The Receptor AT1 Appears to Be Important for the Maintenance of Bone Mass and AT2 Receptor Function in Periodontal Bone Loss Appears to Be Regulated by AT1 Receptor

A large number of experimental studies has demonstrated that angiotensin II (Ang II) is involved in key events of the inflammatory process. This study aimed to evaluate the role of Ang II type 1 (AT1) and Ang II type 2 (AT2) receptors on periodontitis. Methods: Experimental periodontitis was induced by placing a 5.0 nylon thread ligature around the second upper left molar of AT1 mice, no-ligature or ligature (AT1-NL and AT1-L), AT2 (AT2-NL or AT2-L) and wild type (WT-NL or L). Alveolar bone loss was scanned using Micro-CT. Cytokines, peptides and enzymes were analyzed from gingival tissues by Elisa and RT-PCR. Results: The blockade of AT1 receptor resulted in bone loss, even in healthy animals. Ang II receptor blockades did not prevent linear bone loss. Ang II and Ang 1-7 levels were significantly increased in the AT2-L (p < 0.01) group compared to AT2-NL and AT1-L. The genic expression of the Mas receptor was significantly increased in WT-L and AT2-L compared to (WT-NL and AT2-NL, respectively) and in AT1-L. Conclusions: Our data suggest that the receptor AT1 appears to be important for the maintenance of bone mass. AT2 receptor molecular function in periodontitis appears to be regulated by AT1.

Abstract P250: A High-throughput Nitric Oxide Measurement Assay Reveals That Angiotensin-(1-5) Is An AT2 Receptor Agonist

The angiotensin AT2-receptor (AT2R) is a key component within the protective arm of the renin-angiotensin system (RAS), being involved in nitric oxide (NO) production and vasodilation. To this date, no quantitative high-throughput assay is available to identify AT2R agonists in vitro , which may be a reason for the low number of AT2R selective ligands in drug development programs. Objective: To design and validate a high-throughput method for detection of AT2R activation in vitro . Methods and Results: NO release was selected as readout for AT2R activation in AT2R transfected (CHO-AT2) and non-transfected (CHO-NT) CHO cells using DAF-FM (5x10-6 mol/L) as NO probe. Cells were seeded on 96-well plates and stimulated for 15 minutes with C21 or Ang II (established AT2R agonists, 10-6mol/L), Ang-(1-5) (molecule with unknown biological status, 10-6 mol/L) or Ang-(1-7) (Mas-receptor agonist, 10-7 mol/L). After fixation of cells, fluorescence signals were captured by fluorescence microscopy using an automated imaging system (ImageXpress Pico, Molecular Devices, San Jose, USA) and image analysis by ImageJ. In CHO-AT2, C21 (+34.78 ± 12.09%), Ang II (+28.76 ± 17.65%) and Ang-(1-5) (+78.00 ± 23.82%) increased NO release (one-way ANOVA, p < 0.05 vs control, at least 3 independent experiments), while Ang-(1-7) had no effect (+ 0.13 ± 4.74%). In CHO-NT, none of the compounds stimulated release of NO (C21 -4.91 ±10.24%; Ang II -4.79 ± 12.44%; Ang (1-5) -8.88 ± 18.16%; Ang-(1-7) -11.57 ± 19.95%) indicating that the responses in CHO-AT2 were AT2R specific. Conclusion: Measurement of NO release from AT2R transfected CHO cells by DAF-FM fluorescence in an automated way is suitable as high-throughput assay for the identification of AT2R-agonistic compounds in vitro , Application of the assay revealed that Ang-(1-5), which is commonly regarded as an inactive metabolite of Ang II, has AT2R agonistic properties.

Valsartan prevented neointimal hyperplasia and inhibited SRSF1 expression and the TLR4–iNOS–ERK–AT1 receptor pathway in the balloon-injured rat aorta

Valsartan has the potential to attenuate neointimal hyperplasia and to suppress the inflammatory response. This study aimed to evaluate the role of valsartan in neointimal hyperplasia and the toll-like receptor 4 (TLR4)-nitric oxide synthase (NOS) pathway in the balloon-injured rat aorta. Forty-eight Wistar rats were randomly allocated to three groups: sham control (control), balloon-injured group (surgery), and balloon-injured+valsartan-treated group (valsartan). Rats were killed at 14 and 28 days after balloon-injury, and then the aortic tissues were collected for morphometric analysis as well as for measurements of the mRNA or protein expression of angiotensin II, angiotensin II type 1 (AT1) receptor, angiotensin II type 2 (AT2) receptor, TLR4, endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), serine/arginine-rich splicing factor 1(SRSF1) and extracellular signal regulated kinase (ERK). Valsartan at a dose of 20 mg/kg/day markedly decreased neointimal hyperplasia in the aorta of balloon-injured rats, and significantly reduced the mRNA or protein expression of TLR4, AT1 receptor, SRSF1 and phosphorylated-ERK (p-ERK) as well as the aortic levels of iNOS (all p<0.05). Moreover, valsartan increased the eNOS level and AT2 receptor mRNA and protein expression levels (all p<0.05). Valsartan prevented neointimal hyperplasia and inhibited SRSF1 expression and the TLR4-iNOS-ERK-AT1 receptor pathway in the balloon-injured rat aorta.

Angiotensinergic Neurotransmissions in the Medial Amygdala Nucleus Modulate Behavioral Changes in the Forced Swimming Test Evoked by Acute Restraint Stress in Rats

We investigated the role of angiotensin II type 1 (AT1 receptor) and type 2 (AT2 receptor) and MAS receptors present in the medial amygdaloid nucleus (MeA) in behavioral changes in the forced swimming test (FST) evoked by acute restraint stress in male rats. For this, rats received bilateral microinjection of either the selective AT1 receptor antagonist losartan, the selective AT2 receptor antagonist PD123319, the selective MAS receptor antagonist A-779, or vehicle 10 min before a 60 min restraint session. Then, behavior in the FST was evaluated immediately after the restraint (15 min session) and 24 h later (5 min session). The behavior in the FST of a non-stressed group was also evaluated. We observed that acute restraint stress decreased immobility during both sessions of the FST in animals treated with vehicle in the MeA. The decreased immobility during the first session was inhibited by intra-MeA administration of PD123319, whereas the effect during the second session was not identified in animals treated with A-779 into the MeA. Microinjection of PD123319 into the MeA also affected the pattern of active behaviors (i.e., swimming and climbing) during the second session of the FST. Taken together, these results indicate an involvement of angiotensinergic neurotransmissions within the MeA in behavioral changes in the FST evoked by stress.

AT1 and AT2 Receptor Knockout Changed Osteonectin and Bone Density in Mice in Periodontal Inflammation Experimental Model

Background: The aim of this study was to evaluate the role of AT1 and AT2 receptors in a periodontal inflammation experimental model. Methods: Periodontal inflammation was induced by LPS/Porphyromonas gingivalis. Maxillae, femur, and vertebra were scanned using Micro-CT. Maxillae were analyzed histopathologically, immunohistochemically, and by RT-PCR. Results: The vertebra showed decreased BMD in AT1 H compared with WT H (p < 0.05). The femur showed increased Tb.Sp for AT1 H and AT2 H, p < 0.01 and p < 0.05, respectively. The Tb.N was decreased in the vertebra (WT H-AT1 H: p < 0.05; WT H-AT2 H: p < 0.05) and in the femur (WT H-AT1 H: p < 0.01; WT H-AT2 H: p < 0.05). AT1 PD increased linear bone loss (p < 0.05) and decreased osteoblast cells (p < 0.05). RANKL immunostaining was intense for AT1 PD and WT PD (p < 0.001). OPG was intense in the WT H, WT PD, and AT2 PD when compared to AT1 PD (p < 0.001). AT1 PD showed weak immunostaining for osteocalcin compared with WT H, WT PD, and AT2 PD (p < 0.001). AT1 H showed significantly stronger immunostaining for osteonectin in fibroblasts compared to AT2 H (p < 0.01). Conclusion: AT1 receptor knockout changed bone density, the quality and number of bone trabeculae, decreased the number of osteoblast cells, and increased osteonectin in fibroblasts.