isolation-of-mouse-pancreatic-endothelial-cells

Isolation of Mouse Pancreatic Endothelial Cells

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and ...

City of Hope

SHA is an l-rhamnose- and d-galactose-binding lectin that agglutinates human group B erythrocytes and was first purified almost 50 years ago. Although the original SHA-producing  strain was lost, the primary structure of SHA was more recently solved by mass spectrometry of the archived protein, which matched it to a similar sequence in the Streptomyces lavendulae genome. Using genomic and protein biochemical analyses, this study aimed to identify SHA-secreting  strains to further investigate the expression and binding activities of these putative proteins. Of 67 strains genetically related to , 17 secreted pro-SHAs in culture. Seven SHA homologues were purified to homogeneity and then subjected to liquid chromatography-high-resolution multistage mass spectrometry (LC-MS/MS) and hemagglutination (HA) assays. Processing of pro-SHAs occurred during and after purification, indicating that associated proteases converted pro-SHAs into mature SHAs with molecular masses and HA activities similar to that of the archived SHA. Previously, the SHA monomer was shown to have two carbohydrate binding sites. The present study, however, found no HA activity in pro-SHAs, suggesting that pro-SHAs have only one binding site. Genetically, the SHA gene resides in conserved syntenic regions. The published genomes of 1,234  strains were analyzed, revealing 18 strains with SHA genes, 16 of which localized to a unique syntenic region. The SHA syntenic region consists of ∼17 open reading frames (ORFs) and is specific to -related strains. Notably, a lipoprotein gene excludes SHA from the synteny in some strains, suggesting that horizontal gene transfer events during the course of evolution shaped the distribution of SHA genes.  Lectins are extremely useful molecules for the study of glycans and carbohydrates. Here, we show that homologous genes encoding the l-rhamnose- and d-galactose-binding lectins, SHAs, are present in multiple bacterial strains, genetically related to Streptomyces lavendulae. SHA genes are expressed as precursor pro-SHA proteins that are truncated and mature into fully active lectins with two carbohydrate binding sites, which exhibit hemagglutination activity for type B red blood cells. The SHA gene is located within a conserved syntenic region, hinting at specific but yet-to-be-discovered biological roles of this carbohydrate-binding protein for its soil-dwelling microbial producer.

Proteolytic Processing, Maturation, and Unique Synteny of the  Hemagglutinin SHA.

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bβ but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.

Phosphorylation of human CEACAM1-LF by PKA and GSK3β promotes its interaction with β-catenin.

Vascular endothelial cells (ECs) play a pivotal role in whole body homeostasis. Recent advances have revealed enhancer-associated long non-coding RNAs (lncRNAs) as essential regulators in EC function. We investigated LINC00607, a super enhancer-derived lncRNA (SE-lncRNA) in human arteries with an emphasis on ECs. Based on public databases and our single cell RNA-sequencing (scRNA-seq) data from human arteries collected from healthy and diabetic donors, we found that LINC00607 is abundantly expressed in the arteries and its level is increased in diabetic humans. Using RNA-sequencing, we characterized the transcriptomes regulated by LINC00607 in ECs and vascular smooth muscle cells (VSMCs) and in basal and diabetic conditions in ECs. Furthermore, through transcriptomic and promoter analysis, we identified c-Myc as an upstream transcription factor of LINC00607. Finally, using scRNA-seq, we demonstrated that modified antisense oligonucleotide inhibitor of LINC00607 can reverse dysfunctional changes induced by high glucose and TNFα in ECs. Collectively, our study demonstrates a multi-pronged approach to characterize LINC00607 in vascular cells and its gene regulatory networks in ECs and VSMCs. Our findings provide new insights into the regulation and function of SE-derived lncRNAs in both vascular homeostasis and dysfunction in a cell-type and context-dependent manner, which could have a significant impact on our understanding of epigenetic regulation implicated in cardiovascular health and diseases like diabetes.

Vascular Regulation by Super Enhancer-Derived LINC00607.

Impaired angiogenesis in diabetes is a key process contributing to ischemic diseases such as peripheral arterial disease. Epigenetic mechanisms, including those mediated by long noncoding RNAs (lncRNAs), are crucial links connecting diabetes and the related chronic tissue ischemia. Here we identify the lncRNA that enhances endothelial nitric oxide synthase (eNOS) expression (LEENE) as a regulator of angiogenesis and ischemic response. LEENE expression was decreased in diabetic conditions in cultured endothelial cells (ECs), mouse hind limb muscles, and human arteries. Inhibition of LEENE in human microvascular ECs reduced their angiogenic capacity with a dysregulated angiogenic gene program. Diabetic mice deficient in Leene demonstrated impaired angiogenesis and perfusion following hind limb ischemia. Importantly, overexpression of human LEENE rescued the impaired ischemic response in Leene-knockout mice at tissue functional and single-cell transcriptomic levels. Mechanistically, LEENE RNA promoted transcription of proangiogenic genes in ECs, such as KDR (encoding VEGFR2) and NOS3 (encoding eNOS), potentially by interacting with LEO1, a key component of the RNA polymerase II-associated factor complex and MYC, a crucial transcription factor for angiogenesis. Taken together, our findings demonstrate an essential role for LEENE in the regulation of angiogenesis and tissue perfusion. Functional enhancement of LEENE to restore angiogenesis for tissue repair and regeneration may represent a potential strategy to tackle ischemic vascular diseases.

Long noncoding RNA LEENE promotes angiogenesis and ischemic recovery in diabetes models.

The increasing prevalence of the metabolic syndrome (MetS) is a threat to global public health due to its lethal complications. Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the MetS characterized by hepatic steatosis, which is potentially progressive to the inflammatory and fibrotic nonalcoholic steatohepatitis (NASH). The adipose tissue (AT) is also a major metabolic organ responsible for the regulation of whole-body energy homeostasis, and thereby highly involved in the pathogenesis of the MetS. Recent studies suggest that endothelial cells (ECs) in the liver and AT are not just inert conduits but also crucial mediators in various biological processes via the interaction with other cell types in the microenvironment both under physiological and pathological conditions. Herein, we highlight the current knowledge of the role of the specialized liver sinusoidal endothelial cells (LSECs) in NAFLD pathophysiology. Next, we discuss the processes through which AT EC dysfunction leads to MetS progression, with a focus on inflammation and angiogenesis in the AT as well as on endothelial-to-mesenchymal transition of AT-ECs. In addition, we touch upon the function of ECs residing in other metabolic organs including the pancreatic islet and the gut, the dysregulation of which may also contribute to the MetS. Finally, we highlight potential EC-based therapeutic targets for human MetS, and NASH based on recent achievements in basic and clinical research and discuss how to approach unsolved problems in the field.

Endotheliopathy in the metabolic syndrome: Mechanisms and clinical implications.

(1) Background: Hypertension is a complex, multifactorial disease that is caused by genetic and environmental factors. Apart from genetic predisposition, the mechanisms involved in this disease have yet to be fully understood. We previously reported that LEENE (lncRNA enhancing endothelial nitric oxide expression, transcribed from  in the human genome) regulates endothelial cell (EC) function by promoting the expression of endothelial nitric oxide synthase (eNOS) and vascular growth factor receptor 2 (VEGFR2). Mice with genetic deletion of the  homologous region exhibited impaired angiogenesis and tissue regeneration in a diabetic hindlimb ischemia model. However, the role of LEENE in blood pressure regulation is unknown. (2) Methods: We subjected mice with genetic ablation of  and wild-type littermates to Angiotensin II (AngII) and monitored their blood pressure and examined their hearts and kidneys. We used RNA-sequencing to identify potential -regulated molecular pathways in ECs that contributed to the observed phenotype. We further performed in vitro experiments with murine and human ECs and ex vivo experiments with murine aortic rings to validate the select mechanism. (3) Results: We identified an exacerbated hypertensive phenotype of -KO mice in the AngII model, evidenced by higher systolic and diastolic blood pressure. At the organ level, we observed aggravated hypertrophy and fibrosis in the heart and kidney. Moreover, the overexpression of human LEENE RNA, in part, restored the signaling pathways impaired by  deletion in murine ECs. Additionally, Axitinib, a tyrosine kinase inhibitor that selectively inhibits VEGFR suppresses LEENE in human ECs. (4) Conclusions: Our study suggests LEENE as a potential regulator in blood pressure control, possibly through its function in ECs.

Genetic Deletion of the  Homolog in Mouse Aggravates Angiotensin II-Induced Hypertension.

Chromatin-associated RNAs (caRNAs) form a relatively poorly recognized layer of the epigenome. The caRNAs reported to date are transcribed from the nuclear genome. Here, leveraging a recently developed assay for detection of caRNAs and their genomic association, we report that mitochondrial RNAs (mtRNAs) are attached to the nuclear genome and constitute a subset of caRNA, thus termed mt-caRNA. In four human cell types analyzed, mt-caRNAs preferentially attach to promoter regions. In human endothelial cells (ECs), the level of mt-caRNA-promoter attachment changes in response to environmental stress that mimics diabetes. Suppression of a non-coding mt-caRNA in ECs attenuates stress-induced nascent RNA transcription from the nuclear genome, including that of critical genes regulating cell adhesion, and abolishes stress-induced monocyte adhesion, a hallmark of dysfunctional ECs. Finally, we report increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors, suggesting many mtRNA translocate to the nucleus in a cell stress and disease-dependent manner. These data nominate mt-caRNAs as messenger molecules responsible for mitochondrial-nuclear communication and connect the immediate product of mitochondrial transcription with the transcriptional regulation of the nuclear genome.

Article	Year
Proteolytic Processing, Maturation, and Unique Synteny of the Hemagglutinin SHA. Microbiology spectrum\| PubMed ID: 34468183	2021
Phosphorylation of human CEACAM1-LF by PKA and GSK3β promotes its interaction with β-catenin. The Journal of biological chemistry\| PubMed ID: 34656562	2021
Vascular Regulation by Super Enhancer-Derived LINC00607. Frontiers in cardiovascular medicine\| PubMed ID: 35837599	2022
Long noncoding RNA LEENE promotes angiogenesis and ischemic recovery in diabetes models. The Journal of clinical investigation\| PubMed ID: 36512424	2023
Endotheliopathy in the metabolic syndrome: Mechanisms and clinical implications. Pharmacology & therapeutics\| PubMed ID: 36894027	2023
Genetic Deletion of the Homolog in Mouse Aggravates Angiotensin II-Induced Hypertension. Non-coding RNA\| PubMed ID: 37218991	2023
Regulation of nuclear transcription by mitochondrial RNA in endothelial cells. eLife\| PubMed ID: 38251974	2024

Alonso Tapia

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