Relaxin attenuates organ fibrosis via an angiotensin-type 2 receptor mechanism in aged hypertensive female rats

Background: The anti-fibrotic effects of recombinant human relaxin (RLX) in the kidney are dependent on an interaction between its cognate receptor (RXFP1) and the angiotensin type 2 receptor (AT2R) in male models of disease. Whether RLX has therapeutic effects, which are also mediated via the AT2R, in hypertensive adult and aged/reproductively senescent females is unknown. Thus, we determined whether treatment with RLX provides cardiorenal protection, via an AT2R-dependent mechanism, in adult and aged female stroke prone-spontaneously hypertensive rats (SHRSP). Methods: In 6- (6MO) and 15-month-old (15MO; reproductively senescent) female SHRSP, systolic blood pressure (SBP), glomerular filtration rate (GFR) and proteinuria were measured before and after 4 weeks treatment with vehicle (Veh), RLX (0.5 mg/kg/day s.c.) or RLX+PD123319 (AT2R antagonist; 3 mg/kg/day s.c.). Aortic endothelium-dependent relaxation and fibrosis of the kidney, heart and aorta were assessed. Results: In 6MO SHRSP, RLX significantly enhanced GFR by ~25% (P=0.001) and reduced cardiac fibrosis (P=0.01) as compared to vehicle-treated counterparts. These effects were abolished or blunted by PD123319 co-administration. In 15MO females, RLX reduced interstitial renal (P=0.02) and aortic (P=0.003) fibrosis, and lowered SBP (13±3 mmHg; P=0.04) relative to controls. These effects were also blocked by PD123319 co-treatment (all p<0.05 versus RLX treatment alone). RLX also markedly improved vascular function by ~40% (P<0.0001) in 15MO SHRSP, but this was not modulated by PD123319 co-treatment. Conclusion: The anti-fibrotic and organ-protective effects of RLX, when administered to a severe model of hypertension, conferred cardiorenal protection in adult and reproductively senescent female rats, to a great extent via an AT2R-mediated mechanism.

In Aged Females, the Enhanced Pressor Response to Angiotensin II Is Attenuated By Estrogen Replacement via an Angiotensin Type 2 Receptor-Mediated Mechanism

Loss of ovarian hormones following menopause contributes to the rise in cardiovascular risk with age. Estrogen plays a protective role against hypertension and end-organ damage by modulating the depressor actions of the AT 2 R (angiotensin type 2 receptor). Our aim was to determine whether estrogen replacement in aged female mice can lower arterial pressure, improve endothelial function, and reduce organ fibrosis via an AT 2 R-mediated mechanism. Mean arterial pressure was measured via radiotelemetry in ovary-intact adult (3–4-month-old), aged (16–18-month-old; reproductively senescent) and aged–17β-estradiol (E 2 )–treated (3 µg/day SC) female mice, which were administered vehicle, Ang II (angiotensin II; 600 ng/[kg·min] SC) or Ang II+PD123319 (AT 2 R antagonist; 3 mg/[kg·day SC). On day 21 of treatment, aortic endothelium-dependent relaxation and cardiac and renal tissue (fibrosis and gene expression) were analyzed. Basal mean arterial pressure was lower in E 2 -treated aged mice (89±1 mm Hg, n=20) relative to aged controls (94±1 mm Hg; n=18, P =0.002). The Ang II pressor response was enhanced by ≈20 mm Hg in aged compared with adult females ( P =0.01). E 2 -treatment reduced the Ang II pressor response in aged females ( P =0.002), an effect that was reversed by PD123319 in the aged E 2 –Ang II group ( P =0.0009). E 2 -treatment increased renal AT 2 R (≈6-fold; P <0.0001) and MasR (Mas oncoreceptor; 2–3-fold, P <0.05) gene expression in aged females. However, neither Ang II–induced endothelial dysfunction nor the age-related increase in renal and cardiac fibrosis was restored by E 2 -treatment in aged female mice. In conclusion, estrogen replacement in aged females may reduce arterial pressure to levels observed in adult females, via an AT 2 R-mediated renal mechanism.

Angiotensin type 2 receptor activation limits kidney injury during the early phase and induces Treg cells during the late phase of renal ischemia

The angiotensin II type 2 receptor agonist C21 has been known to have a renoprotective role in various kidney pathologies. C21 treatment (before renal ischemia) attenuated postischemic kidney injury, kidney dysfunction, and immune cell infiltration during the injury phase. Also, C21 treatment modulated the kidney microenvironment by enhancing anti-inflammatory responses mainly mediated by IL-10. During the repair phase, C21 treatment enhanced IL-10-secreting CD4 T cells and FoxP3-secreting regulatory T cells in Sprague–Dawley rats.

C21 preserves endothelial function in the thoracic aorta from DIO mice: role for AT2, Mas and B2 receptors

Compound 21 (C21), a selective agonist of angiotensin type 2 receptor (AT2R), induces vasodilation through NO release. Since AT2R seems to be overexpressed in obesity, we hypothesize that C21 prevents the development of obesity-related vascular alterations. The main goal of this study was to assess the effect of C21on thoracic aorta endothelial function in a model of diet-induced obesity and to elucidate the potential crosstalk between AT2R, MasR and/or B2R in this response. 5-week-old male C57BL6J mice were fed a standard (CHOW) or a high-fat diet (HF) for 6 weeks and treated daily with C21 (1mg/Kg p.o) or vehicle, generating four groups: CHOW-C, CHOW-C21, HF-C, HF-C21. Vascular reactivity experiments were performed in thoracic aorta rings. Human endothelial cells (EA.hy926) were used to elucidate the signaling pathways, both at receptor and intracellular levels.Arteries from HF mice exhibited increased contractions to Ang II than CHOW mice, effect that was prevented by C21. PD123177, A779 and HOE-140 (AT2R, Mas and B2R antagonists) significantly enhanced Ang II-induced contractions in CHOW but not in HF-C rings, suggesting a lack of functionality of those receptors in obesity. C21 prevented those alterations and favoured the formation of AT2R/MasR and MasR/B2R heterodimers. HF mice also exhibited impaired relaxations to acetylcholine due to a reduced NO availability. C21 preserved NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways. In conclusion, C21 favours the interaction between AT2R, MasR and B2R and prevents the development of obesity-induced endothelial dysfunction by stimulating NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways.

Activation of angiotensin type 2 receptor attenuates testosterone-induced hypertension and uterine vascular resistance in pregnant rats

Preeclampsia is a pregnancy-related hypertensive disorder with unclear mechanisms. While hypersensitivity to angiotensin II via vasoconstrictive angiotensin type-1 receptor (AT1R) is observed in preeclampsia, the importance of vasodilatory angiotensin type-2 receptor (AT2R) in the control of vascular dysfunction is less clear. We assessed whether AT1R, AT2R and eNOS expression is altered in placental vessels of preeclamptic women and tested if ex vivo incubation with AT2R agonist Compound 21 (C21; 1 μM) could restore AT1R, AT2R and eNOS balance. Further, using a rat model of gestational hypertension induced by elevated testosterone, we examined whether C21 (1 μg·kg−1·day−1, oral) could preserve AT1R and AT2R balance and improve blood pressure, uterine artery blood flow, and vascular function. Western blots revealed that AT1R protein level was higher while AT2R and eNOS protein were reduced in preeclamptic placental vessels, and AT2R agonist C21 decreased AT1R and increased AT2R and eNOS protein levels in preeclamptic vessels. In testosterone-dams, blood pressure was higher, and uterine artery blood flow was reduced, and C21 treatment reversed these levels similar to those in controls dams. C21 attenuated the exaggerated Ang II contraction and improved endothelium-dependent vasorelaxation in uterine arteries of testosterone-dams. These C21-mediated vascular effects were associated with decreased AT1R and increased AT2R and eNOS protein levels. C21 also increased serum nitrate/nitrite and bradykinin production in testosterone-dams and attenuated the feto-placental growth restriction. Thus, AT1R upregulation and AT2R downregulation is observed in preeclampsia and testosterone-model, and increasing AT2R activity could help restore AT1R and AT2R balance and improve gestational vascular function.

Abstract 43: Endovascular Delivery of Low Dose Compound 21 is Neuroprotective Independent of Reperfusion in Ischemic Stroke

Background: Intracerebroventricular (ICV) injection of angiotensin type 2 receptor (AT2R) agonists is neuroprotective in stroke. However, ICV delivery of a blood brain barrier impermeable AT2R agonists has limited utility in clinical stroke treatment. We investigated whether low dose endovascular (EV) delivery of a selective AT2R agonist, Compound 21 (C21), after stroke confers neuroprotection under various states of reperfusion. Methods: Aged male mice (12 mos) underwent either permanent middle cerebral artery ligation (pMCAL)(n=12) or spontaneously reperfusing embolic clot MCA occlusion (eMCAo)(n=10). Mice were randomized to EV-delivery of either C21 (1 μg/kg) or 10μL saline at the site of occlusion 2h post-stroke. Statistical significance was determined at p<0.05. Results: C21 treatment following pMCAL prevented infarct progression and significantly reduced stroke injury by >30% at 48h post pMCAL (p<0.02). In eMCAo stroke, C21 was effective in reducing both infarction volume and edema by >25% (p<0.01). Conclusions: We demonstrate that EV-delivery of C21 is effective in both non-reperfused and spontaneously reperfusing stroke. This site-specific delivery may have potential benefits as an adjuvant neuroprotectant combined with endovascular thrombectomy, while averting possible blood-pressure lowering risks associated with AT2R agonism. Of additional translational interest, the current COVID19 pandemic and related ongoing recombinant huACE2-trial therapies will eventually affect ATRs physiology. Given the prominence of thrombotic strokes in COVID19 patients, C21 as an AT2R agonist may be a potential therapy to test in COVID19-related thrombotic stroke models.

The angiotensin type 2 receptor agonist C21 restores respiratory function in COVID19 - a double-blind, randomized, placebo-controlled Phase 2 trial

ABSTRACTBackgroundAlthough several therapies have been evaluated for treatment of COVID-19, the morbidity and mortality in COVID-19 are still significant, and the need for safe and effective drugs remains high even after launch of vaccine programs.MethodsWe conducted a double-blind, randomized, placebo-controlled trial with the novel oral angiotensin II type 2 receptor agonist C21 in hospitalized COVID-19 patients with C-reactive protein 50-150 mg/L but not needing mechanical ventilation. Patients were randomly assigned to oral C21 (100 mg twice daily) or placebo for 7 days in addition to standard of care, including glucocorticoids and remdesivir.Results106 patients underwent randomization (51 in the C21 group and 55 in the placebo group). At day 14 after start of treatment, the proportion of patients still requiring supplemental oxygen was significantly reduced by 90% in the C21 group compared to the placebo group (p=0.003). Moreover, fewer patients required mechanical ventilation (one C21 patient and four placebo patients), and C21 was associated with a numerical reduction in the mortality rate (one and three deaths in the C21 and placebo group, respectively). Treatment with C21 was safe and well tolerated.ConclusionsAs studied in hospitalized COVID-19 patients, C21 on top of standard of care led to a clinically beneficial improvement in respiratory function compared to placebo, paving the way for a pivotal randomised controlled trial.This study is registered at ClinicalTrials.gov with identifier NCT04452435.

Angiotensin Type 2 Receptors: Painful, or Not?

Pain in response to various types of acute injury can be a protective stimulus to prevent the organism from using the injured part and allow tissue repair and healing. On the other hand, neuropathic pain, defined as ‘pain caused by a lesion or disease of the somatosensory nervous system’, is a debilitating pathology. The TRPA1 neurons in the Dorsal Root Ganglion (DRG) respond to reactive oxygen species (ROS) and induce pain. In acute nerve injury and inflammation, macrophages infiltrating the site of injury undergo an oxidative burst, and generate ROS that promote tissue repair and induce pain via TRPA1. The latter discourages using the injured limb, with a lack of movement helping wound healing. In chronic inflammation caused by diabetes, cancer etc., ROS levels increase systemically and modulate TRPA1 neuronal functions and cause debilitating neuropathic pain. It is important to distinguish between drug targets that elicit protective vs. debilitating pain when developing effective drugs for neuropathic pain. In this context, the connection of the Angiotensin type 2 receptor (AT2R) to neuropathic pain presents an interesting dilemma. Several lines of evidence show that AT2R activation promotes anti-inflammatory and anti-nociceptive signaling, tissue repair, and suppresses ROS in chronic inflammatory models. Conversely, some studies suggest that AT2R antagonists are anti-nociceptive and therefore AT2R is a drug target for neuropathic pain. However, AT2R expression in nociceptive neurons is lacking, indicating that neuronal AT2R is not involved in neuropathic pain. It is also important to consider that Novartis terminated their phase II clinical trial (EMPHENE) to validate that AT2R antagonist EMA401 mitigates post-herpetic neuralgia. This trial, conducted in Australia, United Kingdom, and a number of European and Asian countries in 2019, was discontinued due to pre-clinical drug toxicity data. Moreover, early data from the trial did not show statistically significant positive outcomes. These facts suggest that may AT2R not be the proper drug target for neuropathic pain in humans and its inhibition can be harmful.

The COVID-19 Code – Cracked – WHO can Save The World (?)!

The first step in COVID-19 pathogenesis is the viral spike protein priming by Trans Membrane Peptide Receptor Serine S2 (TMPRSS2). TMPRSS2 promotes viral entry, cell to cell transmission, evasion of host immune response, and Angiotensin-Converting Enzyme 2 (ACE2) downregulation. Androgen through Androgen Receptor (AR) increases TMPRSS2 gene expression. Blocking AR may prevent viral entry and other TMPRSS2 mediated actions. ACE2 acts as an entry point for COVID-19 and as the counter regulator in Renin-Angiotensin-Aldosterone System (RAAS). RAAS maintains homeostasis of blood pressure, salt and water, inflammation, and immune response – through its two arms called “killer” and “protective pathways.” The balance between these two pathways determines life or death in disease states. ACE2 converts Angiotensin II to Angiotensin (1-7), which through Mas receptors mediates antiinflammatory, immune-modulatory, and anti-fibrotic actions. Angiotensin II also acts on Angiotensin type 2 Receptor (AT2R) to produce similar actions, called a "protective pathway." Further, Angiotensin II acts through its primary Angiotensin type 1 Receptor (AT1R), causing inflammatory, cytokine storm, and profibrotic response – called "Killer pathway." In COVID, down-regulated ACE2 leads to unabated Angiotensin II/AT1R – "Killer pathway" – actions producing a vicious cycle of "hyper-inflammatory state," resulting in ALI, ARDS, and death. AT1R activation further stimulates the secretion of aldosterone, which through Mineralocorticoid Receptor (MR), augments AT1R mediated 'killer pathway”. None of the COVID guideline drugs modulate this pathogenic mechanism. We examine the first time in history the scientific rationale for combined AR/AT1R/MR blockade for COVID-19 treatment and prevention.