isolation-endothelial-cells-from-lumen-mouse-carotid-arteries-for

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...

Maintenance of vascular integrity in the adult animal is needed for survival, and it is critically dependent on the endothelial lining, which controls barrier function, blood fluidity, and flow dynamics. However, nodal regulators that coordinate endothelial identity and function in the adult animal remain poorly characterized. Here, we show that endothelial KLF2 and KLF4 control a large segment of the endothelial transcriptome, thereby affecting virtually all key endothelial functions. Inducible endothelial-specific deletion of Klf2 and/or Klf4 reveals that a single allele of either gene is sufficient for survival, but absence of both (EC-DKO) results in acute death from myocardial infarction, heart failure, and stroke. EC-DKO animals exhibit profound compromise in vascular integrity and profound dysregulation of the coagulation system. Collectively, these studies establish an absolute requirement for KLF2/4 for maintenance of endothelial and vascular integrity in the adult animal.

KLF2 and KLF4 control endothelial identity and vascular integrity.

Atherosclerosis is an inflammatory disease with multiple underlying metabolic and physical risk factors. Bone morphogenic protein 4 (BMP4) expression is increased in endothelium in atherosclerosis-prone regions and is known to induce endothelial inflammation, endothelial dysfunction, and hypertension. BMP actions are mediated by 2 different types of BMP receptors (BMPRI and BMPRII). Here, we show a surprising finding that loss of BMPRII expression causes endothelial inflammation and atherosclerosis.

Anti-inflammatory and antiatherogenic role of BMP receptor II in endothelial cells.

Oxidative stress occurs with disturbed blood flow, inflammation and cardiovascular disease (CVD), yet free-radical scavenging antioxidants have shown limited benefit in human CVD. Thioredoxin-1 (Trx1) is a thiol antioxidant protecting against non-radical oxidants by controlling protein thiol/disulfide status; Trx1 translocates from cytoplasm to cell nuclei due to stress signaling, facilitates DNA binding of transcription factors, e.g., NF-κB, and potentiates inflammatory signaling. Whether increased nuclear Trx1 contributes to proatherogenic signaling is unknown.

Disturbed flow enhances inflammatory signaling and atherogenesis by increasing thioredoxin-1 level in endothelial cell nuclei.

Disturbed blood flow (d-flow) induces atherosclerosis by altering the expression of mechanosensitive genes in the arterial endothelium. Previously, we identified >580 mechanosensitive genes in the mouse arterial endothelium, but their role in endothelial inflammation is incompletely understood. From this set, we obtained 84 Drosophila RNAi lines that silences the target gene under the control of upstream activation sequence (UAS) promoter. These lines were crossed with C564-GAL4 flies expressing GFP under the control of drosomycin promoter, an NF-κB target gene and a marker of pathogen-induced inflammation. Silencing of psmd12 or ERN1 decreased infection-induced drosomycin expression, while Bap60 silencing significantly increased the drosomycin expression. Interestingly, knockdown of Bap60 in adult flies using temperature-inducible Bap60 RNAi (C564-GAL4-Bap60-RNAi) enhanced drosomycin expression upon Gram-positive bacterial challenge but the basal drosomycin expression remained unchanged compared to the control. In the mammalian system, smarcd3 (mammalian ortholog of Bap60) expression was reduced in the human- and mouse aortic endothelial cells exposed to oscillatory shear in vitro as well as in the d-flow regions of mouse arterial endothelium in vivo. Moreover, siRNA-mediated knockdown of smarcd3 induced endothelial inflammation. In summary, we developed an in vivo Drosophila RNAi screening method to identify flow-sensitive genes that regulate endothelial inflammation.

Functional screening of mammalian mechanosensitive genes using Drosophila RNAi library- Smarcd3/Bap60 is a mechanosensitive pro-inflammatory gene.

Studying the role of a particular gene in atherosclerosis typically requires a time-consuming and often difficult process of generating double knockouts or transgenics on ApoE or LDL receptor (LDLR) background. Recently, it was reported that adeno-associated-virus-8 (AAV8)-mediated overexpression of PCSK9 (AAV8-PCSK9) rapidly induced hyperlipidemia. However, using this method in C57BL6 wild-type (C57) mice, it took ~3 months to develop atherosclerosis. Our partial carotid ligation model is used to rapidly develop atherosclerosis by inducing disturbed flow in the left common carotid artery within 2 weeks in ApoE or LDLR mice. Here, we combined these two approaches to develop an accelerated model of atherosclerosis in C57 mice. C57 mice were injected with AAV9-PCSK9 or AAV9-luciferase (control) and high-fat diet was initiated. A week later, partial ligation was performed. Compared to the control, AAV-PCSK9 led to elevated serum PCSK9, hypercholesterolemia, and rapid atherosclerosis development within 3 weeks as determined by gross plaque imaging, and staining with Oil-Red-O, Movat's pentachrome, and CD45 antibody. These plaque lesions were comparable to the atherosclerotic lesions that have been previously observed in ApoE or LDLR mice that were subjected to partial carotid ligation and high-fat diet. Next, we tested whether our method can be utilized to rapidly determine the role of a particular gene in atherosclerosis. Using eNOS and NOX1 mice on C57 background, we found that the eNOS mice developed more advanced lesions, while the NOX1 mice developed less atherosclerotic lesions as compared to the C57 controls. These results are consistent with the previous findings using double knockouts (eNOS_ApoE and NOX1_ApoE). AAV9-PCSK9 injection followed by partial carotid ligation is an effective and time-saving approach to rapidly induce atherosclerosis. This accelerated model is well-suited to quickly determine the role of gene(s) interest without generating double or triple knockouts.

Accelerated atherosclerosis development in C57Bl6 mice by overexpressing AAV-mediated PCSK9 and partial carotid ligation.

Arterial stiffness and wall shear stress are powerful determinants of cardiovascular health, and arterial stiffness is associated with increased cardiovascular mortality. Low and oscillatory wall shear stress, termed disturbed flow (d-flow), promotes atherosclerotic arterial remodeling, but the relationship between d-flow and arterial stiffness is not well understood. The objective of this study was to define the role of d-flow on arterial stiffening and discover the relevant signaling pathways by which d-flow stiffens arteries.

Disturbed Flow Promotes Arterial Stiffening Through Thrombospondin-1.

Endothelial cells (ECs) are an important target for therapy in a wide range of diseases, most notably atherosclerosis. Developing efficient nanoparticle (NP) systems that deliver RNA interference (RNAi) drugs specifically to dysfunctional ECs in vivo to modulate their gene expression remains a challenge. To date, several lipid-based NPs are developed and shown to deliver RNAi to ECs, but few of them are optimized to specifically target dysfunctional endothelium. Here, a novel, targeted poly(β-amino ester) (pBAE) NP is demonstrated. This pBAE NP is conjugated with VHPK peptides that target vascular cell adhesion molecule 1 protein, overexpressed on inflamed EC membranes. To test this approach, the novel NPs are used to deliver anti-microRNA-712 (anti-miR-712) specifically to inflamed ECs both in vitro and in vivo, reducing the high expression of pro-atherogenic miR-712. A single administration of anti-miR-712 using the VHPK-conjugated-pBAE NPs in mice significantly reduce miR-712 expression, while preventing the loss of its target gene, tissue inhibitor of metalloproteinase 3 (TIMP3) in inflamed endothelium. miR-712 and TIMP3 expression are unchanged in non-inflamed endothelium. This novel, targeted-delivery platform may be used to deliver RNA therapeutics specifically to dysfunctional endothelium for the treatment of vascular disease.

Article	Year
KLF2 and KLF4 control endothelial identity and vascular integrity. JCI insight\| PubMed ID: 28239661	2017
Anti-inflammatory and antiatherogenic role of BMP receptor II in endothelial cells. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 23559633	2013
Disturbed flow enhances inflammatory signaling and atherogenesis by increasing thioredoxin-1 level in endothelial cell nuclei. PloS one\| PubMed ID: 25265386	2014
Functional screening of mammalian mechanosensitive genes using Drosophila RNAi library- Smarcd3/Bap60 is a mechanosensitive pro-inflammatory gene. Scientific reports\| PubMed ID: 27819340	2016
Accelerated atherosclerosis development in C57Bl6 mice by overexpressing AAV-mediated PCSK9 and partial carotid ligation. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 28504688	2017
Disturbed Flow Promotes Arterial Stiffening Through Thrombospondin-1. Circulation\| PubMed ID: 28778947	2017
Delivery of Anti-microRNA-712 to Inflamed Endothelial Cells Using Poly(β-amino ester) Nanoparticles Conjugated with VCAM-1 Targeting Peptide. Advanced healthcare materials\| PubMed ID: 33448151	2021

Dong-Won Kang

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