Alamandine, a protective component of the renin-angiotensin system, reduces cellular proliferation and interleukin-6 secretion in human macrophages through MasR-MrgDR heteromerization.

Alamandine (ALA) exerts protective effects similar to angiotensin (Ang) (1-7) through Mas-related G protein-coupled receptor type D receptor (MrgDR) activation, distinct from Mas receptor (MasR). ALA induces anti-inflammatory effects in mice but its impact in human macrophages remains unclear. We aimed to investigate the anti-inflammatory effects of ALA in human macrophages. Interleukin (IL)-6 and IL-1ß were measured by ELISA in human THP-1 macrophages and human monocyte-derived macrophages exposed to lipopolysaccharide (LPS). Consequences of MasR-MrgDR heteromerization were investigated in transfected HEK293T cells. ALA decreased IL-6 and IL-1ß secretion in LPS-activated THP-1 macrophages. The ALA-induced decrease in IL-6 but not in IL-1ß was prevented by MasR blockade and MasR downregulation, suggesting MasR-MrgDR interaction. In human monocyte-derived M1 macrophages, ALA decreased IL-1ß secretion independently of MasR. MasR-MrgDR interaction was confirmed in THP-1 macrophages, human monocyte-derived macrophages, and transfected HEK293T cells. MasR and MrgDR formed a constitutive heteromer that was not influenced by ALA. ALA promoted Akt and ERK1/2 activation only in cells expressing MasR-MrgDR heteromers, and this effect was prevented by MasR blockade. While Ang-(1-7) reduced cellular proliferation in MasR -but not MrgDR- expressing cells, ALA antiproliferative effect was elicited in cells expressing MasR-MrgDR heteromers. ALA also induced an antiproliferative response in THP-1 cells and this effect was abolished by MasR blockade, reinforcing MasR-MrgDR interaction. MasR-MrgDR heteromerization is crucial for ALA-induced anti-inflammatory and antiproliferative responses in human macrophages. This study broaden our knowledge of the protective axis of the RAS, thus enabling novel therapeutic approaches in inflammatory-associated diseases.

Effect of age on human ACE2 and ACE2-expressing alveolar type II cells levels.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) is the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes COVID-19. Viral entry requires ACE2 and transmembrane protease serine 2 (TMPRSS2). Transcriptomic studies showed that children display lower ACE2 than adults, though gene expression levels do not always correlate with protein levels. We investigated the effect of age on ACE2 and TMPRSS2 protein expression in alveolar type II (AT2) cells in the lungs of children compared to adults. We also analysed the ratio of Ang-(1-7) to Ang II as a surrogate marker of ACE2 activity in the subjects' lung parenchyma. METHODS: Ang II and Ang-(1-7) levels and ACE2 and TMPRSS2 protein expression were measured by radioimmunoassay and immunohistochemistry, respectively. RESULTS: The amount of ACE2-expressing AT2 cells and ACE2 protein content were lower in children than in adults. Ang II levels were higher in children compared to adults and inversely correlated with the amount of ACE2-expressing AT2 cells. Children presented lower Ang-(1-7)/Ang II ratio than adult suggesting lower ACE2 activity in children. TMPRSS2 protein expression was not influenced by age. CONCLUSIONS: These results expand on previous transcriptomic studies and may partially explain the low susceptibility of children to SARS-CoV-2 infection. CATEGORY OF STUDY: Clinical original research IMPACT: Children display lower ACE2 protein content and activity compared to adults. Ang II levels were higher in children compared to adults and inversely correlated with the amount of ACE2-expressing AT2 cells TMPRSS2 protein expression was not influenced by age. These results expand on previous transcriptomic studies and may partially explain the low susceptibility of children to SARS-CoV-2 infection.

Plasmatic renin-angiotensin system in normotensive and hypertensive patients hospitalized with COVID-19.

BACKGROUND: Besides its counterbalancing role of the renin-angiotensin system (RAS), angiotensin-converting enzyme (ACE) 2 is the receptor for the type 2 coronavirus that causes severe acute respiratory syndrome, the etiological agent of COVID-19. COVID-19 is associated with increased plasmatic ACE2 levels, although conflicting results have been reported regarding angiotensin (Ang) II and Ang-(1-7) levels. We investigated plasmatic ACE2 protein levels and enzymatic activity and Ang II and Ang-(1-7) levels in normotensive and hypertensive patients hospitalized with COVID-19 compared to healthy subjects. METHODS: Ang II and Ang-(1-7), and ACE2 activity and protein levels were measured in 93 adults (58 % (n = 54) normotensive and 42 % (n = 39) hypertensive) hospitalized with COVID-19. Healthy, normotensive (n = 33) and hypertensive (n = 7) outpatient adults comprised the control group. RESULTS: COVID-19 patients displayed higher ACE2 enzymatic activity and protein levels than healthy subjects. Within the COVID-19 group, ACE2 activity and protein levels were not different between normotensive and hypertensive-treated patients, not even between COVID-19 hypertensive patients under RAS blockade treatment and those treated with other antihypertensive medications. Ang II and Ang-(1-7) levels significantly decreased in COVID-19 patients. When COVID-19 patients under RAS blockade treatment were excluded from the analysis, ACE2 activity and protein levels remained higher and Ang II and Ang-(1-7) levels lower in COVID-19 patients compared to healthy people. CONCLUSIONS: Our results support the involvement of RAS in COVID-19, even when patients under RAS blockade treatment were excluded. The increased circulating ACE2 suggest higher ACE2 expression and shedding.

Renin-angiotensin system blockade on angiotensin-converting enzyme 2 and TMPRSS2 in human type II pneumocytes.

AIMS: Angiotensin-converting enzyme (ACE) 2 is the receptor for severe acute respiratory syndrome coronavirus 2 which causes coronavirus disease 2019 (COVID-19). Viral cellular entry requires ACE2 and transmembrane protease serine 2 (TMPRSS2). ACE inhibitors (ACEIs) or angiotensin (Ang) receptor blockers (ARBs) influence ACE2 in animals, though evidence in human lungs is lacking. We investigated ACE2 and TMPRSS2 in type II pneumocytes, the key cells that maintain lung homeostasis, in lung parenchymal of ACEI/ARB-treated subjects compared to untreated control subjects. MAIN METHODS: Ang II and Ang-(1-7) levels and ACE2 and TMPRSS2 protein expression were measured by radioimmunoassay and immunohistochemistry, respectively. KEY FINDINGS: We found that the ratio Ang-(1-7)/Ang II, a surrogate marker of ACE2 activity, as well as the amount of ACE2-expressing type II pneumocytes were not different between ACEI/ARB-treated and untreated subjects. ACE2 protein content correlated positively with smoking habit and age. The percentage of TMPRSS2-expressing type II pneumocytes was higher in males than females and in subjects under 60 years of age but it was not different between ACEI/ARB-treated and untreated subjects. However, there was a positive association of TMPRSS2 protein content with age and smoking in ACEI/ARB-treated subjects, with high TMPRSS2 protein levels most evident in ACEI/ARB-treated older adults and smokers. SIGNIFICANCE: ACEI/ARB treatment influences human lung TMPRSS2 but not ACE2 protein content and this effect is dependent on age and smoking habit. This finding may help explain the increased susceptibility to COVID-19 seen in smokers and older patients with treated cardiovascular-related pathologies.

Interaction Between the Angiotensin-(1-7) Mas Receptor and the Dopamine D2 Receptor: Implications in Inflammation.

[Figure: see text].

Angiotensin Receptors Heterodimerization and Trafficking: How Much Do They Influence Their Biological Function?

G-protein-coupled receptors (GPCRs) are targets for around one third of currently approved and clinical prescribed drugs and represent the largest and most structurally diverse family of transmembrane signaling proteins, with almost 1000 members identified in the human genome. Upon agonist stimulation, GPCRs are internalized and trafficked inside the cell: they may be targeted to different organelles, recycled back to the plasma membrane or be degraded. Once inside the cell, the receptors may initiate other signaling pathways leading to different biological responses. GPCRs' biological function may also be influenced by interaction with other receptors. Thus, the ultimate cellular response may depend not only on the activation of the receptor from the cell membrane, but also from receptor trafficking and/or the interaction with other receptors. This review is focused on angiotensin receptors and how their biological function is influenced by trafficking and interaction with others receptors.

Mas receptor is translocated to the nucleus upon agonist stimulation in brainstem neurons from spontaneously hypertensive rats but not normotensive rats.

AIMS: Activation of the angiotensin (Ang)-(1-7)/Mas receptor (R) axis protects from sympathetic overactivity. Endocytic trafficking is an essential process that regulates receptor (R) function and its ultimate cellular responses. We investigated whether the blunted responses to Ang-(1-7) in hypertensive rats are associated to an alteration in MasR trafficking. METHODS AND RESULTS: Brainstem neurons from Wistar-Kyoto (WKY) or spontaneously hypertensive rats (SHRs) were investigated for (i) Ang-(1-7) levels and binding and MasR expression, (ii) Ang-(1-7) responses (arachidonic acid and nitric oxide release and Akt and ERK1/2 phosphorylation), and (iii) MasR trafficking. Ang-(1-7) was determined by radioimmunoassay. MasR expression and functionality were evaluated by western blot and binding assays. MasR trafficking was evaluated by immunofluorescence. Ang-(1-7) treatment induced an increase in nitric oxide and arachidonic acid release and ERK1/2 and Akt phosphorylation in WKY neurons but did not have an effect in SHR neurons. Although SHR neurons showed greater MasR expression, Ang-(1-7)-elicited responses were substantially diminished presumably due to decreased Ang-(1-7) endogenous levels concomitant with impaired binding to its receptor. Through immunocolocalization studies, we evidenced that upon Ang-(1-7) stimulation MasRs were internalized through clathrin-coated pits and caveolae into early endosomes and slowly recycled back to the plasma membrane. However, the fraction of internalized MasRs into early endosomes was larger and the fraction of MasRs recycled back to the plasma membrane was smaller in SHR than in WKY neurons. Surprisingly, in SHR neurons but not in WKY neurons, Ang-(1-7) induced MasR translocation to the nucleus. Nuclear MasR expression and Ang-(1-7) levels were significantly greater in the nuclei of Ang-(1-7)-stimulated SHR neurons, indicating that the MasR is translocated with its ligand bound to it. CONCLUSION: MasRs display differential trafficking in brainstem neurons from SHRs, which may contribute to the impaired responses to Ang-(1-7).

The depressor axis of the renin-angiotensin system and brain disorders: a translational approach.

All the components of the classic renin-angiotensin system (RAS) have been identified in the brain. Today, the RAS is considered to be composed mainly of two axes: the pressor axis, represented by angiotensin (Ang) II/angiotensin-converting enzyme/AT1 receptors, and the depressor and protective one, represented by Ang-(1-7)/ angiotensin-converting enzyme 2/Mas receptors. Although the RAS exerts a pivotal role on electrolyte homeostasis and blood pressure regulation, their components are also implicated in higher brain functions, including cognition, memory, anxiety and depression, and several neurological disorders. Overactivity of the pressor axis of the RAS has been implicated in stroke and several brain disorders, such as cognitive impairment, dementia, and Alzheimer or Parkinson's disease. The present review is focused on the role of the protective axis of the RAS in brain disorders beyond its effects on blood pressure regulation. Furthermore, the use of drugs targeting centrally RAS and its beneficial effects on brain disorders are also discussed.

1,2,3-triazole-, arylamino- and thio-substituted 1,4-naphthoquinones: potent antitumor activity, electrochemical aspects, and bioisosteric replacement of C-ring-modified lapachones.

1,2,3-Triazole-, arylamino- and thio-substituted naphthoquinones (24, 8, and 2 representatives, respectively) were synthesized in moderate yields and evaluated against several human cancer cell lines (blood, ovarian, breast, central nervous system, colon, and prostate cancers and melanoma), showing, for some of them, IC50 values below 2 µM. The cytotoxic potential of the tested naphthoquinones was also assayed on non-tumor cells such as human peripheral blood mononucluear cells (PBMC) and two murine fibroblast lines (L929 and V79 cells). α-Lapachone- and nor-α-lapachone-based 1,2,3-triazoles and arylamino-substituted naphthoquinones showed potent cytotoxicity against different cancer cell lines. The compounds may represent promising new lead derivatives for anticancer drug development. The electrochemical properties of selected compounds were evaluated in an attempt to correlate them with antitumor activity.

Synthesis and anti-Trypanosoma cruzi activity of naphthoquinone-containing triazoles: electrochemical studies on the effects of the quinoidal moiety.

In our continued search for novel trypanocidal compounds, twenty-six derivatives of para- and ortho-naphthoquinones coupled to 1,2,3-triazoles were synthesized. These compounds were evaluated against the infective bloodstream form of Trypanosoma cruzi, the etiological agent of Chagas disease. Compounds 17-24, 28-30 and 36-38 are described herein for the first time. Three of these novel compounds (28-30) were found to be more potent than the standard drug benznidazole, with IC50/24h values between 6.8 and 80.8µM. Analysis of the toxicity to heart muscle cells led to LC50/24h of <125, 63.1 and 281.6µM for 28, 29 and 30, respectively. Displaying a selectivity index of 34.3, compound 30 will be further evaluated in vivo. The electrochemical properties of selected compounds were evaluated in an attempt to find correlations with trypanocidal activity, and it was observed that more electrophilic quinones were generally more potent.