Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis.
Introduction Endothelial inflammation plays a pivotal role in atherosclerosis. Many inflammatory and metabolic signals converge upon mechanistic target of rapamycin (mTOR), and inhibition of mTOR has been shown to reduce atherosclerosis. However, clinical use of mTOR-inhibitors is limited by serious adverse effects, of which insulin resistance and dyslipidemia are particularly troubling in the context of atherosclerosis. In that respect, targeting PRAS40, an endogenous modulator of mTOR complex 1 (mTORC1) with highly cell type-specific effects on mTOR signaling, may be a more promising approach. In fact, we have previously demonstrated that, in contrast to conventional mTOR inhibitors, PRAS40 gene therapy substantially improves metabolic profile in obese mice. However, the function of PRAS40 in endothelial cells and its role in atherosclerosis have never been investigated. Methods and results To define the impact of PRAS40 on endothelial mTORC1-signaling in this context, cultured human umbilical vein endothelial cells (HUVECs) were exposed to the atherogenic cytokine TNFα. TNFα induced mTOR signaling as evidenced by increased phosphorylation of S6 kinase and ribosomal S6 protein. Interestingly, this effect was strongly augmented upon siRNA-mediated knock-down of PRAS40, indicating a negative regulation of mTORC1 by PRAS40 in endothelial cells. Moreover, PRAS40-knockdown promoted TNFα-induced inflammatory signaling as reflected by increased proliferative activity, upregulation of atherogenic markers like CCL2 and VCAM-1, as well as enhanced monocyte recruitment in the THP-1 adhesion assay. In contrast, PRAS40-overexpression blocked TNFα-induced activation of mTORC1 and consistently suppressed all of these measures of inflammatory activation. All effects of PRAS40-overexpression could be reproduced by the mTORC1 inhibitors rapamycin and torin1. Thus, our in vitro studies suggest that in endothelial cells PRAS40 exerts anti-atherogenic effects by negative regulation of mTORC1. To validate these findings in vivo in the context of atherosclerosis we created transgenic mice with tamoxifen-inducible endothelium-specific PRAS40-deficiency (EC-PRAS40-KO). These mice were exposed to a model of accelerated atherosclerosis based on western diet and partial carotid ligation: Four weeks after partial carotid ligation, neointimal and atherosclerotic lesion formation was strongly enhanced in EC-PRAS40-KO mice. Moreover, mTORC1 activity as well as CCL2 and VCAM-1 expression were markedly increased compared to control mice. Conclusion Our data indicate that PRAS40 suppresses atherosclerosis via inhibition of mTORC1-mediated inflammatory signaling in endothelial cells. In conjunction with its favourable effects on metabolic homeostasis, the overall therapeutic profile of PRAS40-treatment appears to be beneficial compared to conventional mTOR-inhibitors. Taken together PRAS40 may qualify as a promising therapeutic target for the treatment of atherosclerosis. Acknowledgement/Funding German Federal Ministry of Education and Research, DZHK (German Centre for Cardiovascular Research)
Introduction Antagonists of the angiotensin II receptor type 1 (AT1) belong to the most successful treatments of cardiovascular disease. Besides G-protein-independent β-arrestin-mediated signaling, AT1 preferencially signals via G-proteins of the families Gq and G13. Conventional AT1-antagonists equally inhibit all of these pathways, even though the beneficial effects are generally attributed to the inhibition of Gq only. While data on β-arrestin signaling in the vascular disease context are conflicting, we found an excessive pathological remodeling and dramatic exacerbation of atherosclerosis upon interference with G13-mediated signaling. Against this background, selective inhibition of the Gq-pathway seems desirable. Thus, based on the novel concept of ligand-biased signaling, we aimed to identify biased AT1-antagonists that selectively inhibit the Gq-pathway, while allowing for continued signaling through G13. Methods and results We systematically profiled known AT1-ligands and identified the angiotensin analogue Sar-Arg-Val-Tyr-Lys-His-Pro-Ala-OH (TRV023) as a Gq-selective AT1-antagonist: TRV023 has recently been described as a β-arrestin-biased ligand; however its signaling properties regarding distinct G-protein isoforms have never been defined. Indeed, using bioluminescence reporter assays, we found that TRV023 inhibits Gq-signaling at least as potently as losartan and telmisartan, but unlike the conventional antagonists, it does not impair β-arrestin-signaling (Fig. 1A). Intriguingly, while the conventional antagonists effectively inhibited induction of a G13 luciferase reporter, TRV023 did not significantly interfere with G13-mediated signaling, indicating Gq-selective antagonistic effects. According to our hypothesis, these G-protein-biased signaling properties could render TRV023 superior to conventional antagonists in the treatment of vascular disease. In order to test this hypothesis, we exposed ApoE-deficient mice to one of two different atherosclerosis models: A novel model for accelerated atherosclosis based on partial carotid ligation and western diet, or a conventional atherosclerosis model based on 12 weeks of western diet. Using osmotic pumps, mice were continuously treated with vehicle control, TRV023 or losartan in doses that proved equipotent with respect to blood pressure lowering effects. In line with our hypothesis, TRV023 but not losartan significantly attenuated plaque development in both atherosclerosis models (Fig. 1B). Figure 1 Conclusion Our studies have identified TRV023 as a Gq-biased AT1-antagonist and indicate that selective inhibition of AT1-dependent Gq-mediated signaling by biased ligands may be a promising approach for the treatment of atherosclerosis. Hence, TRV023 qualifies as a drug candidate, particularly since it has already proven to be safe and well-tolerable in clinical phase I and II trials in the context of acute heart failure. Acknowledgement/Funding Else Kröner-Fresenius-Foundation
Aortic valve stenosis (AVS) is the most frequent valvular heart disease in industrialized countries, presenting with very high mortality if left untreated. While drug treatment can sometimes alleviate symptoms, it fails to stop progression or cure the underlying disease. Until the first decade of this millennium, surgical aortic valve replacement (SAVR) remained the only available therapy option with a positive impact on mortality and morbidity. Even though several studies reported highly positive effects of SAVR regarding the improved quality of life and better physical performance, SAVR remained an intervention that, due to its remarkable complexity and the need for heart-lung machine and cardioplegia, was limited by the patients' comorbid profile. While unsatisfying hemodynamic results after transcatheter aortic balloon valvuloplasty in high-risk surgical patients limited its adoption as an alternative treatment, it provided the impetus for further interventional approaches to the therapy of AVS. This review considers the invention and development of transcatheter aortic valve implantation (TAVI), which established itself as a catheter-based, minimally invasive procedure over the past decade, and has become an equivalent treatment method for high-risk surgical patients. For that matter, early TAVI concepts, their amendments, and the associated pioneers are recognized for paving the way to a revolutionary diversification in AVS treatment.
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