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Pulmonary arterial hypertension (PAH) is a progressive disease with a complex aetiology and high mortality. Functional and structural changes in the small pulmonary arteries lead to elevated pulmonary arterial pressure, resulting in right heart failure. The pathobiology of PAH is not fully understood, and novel treatment targets in PAH are desperately needed. The renin-angiotensin system is critical for maintaining homeostasis of the cardiovascular system. The system consists of the angiotensin converting enzyme (ACE)-angiotensin (Ang) II-angiotensin type 1 receptor (AT1R) axis and the ACE2-Ang-(1–7)-Mas receptor axis. The former, the ACE-Ang II-AT1R axis, is involved in vasoconstrictive and hypertensive actions along with cardiac and vascular remodelling. The latter, the ACE2-Ang-(1–7)-Mas axis, generally mediates counterbalancing effects against those mediated by the ACE-Ang II-AT1R axis. Based on established functions, the ACE2-Ang-(1–7)-Mas axis may represent a novel target for the treatment of PAH. This review focuses on recent advances in pulmonary circulation science and the role of the ACE2-Ang-(1–7)-Mas axis in PAH.

Activation of transient receptor potential vanilloid 4 (TRPV4) induces neuronal injury. TRPV4 activation enhances inflammatory response and promotes the proinflammatory cytokine release in various types of tissue and cells. Hyperneuroinflammation contributes to neuronal damage in epilepsy. Herein, we examined the contribution of neuroinflammation to TRPV4-induced neurotoxicity and its involvement in the inflammation and neuronal damage in pilocarpine model of temporal lobe epilepsy in mice. Icv. injection of TRPV4 agonist GSK1016790A (GSK1016790A-injected mice) increased ionized calcium binding adapter molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) protein levels and Iba-1-positive (Iba-1+) and GFAP-positive (GFAP+) cells in hippocampi, which indicated TRPV4-induced microglial cell and astrocyte activation. The protein levels of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome components NLRP3, apoptosis-related spotted protein (ASC) and cysteinyl aspartate-specific protease-1 (caspase-1) were increased in GSK1016790A-injected mice, which indicated NLRP3 inflammasome activation. GSK1016790A also increased proinflammatory cytokine IL-1β, TNF-α and IL-6 protein levels, which were blocked by caspase-1 inhibitor Ac-YVAD-cmk. GSK1016790A-induced neuronal death was attenuated by Ac-YVAD-cmk. Icv. injection of TRPV4-specific antagonist HC-067047 markedly increased the number of surviving cells 3 d post status epilepticus in pilocarpine model of temporal lobe epilepsy in mice (pilocarpine-induced status epilepticus, PISE). HC-067047 also markedly blocked the increase in Iba-1 and GFAP protein levels, as well as Iba-1+ and GFAP+ cells 3 d post-PISE. Finally, the increased protein levels of NLRP3, ASC and caspase-1 as well as IL-1β, TNF-α and IL-6 were markedly blocked by HC-067047. We conclude that TRPV4-induced neuronal death is mediated at least partially by enhancing the neuroinflammatory response, and this action is involved in neuronal injury following status epilepticus.

Background. Although accumulating evidence suggested that a molecular signature panel may be more effective for the prognosis prediction than routine clinical characteristics, current studies mainly focused on colorectal or colon cancers. No reports specifically focused on the signature panel for rectal cancers (RC). Our present study was aimed at developing a novel prognostic signature panel for RC. Methods. Sequencing (or microarray) data and clinicopathological details of patients with RC were retrieved from The Cancer Genome Atlas (TCGA-READ) or the Gene Expression Omnibus (GSE123390, GSE56699) database. A weighted gene coexpression network was used to identify RC-related modules. The least absolute shrinkage and selection operator analysis was performed to screen the prognostic signature panel. The prognostic performance of the risk score was evaluated by survival curve analyses. Functions of prognostic genes were predicted based on the interaction proteins and the correlation with tumor-infiltrating immune cells. The Human Protein Atlas (HPA) tool was utilized to validate the protein expression levels. Results. A total of 247 differentially expressed genes (DEGs) were commonly identified using TCGA and GSE123390 datasets. Brown and yellow modules (including 77 DEGs) were identified to be preserved for RC. Five DEGs (ASB2, GPR15, PRPH, RNASE7, and TCL1A) in these two modules constituted the optimal prognosis signature panel. Kaplan-Meier curve analysis showed that patients in the high-risk group had a poorer prognosis than those in the low-risk group. Receiver operating characteristic (ROC) curve analysis demonstrated that this risk score had high predictive accuracy for unfavorable prognosis, with the area under the ROC curve of 0.915 and 0.827 for TCGA and GSE56699 datasets, respectively. This five-mRNA classifier was an independent prognostic factor. Its predictive accuracy was also higher than all clinical factor models. A prognostic nomogram was developed by integrating the risk score and clinical factors, which showed the highest prognostic power. ASB2, PRPH, and GPR15/TCL1A were predicted to function by interacting with CASQ2/PDK4/EPHA67, PTN, and CXCL12, respectively. TCL1A and GPR15 influenced the infiltration levels of B cells and dendritic cells, while the expression of PRPH was positively associated with the abundance of macrophages. HPA analysis supported the downregulation of PRPH, RNASE7, CASQ2, EPHA6, and PDK4 in RC compared with normal controls. Conclusion. Our immune-related signature panel may be a promising prognostic indicator for RC.

PurposeSalusin-β, a multifunctional vasoactive peptide, has a potentially important function in the pathological development of hypertension. However, the exact functional role of salusin-β and the underlying mechanism in this process are still not fully understood. The current study aimed to investigate the effects of silencing salusin-β on vascular function and vascular remodeling, as well as its signaling pathways in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY).MethodsSilencing salusin-β was performed by caudal vein injection of adenovirus expressing salusin-β short hairpin RNA (shRNA). Acetylcholine (ACh)-induced endothelium-dependent relaxation was used to evaluate vasodilator function, and high K+ solution-induced constriction was used to evaluate vasoconstriction function.ResultsSalusin-β levels in plasma and its protein expression in mesenteric artery (MA), coronary artery (CA), and pulmonary artery (PA) of SHR were higher than those in WKY. The salusin-β level and expression were decreased effectively by salusin-β shRNA. Knockdown of salusin-β decreased arterial blood pressure (ABP) and high K+ solution-induced vascular constrictions, and improved the endothelium-dependent relaxation and vascular remodeling in SHR. The improved effect of silencing salusin-β on ACh-induced relaxation in SHR was almost blocked by the nitric oxide synthase (NOS) inhibitor L-NAME. Compared to WKY, the endothelial NOS (eNOS) activity and level, and nitric oxide (NO) level were decreased, while NAD(P)H oxidase activity and reactive oxygen species (ROS) levels in MA, CA, and PA of SHR were increased, which were all redressed by salusin-β knockdown.ConclusionThese results indicate that knockdown of salusin-β improves endothelium-dependent vascular relaxation and vascular remodeling and decreases ABP and vasoconstriction in SHR, which might be accomplished by increasing eNOS activation and NO release while inhibiting NAD(P)H oxidase derived-ROS generation. Scavenging salusin-β improves vascular function and then prevents the development and progression of vasculopathy of hypertension.

Attenuated vasodilatation of small arteries is a hallmark feature of hypertension. Salusin-β, which is a TOR2A gene product and an important vasoactive peptide, has a close relationship with cardiovascular disease. This study aimed to determinate the roles of salusin-β in vasodilatation, and its signal pathways in Wistar–Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Isometric tension experiments were performed. Vasodilatation was induced by acetylcholine (ACh) or sodium nitroprusside (SNP). Plasma salusin-β levels and their protein expressions in coronary artery (CA), mesenteric artery (MA), and pulmonary artery (PA) of SHR were much higher than that of WKY. Intravenous injection of salusin-β increased arterial blood pressure in SHR, while anti-salusin-β IgG decreased it. Salusin-β further deteriorated, while anti-salusin-β IgG improved, the attenuated ACh-induced relaxation, the decreased nitric oxide (NO) level, and endothelial nitric oxide synthase (eNOS) activity in arteries of SHR, and salusin-β had no significant effect on SNP-induced relaxation. The NAD(P)H oxidase activity and reactive oxygen species (ROS) level in arteries of SHR were much higher than that of WKY, which was further increased by salusin-β but reduced by anti-salusin-β IgG. ROS scavenger NAC or antioxidant apocynin significantly inhibited, while SOD inhibitor DETC aggravated, the effects of salusin-β, and the eNOS inhibitor L-NAME inhibited the effects of anti-salusin-β IgG. These results indicated that enhanced salusin-β activity is involved in attenuated endothelium-dependent vasodilatation pathogenesis in SHR by activating NAD(P)H oxidase derived ROS generation and inhibiting eNOS activation and NO release.

Key points Chronic subcutaneous infusion of Ang II causes a progressive increase in blood pressure (BP) associated with significant increases in angiotensin type 1 receptor (AT1R) and mineralocorticoid receptor (MR) expression in hypothalamic nuclei. Intra-paraventricular nucleus (PVN) infusion of AAV- AT1aR-siRNA or of AAV-MR-siRNA markedly knockdown AT1a-R or MR expression in the PVN but not in the subfornical organ; or supraoptic nucleus, and prevent most of the increase in BP. These findings indicate that increased MR and AT1R activation in the PVN play a critical role in Ang II-induced hypertension in rats on regular salt intake. Abstract Circulating Ang II activates an aldosterone-mineralocorticoid receptor (MR) – angiotensin II (Ang II) – angiotensin type 1 receptor (AT1R) pathway in the hypothalamus. To obtain insights into the actual neuronal projections involved, adeno-associated virus carrying small interfering RNA against either AT1aR (AAV-AT1aR-siRNA) or MR (AAV-MR-siRNA) were infused into the paraventricular nucleus (PVN) in Wistar rats. Intra-PVN infusion of AAV-AT1aR-siRNA or AAV-MR-siRNA decreased AT1R or MR expression in the PVN but not in the subfornical organ (SFO) or supraoptic nucleus (SON). Subcutaneous infusion of Ang II at 500 ng kg−1 min−1 for 2 weeks increased mean arterial pressure by 60–70 mmHg, and increased AT1R and MR expression in the SFO, SON and PVN. Intra-PVN AT1aR-siRNA prevented the Ang II-induced increase in AT1R but not MR expression in the PVN, and MR-siRNA prevented MR but not AT1R expression in the PVN. The increases in AT1R and MR expression in both the SFO and the SON were not changed by the two AAV-siRNAs. Specific knockdown of AT1R or MR in the PVN by AAV-siRNA each prevented most of the Ang II-induced hypertension. Prevention of the subcutaneous Ang II-induced increase in MR but not the increase in AT1R by knockdown of MR and vice versa suggests an independent regulation of MR and AT1R expression in the PVN. Both AT1R and MR activation in the PVN play a critical role in Ang II-induced hypertension in rats.

Key points Central mineralocorticoid receptor (MR) and angiotensin II type 1 receptor (AT1R) activation play a critical role in sympathetic hyperactivity and progressive left ventricle (LV) remodelling and dysfunction after a myocardial infarction (MI). Intra-paraventricular nucleus (PVN) infusion of adeno-associated virus (AAV) carrying small interfering RNA (siRNA) against MR (AAV-MR-siRNA) markedly decreases both MR and AT1R expression in the PVN post MI, whereas AAV-AT1aR-siRNA only decreases AT1R expression. Both AAVs largely prevent sympathetic hyperactivity and inhibit part of LV remodelling and dysfunction post MI. These findings indicate that enhanced MR–AT1R signalling in the PVN is critical for sympathetic hyperactivity post MI, and contributes to part of LV dysfunction post MI. Abstract Intracerebroventricular infusion of a mineralocorticoid receptor (MR) or angiotensin II type 1 receptor (AT1R) blocker in rats attenuates sympathetic hyperactivity and progressive left ventricular (LV) dysfunction post myocardial infarction (MI). The present study examined whether knockdown of MRs or AT1Rs specifically in the paraventricular nucleus (PVN) contributes to these effects, and compared cardiac effects with those of systemic treatment with the β1-adrenergic receptor blocker metoprolol. The PVN of rats was infused with adeno-associated virus carrying small interfering RNA against either MR (AAV-MR-siRNA) or AT1R (AAV-AT1R-siRNA), or as control scrambled siRNA. At 4 weeks post MI, AT1R but not MR expression was increased in the PVN, excitatory renal sympathetic nerve activity and pressor responses to air stress were enhanced, and arterial baroreflex function was impaired; LV end-diastolic pressure (LVEDP) was increased and LV peak systolic pressure (LVPSP), ejection fraction (EF) and dP/dtmax decreased. AAV-MR-siRNA and AAV-AT1R-siRNA both normalized AT1R expression in the PVN, similarly ameliorated sympathetic and pressor responses to air stress, largely prevented baroreflex desensitization, and improved LVEDP, EF and dP/dtmax as well as cardiac interstitial (but not perivascular) fibrosis. In a second set of rats, metoprolol at 70 or 250 mg kg−1 day−1 in the drinking water for 4 weeks post MI did not improve LV function except for a decrease in LVEDP at the lower dose. These results suggest that in rats MR-dependent upregulation of AT1Rs in the PVN contributes to sympathetic hyperactivity, and LV dysfunction and remodelling post MI. In rats, normalizing MR–AT1R signalling in the PVN is a more effective strategy to improve LV dysfunction post MI than systemic β1 blockade.

Rheumatoid arthritis (RA) is the most common chronic inflammatory disease with unknown causes and unknown cures in Western medicine. This double-blinded study aimed to investigate the efficacy and safety of a widely used traditional Chinese medicine (Paeoniflorin (PAE) plus cervus and cucumis polypeptide injection (CCPI) using disease-modifying antirheumatic drugs (DMARD) as a control (methotrexate (MTX) plus leflunomide (LEF)). Patients were randomly assigned to one of the three groups: PAE + CCPI, MTX + LEF, and MTX + LEF + CCPI. The primary end point was the American College of Rheumatology 20% improvement response criteria (ACR20). The secondary end point was that of adverse effect frequencies and the speed of onset action. Our results showed that more patients in the CCPI-containing groups responded to the ACR20 during early treatment. After six months, ACR20 showed no significant difference among the three treatments. The maximum improvement in the two DMARD groups was significantly higher than that in the PAE + CCPI group (p < 0.01). CCPI made the onset action of the DMARD therapy 4.6 times faster. PAE + CCPI had significantly lower adverse event incidences than the two DMARD groups. These results indicate that PAE + CCPI appear to be a more acceptable alternative to DMARDs when patients cannot use DMARDs. CCPI appears to be a beneficial add-on to DMARDs that makes the onset of action faster, especially when patients need to relieve RA symptoms as soon as possible. Although not as effective as DMARDs, PAE appears to be a safer option to substitute DMARDs for long-term RA treatment when DMARD toxicity is an issue.

Activation of angiotensinergic pathways by central aldosterone (Aldo)-mineralocorticoid receptor (MR) pathway plays a critical role in angiotensin II (Ang II)-induced hypertension. The subfornical organ (SFO) contains both MR and angiotensin II type 1 receptors (AT1R) and can relay the signals of circulating Ang II to downstream nuclei such as the paraventricular nucleus (PVN), supraoptic nucleus (SON) and rostral ventrolateral medulla (RVLM). In Wistar rats, subcutaneous (sc) infusion of Ang II at 500ng/min/kg for 1 or 2weeks increased reactive oxygen species (ROS) as measured by dihydroethidium (DHE) staining in a nucleus - specific pattern. Intra-SFO infusion of AAV-MR- or AT1aR-siRNA prevented the Ang II-induced increase in AT1R mRNA expression in the SFO and decreased MR mRNA. Both MR- and AT1aR-siRNA prevented increases in ROS in the PVN and RVLM. MR- but not AT1aR-siRNA in the SFO prevented the Ang II-induced ROS in the SON. Both MR- and AT1aR-siRNA in the SFO prevented most of the Ang II-induced hypertension as assessed by telemetry. These results indicate that Aldo-MR signaling in the SFO is needed for the activation of Ang II-AT1R-ROS signaling from the SFO to the PVN and RVLM. Activation of Aldo-MR signaling from the SFO to the SON may enhance AT1R dependent activation of pre-sympathetic neurons in the PVN.

Central blockade of mineralocorticoid receptors (MRs) or angiotensin II type 1 receptors (AT1Rs) attenuates aldosterone (aldo)-salt induced hypertension. We examined the role of the subfornical organ (SFO), aldo synthesized locally in the brain, and MR and AT1R specifically in the paraventricular nucleus (PVN) in aldo-salt hypertension. Wistar rats were treated with subcutaneous aldo (1 μg/h) plus saline as drinking fluid, and gene expression was assessed by real-time qPCR. Other sets of rats received chronic intra-cerebroventricular (icv) infusion of aldo synthase (AS) inhibitor FAD286, MR blocker eplerenone or vehicle, electrolytic or sham lesions of the SFO, or intra-PVN infusion of AAV-MR-siRNA or AAV-AT1aR-siRNA. Infusion of aldo had no effect on 11βHSD2, MR and AT1R mRNA in different nuclei but increased CYP11B2 mRNA in the SFO, and serum and glucocorticoid-kinase 1 (Sgk1) and epithelial sodium channel (ENaC) γ subunit mRNA in the SFO and supraoptic nucleus (SON). MR-siRNA decreased both MR and AT1R mRNA in the PVN by ∼ 60%, but AT1aR-siRNA only decreased AT1R mRNA. SFO lesion, blockade of brain AS or MR, or knockdown of MR or AT1R in the PVN similarly attenuated aldosterone-induced saline intake by ∼ 50% and hypertension by ∼ 70%. These results suggest that an increase in circulating aldosterone may via MR and AT1R in the SFO increase local aldosterone production in hypothalamic nuclei such as the SON and PVN, and via MR enhance AT1R signaling in the PVN. This central aldosterone-MR-AT1R neuro-modulatory pathway appears to play a major role in the progressive hypertension.

Activation of angiotensinergic pathways and central aldosterone (aldo)-MR-ENaC-endogenous ouabain (EO)-AT 1 R pathway play a critical role in Ang II associated hypertension. The SFO contains both MR and AT 1 R and can relay the signals of circulating Ang II to downstream nuclei such as the PVN, SON and RVLM. We evaluated the effect of knockdown of MR and AT 1 R specific in the SFO on reactive oxygen species (ROS) production in downstream nuclei. Wistar rats were intra SFO infused with AAV-MR- or AT 1a R-siRNA and after 7 days received a sc infusion of Ang II at 500 ng/min/kg for 2 weeks. MR and AT 1 R expression were measured by real-time qPCR and western blotting. ROS was assessed by DHE staining. Ang II increased AT 1 R mRNA expression in the SFO. Both MR- and AT 1 R-siRNA in the SFO prevented this increase. Ang II decreased MR mRNA but increased protein expression in the SFO. Both MR- and AT 1 R-siRNA further decreased MR mRNA expression. Ang II significantly increased ROS in the SFO, magno- and parvocellular parts of the PVN, SON and RVLM. Both MR- and AT 1 R-siRNA in the SFO prevented ROS increases in the PVN and RVLM. In contrast, MR- but not AT 1 R-siRNA in the SFO prevented the Ang II-induced ROS in the SON. Both MR- and AT 1 R-siRNA in the SFO prevented most of the Ang II-induced hypertension. These results suggest that aldo-MR signaling in the SFO is needed for the activation of Ang II-AT 1 R signaling from the SFO to the PVN and RVLM. Considering that only MR-siRNA in the SFO prevents circulating Ang II induced ROS in the SON, activation of aldo-MR signaling from the SFO to the SON may via EO enhance AT 1 R dependent activation of pre-sympathetic neurons in the PVN, and thereby to Ang II-hypertension.

Circulating Ang II increases BP through both peripheral and central mechanisms. Activation of both AT 1 R and MR in the CNS plays a critical role in Ang II-induced hypertension. The subfornical organ (SFO) contains both MR and AT 1 R and can relay the signals of circulating Ang II to downstream nuclei such as the PVN. In this study, we evaluated the effect of intra-SFO infusion of AAV-MR-siRNA or AAV-AT 1a R-siRNA on MR or AT 1 R mRNA expression in the SFO and PVN and Ang II induced hypertension as assessed by telemetry. Two week sc infusion of Ang II at 500 ng/kg/min in Wistar rats on regular salt diet increased BP by 60~65 mmHg. This pressor effect of Ang II was largely prevented by AAV-MR-siRNA or AAV-AT 1 R-siRNA in the SFO (Figure). Ang II increased AT 1 R mRNA expression (1.7± 0.4 vs 0.8 ± 0.04 х10 -1 , p1 R-siRNA in the SFO prevented this increase (1.1 ± 0.2х10 -1 and 1.0± 0.3х10 -1 ). In contrast, Ang II decreased MR mRNA expression (3.2 ± 0.2 vs. 4.1 ± 0.2х10 -2 , p1 R-siRNA further decreased MR mRNA expression (2.7 ± 0.2 х10 -2 and 2.3 ± 0.3 х10 -2 ). In the PVN, Ang II increased AT 1 R mRNA by ~100% and MR mRNA by ~20%. Only AT 1 R-siRNA in the SFO prevented these increases in the PVN. AAV mediated eGFP fluorescence expression was observed in the SFO but not the PVN. These results suggest that circulating Ang II differentially regulates MR and AT 1 R expression in the SFO. Prevention of Ang II induced AT 1 R up-regulation by knockdown of either AT 1 R or MR suggests a MR dependent regulation of AT 1 R in the SFO. MR-AT 1 R signaling in the SFO appears to play a major role in Ang II-induced hypertension in rats. * p

Circulating Ang II activates an aldosterone-MR-AT 1 R pathway in the hypothalamus. To obtain insights into the actual aldosterone-MR- AT 1 R neuronal pathway, the present study examined whether intra-PVN infusion of AAV-AT 1a R-siRNA or AAV-MR-siRNA prevents the Ang II induced increases in AT 1 R and MR expression and BP. Two weeks sc infusion of Ang II at 500ng/kg/min in Wistar rats on regular salt diet increased BP by telemetry by 66 ± 3 mm Hg, and AT 1 R and MR protein in the SFO, SON and PVN. Intra-PVN AT 1a R-siRNA decreased AT 1 R but not MR expression in the PVN by sc Ang II, and MR-siRNA decreased MR but not AT 1 R expression in the PVN. AT 1 R and MR expression in both SFO and SON were not significantly changed by the two AAV-siRNA. Specific knockdown of AT 1 R or MR in the PVN by AAV-siRNA prevented ~80% of the increase in BP induced by sc Ang II. These results suggest that circulating Ang II up-regulates brain Ang II and aldosterone and increases MR and AT 1 R in the SFO, SON and PVN. Prevention of Ang II induced increases in MR but not AT 1 R by knockdown of MR and vice versa suggests an independent regulation of MR and AT 1 R in the PVN. Both AT 1 R and MR in the PVN play a critical role in Ang II-induced hypertension in rats. * p

Central blockade of mineralocorticoid receptor (MR) prevents aldosterone (aldo)-salt induced hypertension. We examined the role of the subfornical organ (SFO), aldo synthesized locally in the brain, and MR specifically in the paraventricular nucleus (PVN) in aldo-salt hypertension. Wistar rats with telemetry probes were treated with chronic intra-cerebroventricular infusion of aldo synthase (AS) inhibitor FAD286 (25 μg/day), MR blocker eplerenone (5 μg/day) or vehicle. After 2 days, subcutaneous (sc) aldo infusion (1 μg/h) was started and saline was provided as drinking water. In a second set of rats with telemetry, electrolytic or sham lesions of the SFO, or bilateral intra-PVN infusion of AAV-MR-siRNA or AAV-SCM-siRNA (1 μl of 5x10 10 genomic particles/ml) at 100ng/min were performed, and sc aldo infusion started 1 week later. AAV-MR-siRNA decreased MR mRNA and protein expression in the PVN by ~60 and 30%, but had no effects on MR expression in other nuclei such as the SFO and SON. SFO lesion, blockade of brain AS or MR, or knockdown of MR in the PVN attenuated aldo-salt hypertension by ~60%. Data=means± SE (n=5-9/group). *p These results suggest that an increase in circulating aldosterone may via MR and AT 1 R in the SFO increase local aldosterone production in hypothalamic nuclei such as the SON and PVN, and via MR in the PVN activate a central aldosterone-MR-AT 1 R neuro-modulatory pathway, which plays a major role in the progressive hypertension. (Supported by grant # FRN:MOP-74432 from the Canadian Institutes of Health Research)