Neurofilament light: a possible prognostic biomarker for treatment of vascular contributions to cognitive impairment and dementia.

BACKGROUND: Decreased cerebral blood flow and systemic inflammation during heart failure (HF) increase the risk for vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer disease-related dementias (ADRD). We previously demonstrated that PNA5, a novel glycosylated angiotensin 1-7 (Ang-(1-7)) Mas receptor (MasR) agonist peptide, is an effective therapy to rescue cognitive impairment in our preclinical model of VCID. Neurofilament light (NfL) protein concentration is correlated with cognitive impairment and elevated in neurodegenerative diseases, hypoxic brain injury, and cardiac disease. The goal of the present study was to determine (1) if treatment with Ang-(1-7)/MasR agonists can rescue cognitive impairment and decrease VCID-induced increases in NfL levels as compared to HF-saline treated mice and, (2) if NfL levels correlate with measures of cognitive function and brain cytokines in our VCID model. METHODS: VCID was induced in C57BL/6 male mice via myocardial infarction (MI). At 5 weeks post-MI, mice were treated with daily subcutaneous injections for 24 days, 5 weeks after MI, with PNA5 or angiotensin 1-7 (500 microg/kg/day or 50 microg/kg/day) or saline (n = 15/group). Following the 24-day treatment protocol, cognitive function was assessed using the Novel Object Recognition (NOR) test. Cardiac function was measured by echocardiography and plasma concentrations of NfL were quantified using a Quanterix Simoa assay. Brain and circulating cytokine levels were determined with a MILLIPLEX MAP Mouse High Sensitivity Multiplex Immunoassay. Treatment groups were compared via ANOVA, significance was set at p < 0.05. RESULTS: Treatment with Ang-(1-7)/MasR agonists reversed VCID-induced cognitive impairment and significantly decreased NfL levels in our mouse model of VCID as compared to HF-saline treated mice. Further, NfL levels were significantly negatively correlated with cognitive scores and the concentrations of multiple pleiotropic cytokines in the brain. CONCLUSIONS: These data show that treatment with Ang-(1-7)/MasR agonists rescues cognitive impairment and decreases plasma NfL relative to HF-saline-treated animals in our VCID mouse model. Further, levels of NfL are significantly negatively correlated with cognitive function and with several brain cytokine concentrations. Based on these preclinical findings, we propose that circulating NfL might be a candidate for a prognostic biomarker for VCID and may also serve as a pharmacodynamic/response biomarker for therapeutic target engagement.

Angiotensin-(1-7) receptor Mas antagonist (A779) influenced gliosis and reduced synaptic density in the spinal cord after peripheral axotomy.

The peptide angiotensin-(1-7) [Ang (1-7)] and its receptor Mas are involved in controlling arterial pressure and display actions on the nervous system. In a previous study, our laboratory showed that A779 [(peptidyl antagonist of the Ang-(1-7)] treatment had a negative effect following a lesion of the sciatic nerve, possibly by delaying the responses of Schwann cells, resulting in a decreased axonal organization along with a slowed functional return. In the present work, we investigated the central cellular changes after sciatic nerve injury in rodents treated with A779 after two weeks. In the lumbar spinal cords, where the neuronal bodies that make up the sciatic are, the treatment with A779 showed reduced reactivity of astrocytes (p = 0.004, Mann-Whitney U test) and less synaptic density (p = 0.004, Mann-Whitney U test) after injury. Also, the treatment upregulated microglia activity in both sides (p = 0.004, Mann-Whitney U test), ipsilateral and contralateral to the lesion, of the spinal cord. In addition, the Mas expression in spine neurons was increased in response to axotomy especially after two weeks (p = 0.03, Mann-Whitney U test) following the nerve lesion in comparison to earlier stages after injury. Therefore, we can conclude that Ang-(1-7)/Mas axis plays a role during spinal cord recovery after peripheral nerve injury.

Angiotensin-(1-7) in human follicular fluid correlates with oocyte maturation.

STUDY QUESTION: Do angiotensin (Ang)-(1-7) levels in human ovarian follicular fluid (FF) correlate with the number and proportion of mature oocytes obtained for IVF? SUMMARY ANSWER: The present study shows for the first time that Ang-(1-7) levels in human FF correlate with the proportion of mature oocytes collected upon ovarian stimulation for IVF. WHAT IS KNOWN ALREADY: Ang-(1-7) is an active peptide of the renin-angiotensin system that stimulates oocyte maturation in isolated rabbit and rat ovaries. However, its role in human ovulation remains unexplored. STUDY DESIGN, SIZE, DURATION: This was a prospective cohort study including 64 participants from a single IVF center. Sample size was calculated to achieve a statistical power of 80% in detecting 20% differences in the proportion of mature oocytes between groups. The participants were enrolled in the study during six consecutive months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Plasma samples were obtained from all subjects at Day 21 of the last menstrual cycle before starting pituitary blockade and controlled ovarian stimulation (COS). Plasma and FF samples were quickly mixed with a protease inhibitor cocktail and stored at -80°C. Ang-(1-7) was quantified in plasma and FF samples by a highly sensitive and specific radioimmunoassay, which was preceded by solid phase extraction, speed vacuum concentration and sample reconstitution in assay buffer. FF Ang-(1-7) levels were stratified into tertiles and the patients of each tertile were compared for COS/IVF outcomes using Kruskal-Wallis ANOVA. Multiple regression analysis was used to adjust correlations for potential confounders. The mRNA encoding for Mas, a receptor for Ang-(1-7), was investigated by real-time PCR in luteinized granulosa cells purified from the FF. MAIN RESULTS AND THE ROLE OF CHANCE: There was a four-fold increase in plasma Ang-(1-7) after ovulation induction (median 160.9 vs 41.4 pg/ml, P < 0.0001). FF Ang-(1-7) levels were similar to (169.9 pg/ml) but did not correlate with plasma Ang-(1-7) levels (r = -0.05, P = 0.665). Patients at the highest FF Ang-(1-7) tertile had a higher proportion of mature oocytes compared to patients at the lower FF Ang-(1-7) tertile (median 100% vs 70%, P < 0.01). There was a linear correlation between FF Ang-(1-7) and the proportion of mature oocytes (r = 0.380, P < 0.01), which remained significant after adjustment for age and duration of infertility (r = 0.447, P < 0.001). The luteinized granulosa cells expressed Mas receptor mRNA, which was positively correlated to the number of mature oocytes in women with more than three mature oocytes retrieved (r = 0.42, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This is an observational study, therefore, no causal relationship can be established between Ang-(1-7) and human oocyte maturation. Mas protein expression was not quantified due to limited availability of granulosa cells. WIDER IMPLICATIONS OF THE FINDINGS: Since this peptide promotes oocyte maturation in other species, it deserves further investigation as a potential maturation factor to human oocytes. STUDY FUNDING AND COMPETING INTEREST(S): Research supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). The authors have nothing to disclose.

ACE inhibition, ACE2 and angiotensin-(1-7) axis in kidney and cardiac inflammation and fibrosis.

The Renin Angiotensin System (RAS) is a pivotal physiological regulator of heart and kidney homeostasis, but also plays an important role in the pathophysiology of heart and kidney diseases. Recently, new components of the RAS have been discovered, including angiotensin converting enzyme 2 (ACE2), Angiotensin(Ang)-(1-7), Mas receptor, Ang-(1-9) and Alamandine. These new components of RAS are formed by the hydrolysis of Ang I and Ang II and, in general, counteract the effects of Ang II. In experimental models of heart and renal diseases, Ang-(1-7), Ang-(1-9) and Alamandine produced vasodilation, inhibition of cell growth, anti-thrombotic, anti-inflammatory and anti-fibrotic effects. Recent pharmacological strategies have been proposed to potentiate the effects or to enhance the formation of Ang-(1-7) and Ang-(1-9), including ACE2 activators, Ang-(1-7) in hydroxypropyl ß-cyclodextrin, cyclized form of Ang-(1-7) and nonpeptide synthetic Mas receptor agonists. Here, we review the role and effects of ACE2, ACE2 activators, Ang-(1-7) and synthetic Mas receptor agonists in the control of inflammation and fibrosis in cardiovascular and renal diseases and as counter-regulators of the ACE-Ang II-AT1 axis. We briefly comment on the therapeutic potential of the novel members of RAS, Ang-(1-9) and alamandine, and the interactions between classical RAS inhibitors and new players in heart and kidney diseases.

Baicalin attenuates angiotensin II-induced endothelial dysfunction.

Angiotensin II (Ang II) has been shown to activate multiple downstream pathways resulting in endothelial dysfunction and oxidative stress. Baicalin, a natural flavone, exerts anti-oxidant and anti-apoptotic effects in cardiovascular diseases. In the present study, we hypothesized that baicalin has beneficial effects in Ang II-induced endothelial cells injury. Here, we shown that baicalin improved endothelial fuction impaired by Ang II through promoting endothelial-dependent vasodilation and suppressing the apoptosis of HUVECs in which baicalin decreased the expression of bax and cleaved caspase-3, and increased bcl-2 expression. Additionally, baicalin significantly conversed Ang II to angiotensin-1-7 [Ang-(1-7)] by activating angiotensin-converting enzyme 2 (ACE2) and Mas receptor mRNA expression and protein expression. Moreover, treatment with baicalin significantly reduced cell oxidative damage induced by Ang II through MDA/ROS decrease and NO/T-AOC increase. This antioxidant capacity was related to the increases of PI3K, phosphor-AKT (Ser-473) and phosphor-eNOS (Ser-1177). In conclusion, our results implicate that baicalin could protect endothelial cells from Ang II-induced endothelial dysfunction and oxidative stress via modulating the expression of bax, bcl-2 and cleaved caspase-3, activating ACE2/Ang-(1-7)/Mas axis and up-regulating PI3K/AKT/eNOS pathway.

Olmesartan potentiates the anti-angiogenic effect of sorafenib in mice bearing Ehrlich's ascites carcinoma: role of angiotensin (1-7).

Local renin-angiotensin systems exist in various malignant tumor tissues; this suggests that the main effector peptide, angiotensin II, could act as a key factor in tumor growth. The underlying mechanisms for the anti-angiogenic effect of angiotensin II type 1 receptor blockers need to be further evaluated. The present study was carried out to investigate the anti-angiogenic effect of olmesartan alone or in combination with sorafenib, an angiotensin (1-7) agonist or an angiotensin (1-7) antagonist in Ehrlich's ascites carcinoma-bearing mice. The tumor was induced by intradermal injection of Ehrlich's ascites carcinoma cells into mice. Tumor discs were used to evaluate the microvessel density; the serum levels of vascular endothelial growth factor (VEGF) and serum insulin-like growth factor I (IGF-I); and their intratumoral receptors, VEGF receptor-2 and IGF-I receptor, respectively. All parameters were determined following the treatment course, which lasted for 21 days post-inoculation. Monotherapy with olmesartan and its combination with sorafenib resulted in a significant reduction in microvessel density and serum levels of VEGF and IGF-I, as well as their intratumoral receptors. In addition, the combination of olmesartan (30 mg/kg) with an angiotensin (1-7) agonist reduced the microvessel density, IGF-I serum levels and the levels of its intratumoral receptor. In conclusion, olmesartan reduced the levels of the angiogenesis markers IGF-I and VEGF and down-regulated the intratumoral expression of their receptors in a dose-dependent manner, and these effects were dependent on the angiotensin (1-7) receptor. These results suggest that olmesartan is a promising adjuvant to sorafenib in the treatment of cancer.

The influence of angiotensin-(1-7) peptidomimetic (AVE 0991) and nebivolol on angiotensin I metabolism in aorta of apoE-knockout mice.

The detrimental role of over activation of renin-angiotensin system (RAS) in atherogenesis is widely recognized. Recently, we have demonstrated that Ang-(1-7) peptidomimetic - AVE0991, as well as known beta-adrenolytic agent nebivolol, exert anti-atherogenic actions in mouse model of atherosclerosis - apoE-knockout mice. Here, using LC-ESI-MS ex vivo system, we tested whether prolonged treatment of apoE-knockout mice by these drugs can influence RAS in aorta of apoE-knockout mice in regard to generation of most active metabolites of Ang I-Ang II and Ang-(1-7). As compared to wild type animals there was increased generation of Ang II in aorta of apoE-knockout mice, while the formation of Ang-(1-7) did not differ between both groups. Either treatment with AVE0991 or nebivolol resulted in significant attenuation of Ang II production in aorta of apoE-knockout mice. In conclusion, for the first time we directly demonstrated that there is increase in ability of aortic tissue to generate Ang II in mouse model of atherosclerosis of apoE knockout mice, and that such effect could be efficiently attenuated either by treatment of nebivolol or Ang-(1-7) peptidomimetic - AVE0991. The exact mechanism(s) responsible for interference of both drugs with RAS require further investigation.

Angiotensin-(1-7) receptor Mas agonist ameliorates progress of atherosclerosis in apoE-knockout mice.

Our interest focused on an open question whether AT-(1-7), nonpeptide receptor agonist: AVE 0991, is able to ameliorate atherosclerosis. We used an apolipoprotein E (apoE) - knockout mice model of atherosclerosis. Experimental groups received the same diet as control, mixed with: AVE 0991 at a dose of 0.58 µmol/kg b.w./day, perindopril at a dose of 0.4 mg/kg b.w./day or with tiorphan at a dose of 2.5 mg/kg b.w./day. A-779 [(D-alanine)-angiotensin (1-7)] was given at a dose of 3.3 mg/kg b.w., 3 times a week i.p. Measured by "en face" method, the percentage of occupied by Sudan IV-stained surfaces were as follows: 14.2±1.9 % in control group, whereas in AVE 0991-treated as well as in perindopril-treated groups percentages were statistically significantly lower. In tiorphan group there was no change comparing to control group, whereas in A-779 group percentage was statistically significantly higher. "Cross-section" of aortic roots revealed also the difference in atherosclerotic lesions. The mean surfaces, occupied by oil red O-stained changes were: 91.213±8.123 µm(2) in control group, while in AVE 0991-treated as well as in perindopril-treated groups lesions were statistically significantly lower. In tiorphan group there was no change; however, in A-779 group lesions were statistically significantly higher. Measured by real time RT-PCR relative p22phox (submit of NADPH oxidase) expression was significantly decreased in AVE 0991-treated mice. As revealed by flow cytometry, the expression of co-stimulatory molecules: CD86, CD80 and CD40 on both dendritic cells (CD11c+) and macrophages (F4/80+) was reduced in AVE 0991-treated group, which correlated with decreased expression of CD69 activation marker on CD4+T cells. In our report we showed the beneficial effect of AVE 0991 on atherogenesis in gene-targeted mice.

Nonpeptide AVE 0991 is an angiotensin-(1-7) receptor Mas agonist in the mouse kidney.

It has been described recently that the nonpeptide AVE 0991 (AVE) mimics the effects of angiotensin-(1-7) [Ang-(1-7)] in bovine endothelial cells. In this study, we tested the possibility that AVE is an agonist of the Ang-(1-7) receptor Mas, in vitro and in vivo. In water-loaded C57BL/6 mice, AVE (0.58 nmol/g body weight) produced a significant reduction in urinary volume (0.06+/-0.03 mL/60 min [n=9] versus 0.27+/-0.05 [n=9]; P<0.01), associated with an increase in urinary osmolality. The Ang-(1-7) antagonist A-779 completely blocked the antidiuretic effect of AVE. As observed previously for Ang-(1-7), the antidiuretic effect of AVE after water load was blunted in Mas-knockout mice (0.37+/-0.10 mL/60 min [n=9] versus 0.27+/-0.03 mL/60 min [n=11] AVE-treated mice). In vitro receptor autoradiography in C57BL/6 mice showed that the specific binding of 125I-Ang-(1-7) to mouse kidney slices was displaced by AVE, whereas no effects were observed in the binding of 125I-angiotensin II or 125I-angiotensin IV. Furthermore, AVE displaced the binding of 125I-Ang-(1-7) in Mas-transfected monkey kidney cells (COS) cells (IC50=4.75x10(-8) mol/L) and of rhodamine-Ang-(1-7) in Mas-transfected Chinese hamster ovary (CHO) cells. It also produced NO release in Mas-transfected CHO cells blocked by A-779 but not by angiotensin II type-1 (AT1) and AT2 antagonists. Contrasting with these data, the antidiuretic effect of AVE was totally blocked by AT2 antagonists and partially blocked (approximately 60%) by AT1 antagonists. The binding data, the results obtained in Mas-knockout mice and in Mas-transfected cells, show that AVE is a Mas receptor agonist. Our data also suggest the involvement of AT2/AT1-related mechanisms, including functional antagonism, oligomerization or cross-talk, in the renal responses to AVE.

Effects of electroacupuncture on pressor response to angiotensin-(1-7) by amino acid release in the rostral ventrolateral medulla.

Unilateral microinjection of Angiotensin-(1-7)[Ang-(1-7)] into the rostral ventrolateral medulla (RVLM) of anesthetized rats caused an increase in mean arterial pressure (MAP) accompanied by an increased release of excitatory amino acid (EAA) glutamate. In contrast, microinjection of Ang779, a selective antagonist of Ang-(1-7) receptor, into the RVLM caused a decrease in MAP accompanied by a deceased release of EAA glutamate as well as an increased release of inhibitory amino acid (IAA) glycine, taurine and gamma-aminobutyric acid. After electroacupuncture (EA) stimulation at "Zusanli"(St.36) for 20 min, the above effects of Ang-(1-7) or Ang779 attenuated. These results suggest that attenuation of EA on the pressor effect of Ang-(1-7) or the depressor effect of Ang779 may be through regulating the corresponding amino acid neurotransmitter release in the RVLM.