Welcome to the EAS 2022 Interactive Program
The congress will officially run on CET time zone (Central European Time, Milano)
Cell metabolism in endothelial cells
Genome-wide regulation of microRNAs in endothelial cells
ROLE OF PROTRUDIN IN ENDOTHELIAL CELL FUNCTION
Abstract
Background and Aims
During angiogenesis, endothelial cells form protrusive sprouts and migrate towards the angiogenic stimulus. Protrudin/ZFYVE27, an endoplasmic reticulum (ER)-anchored protein, is essential for neurite outgrowth. It also promotes invadopodia formation and invasion of breast cancer cells. However, the role of Protrudin in endothelial cell function is not known. In the present study we investigate the role of Protrudin in endothelial cell migration and angiogenesis.
Methods
Various methodologies like in vitro tube formation assay, RNA sequencing, western immunoblotting and confocal imaging were adopted to elucidate the role of Protrudin in endothelial cells.
Results
Our results demonstrate that Protrudin promotes angiogenic tube formation in primary endothelial cells, Human umbilical vein endothelial cells (HUVECs). Analysis and validation of RNA sequencing data revealed cell migration as a prominent cellular function affected in HUVECs upon Protrudin knockdown. Furthermore, knockdown of Protrudin inhibits focal adhesion kinase (FAK) activation in HUVECs and human aortic endothelial cells (HAECs). This is associated with loss of polarized FAK distribution. Protrudin inhibition results in perinuclear accumulation of mTOR and inhibits S6K activation. Furthermore, C57Bl/6J mice with global deletion of Protrudin demonstrate reduced retinal vascular progression at post-natal day 7 (n=5, *p<0.05).
Conclusions
Overall, our results demonstrate that Protrudin regulates FAK and mTOR/S6K signaling pathways in endothelial cells and provide evidence for a key role of Protrudin in angiogenesis in vitro and in vivo. As Protrudin has been previously known to promote cancer cell invasion, it will be intriguing to further study its role in tumor angiogenesis and neovascularization of arthesclerotic plaques.
ENDOTHELIAL TKK1 DEFICIENCY REDUCES ENOS MRNA LEVELS, PROMOTING ENDOTHELIAL DYSFUNCTION AND ATHEROGENESIS
Abstract
Background and Aims
Deprivation of optimal endothelial nitric oxide synthase (eNOS) expression under athero-prone conditions leads to endothelial dysfunction, however little is known about the protector of eNOS under diseased condition. The aim of this study is to identify the role of endothelial TKK1 as homeostatic regulator for vascular inflammatory disease like atherosclerosis.
Methods
The expression levels of human or murine TKK1 and eNOS were confirmed in the endothelium of aortas. Lesion formation with measurement of blood pressure and endothelial wall shear stress was evaluated in vascular or whole-body deficiency of Tkk1 in apolipoprotein e-deficient (Apoe-/-) mice. Atherosclerotic aortas or HUVECs were used for flow cytometry, analysis of inflammation-associated gene expression and various RNA sequencing (RNA-seq) for identifying mechanistic features.
Results
Human and murine TKK1 were localized to ECs in athero-prone sites and both Apoe-/-mice lacking Tkk1 throughout the body and specifically in ECs promoted plaque progression, an effect preceded by endothelial dysfunction and elevated leukocyte infiltration. Endothelial Tkk1 deletion or TKK knockdown caused an enhanced inflammatory response accompanied by decreased NO bioavailability, reflecting reduced eNOS expression under athero-prone conditions. Mechanistically, knockdown of PI3K/Akt signaling-dependent TKK expression in HUVECs decreased the NOS3 mRNA levels following up-regulation of the negative regulatory micro-RNA, miR-584 and induced Erk1/2 and NF-κB activation. Treatment of miR-584 mimetics on HUVECs decreased NOS3 mRNA stability, while miR-584 inhibitor reversed TKK knockdown effects.
Conclusions
TKK1 is a novel regulator for NOS3 mRNA expression under athero-prone conditions, and endothelial miR-584 antagonism could be a feasible therapeutic target mechanism for maintaining eNOS in inflammatory vascular disorders.
ENDOTHELIAL CANNABINOID RECEPTOR CB1 DEFICIENCY DECREASES OXLDL UPTAKE AND ATTENUATES VASCULAR INFLAMMATION IN ATHEROSCLEROSIS
Abstract
Background and Aims
The endocannabinoid system has emerged as an important lipid signaling system in atherosclerosis. Since endothelial dysfunction plays a critical role in atherosclerosis, we aimed to study the endothelial cell-specific role of CB1 in atherosclerotic plaque development.
Methods
We generated endothelial cell-specific CB1 knockout mice on ApoE-/- background and treated the mice with Western Diet (WD).
Results
Endothelial CB1 deficiency (EC-CB1-KO) attenuated plaque development in the aortic roots and abdominal aortas of female mice, accompanied by more stable plaque phenotype with increased collagen at advanced stage (16 weeks WD). However, the effect was not observed in male mice, suggesting that endothelial CB1 affects atherosclerosis in a sex-specific manner. Female EC-CB1-KO mice also had significantly lower aortic adhesion molecule ICAM-1 and VCAM-1 expression. Moreover, ex vivo imaging of perfused carotid arteries revealed less DIL-oxLDL uptake by CB1-deficient endothelium. Immunofluorescence staining revealed a significant reduction of aortic endothelial caveolin-1 (CAV-1) expression in female EC-CB1-KO mice, which might provide an explanation for reduced endothelial lipid uptake in EC-CB1-KO mice. Treatment of atherosclerotic mice with the peripheral CB1 antagonist JD-5037 reduced plaque progression, endothelial adhesion molecule and CAV-1 expression again only observed in females, but not males. Our in vivo findings are supported by ongoing mechanistic in vitro experiments using siRNA-CB1 silencing, CB1 agonists and antagonists in primary aortic endothelial cells.
Conclusions
In conclusion, our results indicate that endothelial CB1 promotes atherosclerosis by modulating endothelial oxLDL uptake and vascular inflammation, linked to a sex-specific regulation of CAV-1 expression.
ENDOTHELIAL ACKR3 DRIVES ATHEROSCLEROSIS BY PROMOTING IMMUNE CELL ADHESION TO VASCULAR ENDOTHELIUM
Abstract
Background and Aims
Genome wide association studies revealed a strong association between the CXCL12 gene locus and cardiovascular diseases, highlighting its receptors CXCR4 and atypical chemokine receptor-3 (ACKR3) as targets for CVD research. Although vascular CXCR4 is well studied in atherosclerosis, the exact role of ACKR3 remains elusive. Therefore, the aim of this project is to decipher the role of vascular ACKR3 in atherosclerosis.
Methods
Vascular endothelium (Bmx-cre) specific Ackr3 deficient mice on Apoe-/- background were fed with a cholesterol-rich diet. Lesions in the aortic roots and arches were analyzed via H&E staining. The phenotype was further characterized employing ex-vivo perfusion and intravital microscopy for cell adhesion and Evans-blue injection for endothelial permeability. Signaling studies are performed in human coronary artery endothelial cells (HCAECs).
Results
Endothelial Ackr3 deficiency resulted in significantly smaller atherosclerotic lesions, accompanied by a more stable plaque phenotypes (more lesional collagen and smooth muscle cell content, smaller necrotic cores). Endothelial-Ackr3-/- mice revealed a reduction in plaque macrophage content and ICAM positive endothelial cells in atherosclerotic lesions. Moreover, immune cell adhesion on carotid arteries lacking endothelial ACKR3 was decreased whilst endothelial permeability was not affected. ACKR3 silencing in TNF-stimulated HCAECs decreased adhesion molecule expression and downregulated MAPK signaling and NF-kB p65 phosphorylation. Endothelial cells in atherosclerotic lesions also revealed decreased phospho-NF-kB p65 expression in Ackr3 deficient mice.
Conclusions
Collectively, our findings indicate that arterial endothelial ACKR3 fuels atherosclerosis by mediating endothelium-immune cell adhesion, most likely through inflammatory MAPK and NF-kB pathways resulting in increased adhesion molecule expression.
ACETATE REVERSES THE GUT MICROBIOTA METABOLITE PHENYLACETYL GLUTAMINE (PAG)-INDUCED ENDOTHELIAL SENESCENCE BY EPIGENETIC AND SASP MODULATION
Abstract
Background and Aims
The gut microbiota metabolite PAG is clinically linked to CAD and arterial thrombosis. Yet, very little is known about the role and mechanisms of PAG in vascular cells. We sought to investigate 1) effects of PAG on metabolic/epigenetic state and senescence-associated secretory phenotype (SASP) in ECs; 2) reversal effects of acetate on PAG-induced endothelial senescence and dysfunction.
Methods
We examined the effects of PAG (100 μM; according to our preliminary studies) with or without acetate (500 μM) on cellular senescence (via SA-β-galactosidase staining) and function (via in vitro angiogenesis and migration assays), and epigenetic and SASP characterization (via biochemical assays) in proliferating human aortic EC (PEC; passage 5).
Results
We found that PAG increases mitochondrial ROS production accompanied by downregulated isocitrate dehydrogenase-2 (Idh2), a marker of mitochondrial function. PAG-treated cells revealed a significant reduction in angiogenesis and cell migration accompanied by decreased phosphorylation of eNOSS1177 and AMPKT172 and activation of acetyl-coA carboxylase (ACC) that leads to the loss of acetyl-coA, leading to decreased histone 3 (H3) acetylation (p<0.001, n=6). Importantly, PAG significantly induced senescence, as represented by increased SA-β-gal-positive cells (p<0.01), and upregulated SASP (TNF-α and VCAM-1; p<0.001). Conversely, acetate intriguingly increased phosphorylation of AMPKT172 and eNOSS1177 and H3 acetylation accompanied by reduced SA-β-gal-positive cells, downregulated SASP and improved mitochondrial function. Additionally, acetate restored angiogenesis and migration in PAG-treated cells to the magnitude seen in PEC.
Conclusions
We conclude that PAG causes endothelial dysfunction via metabolic/epigenetic and SASP modulation and that acetate represent new possibilities for rejuvenating senescent ECs and preventing aging-related CVD.