Endothelial cell-specific overexpression of developmental endothelial locus-1 does not influence atherosclerosis development in ApoE-/- mice.
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ABSTRACT:
SUBMITTER: Subramanian P
PROVIDER: S-EPMC5629113 | biostudies-literature | 2017 Oct
REPOSITORIES: biostudies-literature
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Endothelial cell-specific overexpression of developmental endothelial locus-1 does not influence atherosclerosis development in ApoE<sup>-/-</sup> mice.
Subramanian Pallavi P Prucnal Marta M Gercken Bettina B Economopoulou Matina M Hajishengallis George G Chavakis Triantafyllos T
Thrombosis and haemostasis 20170810 10
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Project description:The development of atherosclerotic plaques is the result of a chronic inflammatory response coordinated by stromal and immune cellular components of the vascular wall. While endothelial cells and leukocytes are well-recognised mediators of inflammation in atherosclerosis, the role of smooth muscle cells (SMCs) remains incompletely understood. Here we aimed to address the role of canonical NF-κB signalling in SMCs in the development of atherosclerosis. We investigated the role of NF-κB signalling in SMCs in atherosclerosis by employing SMC-specific ablation of NEMO, an IKK complex subunit that is essential for canonical NF-κB activation, in ApoE-/- mice. We show that SMC-specific ablation of NEMO (NEMOSMCiKO) inhibited high fat diet induced atherosclerosis in ApoE-/- mice. NEMOSMCiKO/ApoE-/- mice developed less and smaller atherosclerotic plaques, which contained fewer macrophages, decreased numbers of apoptotic cells and smaller necrotic areas and showed reduced inflammation compared to the plaques of ApoE-/- mice. In addition, the plaques of NEMOSMCiKO/ApoE-/- mice showed higher expression of α-SMA and lower expression of the transcriptional factor KLF4 compared to those of ApoE-/- mice. Consistently, in vitro, NEMO-deficient SMCs exhibited reduced proliferation and migration, as well as decreased KLF4 expression and lower production of IL-6 and MCP-1 upon inflammatory stimulus (TNF or LPS) compared to NEMO-expressing SMCs. In conclusion, NEMO-dependent activation of NF-κB signalling in SMCs critically contributes to the pathogenesis of atherosclerosis by regulating SMC proliferation, migration and phenotype switching in response to inflammatory stimuli.
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Project description:AimsVascular disease states are associated with endothelial dysfunction and increased production of reactive oxygen species derived from NADPH oxidases. However, it remains unclear whether a primary increase in superoxide production specifically in the endothelium alters the initiation or progression of atherosclerosis.Methods and resultsMice overexpressing Nox2 specifically in the endothelium (Nox2-Tg) were crossed with ApoE(-/-) mice to produce Nox2-Tg ApoE(-/-) mice and ApoE(-/-) littermates. Endothelial overexpression of Nox2 in ApoE(-/-) mice did not alter blood pressure, but significantly increased vascular superoxide production compared with ApoE(-/-) littermates, measured using both lucigenin chemiluminescence and 2-hydroxyethidium production (ApoE(-/-), 19.9 ± 6.3 vs. Nox2-Tg ApoE(-/-), 47.0 ± 7.0 nmol 2-hydroxyethidium/aorta, P< 0.05). Increased endothelial superoxide production increased endothelial levels of vascular cell adhesion protein 1 and enhanced macrophage recruitment in early lesions in the aortic roots of 9-week-old mice, indicating increased atherosclerotic plaque initiation. However, endothelial-specific Nox2 overexpression did not alter native or angiotensin II-driven atherosclerosis in either the aortic root or the descending aorta.ConclusionEndothelial-targeted Nox2 overexpression in ApoE(-/-) mice is sufficient to increase vascular superoxide production and increase macrophage recruitment possible via activation of endothelial cells. However, this initial increase in macrophage recruitment did not alter the progression of atherosclerosis. These results indicate that Nox-mediated reactive oxygen species signalling has important cell-specific and distinct temporal roles in the initiation and progression of atherosclerosis.
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Project description:Adropin, a secreted protein, coded by energy homeostasis-associated gene (Enho), is recently reported to modulate atherogenesis, with endothelial-to-mesenchymal transition (EndMT) involved in the early process. We explored whether adropin may alleviate atherosclerosis by regulating EndMT. We found that an intraperitoneal injection of adropin [105 μg/(kg·d) for 13 weeks] inhibited the progression of high-fat diet (HFD)-induced aortic atherosclerosis in apolipoprotein E-deficient mice (ApoE-/-) and those with double gene deletion (ApoE-/-/Enho-/-), as detected by Oil Red O and haematoxylin-eosin staining. In the aortas of ApoE-/- mouse, adropin treatment ameliorated the decrease in the mRNA expression of endothelial cell markers (leukocyte differentiation antigen 31, CD31, and vascular endothelial cadherin, VE-cadherin), but increased that of EndMT markers (alpha smooth muscle actin, α-SMA, and fibroblasts specific protein-1). In vitro, an adropin treatment (30 ng/ml) arrested the hydrogen peroxide (H2O2)-induced EndMT in human umbilical vein endothelial cells (HUVECs), attenuated the morphological changes of HUVECs, reduced the number of immunofluorescence-positive α-SMA, increased the mRNA and protein expressions of CD31 and VE-cadherin, and decreased those of α-SMA. Furthermore, the adropin treatment decreased the mRNA and protein expressions of transforming growth factor (TGF)-β1 and TGF-β2, and suppressed the phosphorylation of downstream signal protein Smad2/3 in HUVECs. These mitigative effects of adropin on H2O2-induced EndMT were reversed by the transfection of TGF-β plasmid. The findings signify that adropin treatment may alleviate the atherosclerosis in ApoE-/-/Enho-/- mice by inhibiting EndMT via the TGF-β/Smad2/3 signaling pathway.
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Project description:Background: Atherosclerosis (AS) is still the major cause of cardiovascular disease (CVD) as well as stroke. Endothelial metabolic disorder has been found to be activated and then promote endothelial cells (ECs) injury, which is regarded to initiate AS progression. N-acetylneuraminic acid (Neu5Ac), a metabolite produced by hexosamine-sialic acid pathway branching from glucose metabolism, was presented as a notable biomarker of CVD and is positively correlated with ECs function. However, few studies explain whether Neu5Ac regulate AS progression by affecting EC function as well as its involved mechanisms are still unknown. Methods: Here, we mimicked an animal model in ApoE-/- mice which displaying similar plasma Neu5Ac levels with AS model to investigate its effect on AS progression. Results: We found that Neu5Ac exacerbated plaques area and increased lipids in plasma in absence of HFD feeding, and ECs inflammatory injury was supposed as the triggering factor upon Neu5Ac treatment with increasing expression of IL-1β, ICAM-1, and promoting ability of monocyte adhesion to ECs. Mechanistic studies showed that Neu5Ac facilitated SLC3A2 binding to ubiquitin and then triggered P62 mediated degradation, further leading to accumulation of lipid peroxidation in ECs. Fer-1 could inhibit ECs injury and reverse AS progression induced by Neu5Ac in ApoE-/- mice. Interestingly, mitochondrial dysfunction was also partly participated in ECs injury after Neu5Ac treatment and been reversed by Fer-1. Conclusions: Together, our study unveils a new mechanism by which evaluated metabolite Neu5Ac could promote SLC3A2 associated endothelial ferroptosis to activate ECs injury and AS plaque progression, thus providing a new insight into the role of Neu5Ac-ferroptosis pathway in AS. Also, our research revealed that pharmacological inhibition of ferroptosis may provide a novel therapeutic strategy for premature AS.
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Project description:BackgroundNuclear erythroid 2-related factor 2 (Nrf2), a transcription factor, is critically involved in the regulation of oxidative stress and inflammation. However, the role of endothelial Nrf2 in atherogenesis has yet to be defined. In addition, how endothelial Nrf2 is activated and whether Nrf2 can be targeted for the prevention and treatment of atherosclerosis is not explored.MethodsRNA-sequencing and single-cell RNA sequencing analysis of mouse atherosclerotic aortas were used to identify the differentially expressed genes. In vivo endothelial cell (EC)-specific activation of Nrf2 was achieved by injecting adeno-associated viruses into ApoE-/- mice, while EC-specific knockdown of Nrf2 was generated in Cdh5CreCas9floxed-stopApoE-/- mice.ResultsEndothelial inflammation appeared as early as on day 3 after feeding of a high cholesterol diet (HCD) in ApoE-/- mice, as reflected by mRNA levels, immunostaining and global mRNA profiling, while the immunosignal of the end-product of lipid peroxidation (LPO), 4-hydroxynonenal (4-HNE), started to increase on day 10. TNF-α, 4-HNE, and erastin (LPO inducer), activated Nrf2 signaling in human ECs by increasing the mRNA and protein expression of Nrf2 target genes. Knockdown of endothelial Nrf2 resulted in augmented endothelial inflammation and LPO, and accelerated atherosclerosis in Cdh5CreCas9floxed-stopApoE-/- mice. By contrast, both EC-specific and pharmacological activation of Nrf2 inhibited endothelial inflammation, LPO, and atherogenesis.ConclusionsUpon HCD feeding in ApoE-/- mice, endothelial inflammation is an earliest event, followed by the appearance of LPO. EC-specific activation of Nrf2 inhibits atherosclerosis while EC-specific knockdown of Nrf2 results in the opposite effect. Pharmacological activators of endothelial Nrf2 may represent a novel therapeutic strategy for the treatment of atherosclerosis.
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