Disruption of vascular endothelial homeostasis in systemic juvenile idiopathic arthritis-associated macrophage activation syndrome: The dynamic roles of angiopoietin-1 and -2

To assess the role of angiopoietin (Ang)-1 and Ang-2 and to investigate the clinical significance of serum levels of them in systemic juvenile idiopathic arthritis (s-JIA)-associated macrophage activation syndrome (MAS), we determined these levels in 51 patients with s-JIA, 11 patients with polyarticular JIA (poly-JIA), 12 patients with virus associated hemophagocytic syndrome (VAHS), 12 patients with Kawasaki disease (KD), and 15 age-matched healthy controls (HC). The results were compared with clinical features of MAS. During the MAS phase, serum Ang-1 levels were significantly decreased compared with those during the active and inactive phases. Serum Ang-2/1 ratio were significantly elevated during the MAS phase, compared with those during the active and inactive phases. There was a rapid increase in the Ang-2/1 ratio at the onset of MAS. Serum Ang-1 and the Ang-2/1 ratio significantly correlated with measures of disease activity, including AST and LDH. Ang-2/1 dysregulation was also observed in patients with VAHS, whereas not observed in most cases of KD. The homeostasis of vascular endothelial function by Ang-1 and Ang-2 is disrupted in MAS. Serum Ang-1 levels and the Ang-2/1 ratio might represent promising indicators of disease activity for MAS.     

Regulation of placental villous angiopoietin-1 and -2 expression by estrogen during baboon pregnancy

We recently showed an increase in vascular endothelial growth factor (VEGF), decrease in angiopoietin-1 (Ang-1) and unaltered Ang-2 expression by the villous placenta with advancing baboon pregnancy. Moreover, placental VEGF expression was increased by estrogen in early pregnancy. In the present study, we determined whether placental Ang-1 and Ang-2 are regulated by estrogen. Ang-1 and Ang-2 mRNA and protein were determined by RT-PCR and immunocytochemistry in the placenta of baboons on Day 60 of gestation (term is 184 days) after administration of estrogen precursor androstenedione on Days 25-59 or on Day 54 after acute estradiol administration. Chronic androstenedione treatment increased serum estradiol levels three-fold (P < 0.001) and decreased (P < 0.05) villous cytotrophoblast Ang-1 mRNA to a level (0.36 +/- 0.08 relative to 18S rRNA) that was one-third of that in untreated animals (0.98 +/- 0.26). Within 2 hr of estradiol administration, cytotrophoblast Ang-1 mRNA was decreased to a level (0.24 +/- 0.05) one-fifth (P < 0.05) of that in untreated animals (1.14 +/- 0.23). However, Ang-2 mRNA levels were unaltered. Ang-1, Ang-2 and estrogen receptors alpha and beta protein were localized within villous cytotrophoblasts providing a mechanism for estrogen action at this site. In summary, estrogen increased VEGF, decreased Ang-1, and had no effect on Ang-2 expression within placental cytotrophoblasts during early baboon pregnancy. We propose that the estrogen-dependent differential regulation of these angioregulatory factors underpins the unique pattern of neovascularization established within the villous placenta during primate pregnancy.     

Regulation of angiopoietin expression by bacterial lipopolysaccharide

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-kappaB. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.     

Levels of angiopoietins 1 and 2 in induced sputum supernatant in patients with COPD

Pathological features of chronic obstructive pulmonary disease (COPD) include lung vascular remodeling and angiogenesis. Angiopoietin-1 (Ang-1), is an essential mediator of angiogenesis by establishing vascular integrity, whereas angiopoietin-2 (Ang-2) acts as its natural inhibitor. We determined the levels of angiopoietins in sputum supernatants of patients with COPD and investigated their possible association with mediators and cells involved in the inflammatory and remodeling process. Fifty-nine patients with COPD, 25 healthy smokers and 20 healthy non-smokers were studied. All subjects underwent lung function tests, sputum induction for cell count identification and Ang-1, Ang-2, VEGF, TGF-β1, MMP-2, LTB4, IL-8, albumin measurement in sputum supernatants. Airway vascular permeability (AVP) index was also assessed. Ang-2 levels were significantly higher in patients with COPD compared to healthy smokers and healthy non-smokers [median, interquartile ranges pg/ml, 267 (147-367) vs. 112 (67-171) and 98 (95-107), respectively; p<0.001]. Regression analysis showed a significant association between Ang-2 levels and AVP index, VEGF, IL-8 and MMP-2 levels in COPD, the strongest being with VEGF. Our results indicate that induced sputum Ang-2 levels are higher in COPD compared to healthy smokers and healthy non-smokers. Moreover, Ang-2 is associated with AVP, IL-8, MMP-2, and VEGF, indicating a possible role for Ang-2 in the pathogenesis of the disease.     

Angiopoietin-2 and Angiopoietin-2/Angiopoietin-1 Ratio as Indicators of Potential Severity of Plasmodium vivax Malaria in Patients with Thrombocytopenia

Introduction

Angiogenic factors such as angiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2) are biomarkers produced during activation and dysfunction of the vascular endothelium in several infectious diseases. The aim of this study was to determine the serum levels of Ang-1 and Ang-2 and to establish their relationship with the main indicators of worst-case prognosis in patients with P. vivax malaria.

Methods

This is a retrospective case-control study nested within a cohort of symptomatic malaria patients. A potentially severe case was defined as a patient that presented at least one of the main indicators of the worst-case prognosis for falciparum malaria, as established by the World Health Organization. Ang-2 and Ang-1 and the Ang-2/Ang-1 ratio were used to analyze the role of angiopoietins as biomarkers in signaling potentially severe vivax malaria. ROC curves were generated to identify a cut-off point discriminating between the angiopoietin concentrations that were most strongly associated with potential infection severity.

Results

The serum levels of Ang-2 and the Ang-2/Ang-1 ratio were higher in the case group. In contrast, the serum levels of Ang-1 were lower in the cases than in the control patients. The blood count for platelets showed a positive correlation with Ang-1 and a negative correlation with Ang-2 and with the Ang-2/Ang-1 ratio. The area under the ROC curve (AUC) for serum angiopoietins, as an indicator of worst-case prognosis in a potentially severe P. vivax malarial infection, was larger in the subgroup of patients with platelet counts <75,000/µL.

Conclusion

This study showed that patients with predictors of worst-case prognoses for P. vivax malaria have lower Ang-1 and higher Ang-2 serum levels (and higher values for the Ang-2/Ang-1 ratio) than controls. Elevated serum levels of Ang-2 and high values for the Ang-2/Ang-1 ratio may potentially be used as predictors of worst-case prognoses for P. vivax malaria, especially in patients with thrombocytopenia.     

Angiopoietin-2 deficiency decelerates age-dependent vascular changes in the mouse retina.

Retinae of aged humans show signs of vascular regression. Vascular regression involves a mismatch between Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) expression. We used heterozygous Ang-2 deficient (Ang2LacZ) mice to evaluate murine retinal vascular changes and gene expression of growth factors. Vascular changes were assessed by quantitative retinal morphometry and gene expression levels of growth factors were measured by quantitative PCR. The numbers of endothelial cells and pericytes did not change in the Ang2LacZ retinae with age, whereas they decreased throughout the age spectrum studied in the wild type retinae. Moreover, vascular regression significantly decelerated in the heterozygous Ang2LacZ retinae (200% to 1 month), while the formation of acellular capillaries was significantly increased at 13 months in the wild type retinae (340% to 1 month). Gene expression analysis revealed that VEGF, Ang-1, PDGF-B and Ang2 mRNA levels were decreased in the wild type retinae at 9 month of age. However, the decrease of Ang-2 was smaller compared with other genes. While VEGF levels dropped in wild type mice up to 60% compared to 1 month, VEGF increased in heterozygous Ang-2 deficient retinae at an age of 9 months (141% to 1 month). Similarly, Ang-1 levels decreased in wild type mice (45% to 1 month), but remained stable in Ang2LacZ mice. These data suggest that Ang-2 gene dose reduction decelerates vasoregression in the retina with age. This effect links to higher levels of survival factors such as VEGF and Ang-1, suggesting that the ratio of these factors is critical for capillary cell survival.     

Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

As angiotensin (Ang) (1-7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang-(1-7) on NE neuronal uptake in spontaneously hypertensive rats. [(3)H]-NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western-blot. Ang-(1-7) lacked an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3-kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3-kinase/Akt and MEK1/2-ERK1/2 pathways in the Ang-(1-7)-stimulated NET expression. Ang-(1-7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang-(1-7) stimulation of NET expression suggesting that Ang-(1-7) stimulates NET synthesis. In fact, Ang-(1-7) increased NET mRNA levels. Thus, we evaluated the long-term effect of Ang-(1-7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang-(1-7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti-Ang-(1-7) antibody. Ang-(1-7) induces a chronic stimulatory effect on NET expression. In this way, Ang-(1-7) may regulate a pre-synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.     

Dynamic changes in serum angiopoietin-1, angiopoietin-2, and angiopoietin-2/angiopoietin-1 ratio in acute myocardial infarction patients treated with primary percutaneous coronary intervention

Context: Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) play divergent roles in myocardial ischemia and reperfusion injury.

Objective: To investigate serum Ang-1 and Ang-2 levels in ST-segment elevation myocardial infarction (STEMI) patients treated with primary percutaneous coronary intervention (PCI).

Methods: Serum Ang-1 and Ang-2 were measured in 85 STEMI patients in the first week after PCI.

Results: Ang-1, Ang-2 and Ang-2/Ang-1 ratio (Ang-2/1) were all increased at admission, and had dynamic changes after PCI. Ang-2 and Ang-2/1 at admission and 2 h after PCI were positively correlated with peak cardiac troponin T levels.

Conclusion: The extent of myocardial damage may be linked to circulating Ang-2 and Ang-2/1.     

Effect of hypertension on angiotensin-(1-7) levels in rats with different angiotensin-I converting enzyme polymorphism

To determine circulating angiotensin-(1-7) [Ang-(1,7)] levels in rats with different angiotensin converting enzyme (ACE) genotypes and to evaluate the effect of hypertension on levels of this heptapeptide, plasma levels of angiotensin II (Ang II) and Ang-(1-7) were determined by HPLC and radioimmunoassay in (a) normotensive F0 and F2 homozygous Brown Norway (BN; with high ACE) or Lewis (with low ACE) rats and (b) in hypertensive F2 homozygous male rats (Goldblatt model). Genotypes were characterized by PCR and plasma ACE activity measured by fluorimetry. Plasma ACE activity was 2-fold higher (p < 0.05) in homozygous BN compared to homozygous Lewis groups. In the Goldblatt groups, a similar degree of hypertension and left ventricular hypertrophy was observed in rats with both genotypes. Plasma Ang II levels were between 300-400% higher (p < 0.05) in the BN than in the Lewis rats, without increment in the hypertensive animals. Plasma Ang-(1-7) levels were 75-87% lower in the BN rats (p < 0.05) and they were significantly higher (p < 0.05) in the hypertensive rats from both genotypes. Plasma levels of Ang II and Ang-(1-7) levels were inversely correlated in the normotensive rats (r = -0.64; p < 0.001), but not in the hypertensive animals. We conclude that there is an inverse relationship between circulating levels of Ang II and Ang-(1-7) in rats determined by the ACE gene polymorphism. This inverse relation is due to genetically determined higher ACE activity. Besides, plasma levels of Ang-(1-7) increase in renovascular hypertension.     

Endothelium-based biomarkers are associated with cerebral malaria in Malawian children: a retrospective case-control study

Background

Differentiating cerebral malaria (CM) from other causes of serious illness in African children is problematic, owing to the non-specific nature of the clinical presentation and the high prevalence of incidental parasitaemia. CM is associated with endothelial activation. In this study we tested the hypothesis that endothelium-derived biomarkers are associated with the pathophysiology of severe malaria and may help identify children with CM.

Methods and Findings

Plasma samples were tested from children recruited with uncomplicated malaria (UM; n = 32), cerebral malaria with retinopathy (CM-R; n = 38), clinically defined CM without retinopathy (CM-N; n = 29), or non-malaria febrile illness with decreased consciousness (CNS; n = 24). Admission levels of angiopoietin-2 (Ang-2), Ang-1, soluble Tie-2 (sTie-2), von Willebrand factor (VWF), its propeptide (VWFpp), vascular endothelial growth factor (VEGF), soluble ICAM-1 (sICAM-1) and interferon-inducible protein 10 (IP-10) were measured by ELISA. Children with CM-R had significantly higher median levels of Ang-2, Ang-2:Ang-1, sTie-2, VWFpp and sICAM-1 compared to children with CM-N. Children with CM-R had significantly lower median levels of Ang-1 and higher median concentrations of Ang-2:Ang-1, sTie-2, VWF, VWFpp, VEGF and sICAM-1 compared to UM, and significantly lower median levels of Ang-1 and higher median levels of Ang-2, Ang-2:Ang-1, VWF and VWFpp compared to children with fever and altered consciousness due to other causes. Ang-1 was the best discriminator between UM and CM-R and between CNS and CM-R (areas under the ROC curve of 0.96 and 0.93, respectively). A comparison of biomarker levels in CM-R between admission and recovery showed uniform increases in Ang-1 levels, suggesting this biomarker may have utility in monitoring clinical response.

Conclusions

These results suggest that endothelial proteins are informative biomarkers of malarial disease severity. These results require validation in prospective studies to confirm that this group of biomarkers improves the diagnostic accuracy of CM from similar conditions causing fever and altered consciousness.     

Angiotensin-(1–7) attenuates collagen-induced arthritis via inhibiting oxidative stress in rats

The present study was designed to investigate the anti-rheumatic effects and the mechanism of angiotensin (Ang)-(1–7) in rat models with collagen-induced arthritis (CIA). The CIA model was established using male Wistar rats by intradermal injection of bovine collagen-II in complete Freund's adjuvant at the base of the tail. The levels of angiotensin converting enzyme 2 (ACE2)/Ang-(1–7)/Mas receptor (MasR) were reduced in CIA rats. The attenuation of paw swelling and arthritis scores and improvement of indexes of spleen and thymus were done by Ang-(1–7) injection in CIA rats. The increased levels of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the serum and hind paw were blocked by Ang-(1–7) administration. In addition, enhanced NADPH oxidase (Nox) activity, increased levels of superoxide anions and malondialdehyde (MDA), and weakened superoxide dismutase (SOD) activity, were all reversed by treatment with Ang-(1–7). Nox1 overexpression reversed the suppressing effects of Ang-(1–7) on paw swelling and arthritis scores in CIA rats. The Ang-(1–7)-induced improvement in spleen and thymus indexes in CIA rats was abolished by Nox1 overexpression. Nox1 overexpression reversed the inhibitory effects of Ang-(1–7) by increasing IL-1β, IL-6, TNF-α, and IFN-γ levels in the serum and hind paw of CIA rats. These results demonstrated that Nox1 increased the oxidative stress in arthritis, and Ang-(1–7) improved rheumatism in arthritis via inhibiting oxidative stress.

     

Role of PGI2 and effects of ACE inhibition on the bradykinin potentiation by angiotensin-(1-7) in resistance vessels of SHR

The present study determined the participation of PGI2 in the angiotensin-(1-7) [Ang-(1-7)]/bradykinin (BK) interaction, in the presence and absence of Angiotensin Converting Enzyme (ACE) inhibition, trying to correlate it with tissue levels of both peptides. The isolated mesenteric arteriolar bed of Spontaneously Hypertensive Rats (SHR) was perfused with Krebs or Krebs plus enalaprilat (10 nM), and drugs were injected alone or in association. BK (10 ng)-induced relaxation was potentiated by Ang-(1-7) (2.2 microg) in the presence or absence of enalaprilat. Ang-(1-7) receptor blockade [A-779 (4.8 microg)] did not interfere with the BK effect in preparations perfused with normal Krebs, but reversed the increased BK relaxation observed after ACE inhibition. PGI2 release by mesenteric vessels was not altered by BK or Ang-(1-7) alone, but was increased when both peptides were injected in association, in the absence or in the presence of enalaprilat. ACE inhibition caused a 2-fold increase in the BK tissue levels, and a significant decrease in the Ang-(1-7) values. We conclude that endogenous Ang-(1-7) has an important contribution to the effect of ACE inhibitors participating in the enhancement of BK response. The mechanism of Ang-(1-7) potentiating effect probably involves an increased production of PGI2. Our results suggest that a different enzymatic pathway (non-related to ACE) is involved in the local Ang-(1-7) metabolism.     

Chronic infusion of angiotensin-(1-7) reduces heart angiotensin II levels in rats

We tested the hypothesis that the actions of Angiotensin (Ang)-(1-7) in the heart could involve changes in tissue levels of Ang II. This possibility was addressed by determining the effect of chronic infusion of Ang-(1-7) on plasma and tissue angiotensins. Ang-(1-7) was infused subcutaneously (osmotic minipumps) in Wistar rats. Angiotensins were determined by radioimmunoassay (RIA) in plasma, heart, and kidney. Tissue and plasma angiotensin-converting enzyme (ACE) activity and plasma renin activity (PRA) were also measured. Cardiac and renal ACE2 mRNA levels and cardiac angiotensinogen mRNA levels were assessed by semi-quantitative polymerase chain reaction (PCR). AT1 receptor number was evaluated by autoradiograph. Chronic infusion of Ang-(1-7) (2 microg/h, 6 days) produced a marked decrease of Ang II levels in the heart. A less pronounced but significant decrease of Ang-(1-7) was also observed. No significant changes were observed for Ang I. Ang II was not altered in the kidney. In this tissue, a significant increase of Ang-(1-7) and Ang I concentration was observed. A significant increase of plasma Ang-(1-7) and Ang II was also observed. Ang-(1-7) infusion did not change ACE activity or PRA. A selective slight significant increase in ACE2 expression in the heart was observed. Heart angiotensinogen mRNA as well as the number of Ang II binding sites did not change. These results suggest that AT1 receptors-independent changes in heart Ang II concentration might contribute for the beneficial effects of Ang-(1-7) in the heart. Moreover, these results reinforce the hypothesis that this angiotensin plays an important site-specific role within the renin-angiotensin system.     

Circulating angiopoietin-2 as a biomarker in ANCA-associated vasculitis

The endothelial-specific Angiopoietin-Tie2 ligand-receptor system is an important regulator of endothelial activation. Binding of angiopoietin-2 (Ang-2) to Tie2 receptor renders the endothelial barrier responsive to pro-inflammatory cytokines. We previously showed that circulating Ang-2 correlated with disease severity in a small cohort of critically ill patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis. The current study reassessed Ang-2 as a biomarker of disease activity and relapse in AAV. Circulating Ang-2 was measured in 162 patients with severe AAV (BVAS/WG≥3, with or without glomerulonephritis) in a clinical trial. Ang-2 levels during active AAV were compared to levels in the same patients during remission (BVAS/WG = 0). Levels in clinical subsets of AAV were compared, and association with future disease course was assessed. Ang-2 levels were elevated in severe disease (median 3.0 ng/ml, interquartile range 1.9–4.4) compared to healthy controls (1.2, 0.9–1.5). However, they did not reliably decline with successful treatment (median 2.6 ng/ml, interquartile range 1.9–3.8, median change −0.1). Ang-2 correlated weakly with BVAS/WG score (r = 0.17), moderately with markers of systemic inflammation (r = 0.25–0.41), and inversely with renal function (r = −0.36). Levels were higher in patients with glomerulonephritis, but levels adjusted for renal dysfunction were no different in patients with or without glomerulonephritis. Levels were higher in patients with newly diagnosed AAV and lower in patients in whom treatment had recently been started. Ang-2 levels during active disease did not predict response to treatment, and Ang-2 levels in remission did not predict time to flare. Thus, Ang-2 appears to have limited practical value in AAV as a biomarker of disease activity at time of measurement or for predicting future activity.     

Regulation of insulin sensitivity,insulin production,and pancreatic β cell survival by angiotensin-(1-7) in a rat model of streptozotocin-induced diabetes mellitus

The aim of this study is to determine the antidiabetic activity of Ang-(1-7), an important component of the renin–angiotensin system, in a rat model of streptozotocin (STZ)-induced type 2 diabetes mellitus (DM). A total of 36 male Wistar rats were randomly divided into 3 groups: control group fed standard laboratory diet, DM group fed high-fat diet and injected with STZ, and Ang-(1-7) group receiving injection of STZ followed by Ang-(1-7) treatment. Body weight, blood glucose levels, fasting serum Ang II and insulin levels, and homeostasis model assessment of insulin resistance (HOMA-IR) were measured. The pancreas was collected for histological examination and gene expression analysis. Notably, the Ang-(1-7) group showed a significant decrease in fasting blood glucose and serum Ang II levels and HOMA-IR values and increase in fasting serum insulin levels. Pancreatic β cells in the control and Ang-(1-7) groups were normally distributed in the center of pancreatic islets with large clear nuclei. In contrast, pancreatic β cells in the DM group had a marked shrinkage of the cytoplasm and condensation of nuclear chromatin. Ang-(1-7) treatment significantly facilitated insulin production by β cells in diabetic rats. The DM-associated elevation of inducible nitric oxide synthase (iNOS), caspase-3, caspase-9, caspase-8, and Bax and reduction of Bcl-2 was significantly reversed by Ang-(1-7) treatment. Taken together, Ang-(1-7) protects against STZ-induced DM through improvement of insulin resistance, insulin secretion, and pancreatic β cell survival, which is associated with reduction of iNOS expression and alteration of the Bcl-2 family.     

Expression and regulation of ang-2 in murine ovaries during sexual maturation and development of corpus luteum

The aim of this study was to examine the expression and regulation of angiopoietin-2 (Ang-2) in murine ovaries during sexual maturation, gonadotropin treatment and luteal development by in situ hybridization and RT-PCR. By in situ hybridization Ang-2 mRNA was mainly localized in granulosa cells, thecal cells and corpus luteum, otherwise in oocytes. Moreover, Ang-2 mRNA was highly expressed in corpus luteum and granulosa cells of atretic follicles. According to RT-PCR data, Ang-2 mRNA was lowly expressed on day 10 after birth, then expression levels gradually increased and reached their highest values on day 25 after birth. In the superovulated model of immature mice, Ang-2 expression was strongly induced by equine chorionic gonadotropin (eCG) 48 h post the eCG injection, and was high from 0.5 to 13 h after hCG treatment. in situ hybridization showed that Ang-2 mRNA was highly expressed in corpus luteum from day 2 to 9 post the hCG injection, then the expression levels gradually declined on days 11 and 13 after hCG treatment. According to RT-PCR data, the levels of Ang-2 mRNA expression showed a decline after the hCG injection, with a nadir on day 3, followed by an increase, reaching the highest level on day 9 post-hCG injection. Then again Ang-2 expression gradually declined from day 11 to 15 after hCG injection. These results suggest that Ang-2 may be involved in follicular development, atresia, ovulation, and corpus luteum formation and regression.     

Angiotensin-(3-4) counteracts the Angiotensin II inhibitory action on renal Ca2+-ATPase through a cAMP/PKA pathway

We recently demonstrated that Angiotensin-(3-4) [Ang-(3-4)], an Ang II-derived dipeptide, overcomes inhibition of plasma membrane Ca(2+)-ATPase promoted by nanomolar concentrations of Ang II in basolateral membranes of renal proximal tubule cells, with involvement of a so far unknown AT(2)R-dependent and NO-independent mechanism. The present study investigates the signaling pathway triggered by Ang-(3-4) that is responsible for counteracting the inhibitory effect of Ang II, and attempts to elucidate the functional interaction of the dipeptide with Ang II at the level of AT(2)R. Stimulation by cholera toxin of G(s)α protein structurally linked to AT(2)R--as revealed by their co-immunoprecipitation--mimicked the effect of Ang-(3-4) on Ca(2+)-ATPase activity. Furthermore, addition of dibutyril-cAMP (db-cAMP) mimicked Ang-(3-4), whereas the specific PKA inhibitor, PKAi(5-24) peptide, suppressed the counter-regulatory effect of Ang-(3-4) and the AT(2)R agonist, CGP42112A. Membrane-associated PKA activity was stimulated by Ang-(3-4) or CGP42112A to comparable levels as db-cAMP, and the Ang-(3-4) effect was abrogated by the AT(2)R antagonist PD123319, whereas the AT(1)R antagonist Losartan had no effect. Ang-(3-4) stimulated PKA-mediated phosphorylation of Ca(2+)-ATPase and activated PKA to comparable levels. Binding assays demonstrated that Ang-(3-4) could not displace (3)H-Ang II from HEK 293T cells expressing AT(2)R, but 10(-10) mol/L Ang-(3-4) resulted in the appearance of a probable higher-affinity site (picomolar range) for Ang II. The results presented herein demonstrate that Ang-(3-4), acting as an allosteric enhancer, suppresses Ang II-mediated inhibition of Ca(2+)-ATPase through an AT(2)R/cAMP/PKA pathway, after inducing conformational changes in AT(2)R that results in generation of higher-affinity sites for Ang II.     

Antiangiogenic effect of Lygodium flexuosum against N-nitrosodiethylamine-induced hepatotoxicity in rats

The antiangiogenic effect of Lygodium flexuosum extract was evaluated in Wistar rats intoxicated with N-nitrosodiethylamine (NDEA) in preventive and curative models. In preventive groups, NDEA was administered for 20 weeks. Daily doses of L. flexuosumn-hexane extract (200mg/kg) started 1 week before the onset of NDEA intoxication and continued for 20 weeks. In curative animals, NDEA was administered for 20 weeks followed by treatment with the n-hexane extract of L. flexuosum for 28 days. Rats intoxicated with NDEA had elevated levels of serum gamma-GT, AST, ALT, LDH levels and hepatic MDA and decreased levels of hepatic GSH. When treated with L. flexuosum extract had normal levels of gamma-GT, AST, ALT, LDH levels, hepatic MDA and GSH. NDEA administered rat liver showed an overexpressed levels of angiopoietins 1 (Ang-1) and 2 (Ang-2) and its receptor Tie-2 mRNA. L. flexuosum extract treatment significantly (p相似文献   

Lack of Renoprotective Effect of Chronic Intravenous Angiotensin-(1-7) or Angiotensin-(2-10) in a Rat Model of Focal Segmental Glomerulosclerosis

Unopposed angiotensin (Ang) II-mediated cellular effects may lead to progressive glomerulosclerosis. While Ang-II can be locally generated in the kidneys, we previously showed that glomerular podocytes primarily convert Ang-I, the precursor of Ang-II, to Ang-(1-7) and Ang-(2-10), peptides that have been independently implicated in biological actions opposing those of Ang-II. Therefore, we hypothesized that Ang-(1-7) and Ang-(2-10) could be renoprotective in the fawn-hooded hypertensive rat, a model of focal segmental glomerulosclerosis. We evaluated the ability of 8–12 week-long intravenous administration of either Ang-(1-7) or Ang-(2-10) (100–400 ng/kg/min) to reduce glomerular injury in uni-nephrectomized fawn-hooded hypertensive rats, early or late in the disease. Vehicle-treated rats developed hypertension and lesions of focal segmental glomerulosclerosis. No reduction in glomerular damage was observed, as measured by either 24-hour urinary protein excretion or histological examination of glomerulosclerosis, upon Ang-(1-7) or Ang-(2-10) administration, regardless of peptide dose or disease stage. On the contrary, when given at 400 ng/kg/min, both peptides induced a further increase in systolic blood pressure. Content of Ang peptides was measured by parallel reaction monitoring in kidneys harvested at sacrifice. Exogenous administration of Ang-(1-7) and Ang-(2-10) did not lead to a significant increase in their corresponding intrarenal levels. However, the relative abundance of Ang-(1-7) with respect to Ang-II was increased in kidney homogenates of Ang-(1-7)-treated rats. We conclude that chronic intravenous administration of Ang-(1-7) or Ang-(2-10) does not ameliorate glomerular damage in a rat model of focal segmental glomerulosclerosis and may induce a further rise in blood pressure, potentially aggravating glomerular injury.