Background and Purpose Nitrite (NO2−) has recently been shown to represent a potential source of NO, in particular under hypoxic conditions. The aim of the current study was to compare the haemodynamic effects of NO2− in healthy volunteers and patients with stable congestive heart failure (CHF). Experimental Approach The acute haemodynamic effects of brachial artery infusion of NO2− (0.31 to 7.8 μmol·min−1) was assessed in normal subjects (n = 20) and CHF patients (n = 21). Key Results NO2− infusion was well tolerated in all subjects. Forearm blood flow (FBF) increased markedly in CHF patients at NO2− infusion rates which induced no changes in normal subjects (anova: F = 5.5; P = 0.02). Unstressed venous volume (UVV) increased even with the lowest NO2− infusion rate in all subjects (indicating venodilation), with CHF patients being relatively hyporesponsive compared with normal subjects (anova: F = 6.2; P = 0.01). There were no differences in venous blood pH or oxygen concentration between groups or during NO2− infusion. Venous plasma NO2− concentrations were lower in CHF patients at baseline, and rose substantially less with NO2− infusion, without incremental oxidative generation of nitrate, consistent with accelerated clearance in these patients. Plasma protein‐bound NO concentrations were lower in CHF patients than normal subjects at baseline. This difference was attenuated during NO2− infusion. Prolonged NO2− exposure in vivo did not induce oxidative stress, nor did it induce tolerance in vitro. Conclusions and Implications The findings of arterial hyper‐responsiveness to infused NO2− in CHF patients, with evidence of accelerated transvascular NO2− clearance (presumably with concomitant NO release) suggests that NO2− effects may be accentuated in such patients. These findings provide a stimulus for the clinical exploration of NO2− as a therapeutic modality in CHF.

The prostanoid thromboxane (TX) A 2 is increasingly implicated in neoplastic progression, including prostate cancer (PCa). Mechanistically, we recently identified protein kinase C-related kinase (PRK) 1 as a functional interactant of both the TP` and TPβ isoforms of the human T prostanoid receptor (TP). The interaction with PRK1 was not only essential for TP`/TPβ-induced PCa cell migration but also enabled the TXA 2 -TP axis to induce phosphorylation of histone H3 at Thr11 (H3Thr11), an epigenetic marker both essential for and previously exclusively associated with androgen-induced chromatin remodelling and transcriptional activation. PRK1 is a member of a subfamily of three structurally related kinases comprising PRK1/PKN`, PRK2/PKNγ and PRK3/PKNβ that are widely yet differentially implicated in various cancers. Hence, focusing on the setting of prostate cancer, this study investigated whether TP` and/or TPβ might also complex with PRK2 and PRK3 to regulate their activity and neoplastic responses. While TP` and TPβ were found in immune complexes with PRK1, PRK2 and PRK3 to regulate their activation and signalling, they do so differentially and in a TP agonist-regulated manner dependent on the T-loop activation status of the PRKs but independent of their kinase activity. Furthermore, TXA 2 -mediated neoplastic responses in prostate adenocarcinoma PC-3 cells, including histone H3Thr11 phosphorylation, was found to occur through a PRK1-and PRK2-, but not PRK3-, dependent mechanism. Collectively, these data suggest that TXA 2 acts as both a neoplastic and epigenetic regulator and provides a mechanistic explanation, at least in part, for the prophylactic benefits of Aspirin in reducing the risk of certain cancers.

The prostanoid thromboxane (TX)A signals through the TPα and TPβ isoforms of T Prostanoid receptor (TP) that are transcriptionally regulated by distinct promoters termed Prm1 and Prm3, respectively, within the TBXA2R gene. We recently demonstrated that expression of TPα and TPβ is increased in PCa, differentially correlating with Gleason grade and with altered CpG methylation of the individual Prm1/Prm3 regions within the TBXA2R. The current study sought to localise the sites of CpG methylation within Prm1 and Prm3, and to identify the main transcription factors regulating TPβ expression through Prm3 in the prostate adenocarcinoma PC-3 and LNCaP cell lines. Bisulfite sequencing revealed extensive differences in the pattern and status of CpG methylation of the individual Prm1 and Prm3 regions that regulate TPα and TPβ expression, respectively, within the TBXA2R. More specifically, Prm1 is predominantly hypomethylated while Prm3 is hypermethylated across its entire sequence in PC-3 and LNCaP cells. Furthermore, the tumour suppressors FOXP1 and NKX3.1, strongly implicated in PCa development, were identified as key transcription factors regulating TPβ expression through Prm3 in both PCa cell lines. Specific siRNA-disruption of FOXP1 and NKX3.1 each coincided with up-regulated TPβ protein and mRNA expression, while genetic-reporter and chromatin immunoprecipitation (ChIP) analyses confirmed that both FOXP1 and NKX3.1 bind to cis‑elements within Prm3 to transcriptionally repress TPβ in the PCa lines. Collectively these data identify Prm3/TPβ as a bona fide target of FOXP1 and NKX3.1 regulation, providing a mechanistic basis, at least in part, for the highly significant upregulation of TPβ expression in PCa.

Background and PurposeHypoxic conditions favour the reduction of nitrite to nitric oxide (NO) to elicit vasodilatation, but the mechanism(s) responsible for bioconversion remains ill defined. In the present study, we assess the role of aldehyde dehydrogenase 2 (ALDH2) in nitrite bioactivation under normoxia and hypoxia in the rat and human vasculature.Experimental ApproachThe role of ALDH2 in vascular responses to nitrite was studied using rat thoracic aorta and gluteal subcutaneous fat resistance vessels from patients with heart failure (HF; 16 patients) in vitro and by measurement of changes in forearm blood flow (FBF) during intra-arterial nitrite infusion (21 patients) in vivo. Specifically, we investigated the effects of (i) ALDH2 inhibition by cyanamide or propionaldehyde and the (ii) tolerance-independent inactivation of ALDH2 by glyceryl trinitrate (GTN) on the vasodilator activity of nitrite. In each setting, nitrite effects were measured via evaluation of the concentration–response relationship under normoxic and hypoxic conditions in the absence or presence of ALDH2 inhibitors.Key ResultsBoth in rat aorta and human resistance vessels, dilatation to nitrite was diminished following ALDH2 inhibition, in particular under hypoxia. In humans there was a non-significant trend towards attenuation of nitrite-mediated increases in FBF.Conclusions and ImplicationsIn human and rat vascular tissue in vitro, hypoxic nitrite-mediated vasodilatation involves ALDH2. In patients with HF in vivo, the role of this enzyme in nitrite bioactivation is at the most, modest, suggesting the involvement of other more important mechanisms.

The prostanoid thromboxane (TX) A and its T Prostanoid receptor (the TP) are increasingly implicated in prostate cancer (PCa). Mechanistically, we recently discovered that both TPα and TPβ form functional signalling complexes with members of the protein kinase C-related kinase (PRK) family, AGC- kinases essential for the epigenetic regulation of androgen receptor (AR)-dependent transcription and promising therapeutic targets for treatment of castrate-resistant prostate cancer (CRPC). Critically, similar to androgens, activation of the PRKs through the TXA/TP signalling axis induces phosphorylation of histone H3 at Thr11 (H3Thr11), a marker of androgen-induced chromatin remodelling and transcriptional activation, raising the possibility that TXA-TP signalling can mimic and/or enhance AR-induced cellular changes even in the absence of circulating androgens such as in CRPC. Hence the aim of the current study was to investigate whether TXA/TP-induced PRK activation can mimic and/or enhance AR-mediated cellular responses in the model androgen-responsive prostate adenocarcinoma LNCaP cell line. We reveal that TXA/TP signalling can act as a neoplastic- and epigenetic-regulator, promoting and enhancing both AR-associated chromatin remodelling (H3Thr11 phosphorylation, WDR5 recruitment and acetylation of histone H4 at lysine 16) and AR-mediated transcriptional activation (e.g of the KLK3/prostate-specific antigen and TMPRSS2 genes) through mechanisms involving TPα/TPβ mediated-PRK1 and PRK2, but not PRK3, signalling complexes. Overall, these data demonstrate that TPα/TPβ can act as neoplastic and epigenetic regulators by mimicking and/or enhancing the actions of androgens within the prostate and provides further mechanistic insights into the role of the TXA/TP signalling axis in PCa, including potentially in CRPC.