The thromboxane receptor antagonist NTP42 promotes beneficial adaptation and preserves cardiac function in experimental models of right heart overload.

Background: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary artery pressure leading to right ventricular (RV) failure. While current PAH therapies improve patient outlook, they show limited benefit in attenuating RV dysfunction. Recent investigations demonstrated that the thromboxane (TX) A2 receptor (TP) antagonist NTP42 attenuates experimental PAH across key hemodynamic parameters in the lungs and heart. This study aimed to validate the efficacy of NTP42:KVA4, a novel oral formulation of NTP42 in clinical development, in preclinical models of PAH while also, critically, investigating its direct effects on RV dysfunction. Methods: The effects of NTP42:KVA4 were evaluated in the monocrotaline (MCT) and pulmonary artery banding (PAB) models of PAH and RV dysfunction, respectively, and when compared with leading standard-of-care (SOC) PAH drugs. In addition, the expression of the TP, the target for NTP42, was investigated in cardiac tissue from several other related disease models, and from subjects with PAH and dilated cardiomyopathy (DCM). Results: In the MCT-PAH model, NTP42:KVA4 alleviated disease-induced changes in cardiopulmonary hemodynamics, pulmonary vascular remodeling, inflammation, and fibrosis, to a similar or greater extent than the PAH SOCs tested. In the PAB model, NTP42:KVA4 improved RV geometries and contractility, normalized RV stiffness, and significantly increased RV ejection fraction. In both models, NTP42:KVA4 promoted beneficial RV adaptation, decreasing cellular hypertrophy, and increasing vascularization. Notably, elevated expression of the TP target was observed both in RV tissue from these and related disease models, and in clinical RV specimens of PAH and DCM. Conclusion: This study shows that, through antagonism of TP signaling, NTP42:KVA4 attenuates experimental PAH pathophysiology, not only alleviating pulmonary pathologies but also reducing RV remodeling, promoting beneficial hypertrophy, and improving cardiac function. The findings suggest a direct cardioprotective effect for NTP42:KVA4, and its potential to be a disease-modifying therapy in PAH and other cardiac conditions.

Efficacy of the thromboxane receptor antagonist NTP42 alone, or in combination with sildenafil, in the sugen/hypoxia-induced model of pulmonary arterial hypertension.

NTP42 is a novel antagonist of the thromboxane A2 receptor (TP) in development for the treatment of pulmonary arterial hypertension (PAH). Recent studies demonstrated that NTP42 and TP antagonism have a role in alleviating PAH pathophysiology. However, the efficacy of NTP42 when used in combination with existing PAH therapies has not yet been investigated. Herein, the Sugen 5416/hypoxia (SuHx)-induced PAH model was employed to evaluate the efficacy of NTP42 when used alone or in dual-therapy with Sildenafil, a PAH standard-of-care. PAH was induced in rats by injection of Sugen 5416 and exposure to hypoxia for 21 days. Thereafter, animals were treated orally twice-daily for 28 days with either vehicle, NTP42 (0.05 mg/kg), Sildenafil (50 mg/kg), or NTP42+Sildenafil (0.05 mg/kg + 50 mg/kg, respectively). While Sildenafil or NTP42 mono-therapy led to non-significant reductions in the SuHx-induced rises in mean pulmonary arterial pressure (mPAP) or right ventricular systolic pressure (RSVP), combined use of NTP42+Sildenafil significantly reduced these increases in mPAP and RVSP. Detailed histologic analyses of pulmonary vessel remodelling, right ventricular hypertrophy and fibrosis demonstrated that while NTP42 and Sildenafil in mono-therapy resulted in significant benefits, NTP42+Sildenafil in dual-therapy showed an even greater benefit over either drug used alone. In summary, combined use of NTP42+Sildenafil in dual-therapy confers an even greater benefit in treating or offsetting key aetiologies underlying PAH. These findings corroborate earlier preclinical findings suggesting that, through antagonism of TP signalling, NTP42 attenuates PAH pathophysiology, positioning it as a novel therapeutic for use alone or in combination therapy regimens.

NTP42, a novel antagonist of the thromboxane receptor, attenuates experimentally induced pulmonary arterial hypertension.

BACKGROUND: NTP42 is a novel antagonist of the thromboxane prostanoid receptor (TP), currently in development for the treatment of pulmonary arterial hypertension (PAH). PAH is a devastating disease with multiple pathophysiological hallmarks including excessive pulmonary vasoconstriction, vascular remodelling, inflammation, fibrosis, in situ thrombosis and right ventricular hypertrophy. Signalling through the TP, thromboxane (TX) A2 is a potent vasoconstrictor and mediator of platelet aggregation. It is also a pro-mitogenic, pro-inflammatory and pro-fibrotic agent. Moreover, the TP also mediates the adverse actions of the isoprostane 8-iso-prostaglandin F2α, a free-radical-derived product of arachidonic acid produced in abundance during oxidative injury. Mechanistically, TP antagonists should treat most of the hallmarks of PAH, including inhibiting the excessive vasoconstriction and pulmonary artery remodelling, in situ thrombosis, inflammation and fibrosis. This study aimed to investigate the efficacy of NTP42 in the monocrotaline (MCT)-induced PAH rat model, alongside current standard-of-care drugs. METHODS: PAH was induced by subcutaneous injection of 60 mg/kg MCT in male Wistar-Kyoto rats. Animals were assigned into groups: 1. 'No MCT'; 2. 'MCT Only'; 3. MCT + NTP42 (0.25 mg/kg BID); 4. MCT + Sildenafil (50 mg/kg BID), and 5. MCT + Selexipag (1 mg/kg BID), where 28-day drug treatment was initiated within 24 h post-MCT. RESULTS: From haemodynamic assessments, NTP42 reduced the MCT-induced PAH, including mean pulmonary arterial pressure (mPAP) and right systolic ventricular pressure (RSVP), being at least comparable to the standard-of-care drugs Sildenafil or Selexipag in bringing about these effects. Moreover, NTP42 was superior to Sildenafil and Selexipag in significantly reducing pulmonary vascular remodelling, inflammatory mast cell infiltration and fibrosis in MCT-treated animals. CONCLUSIONS: These findings suggest that NTP42 and antagonism of the TP signalling pathway have a relevant role in alleviating the pathophysiology of PAH, representing a novel therapeutic target with marked benefits over existing standard-of-care therapies.

Adipocyte Expression of SLC19A1 Links DNA Hypermethylation to Adipose Tissue Inflammation and Insulin Resistance.

Context: Insulin resistance (IR) is promoted by a chronic low-grade inflammation in white adipose tissue (WAT). The latter might be regulated through epigenetic mechanisms such as DNA methylation. The one carbon cycle (1CC) is a central metabolic process governing DNA methylation. Objective: To identify adipocyte-expressed 1CC genes linked to WAT inflammation, IR, and their causal role. Design: Cohort study. Setting: Outpatient academic clinic. Participants: Obese and nonobese subjects. Methods: Gene expression and DNA methylation arrays were performed in subcutaneous WAT and isolated adipocytes. In in vitro differentiated human adipocytes, gene knockdown was achieved by small interfering RNA, and analyses included microarray, quantitative polymerase chain reaction, DNA methylation by enzyme-linked immunosorbent assay and pyrosequencing, protein secretion by enzyme-linked immunosorbent assay, targeted metabolomics, and luciferase reporter and thermal shift assays. Main Outcome Measures: Effects on adipocyte inflammation. Results: In adipocytes from obese individuals, global DNA hypermethylation was associated positively with gene expression of proinflammatory pathways. Among the 1CC genes, IR in vivo and proinflammatory gene expression in WAT were most strongly and inversely associated with SLC19A1, a gene encoding a membrane folate carrier. SLC19A1 knockdown in human adipocytes perturbed intracellular 1CC metabolism, induced global DNA hypermethylation, and increased expression of proinflammatory genes. Several CpG loci linked SLC19A1 to inflammation; validation studies were focused on the chemokine C-C motif chemokine ligand 2 (CCL2) in which methylation in the promoter (cg12698626) regulated CCL2 expression and CCL2 secretion through altered transcriptional activity. Conclusions: Reduced SLC19A1 expression in human adipocytes induces DNA hypermethylation, resulting in increased expression of specific proinflammatory genes, including CCL2. This constitutes an epigenetic mechanism that might link dysfunctional adipocytes to WAT inflammation and IR.

Estrogen receptor ß2 induces proliferation and invasiveness of triple negative breast cancer cells: association with regulation of PHD3 and HIF-1α.

The two estrogen receptor (ER) subtypes, ERα and ERß, belong to the nuclear receptor superfamily. The human ERß variant ERß2 is proposed to be expressed at higher levels than ERß1 in many breast tumors and it has been suggested that ERß2, in contrast to ERß1, is associated with aggressive phenotypes of various cancers. However, the role of endogenous ERß2 in breast cancer cells remains elusive. In this study, we identified that triple negative breast cancer (TNBC) cell lines express endogenous ERß2, but not ERα or ERß1. This allows novel studies of endogenous ERß2 functions independent of ERα and ERß1. We show that overexpression of ERß2 in TNBC cells increased whereas knockdown of endogenous ERß2 decreased cell proliferation and cell invasion. To elucidate the molecular mechanism responsible for these cellular phenotypes, we assayed ERß2 dependent global gene expression profiles. We show that ERß2 decreases prolyl hydroxylase 3 (PHD3) gene expression and further show that this is associated with increased hypoxia inducible factor 1α (HIF-1α) protein levels, thus providing a possible mechanism for the invasive phenotype. These results are further supported by analysing the expression of ERß2 and PHD3 in breast tumor samples where a negative correlation between ERß2 and PHD3 expression was observed. Together, we demonstrate that ERß2 has an important role in enhancing cell proliferation and invasion, beyond modulation of ERß and ERß1 signalling which might contribute to the invasive characteristics of TNBC. The invasive phenotype could potentially be mediated through transcriptional repression of PHD3 and increased HIF-1α protein levels.

Epigenetic Regulation of PLIN 1 in Obese Women and its Relation to Lipolysis.

Increased adipocyte lipolysis links obesity to insulin resistance. The lipid droplet coating-protein Perilipin participates in regulation of lipolysis and is implicated in obesity. In the present study we investigate epigenetic regulation of the PLIN1 gene by correlating PLIN1 CpG methylation to gene expression and lipolysis, and functionally evaluating PLIN1 promoter methylation. PLIN1 CpG methylation in adipocytes and gene expression in white adipose tissue (WAT) was quantified in two cohorts by array. Basal lipolysis in WAT explants and adipocytes was quantified by measuring glycerol release. CpG-methylation of the PLIN1 promoter in adipocytes from obese women was higher as compared to never-obese women. PLIN1 promoter methylation was inversely correlated with PLIN1 mRNA expression and the lipolytic activity. Human mesenchymal stem cells (hMSCs) differentiated in vitro into adipocytes and harboring methylated PLIN1 promoter displayed decreased reporter gene activity as compared to hMSCs harboring unmethylated promoter. Treatment of hMSCs differentiated in vitro into adipocytes with a DNA methyltransferase inhibitor increased levels of PLIN1 mRNA and protein. In conclusion, the PLIN1 gene is epigenetically regulated and promoter methylation is inversely correlated with basal lipolysis in women suggesting that epigenetic regulation of PLIN1 is important for increased adipocyte lipolysis in insulin resistance states.

Agonistic effect of selected isoflavones on arylhydrocarbon receptor in a novel AZ-AhR transgenic gene reporter human cell line.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls the expression of a diverse set of genes. Structurally diverse compounds bind to AhR and act as AhR agonists. Well characterised family of natural AhR ligands are isoflavones, which are compounds found predominantly in soy beans or red clover. In this study we have examined agonistic effect of selected isoflavones (genistein, daidzein, biochanin A, formononetin and equol) on AhR in the novel transgenic gene reporter human cell line AZ-AhR, a stably transfected AhR-responsive cell line allowing rapid and sensitive assessment of AhR transcriptional activity. We demonstrated that biochanin A, formononetin and genistein at concentration 10(-4) mol/l exerted agonistic effects on AhR with fold activation of 309- fold, 108-fold and 27-fold, which is about 84.8%, 29.6% and 7.4%, respectively, of the value attained by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Daidzein and equol did not show any significant effects on AhR.

The relationship between renal cell carcinoma and nuclear retinoid/rexinoid receptors.

BACKGROUND: Renal cell carcinoma (RCC) is a urologic malignancy with a steady rise in incidence and high mortality rate. Between 60 to 70% of patients with renal cell carcinoma can only be cured with surgery but despite advances in early diagnostis, in around 20-30% of cases there is metastasis. For these patients, chemotherapy and radiotherapy are ineffective and hence the prognosis is poor. Retinoids are biologically active compounds of either natural or synthetic origin that are involved in complex physiological and developmental processes in many tissues including cell proliferation and activation of tumour suppression genes. This article reviews the role of retinoids and their cognate nuclear retinoid/rexinoid receptors in relation to renal cell carcinoma. METHODS: A literature search using ScienceDirect and Medline with a focus on the relationship between renal cell carcinoma and nuclear retinoid/rexinoid receptors. RESULTS: Use of retinoids/rexinoids in the treatment of locally advanced and metastatic RCC significantly prolongs median time of tumour progression and overall survival of patients. Combination therapy with other preparations has greater efficacy than treatment with retinoids alone. Patient survival can be predicted on the basis of the expression of different all-trans retinoic acid receptor (RAR) and 9-cis retinoic acid receptor (RXR) subtypes. CONCLUSIONS: Since nuclear retinoid receptors play a crucial role as ligand-activated, DNA binding, trans-acting, transcription-modulating proteins involved in a general molecular mechanism responsible for transcriptional responses in target genes, retinoids might be an alternative approach for the treatment of renal cell carcinoma.

Nuclear receptors - target molecules for isoflavones in cancer chemoprevention.

Breast cancer is the most occurring type of cancer among women. In Slovakia, there are yearly diagnosed about 1900 new cases of this disease. Breast cancer treatment is very expensive, psychic stressful and in some cases ineffective. Therefore, it is essential to search for new and/or alternative methods for breast cancer treatment, since nuclear receptors are considered to be a central goal for maximizing treatment opportunities in breast cancer. Among natural ligands for estrogen receptors (ERα and ERß), which are member of nuclear receptors superfamily, belongs also isoflavones. These natural compounds have similar structure to main female hormon-17ß estradiol. A rich source of isoflavones is soy and its products. Three aglycones form of isoflavones (genistein, daidzein, glycitein) are predominantly found in soybean and red clover. Among other important isoflavones belongs also biochanin A and formononetin.

mRNA expression pattern of retinoic acid and retinoid X nuclear receptor subtypes in human thyroid papillary carcinoma.

Retinoids have shown potential for the inhibition of tumour growth and progression. The objective of this study was to investigate retinoic acid nuclear receptor subtypes RAR/RXR and iodothyronine 5'-deiodinase, type I expression pattern in papillary thyroid tumour tissue of 26 patients in order to compare with those of the non-neoplastic thyroid tissue of the corresponding patients. The expression of selected parameters mRNA was examined by semi-quantitative RT-PCR. Papillary thyroid carcinoma (PTC) expressed RXRγ, when compared to non-neoplastic thyroid tissues of the corresponding patients that were lacking expression of RXRγ or its expression was very low. Moreover, we found significantly increased expression of RARα and RARγ in the overall group of PTC. This increase was detected in cases with positive lymph node metastasis (LNM), but not in cases with negative LNM. RARß was significantly reduced in the subgroup of classic variant (CV). We also detected absence or significantly lower expression of hDIO1 mRNA in tumour tissue when compared to non-neoplastic tissue in both overall PTC cases and in the CV subgroup. However, the significantly decreased levels of hDIO1 mRNA were detected in cases with negative LNM but not in cases with positive LNM when compared to corresponding non-tumour tissue in both overall PTC cases and in the CV subgroup. Differences in RAR and RXR subtype mRNA expression patterns in various PTCs may contribute to the immunochemistry data available, and may thus find exploitation in clinical oncology, particularly in the differential diagnosis of thyroid neoplasms.