. RESULTS: FRAP led to the cleavages of caspase-3, -8, and -9 and the cleavage of poly (ADP-ribose) polymerase (PARP) in HeLa cells. Caspase-3 inhibitor (z-DEVD-fmk), caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD), and broad caspase inhibitor (z-VAD-fmk) significantly suppressed the FRAP-induced accumulation of annexin V positive cells. Furthermore, it was found that FRAP caused a loss of mitochondrial membrane potential (MMP) and the release of cytochrome c to the cytosol. Furthermore, the overexpression of Bcl-xL significantly prevented FRAP-induced apoptosis, MMP changes, and the activations of caspase-3, -8, and -9. Interestingly, pretreatment with caspase-8 inhibitor significantly reduced the FRAP-induced activation of caspase-3 but not that of caspase-9, whereas the caspase-3 inhibitor, z-DEVD-fmk, markedly attenuated the FRAP-induced activation of caspase-8. In BALB/c(nu/nu) mice bearing a HeLa xenograft, FRAP dosed at 25 or 50mg/kg significantly inhibited tumor growth. CONCLUSION: Our results indicate caspase-mediated activation of the mitochondrial death pathway plays a critical role in the FRAP-induced apoptosis of HeLa cells and that FRAP inhibits the in vivo tumor growth of HeLa xenograft mice.