Abstract 462: Expression, Phosphorylation and Interaction Partners of the Transcription Factor Grainyhead-like 3 in the Endothelium

The transcription factor Grainyhead-like 3 (GRHL3) regulates apoptosis, migration and NO-bioavailability and thus, critical functions of endothelial cell, which are impaired in many cardiovascular diseases. However, due to the lack of a good antibody, all experiments concerning the regulation of GRHL3 itself were performed on the RNA level. After establishing a new antibody, we analyzed the GRHL3 protein in aortic sections of ApoE-deficient mice fed a high fat diet, a model for atherosclerosis. The diet resulted in a reduction of GRHL3 levels in the endothelium, which was corroborated ex vivo in endothelial cells treated with LDL. This simulated high fat diet also led to a decrease in endothelial NO-synthase. As the activity of transcription factors is regulated by post-translational modifications and protein-protein interactions, we analyzed the phosphorylation of GRHL3 and identified potential interaction partners. Using a combination of immunoprecipitation and -blotting we demonstrated for the first time that GRHL3 is phosphorylated on tyrosine residues. Furthermore, this phosphorylation was NO-inducible and Src-kinase-dependent. After characterization of the modified residues, we will assess their relevance by determining the impact of phospho-mimetic and non-phosphorylatable mutants on functional parameters of endothelial cells. To identify potential interaction partners of GRHL3 we immunoprecipitated the protein and analyzed the co-precipitated proteins by mass spectrometry. We identified the DBHS-proteins NONO and SFPQ, which have been implicated in the regulation of transcription and alternative splicing. The interaction with these two proteins was validated by co-immunoprecipitation. As a next step, we will overexpress and downregulate these proteins in endothelial cells to evaluate their cross-talk with GRHL3. Taken together our findings demonstrate (i) a downregulation of GRHL3 in a disease setting, (ii) a Src-kinase dependent, NO-inducible phosphorylation and (iii) an interaction with other gene-regulatory proteins. The analysis of the functional consequences of these different aspects of GRHL3 regulation will further shed light on the GRHL3 network in the endothelium and thus, its functions in the vasculature.

Download Full-text

Mineralocorticoid and Estrogen Receptors in Endothelial Cells Coordinately Regulate Microvascular Function in Obese Female Mice

Hypertension ◽

10.1161/hypertensionaha.120.16911 ◽

2021 ◽

Author(s):

Lauren A. Biwer ◽

Brigett V. Carvajal ◽

Qing Lu ◽

Joshua J. Man ◽

Iris Z. Jaffe

Keyword(s):

Endothelial Cells ◽

High Fat Diet ◽

Ex Vivo ◽

Microvascular Dysfunction ◽

Estrogen Receptor Α ◽

No Synthase ◽

Wild Type ◽

High Fat ◽

Obese Female

Obesity impairs endothelial-mediated vasodilation, the earliest step in vascular disease and a contributor to hypertension. We previously demonstrated that endothelial cell MR (mineralocorticoid receptor) deletion prevents obesity-induced microvascular dysfunction in females by increasing nitric oxide (NO)-mediated vasodilation. ERα (Estrogen receptor α) can oppose MR function, therefore, we hypothesized that ERα mediates the benefits of endothelial MR deficiency. Females lacking endothelial MR or wild-type littermates were fed control or high-fat diet for 20 weeks to cause obesity. MR deletion improved mesenteric artery endothelial-dependent vasodilation in obese females, and ex vivo ERα inhibition negated this protective effect. Endothelial MR deletion resulted in significantly more ERα mRNA and protein. In vitro, estrogen increased endothelial NO synthase phosphorylation, and this was inhibited by aldosterone and dependent on MR. Both proteins coimmunoprecipitated with striatin and a mimetic peptide that disrupts ERα-striatin binding also decreased MR-striatin interaction. Finally, removing endothelial MR in obese females restored endothelial function by increasing the NO component of vasodilation. Combined deletion of endothelial ERα negated the benefit of endothelial MR deletion. These results indicate that endothelial ERα prevents the detrimental effects of MR in obesity by increasing NO to rescue vasodilation in females. MR and ERα may compete for striatin binding within endothelial cells to regulate NO. These data identify a novel mechanism that promotes MR antagonism to prevent obesity-induced microvascular dysfunction in females.

Download Full-text

Abstract 14050: The Gut Flora Promotes Endothelial Dysfunction Through Vascular MicroRNA-204-mediated Down-regulation of Endothelial Sirtuin1

Circulation ◽

10.1161/circ.130.suppl_2.14050 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Ajit Vikram ◽

Young-Rae Kim ◽

Santosh Kumar ◽

Julia S Jacobs ◽

Kaikobad Irani

Keyword(s):

Endothelial Cells ◽

Endothelial Dysfunction ◽

Endothelial Function ◽

High Fat Diet ◽

Ex Vivo ◽

Vascular Wall ◽

High Fat ◽

Gut Flora ◽

Atherosclerotic Vascular Disease ◽

Germ Free

The gut flora contributes to development of atherosclerosis. Endothelial dysfunction, one manifestation of which is impaired endothelium-dependent vasorelaxation, accompanies and promotes atherosclerotic vascular disease. Here we show that gut flora impair endothelium-dependent vasorelaxation by remotely up-regulating microRNA-204 (miR-204) which downregulates SIRTUIN1 (SIRT1) in the vascular wall. Microarray analysis in aortas of germ-free mice revealed a set of down-regulated microRNAs, including miR-204, which target SIRT1. Suppression of gut flora in mice with antibiotics in drinking water decreased aortic miR-204, increased aortic SIRT1, and improved endothelium-dependent vasorelaxation, effects that were reversed with discontinuation of antibiotics. In addition, miR-204 mimic impaired endothelium-dependent aortic vasorelaxation ex vivo. Moreover, high-fat diet feeding stimulated aortic miR-204, suppressed SIRT1, and impaired endothelial function, all of which were mitigated by administration of antibiotics, and reversed with stoppage of antibiotics. In contrast, antibiotics did not improve high-fat diet-induced endothelial dysfunction in mice conditionally lacking endothelial SIRT1. In addition, anti-miR-204 delivered systemically prevented high-fat diet-induced endothelial dysfunction and vascular SIRT1 decrease. Finally, serum from mice on antibiotics suppressed miR-204, and increased SIRT1, in endothelial cells, effects that were not observed with serum from mice in which antibiotics were discontinued. Therefore, the gut flora remotely downregulates endothelial SIRT1 through miR-204, leading to impairment of endothelial function.

Download Full-text

The impact of High Fat Diet on bone: potential mechanisms

Food & Function ◽

10.1039/d0fo02664f ◽

2021 ◽

Author(s):

Qiao Jie ◽

Yue-Zhong Ren ◽

Yi-wen Wu

Keyword(s):

Total Energy ◽

High Fat Diet ◽

High Fat ◽

High Fat Diets ◽

Fat Diets ◽

The Impact

High-fat diets(HFD)are defined as lipids accounting for exceeded 30% of total energy in-take, and current research is mostly 45% and 60%. With a view of the tendency that patients who...

Download Full-text