Abstract P075: Angiotensin II Induces Endothelial Dysfunction And Vascular Remodeling Dependent Of Nlrp3 Inflammasome
Stêfany B. A. CauMarcondes da SilvaNathanne dos Santos FerreiraRita C. TostesThiago Bruder‐Nascimento
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The NLRP3 inflammasome is a multimeric protein complex constituted by NLRP3, Asc and Capase-1 (Casp1). It triggers an inflammatory response by releasing the pro-inflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome is expressed in different cells and its activation has been associated with several diseases including atherosclerosis and hypertension. Herein we tested the hypothesis that angiotensin II (AngII) induces vascular damage by activating the NLPR3 inflammasome in the vasculature. C57BL/6J male mice (Ctrl) and Casp-1 deficient mice (Casp1-/-) were treated with AngII (490 ng/min/kg/14 days by osmotic mini pump). In Ctrl mice, AngII treatment impaired the vascular relaxation to acetylcholine in mesenteric arteries, increased aorta media thickness [Ctrl: 49.4 ± 2.5 vs AngII: 62.3 ± 2.3* (μm), *P<0.05] and cross-sectional area [Ctrl: 0.11 ± 0.1 vs AngII: 0.15 ± 0.2* (mm), *P<0.05] and triggered NLRP3 inflammasome activation in aorta and mesenteric arteries, analyzed by caspase-1 cleavage and IL-1B maturation via western blot and casp1 activity - FAM-FLICA assay. Fascinatingly, Casp1-/- mice were protected from AngII-induced endothelial dysfunction and vascular remodeling. Furthermore, AngII (0.1uM) incubation, combined or not with lipopolysaccharide (500 ng.ml –1 ultrapure) or Nigericin (20 μM), elevated Casp1 cleavage and IL-1B maturation in Rat Aortic Smooth Muscle Cells (RASMC). Moreover, AngII elevated PCNA (~2.5-fold) and CyclinD1 (~2.1-fold) protein expression and induced vascular migration and proliferation measured by scratch assay and cell counting kit-8 (CCK-8) assay respectively. Interestingly NLRP3 antagonist incubation (MCC950, 1uM) abolished PCNA expression and attenuated the vascular migration and proliferation produced by AngII incubation. Our data suggest that AngII induces vascular damage by activating NLPR3 inflammasome directly in the vasculature. We place this innate immune receptor as a master regulator of the vascular phenotype and as a target for therapeutic strategies for vascular diseases. Future studies will be helpful providing a better understanding into the molecular mechanism of NLRP3 inflammasome activation and regulation in the control of vascular diseases.Keywords:
Mesenteric arteries
Angiotensin (Ang) II plays vital roles in vascular inflammation and remodeling in hypertension. Phenotypic transformation of vascular smooth muscle cells (VSMCs) is a major initiating factor for vascular remodeling. The present study was designed to determine the roles of NLRP3 inflammasome activation in Ang II-induced VSMC phenotypic transformation and vascular remodeling in hypertension.Primary VSMCs from the aorta of NLRP3 knockout (NLRP3-/-) mice and wild-type (WT) mice were treated with Ang II for 24 h. Subcutaneous infusion of Ang II via osmotic minipump for 2 weeks was used to induce vascular remodeling and hypertension in WT and NLRP3-/- mice.NLRP3 gene deletion attenuates Ang II-induced NLRP3 inflammasome activation, phenotypic transformation from a contractile phenotype to a synthetic phenotype and proliferation in primary mice VSMCs. Ang II-induced hypertension and vascular remodeling in WT mice were attenuated in NLRP3-/- mice. Furthermore, Ang II-induced NLRP3 inflammasome activation, phenotypic transformation and proliferating cell nuclear antigen (PCNA) upregulation were inhibited in the media of aorta of NLRP3-/- mice.NLRP3 inflammasome activation contributes to Ang II-induced VSMC phenotypic transformation and proliferation as well as vascular remodeling and hypertension.
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Angiotensin II is a major effector molecule in the development of cardiovascular disease. In vascular smooth muscle cells (VSMCs), angiotensin II promotes cellular proliferation and extracellular matrix accumulation through the upregulation of plasminogen activator inhibitor-1 (PAI-1) expression. Previously, we demonstrated that small heterodimer partner (SHP) represses PAI-1 expression in the liver through the inhibition of TGF-β signaling pathways. Here, we investigated whether SHP inhibited angiotensin II-stimulated PAI-1 expression in VSMCs. Adenovirus-mediated overexpression of SHP (Ad-SHP) in VSMCs inhibited angiotensin II- and TGF-β-stimulated PAI-1 expression. Ad-SHP also inhibited angiotensin II-, TGF-β- and Smad3-stimulated PAI-1 promoter activity, and angiotensin II-stimulated AP-1 activity. The level of PAI-1 expression was significantly higher in VSMCs of SHP-/- mice than wild type mice. Moreover, loss of SHP increased PAI-1 mRNA expression after angiotensin II treatment. These results suggest that SHP inhibits PAI-1 expression in VSMCs through the suppression of TGF-β/Smad3 and AP-1 activity. Thus, agents that target the induction of SHP expression in VSMCs might help prevent the development and progression of atherosclerosis.
Small heterodimer partner
Plasminogen activator inhibitor-1
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Restenosis after vessel angioplasty due to dedifferentiation of the vascular smooth muscle cells (VSMCs) limits the success of surgical treatment of vascular occlusions. Type 2 diabetes (T2DM) has a major impact on restenosis, with patients exhibiting more aggressive forms of vascular disease and poorer outcomes after surgery. Kv1.3 channels are critical players in VSMC proliferation. Kv1.3 blockers inhibit VSMCs MEK/ERK signalling and prevent vessel restenosis. We hypothesize that dysregulation of microRNAs (miR) play critical roles in adverse remodelling, contributing to Kv1.3 blockers efficacy in T2DM VSMCs.We used clinically relevant in vivo models of vascular risk factors (VRF) and vessels and VSMCs from T2DM patients.Human T2DM vessels showed increased remodelling, and changes persisted in culture, with augmented VSMCs migration and proliferation. Moreover, there were downregulation of PI3K/AKT/mTOR and upregulation of MEK/ERK pathways, with increased miR-126 expression. The inhibitory effects of Kv1.3 blockers on remodelling were significantly enhanced in T2DM VSMCs and in VRF model. Finally, miR-126 overexpression confered "diabetic" phenotype to non-T2DM VSMCs by downregulating PI3K/AKT axis.miR-126 plays crucial roles in T2DM VSMC metabolic memory through activation of MEK/ERK pathway, enhancing the efficacy of Kv1.3 blockers in the prevention of restenosis in T2DM patients.
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Vascular peroxidase 1 (VPO1) is a newly identified haem-containing peroxidase that catalyses the oxidation of a variety of substrates by hydrogen peroxide (H(2)O(2)). Considering the well-defined effects of H(2)O(2) on the vascular remodelling during hypertension, and that VPO1 can utilize H(2)O(2) generated from co-expressed NADPH oxidases to catalyse peroxidative reactions, the aims of this study were to determine the potential role of VPO1 in vascular remodelling during hypertension.The vascular morphology and the expression of VPO1 in arterial tissues of spontaneously hypertensive rats and Wistar-Kyoto rats were assessed. The VPO1 expression was significantly increased concomitantly with definite vascular remodelling assessed by evaluating the media thickness, lumen diameter, media thickness-to-lumen diameter ratio and mean nuclear area in artery media in spontaneously hypertensive rats. In addition, in cultured rat aortic smooth muscle cells we found that the angiotensin II-mediated cell proliferation was inhibited by knockdown of VPO1 using small hairpin RNA. Moreover, the NADPH oxidase inhibitor, apocynin, and the hydrogen peroxide scavenger, catalase, but not the ERK1/2 inhibitor, PD98059, attenuated angiotensin II-mediated up-regulation of VPO1 and generation of hypochlorous acid.VPO1 is a novel regulator of vascular smooth muscle cell proliferation via NADPH oxidase-H(2)O(2)-VPO1-hypochlorous acid-ERK1/2 pathways, which may contribute to vascular remodelling in hypertension.
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In healthy blood vessels, vascular smooth muscle cells (VSMCs) exist in a contractile, quiescent state but can switch phenotype to activate proliferation, migration and remodelling of the extracellular matrix. Phenotypically switched VSMCs contribute most cells within neointimal lesions, characteristic of atherosclerosis and in-stent restenosis, diseases that underlie heart attack and stroke. Using multicolour 'Confetti' VSMC-specific lineage tracing in animal models of vascular disease, we showed that the extensive VSMC contribution to these lesions results from the clonal expansion of few cells. To understand how oligoclonal VSMC lesion contribution arises and to identify the signals activating VSMC proliferation in vivo, we used confocal microscopy to quantify VSMC clonal development over time in two models of vascular disease. We observed that the number and sizes of patches of clonally expanded VSMCs steadily increased, then plateaued post-injury. This suggests VSMC investment results from activation of a small number of VSMCs, rather than clonal competition following general VSMC activation. Selective VSMC activation in plaques was evidenced by the absence of plaques with high numbers of colours at any stage of plaque development. In both models, VSMC activation was associated with vascular regions displaying elastic lamina alterations, medial acellularity and immune cell recruitment, implicating these as proliferation-triggering cues. However, not all VSMCs in these regions formed patches, suggesting that VSMCs must be primed to respond. In culture, few VSMCs gave rise to patches, suggesting cell-autonomous activation. This work supports the targeting of primed VSMCs in the healthy vessel as a therapeutic strategy against vascular lesion development.
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Abstract Hypertension is a common type of cardiovascular disease that remains a major cause of death in the world. Vascular remodelling is an important complication of hypertension, and vascular smooth muscle cells (VSMCs) play a major role in vascular remodelling. Sauchinone is one of the active lignins which has been found to possess vascular protective effects. However, the functional role of sauchinone in hypertension has not been investigated. The aim of this study was to evaluate the role of sauchinone in the angiotensin II (Ang II)‐induced vascular remodelling model in VSMCs. The results showed that treatment of sauchinone inhibited Ang II‐induced VSMCs proliferation and migration in VSMCs. Sauchinone treatment suppressed the reactive oxygen species (ROS) production and NADPH oxidase (NOX) activity in Ang II‐induced VSMCs. The inhibitory effects of Ang II on expressions of VSMCs phenotype markers including α‐smooth muscle actin (α‐SMA), calponin, osteopontin were mitigated by sauchinone treatment. Furthermore, sauchinone inhibited Ang II‐induced over‐activation of TGF‐β1/Smad3 signalling pathway in VSMCs. Taken together, this study identified sauchinone as a potential agent for preventing vascular remodelling in hypertension.
Osteopontin
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AIM: To determine the effects of Angiotensin II(AngII) on migration of rat smooth muscle cells and to investigate the mechanisms underlying Ang II action in the development of injured vascular disease. METHODS: VSMCs isolated from aortic media of Wistar rats and cultured by the modified explant method were adopted. In prersence and absence of AngII, the expression of AngII receptor and reorganization of the actin cytoskeleton of VSMCs were studied by immunocytochemistry technique, fluorocytochemistry technique. The migration assays were performed by a modified Boyden's chamber. And the effects of AT 1R antagonist (CV-11974), AT 2R antagonist (PD123319) on aforementioned target were studied. RESULTS: VSMCs migration was stimulated by addition of AngII. The dynamic reorganization of actin cytoskeleton may be an important mechanism by which AngII facilitates VSMC motility. The expression of AT 1R in VSMCs can be upregulated after treatment with AngII initially, then decreased gradually. The expression of AT 1R was downregulated by AT 1R antagonist. The effect of AngII on VSMCs migration was mediated by AT 1R, while AT 2R had no significant effect. CONCLUSION: The dynamic reorganization of actin cytoskeleton is required for AngII-induced VSMC migration, and this effect is mediated by AT 1R .
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To determine the biotic effects of angiotensin II (Ang II) on the migration of rat smooth muscle cells (VSMCs) and investigate the mechanisms involved in the development of vascular injury.VSMCs isolated from aortic media of Wistar rats and cultured by the modified explant method were adopted. In the presence and absence of Ang II, the expression of Ang II receptor (ATR) and reorganization of the actin cytoskeleton and focal adhesion of VSMCs were studied by an immunocytochemistry technique and fluorocytochemistry technique. Migration assays were performed with a modified Boyden's chamber. The effects of AT(1)R antagonist (CV-11974), AT(2)R antagonist (PD123319) on the aforementioned target were studied.VSMCs migration was stimulated by adding Ang II. The dynamic reorganization of actin cytoskeleton and focal adhesions may be an important mechanism by which Ang II facilitates VSMCs motility. The expression of AT(1)R in VSMCs could be upregulated initially after treatment with Ang II, then decreased gradually. The expression of AT(1)R was downregulated by AT(1)R antagonists. The effect of Ang II on VSMCs migration was mediated by AT(1)R, while AT(2)R had no significant effect.The dynamic reorganization of focal adhesions and the actin cytoskeleton is required for Ang II-induced VSMCs migration. This effect is mediated by AT(1)R.
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