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Inhibition of endothelial Nox2 activation by LMH001 protects mice from angiotensin II-induced vascular oxidative stress, hypertension and aortic aneurysm.

ABSTRACT: Endothelial oxidative stress and inflammation attributable to the activation of a Nox2-NADPH oxidase are key features of many cardiovascular diseases. Here, we report a novel small chemical compound (LMH001, MW = 290.079), by blocking phosphorylated p47^phox interaction with p22^phox, inhibited effectively angiotensin II (AngII)-induced endothelial Nox2 activation and superoxide production at a small dose (IC50 = 0.25 μM) without effect on peripheral leucocyte oxidative response to pathogens. The therapeutic potential of LMH001 was tested using a mouse model (C57BL/6J, 7-month-old) of AngII infusion (0.8 mg/kg/d, 14 days)-induced vascular oxidative stress, hypertension and aortic aneurysm. Age-matched littermates of p47^phox knockout mice were used as controls of Nox2 inhibition. LMH001 (2.5 mg/kg/d, ip. once) showed no effect on control mice, but inhibited completely AngII infusion-induced excess ROS production in vital organs, hypertension, aortic walls inflammation and reduced incidences of aortic aneurysm. LMH001 effects on reducing vascular oxidative stress was due to its inhibition of Nox2 activation and was abrogated by knockout of p47^phox. LMH001 has the potential to be developed as a novel drug candidate to treat oxidative stress-related cardiovascular diseases.

SUBMITTER: Fan LM

PROVIDER: S-EPMC8908273 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Inhibition of endothelial Nox2 activation by LMH001 protects mice from angiotensin II-induced vascular oxidative stress, hypertension and aortic aneurysm.

Fan Lampson M LM Liu Fangfei F Du Junjie J Geng Li L Li Jian-Mei JM

Redox biology 20220303

Endothelial oxidative stress and inflammation attributable to the activation of a Nox2-NADPH oxidase are key features of many cardiovascular diseases. Here, we report a novel small chemical compound (LMH001, MW = 290.079), by blocking phosphorylated p47<sup>phox</sup> interaction with p22<sup>phox</sup>, inhibited effectively angiotensin II (AngII)-induced endothelial Nox2 activation and superoxide production at a small dose (IC50 = 0.25 μM) without effect on peripheral leucocyte oxidative respo ...[more]

PMID: 35276443

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Project description:AimsAngiotensin II (AngII)-induced superoxide (O2(•-)) production by the NADPH oxidases and mitochondria has been implicated in the pathogenesis of endothelial dysfunction and hypertension. In this work, we investigated the specific molecular mechanisms responsible for the stimulation of mitochondrial O2(•-) and its downstream targets using cultured human aortic endothelial cells and a mouse model of AngII-induced hypertension.ResultsWestern blot analysis showed that Nox2 and Nox4 were present in the cytoplasm but not in the mitochondria. Depletion of Nox2, but not Nox1, Nox4, or Nox5, using siRNA inhibits AngII-induced O2(•-) production in both mitochondria and cytoplasm. Nox2 depletion in gp91phox knockout mice inhibited AngII-induced cellular and mitochondrial O2(•-) and attenuated hypertension. Inhibition of mitochondrial reverse electron transfer with malonate, malate, or rotenone attenuated AngII-induced cytoplasmic and mitochondrial O2(•-) production. Inhibition of the mitochondrial ATP-sensitive potassium channel (mitoK(+)ATP) with 5-hydroxydecanoic acid or specific PKCɛ peptide antagonist (EAVSLKPT) reduced AngII-induced H2O2 in isolated mitochondria and diminished cytoplasmic O2(•-). The mitoK(+)ATP agonist diazoxide increased mitochondrial O2(•-), cytoplasmic c-Src phosphorylation and cytoplasmic O2(•-) suggesting feed-forward regulation of cellular O2(•-) by mitochondrial reactive oxygen species (ROS). Treatment of AngII-infused mice with malate reduced blood pressure and enhanced the antihypertensive effect of mitoTEMPO. Mitochondria-targeted H2O2 scavenger mitoEbselen attenuated redox-dependent c-Src and inhibited AngII-induced cellular O2(•-), diminished aortic H2O2, and reduced blood pressure in hypertensive mice.Innovation and conclusionsThese studies show that Nox2 stimulates mitochondrial ROS by activating reverse electron transfer and both mitochondrial O2(•-) and reverse electron transfer may represent new pharmacological targets for the treatment of hypertension.

Project description:ObjectiveThe superoxide-generating Nox2 (NADPH oxidase-2) is expressed in multiple cell types. Previous studies demonstrated distinct roles for cardiomyocyte, endothelial cell, and leukocyte cell Nox2 in ANG II (angiotensin II)-induced cardiovascular remodeling. However, the in vivo role of fibroblast Nox2 remains unclear. Approach and Results: We developed a novel mouse model with inducible fibroblast-specific deficiency of Nox2 (fibroblast-specific Nox2 knockout or Fibro-Nox2KO mice) and investigated the responses to chronic ANG II stimulation. Fibro-Nox2KO mice showed no differences in basal blood pressure or vessel wall morphology, but the hypertensive response to ANG II infusion (1.1 mg/[kg·day] for 14 days) was substantially reduced as compared to control Nox2-Flox littermates. This was accompanied by a significant attenuation of aortic and resistance vessel remodeling. The conditioned medium of ANG II-stimulated primary fibroblasts induced a significant increase in vascular smooth muscle cell growth, which was inhibited by the short hairpin RNA (shRNA)-mediated knockdown of fibroblast Nox2. Mass spectrometric analysis of the secretome of ANG II-treated primary fibroblasts identified GDF6 (growth differentiation factor 6) as a potential growth factor that may be involved in these effects. Recombinant GDF6 induced a concentration-dependent increase in vascular smooth muscle cell growth while chronic ANG II infusion in vivo significantly increased aortic GDF6 protein levels in control mice but not Fibro-Nox2KO animals. Finally, silencing GDF6 in fibroblasts prevented the induction of vascular smooth muscle cell growth by fibroblast-conditioned media in vitro.ConclusionsThese results indicate that fibroblast Nox2 plays a crucial role in the development of ANG II-induced vascular remodeling and hypertension in vivo. Mechanistically, fibroblast Nox2 may regulate paracrine signaling to medial vascular smooth muscle cells via factors, such as GDF6.

Dataset Information

Inhibition of endothelial Nox2 activation by LMH001 protects mice from angiotensin II-induced vascular oxidative stress, hypertension and aortic aneurysm.

Publications

Inhibition of endothelial Nox2 activation by LMH001 protects mice from angiotensin II-induced vascular oxidative stress, hypertension and aortic aneurysm.

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