Publications by authors named "Hanjoong Jo"

150 Publications

Focal Adhesion Kinase Activity and Localization is Critical for TNF-α-Induced Nuclear Factor-κB Activation.

Inflammation 2021 Jun 2;44(3):1130-1144. Epub 2021 Feb 2.

Department of Biochemistry and Molecular Biology, University of South Alabama College of Medicine, 5851 N. USA Drive, Room 2366, Mobile, AL, 36688, USA.

While sustained nuclear factor-κB (NF-κB) activation is critical for proinflammatory molecule expression, regulators of NF-κB activity during chronic inflammation are not known. We investigated the role of focal adhesion kinase (FAK) on sustained NF-κB activation in tumor necrosis factor-α (TNF-α)-stimulated endothelial cells (ECs) both in vitro and in vivo. We found that FAK inhibition abolished TNF-α-mediated sustained NF-κB activity in ECs by disrupting formation of TNF-α receptor complex-I (TNFRC-I). Additionally, FAK inhibition diminished recruitment of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and the inhibitor of NF-κB (IκB) kinase (IKK) complex to TNFRC-I, resulting in elevated stability of IκBα protein. In mice given TNF-α, pharmacological and genetic FAK inhibition blocked TNF-α-induced IKK-NF-κB activation in aortic ECs. Mechanistically, TNF-α activated and redistributed FAK from the nucleus to the cytoplasm, causing elevated IKK-NF-κB activation. On the other hand, FAK inhibition trapped FAK in the nucleus of ECs even upon TNF-α stimulation, leading to reduced IKK-NF-κB activity. Together, these findings support a potential use for FAK inhibitors in treating chronic inflammatory diseases.
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http://dx.doi.org/10.1007/s10753-020-01408-5DOI Listing
June 2021

Delivery of siRNA to Endothelial Cells In Vivo Using Lysine/Histidine Oligopeptide-Modified Poly(β-amino ester) Nanoparticles.

Cardiovasc Eng Technol 2021 Feb 20;12(1):114-125. Epub 2021 Jan 20.

Grup d'Enginyera de Materials (GEMAT), Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017, Barcelona, Spain.

Purpose: Endothelial cell (EC) dysfunction underlies the pathology of multiple disease conditions including cardiovascular and pulmonary diseases. Dysfunctional ECs have a distinctive gene expression profile compared to healthy ECs. RNAi therapy is a powerful therapeutic approach that can be used to silence multiple genes of interests simultaneously. However, the delivery of RNAi to ECs in vivo continues to be a major challenge. Here, we optimized a polymer formulation based on poly(β-amino ester)s (pBAEs) to deliver siRNA to vascular ECs.

Methods: We developed a library of bioinspired oligopeptide-modified pBAE nanoparticles (NPs) with different physicochemical proprieties and screened them for cellular uptake and efficacy of RNAi delivery in vitro using ECs, vascular smooth muscle cells, and THP-1 monocytes. From the screening, the lysine-/histidine-oligopeptide modified pBAE (C6-KH) NP was selected and further tested ex vivo using mouse aorta and in mice to determine efficiency of siRNA delivery in vivo.

Results: The in vitro screening study showed that C6-KH was most efficient in delivering siRNA to ECs. Ex vivo study showed that C6-KH nanoparticles containing siRNAs accumulated in the endothelial layer of mouse aortas. In vivo study showed that C6-KH nanoparticles carrying siICAM2 injected via tail-vein in mice significantly reduced ICAM2 level in the artery endothelium (55%), lung (52%), and kidney (31%), but not in the liver, heart, and thymus, indicating a tissue-specific delivery pattern.

Conclusions: We demonstrate that C6-KH pBAE can used for delivery of siRNAs to the artery endothelium and lung, while minimizing potential side or toxic effects in the liver and heart.
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http://dx.doi.org/10.1007/s13239-021-00518-xDOI Listing
February 2021

Delivery of Anti-microRNA-712 to Inflamed Endothelial Cells Using Poly(β-amino ester) Nanoparticles Conjugated with VCAM-1 Targeting Peptide.

Adv Healthc Mater 2021 Jan 14:e2001894. Epub 2021 Jan 14.

Wallace H. Coulter Department of Biomedical Engineering and Division of Cardiology, Georgia Institute of Technology and Emory University, Atlanta, Georgia, 30332, USA.

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.
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http://dx.doi.org/10.1002/adhm.202001894DOI Listing
January 2021

Recent advances in nanomaterials for therapy and diagnosis for atherosclerosis.

Adv Drug Deliv Rev 2021 03 9;170:142-199. Epub 2021 Jan 9.

Department of Biomedical Engineering, The University of Alabama at Birmingham, Birmingham, AL, United States. Electronic address:

Atherosclerosis is a chronic inflammatory disease driven by lipid accumulation in arteries, leading to narrowing and thrombosis. It affects the heart, brain, and peripheral vessels and is the leading cause of mortality in the United States. Researchers have strived to design nanomaterials of various functions, ranging from non-invasive imaging contrast agents, targeted therapeutic delivery systems to multifunctional nanoagents able to target, diagnose, and treat atherosclerosis. Therefore, this review aims to summarize recent progress (2017-now) in the development of nanomaterials and their applications to improve atherosclerosis diagnosis and therapy during the preclinical and clinical stages of the disease.
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http://dx.doi.org/10.1016/j.addr.2021.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7981266PMC
March 2021

Special Issue on Professor John M. Tarbell's Contribution to Cardiovascular Engineering.

Cardiovasc Eng Technol 2021 Feb 7;12(1):1-8. Epub 2021 Jan 7.

Department of Biomedical Engineering, City College of New York, New York, NY, USA.

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http://dx.doi.org/10.1007/s13239-020-00516-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790314PMC
February 2021

Endothelial Reprogramming by Disturbed Flow Revealed by Single-Cell RNA and Chromatin Accessibility Study.

Cell Rep 2020 12;33(11):108491

Wallace H. Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Emory University, Atlanta, GA, USA. Electronic address:

Disturbed flow (d-flow) induces atherosclerosis by regulating gene expression in endothelial cells (ECs). For further mechanistic understanding, we carried out a single-cell RNA sequencing (scRNA-seq) and scATAC-seq study using endothelial-enriched single cells from the left- and right carotid artery exposed to d-flow (LCA) and stable-flow (s-flow in RCA) using the mouse partial carotid ligation (PCL) model. We find eight EC clusters along with immune cells, fibroblasts, and smooth muscle cells. Analyses of marker genes, pathways, and pseudotime reveal that ECs are highly heterogeneous and plastic. D-flow induces a dramatic transition of ECs from atheroprotective phenotypes to pro-inflammatory cells, mesenchymal (EndMT) cells, hematopoietic stem cells, endothelial stem/progenitor cells, and an unexpected immune cell-like (EndICLT) phenotypes. While confirming KLF4/KLF2 as an s-flow-sensitive transcription factor binding site, we also find those sensitive to d-flow (RELA, AP1, STAT1, and TEAD1). D-flow reprograms ECs from atheroprotective to proatherogenic phenotypes, including EndMT and potentially EndICLT.
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http://dx.doi.org/10.1016/j.celrep.2020.108491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801938PMC
December 2020

Disturbed Flow Induces Atherosclerosis by Annexin A2-Mediated Integrin Activation.

Circ Res 2020 09 24;127(8):1091-1093. Epub 2020 Sep 24.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology (C.D., D.W., H.J.), Emory University, Atlanta.

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http://dx.doi.org/10.1161/CIRCRESAHA.120.317909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732028PMC
September 2020

Deletion of NoxO1 limits atherosclerosis development in female mice.

Redox Biol 2020 10 4;37:101713. Epub 2020 Sep 4.

Institute for Cardiovascular Physiology, Goethe-University, Theodor-Stern Kai 7, 60590, Frankfurt Am Main, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Rhein Main, Theodor-Stern Kai 7, 60590, Frankfurt Am Main, Germany. Electronic address:

Objective: Oxidative stress is a risk factor for atherosclerosis. NADPH oxidases of the Nox family produce ROS but their contribution to atherosclerosis development is less clear. Nox2 promotes and Nox4 rather limits atherosclerosis. Although Nox1 with its cytosolic co-factors are largely expressed in epithelial cells, a role for Nox1 for atherosclerosis development was suggested. To further define the role of this homologue, the role of its essential cytosolic cofactor, NoxO1, was determined for atherosclerosis development with the aid of knockout mice.

Methods And Results: Wildtype (WT) and NoxO1 knockout mice were treated with high fat diet and adeno-associated virus (AAV) overexpressing pro-protein convertase subtilisin/kexin type 9 (PCSK9) to induce hepatic low-density lipoprotein (LDL) receptor loss. As a result, massive hypercholesterolemia was induced and spontaneous atherosclerosis developed within three month. Deletion of NoxO1 reduced atherosclerosis formation in brachiocephalic artery and aortic arch in female but not male NoxO1-/- mice as compared to WT littermates. This was associated with a reduced pro-inflammatory cytokine signature in the plasma of female but not male NoxO1-/- mice. MACE-RNAseq of the vessel did not reveal this signature and the expression of the Nox1/NoxO1 system was low to not detectable.

Conclusions: The scaffolding protein NoxO1 plays some role in atherosclerosis development in female mice probably by attenuating the global inflammatory burden.
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http://dx.doi.org/10.1016/j.redox.2020.101713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502371PMC
October 2020

Conditional Deoxyribozyme-Nanoparticle Conjugates for miRNA-Triggered Gene Regulation.

ACS Appl Mater Interfaces 2020 Aug 17;12(34):37851-37861. Epub 2020 Aug 17.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, United States.

DNA-nanoparticle (NP) conjugates have been used to knockdown gene expression transiently and effectively, making them desirable tools for gene regulation therapy. Because DNA-NPs are constitutively active and are rapidly taken up by most cell types, they offer limited control in terms of tissue or cell type specificity. To take a step toward solving this issue, we incorporate toehold-mediated strand exchange, a versatile molecular programming modality, to switch the DNA-NPs from an inactive state to an active state in the presence of a specific RNA input. Because many transcripts are unique to cell subtype or disease state, this approach could one day lead to responsive nucleic acid therapeutics with enhanced specificity. As a proof of concept, we designed conditional deoxyribozyme-nanoparticles (conditional DzNPs) that knockdown tumor necrosis factor α (TNFα) mRNA upon miR-33 triggering. We demonstrate toehold-mediated strand exchange and restoration of TNFα DNAzyme activity in the presence of miR-33 trigger, with optimization of the preparation, configuration, and toehold length of conditional DzNPs. Our results indicate specific and strong ON/OFF response of conditional DzNPs to the miR-33 trigger in buffer. Furthermore, we demonstrate endogenous miR-33-triggered knockdown of TNFα mRNA in mouse macrophages, implying the potential of conditional gene regulation applications using these DzNPs.
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http://dx.doi.org/10.1021/acsami.0c07609DOI Listing
August 2020

Affinity-Driven Design of Cargo-Switching Nanoparticles to Leverage a Cholesterol-Rich Microenvironment for Atherosclerosis Therapy.

ACS Nano 2020 06 1;14(6):6519-6531. Epub 2020 May 1.

Atherosclerotic plaques exhibit high deposition of cholesterol and macrophages. These are not only the main components of the plaques but also key inflammation-triggering sources. However, no existing therapeutics can achieve effective removal of both components within the plaques. Here, we report cargo-switching nanoparticles (CSNP) that are physicochemically designed to bind to cholesterol and release anti-inflammatory drug in the plaque microenvironment. CSNP have a core-shell structure with a core composed of an inclusion complex of methyl-β-cyclodextrin (cyclodextrin) and simvastatin (statin), and a shell of phospholipids. Upon interaction with cholesterol, which has higher affinity to cyclodextrin than statin, CSNP release statin and scavenge cholesterol instead through cargo-switching. CSNP exhibit cholesterol-sensitive multifaceted antiatherogenic functions attributed to statin release and cholesterol depletion . In mouse models of atherosclerosis, systemically injected CSNP target atherosclerotic plaques and reduce plaque content of cholesterol and macrophages, which synergistically leads to effective prevention of atherogenesis and regression of established plaques. These findings suggest that CSNP provide a therapeutic platform for interfacing with cholesterol-associated inflammatory diseases such as atherosclerosis.
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http://dx.doi.org/10.1021/acsnano.9b08216DOI Listing
June 2020

Targeted Intravenous Nanoparticle Delivery: Role of Flow and Endothelial Glycocalyx Integrity.

Ann Biomed Eng 2020 Jul 18;48(7):1941-1954. Epub 2020 Feb 18.

Department of Chemical Engineering, Northeastern University, 360 Huntington Avenue, 313 Snell Engineering Building, Boston, MA, 02115, USA.

Therapies for atherosclerotic cardiovascular disease should target early disease stages and specific vascular sites where disease occurs. Endothelial glycocalyx (GCX) degradation compromises endothelial barrier function and increases vascular permeability. This initiates pro-atherosclerotic lipids and inflammatory cells to penetrate vessel walls, and at the same time this can be leveraged for targeted drug delivery. In prior cell culture studies, GCX degradation significantly increased endothelial cell uptake of nanoparticle vehicles that are designed for drug delivery, compared to the effects of intact GCX. The present study assessed if the cell culture findings translate to selective nanoparticle uptake in animal vessels. In mice, the left carotid artery (LCA) was partially ligated to disturb blood flow, which induces GCX degradation, endothelial dysfunction, and atherosclerosis. After ligation, the LCA vessel wall exhibited a loss of continuity of the GCX layer on the intima. 10-nm gold nanospheres (GNS) coated with polyethylene glycol (PEG) were delivered intravenously. GCX degradation in the ligated LCA correlated to increased GNS infiltration of the ligated LCA wall. This suggests that GCX dysfunction, which coincides with atherosclerosis, can indeed be targeted for enhanced drug delivery, offering a new approach in cardiovascular disease therapy.
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http://dx.doi.org/10.1007/s10439-020-02474-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025840PMC
July 2020

The histone demethylase JMJD2B regulates endothelial-to-mesenchymal transition.

Proc Natl Acad Sci U S A 2020 02 7;117(8):4180-4187. Epub 2020 Feb 7.

Institute for Cardiovascular Regeneration, Goethe University, 60590 Frankfurt, Germany;

Endothelial cells play an important role in maintenance of the vascular system and the repair after injury. Under proinflammatory conditions, endothelial cells can acquire a mesenchymal phenotype by a process named endothelial-to-mesenchymal transition (EndMT), which affects the functional properties of endothelial cells. Here, we investigated the epigenetic control of EndMT. We show that the histone demethylase JMJD2B is induced by EndMT-promoting, proinflammatory, and hypoxic conditions. Silencing of JMJD2B reduced TGF-β2-induced expression of mesenchymal genes, prevented the alterations in endothelial morphology and impaired endothelial barrier function. Endothelial-specific deletion of JMJD2B in vivo confirmed a reduction of EndMT after myocardial infarction. EndMT did not affect global H3K9me3 levels but induced a site-specific reduction of repressive H3K9me3 marks at promoters of mesenchymal genes, such as Calponin (CNN1), and genes involved in TGF-β signaling, such as AKT Serine/Threonine Kinase 3 (AKT3) and Sulfatase 1 (SULF1). Silencing of JMJD2B prevented the EndMT-induced reduction of H3K9me3 marks at these promotors and further repressed these EndMT-related genes. Our study reveals that endothelial identity and function is critically controlled by the histone demethylase JMJD2B, which is induced by EndMT-promoting, proinflammatory, and hypoxic conditions, and supports the acquirement of a mesenchymal phenotype.
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http://dx.doi.org/10.1073/pnas.1913481117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049147PMC
February 2020

The novel coronary artery disease risk gene JCAD/KIAA1462 promotes endothelial dysfunction and atherosclerosis.

Eur Heart J 2019 08;40(29):2398-2408

Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.

Aims: Recent genome-wide association studies (GWAS) have identified that the JCAD locus is associated with risk of coronary artery disease (CAD) and myocardial infarction (MI). However, the mechanisms whereby candidate gene JCAD confers disease risk remain unclear. We addressed whether and how JCAD affects the development of atherosclerosis, the common cause of CAD.

Methods And Results: By mining data in the Genotype-Tissue Expression (GTEx) database, we found that CAD-associated risk variants at the JCAD locus are linked to increased JCAD gene expression in human arteries, implicating JCAD as a candidate causal CAD gene. We therefore generated global and endothelial cell (EC) specific-JCAD knockout mice, and observed that JCAD deficiency attenuated high fat diet-induced atherosclerosis in ApoE-deficient mice. JCAD-deficiency in mice also improved endothelium-dependent relaxation. Genome-wide transcriptional profiling of JCAD-depleted human coronary artery ECs showed that JCAD depletion inhibited the activation of YAP/TAZ pathway, and the expression of downstream pro-atherogenic genes, including CTGF and Cyr61. As a result, JCAD-deficient ECs attracted fewer monocytes in response to lipopolysaccharide (LPS) stimulation. Moreover, JCAD expression in ECs was decreased under unidirectional laminar flow in vitro and in vivo. Proteomics studies suggest that JCAD regulates YAP/TAZ activation by interacting with actin-binding protein TRIOBP, thereby stabilizing stress fiber formation. Finally, we observed that endothelial JCAD expression was increased in mouse and human atherosclerotic plaques.

Conclusion: The present study demonstrates that the GWAS-identified CAD risk gene JCAD promotes endothelial dysfunction and atherosclerosis, thus highlighting the possibility of new therapeutic strategies for CAD by targeting JCAD.
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http://dx.doi.org/10.1093/eurheartj/ehz303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698662PMC
August 2019

The flagellin-TLR5-Nox4 axis promotes the migration of smooth muscle cells in atherosclerosis.

Exp Mol Med 2019 07 10;51(7):1-13. Epub 2019 Jul 10.

Department of Life Science, Ewha Womans University, Seoul, Korea.

We hypothesized that NADPH oxidase 4 (Nox4) is involved in the formation of neointimal atherosclerotic plaques through the migration of smooth muscle cells (SMCs) in response to flagellin. Here, we demonstrate that TLR5-mediated Nox4 activation regulates the migration of SMCs, leading to neointimal plaque formation in atherosclerosis. To investigate the molecular mechanism by which the TLR5-Nox4 cascade mediates SMC migration, we analyzed the signaling cascade in primary vascular SMCs (VSMCs) from wild-type (WT) or Nox4 KO mice. Stimulation of VSMCs from Nox4 KO mice with flagellin failed to induce HO production and Rac activation compared with stimulation of VSMCs from WT mice. Moreover, the migration of Nox4-deficient VSMCs was attenuated in response to flagellin in transwell migration and wound healing assays. Finally, we performed partial carotid artery ligation in ApoE KO and Nox4ApoE DKO mice fed a high-fat diet (HFD) with or without recombinant FliC (rFliC) injection. Injection of rFliC into ApoE KO mice fed a HFD resulted in significantly increased SMC migration into the intimal layer, whereas SMC accumulation was not detected in Nox4ApoE DKO mice. We conclude that activation of the TLR5-Nox4 cascade plays an important role in the formation of neointimal atherosclerotic plaques.
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http://dx.doi.org/10.1038/s12276-019-0275-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802658PMC
July 2019

Role of Noncoding RNAs in the Pathogenesis of Abdominal Aortic Aneurysm.

Circ Res 2019 02;124(4):619-630

From the Wallace H. Coulter Department of Biomedical Engineering, Emory University, Georgia Institute of Technology, Atlanta (S.K., H.J.).

Abdominal aortic aneurysm (AAA) is a local dilatation of the abdominal aortic vessel wall and is among the most challenging cardiovascular diseases as without urgent surgical intervention, ruptured AAA has a mortality rate of >80%. Most patients present acutely after aneurysm rupture or dissection from a previously asymptomatic condition and are managed by either surgery or endovascular repair. Patients usually are old and have other concurrent diseases and conditions, such as diabetes mellitus, obesity, and hypercholesterolemia making surgical intervention more difficult. Collectively, these issues have driven the search for alternative methods of diagnosing, monitoring, and treating AAA using therapeutics and less invasive approaches. Noncoding RNAs-short noncoding RNAs (microRNAs) and long-noncoding RNAs-are emerging as new fundamental regulators of gene expression. Researchers and clinicians are aiming at targeting these microRNAs and long noncoding RNAs and exploit their potential as clinical biomarkers and new therapeutic targets for AAAs. While the role of miRNAs in AAA is established, studies on long-noncoding RNAs are only beginning to emerge, suggesting their important yet unexplored role in vascular physiology and disease. Here, we review the role of noncoding RNAs and their target genes focusing on their role in AAA. We also discuss the animal models used for mechanistic understanding of AAA. Furthermore, we discuss the potential role of microRNAs and long noncoding RNAs as clinical biomarkers and therapeutics.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.312438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440479PMC
February 2019

miR-214 is Stretch-Sensitive in Aortic Valve and Inhibits Aortic Valve Calcification.

Ann Biomed Eng 2019 Apr 22;47(4):1106-1115. Epub 2019 Jan 22.

School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 387 Technology Circle NW, Technology Enterprise Park, Suite 200, Atlanta, GA, 30313, USA.

miR-214 has been recently found to be significantly downregulated in calcified human aortic valves (AVs). ER stress, especially the ATF4-mediated pathway, has also been shown to be significantly upregulated in calcific AV disease. Since elevated cyclic stretch is one of the major mechanical stimuli for AV calcification and ATF4 is a validated target of miR-214, we investigated the effect of cyclic stretch on miR-214 expression as well as those of ATF4 and two downstream genes (CHOP and BCL2L1). Porcine aortic valve (PAV) leaflets were cyclically stretched at 15% for 48 h in regular medium and for 1 week in osteogenic medium to simulate the early remodeling and late calcification stages of stretch-induced AV disease, respectively. For both stages, 10% cyclic stretch served as the physiological counterpart. RT-qPCR revealed that miR-214 expression was significantly downregulated during the late calcification stage, whereas the mRNA expression of ATF4 and BCL2L1 was upregulated and downregulated, respectively, during both early remodeling and late calcification stages. When PAV leaflets were statically transfected with miR-214 mimic in osteogenic medium for 2 weeks, calcification was significantly reduced compared to the control mimic case. This implies that miR-214 may have a protective role in stretch-induced calcific AV disease.
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http://dx.doi.org/10.1007/s10439-019-02206-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416061PMC
April 2019

Disturbed Flow Increases UBE2C (Ubiquitin E2 Ligase C) via Loss of miR-483-3p, Inducing Aortic Valve Calcification by the pVHL (von Hippel-Lindau Protein) and HIF-1α (Hypoxia-Inducible Factor-1α) Pathway in Endothelial Cells.

Arterioscler Thromb Vasc Biol 2019 03;39(3):467-481

From the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta (J.F.E., N.V.-R., S.K., L.G., W.R.T., A.P.Y., H.J.).

Objective- Calcific aortic valve (AV) disease, characterized by AV sclerosis and calcification, is a major cause of death in the aging population; however, there are no effective medical therapies other than valve replacement. AV calcification preferentially occurs on the fibrosa side, exposed to disturbed flow (d-flow), whereas the ventricularis side exposed to predominantly stable flow remains protected by unclear mechanisms. Here, we tested the role of novel flow-sensitive UBE2C (ubiquitin E2 ligase C) and microRNA-483-3p (miR-483) in flow-dependent AV endothelial function and AV calcification. Approach and Results- Human AV endothelial cells and fresh porcine AV leaflets were exposed to stable flow or d-flow. We found that UBE2C was upregulated by d-flow in human AV endothelial cells in the miR-483-dependent manner. UBE2C mediated OS-induced endothelial inflammation and endothelial-mesenchymal transition by increasing the HIF-1α (hypoxia-inducible factor-1α) level. UBE2C increased HIF-1α by ubiquitinating and degrading its upstream regulator pVHL (von Hippel-Lindau protein). These in vitro findings were corroborated by immunostaining studies using diseased human AV leaflets. In addition, we found that reduction of miR-483 by d-flow led to increased UBE2C expression in human AV endothelial cells. The miR-483 mimic protected against endothelial inflammation and endothelial-mesenchymal transition in human AV endothelial cells and calcification of porcine AV leaflets by downregulating UBE2C. Moreover, treatment with the HIF-1α inhibitor (PX478) significantly reduced porcine AV calcification in static and d-flow conditions. Conclusions- These results suggest that miR-483 and UBE2C and pVHL are novel flow-sensitive anti- and pro-calcific AV disease molecules, respectively, that regulate the HIF-1α pathway in AV. The miR-483 mimic and HIF-1α pathway inhibitors may serve as potential therapeutics of calcific AV disease.
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http://dx.doi.org/10.1161/ATVBAHA.118.312233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393167PMC
March 2019

Role of flow-sensitive microRNAs and long noncoding RNAs in vascular dysfunction and atherosclerosis.

Vascul Pharmacol 2019 03 7;114:76-92. Epub 2018 Oct 7.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, USA; Division of Cardiology, Emory University, Atlanta, USA. Electronic address:

Atherosclerosis is the primary underlying cause of myocardial infarction, ischemic stroke, and peripheral artery disease. The disease preferentially occurs in arterial regions exposed to disturbed blood flow, in part, by altering expression of flow-sensitive coding- and non-coding genes. In this review, we summarize the role of noncoding RNAs, [microRNAs (miRNAs) and long noncoding RNAs(lncRNAs)], as regulators of gene expression and outline their relationship to the pathogenesis of atherosclerosis. While miRNAs are small noncoding genes that post-transcriptionally regulate gene expression by targeting mRNA transcripts, the lncRNAs regulate gene expression by diverse mechanisms, which are still emerging and incompletely understood. We focused on multiple flow-sensitive miRNAs such as, miR-10a, -19a, -23b, -17~92, -21, -663, -92a, -143/145, -101, -126, -712, -205, and -155 that play a critical role in endothelial function and atherosclerosis by targeting inflammation, cell cycle, proliferation, migration, apoptosis, and nitric oxide signaling. Flow-dependent regulation of lncRNAs is just emerging, and their role in vascular dysfunction and atherosclerosis is unknown. Here, we discuss the flow-sensitive lncRNA STEEL along with other lncRNAs studied in the context of vascular pathophysiology and atherosclerosis such as MALAT1, MIAT1, ANRIL, MYOSLID, MEG3, SENCR, SMILR, LISPR1, and H19. Also discussed is the use of these noncoding RNAs as potential biomarkers and therapeutics to reduce and regress atherosclerosis.
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http://dx.doi.org/10.1016/j.vph.2018.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905428PMC
March 2019

Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells.

Nat Commun 2018 06 12;9(1):2292. Epub 2018 Jun 12.

Institute for Cardiovascular Physiology, Goethe University, Frankfurt am Main, 60590, Germany.

Oxidized phospholipids (oxPAPC) induce endothelial dysfunction and atherosclerosis. Here we show that oxPAPC induce a gene network regulating serine-glycine metabolism with the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) as a causal regulator using integrative network modeling and Bayesian network analysis in human aortic endothelial cells. The cluster is activated in human plaque material and by atherogenic lipoproteins isolated from plasma of patients with coronary artery disease (CAD). Single nucleotide polymorphisms (SNPs) within the MTHFD2-controlled cluster associate with CAD. The MTHFD2-controlled cluster redirects metabolism to glycine synthesis to replenish purine nucleotides. Since endothelial cells secrete purines in response to oxPAPC, the MTHFD2-controlled response maintains endothelial ATP. Accordingly, MTHFD2-dependent glycine synthesis is a prerequisite for angiogenesis. Thus, we propose that endothelial cells undergo MTHFD2-mediated reprogramming toward serine-glycine and mitochondrial one-carbon metabolism to compensate for the loss of ATP in response to oxPAPC during atherosclerosis.
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http://dx.doi.org/10.1038/s41467-018-04602-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997752PMC
June 2018

ZBTB46 is a shear-sensitive transcription factor inhibiting endothelial cell proliferation via gene expression regulation of cell cycle proteins.

Lab Invest 2019 03 8;99(3):305-318. Epub 2018 Jun 8.

Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, GA, USA.

ZBTB46 is a transcription factor identified in classical dendritic cells and keeps dendritic cells in a quiescent state. Chromatin immunoprecipitation sequencing in dendritic cells has identified over 1300 potential gene targets of ZBTB46, affecting many processes including cell cycle. Endothelial cells (ECs) also express ZBTB46 and are mostly in a quiescent non-proliferative state. While EC proliferation is a critical process in development, dysregulation of EC proliferation as seen in areas of disturbed flow play an important role in many disease processes such as atherosclerosis, pulmonary hypertension, transplant vasculopathy, neointimal hyperplasia, and in-stent restenosis. We studied the role of ZBTB46 in ECs, hypothesizing that it inhibits EC proliferation. Using a model of disturbed flow in mice, we found that ZBTB46 is expressed in murine arterial ECs in vivo, and is downregulated by disturbed flow. In vitro results using HAECs showed that cell confluence and laminar shear stress, both known physiological conditions promoting EC quiescence, led to upregulation of ZBTB46 expression. Adenoviral-mediated overexpression of ZBTB46 in vitro caused reduced EC proliferation, and increased number of cells in the G/G phase of cell cycle, without affecting apoptosis or senescence, while siRNA knockdown of ZBTB46 negated the known inhibitory role of unidirectional laminar shear stress on EC proliferation. ZBTB46 overexpression also led to a broad suppression of genes involved in cell cycle progression including multiple cyclins and cyclin-dependent kinases, but an increase in the CDK inhibitor CDKN1A. Phosphorylation of the retinoblastoma protein was also decreased as assessed by Western blot. Tube formation on Matrigel was reduced, suggesting an inhibitory role for ZBTB46 in angiogenesis. Further research is required to investigate the potential role of ZBTB46 in specific pathologic conditions and whether it can be targeted in a therapeutic manner.
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http://dx.doi.org/10.1038/s41374-018-0060-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286701PMC
March 2019

Correction to: Editorial: Special Issue on Heart Valve Mechanobiology : New Insights into Mechanical Regulation of Valve Disease and Regeneration.

Cardiovasc Eng Technol 2018 06;9(2):268

Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, GA, USA.

Original article has been updated to include names and affiliations of authors.
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http://dx.doi.org/10.1007/s13239-018-0365-yDOI Listing
June 2018

Editorial: Special Issue on Heart Valve Mechanobiology : New Insights into Mechanical Regulation of Valve Disease and Regeneration.

Cardiovasc Eng Technol 2018 06;9(2):121-125

Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, GA, USA.

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http://dx.doi.org/10.1007/s13239-018-0360-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990480PMC
June 2018

3D Imaging and Quantitative Analysis of Vascular Networks: A Comparison of Ultramicroscopy and Micro-Computed Tomography.

Theranostics 2018 7;8(8):2117-2133. Epub 2018 Mar 7.

Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.

Rationale: Classic histology is the gold standard for vascular network imaging and analysis. The method however is laborious and prone to artefacts. Here, the suitability of ultramicroscopy (UM) and micro-computed tomography (CT) was studied to establish potential alternatives to histology.

Methods: The vasculature of murine organs (kidney, heart and atherosclerotic carotid arteries) was visualized using conventional 2D microscopy, 3D light sheet ultramicroscopy (UM) and micro-CT. Moreover, spheroid-based human endothelial cell vessel formation in mice was quantified. Fluorescently labeled Isolectin GS-IB4 A647 was used for labeling of vasculature for UM analysis, and analyses were performed ex vivo after sample preparation. For CT imaging, animals were perfused postmortem with radiopaque contrast agent.

Results: Using UM imaging, 3D vascular network information could be obtained in samples of animals receiving injection of the fluorescently labeled Isolectin GS-IB4. Resolution was sufficient to measure single endothelial cell integration into capillaries in the spheroid-based matrigel plug assay. Because of the selective staining of the endothelium, imaging of larger vessels yielded less favorable results. Using micro-CT or even nano-CT, imaging of capillaries was impossible due to insufficient X-ray absorption and thus insufficient signal-to-noise ratio. Identification of lumen in murine arteries using micro-CT was in contrast superior to UM.

Conclusion: UM and micro-CT are two complementary techniques. Whereas UM is ideal for imaging and especially quantifying capillary networks and arterioles, larger vascular structures are easier and faster to quantify and visualize using micro-CT. 3D information of both techniques is superior to 2D histology. UM and micro-CT together may open a new field of clinical pathology diagnosis.
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http://dx.doi.org/10.7150/thno.22610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928875PMC
April 2019

Vascular Semaphorin 7A Upregulation by Disturbed Flow Promotes Atherosclerosis Through Endothelial β1 Integrin.

Arterioscler Thromb Vasc Biol 2018 02 21;38(2):335-343. Epub 2017 Dec 21.

From the Cyrus Tang Hematology Center (S.H., Y.L., T.Y., L.X., Y.W., F.L., F.Y., Y.C., Q.W., C.T., L.Z.), Department of Epidemiology, School of Public Health (X.Z., A.W., Y.Z.), Collaborative Innovation Center of Hematology of Jiangsu Province (S.H., Y.L., T.Y., Q.W., C.T., L.Z.), and Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases (X.Z., A.W., Q.W., Y.Z.), Soochow University, Suzhou, China; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta (J.H., H.J.); Einthoven Laboratory for Experimental Vascular Medicine, Division of Nephrology, Department of Internal Medicine, Leiden University Medical Center, the Netherlands (H.Z., J.M.v.G., A.J.v.Z.); and Department of Molecular Cardiology, Cleveland Clinic, OH (Q.W.).

Objective: Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism.

Approach And Results: Using genetically engineered mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of mice on a high-fat diet. A higher level of Sema7A in the atheroprone lesser curvature suggests a correlation of Sema7A with disturbed flow. This notion is supported by elevated Sema7A expression in human umbilical venous endothelial cells either subjected to oscillatory shear stress or treated with the PKA (protein kinase A)/CREB (cAMP response element-binding protein) inhibitor H89 (-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate). Further studies using the partial carotid artery ligation model showed that disturbed flow in the left carotid artery of mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in mice. Further studies showed that blockage of β1 integrin, a known Sema7A receptor, or inhibition of FAK (focal adhesion kinase), MEK1/2 (mitogen-activated protein kinase kinase 1/2), or NF-κB (nuclear factor-κB) significantly reduced the expression of cell adhesion molecules and THP-1 (human acute monocytic leukemia cell line) monocyte adhesion in Sema7A-overexpressing human umbilical venous endothelial cells. Studies using chimeric mice suggest that vascular, most likely endothelial, Sema7A plays a major role in atherogenesis.

Conclusions: Our findings indicate a significant role of Sema7A in atherosclerosis by mediating endothelial dysfunction in a β1 integrin-dependent manner.
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http://dx.doi.org/10.1161/ATVBAHA.117.310491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785426PMC
February 2018

Conserved Gene Microsynteny Unveils Functional Interaction Between Protein Disulfide Isomerase and Rho Guanine-Dissociation Inhibitor Families.

Sci Rep 2017 12 8;7(1):17262. Epub 2017 Dec 8.

Vascular Biology Laboratory, Heart Institute (Incor), University of São Paulo School of Medicine, São Paulo, Brazil.

Protein disulfide isomerases (PDIs) support endoplasmic reticulum redox protein folding and cell-surface thiol-redox control of thrombosis and vascular remodeling. The family prototype PDIA1 regulates NADPH oxidase signaling and cytoskeleton organization, however the related underlying mechanisms are unclear. Here we show that genes encoding human PDIA1 and its two paralogs PDIA8 and PDIA2 are each flanked by genes encoding Rho guanine-dissociation inhibitors (GDI), known regulators of RhoGTPases/cytoskeleton. Evolutionary histories of these three microsyntenic regions reveal their emergence by two successive duplication events of a primordial gene pair in the last common vertebrate ancestor. The arrangement, however, is substantially older, detectable in echinoderms, nematodes, and cnidarians. Thus, PDI/RhoGDI pairing in the same transcription orientation emerged early in animal evolution and has been largely maintained. PDI/RhoGDI pairs are embedded into conserved genomic regions displaying common cis-regulatory elements. Analysis of gene expression datasets supports evidence for PDI/RhoGDI coexpression in developmental/inflammatory contexts. PDIA1/RhoGDIα were co-induced in endothelial cells upon CRISP-R-promoted transcription activation of each pair component, and also in mouse arterial intima during flow-induced remodeling. We provide evidence for physical interaction between both proteins. These data support strong functional links between PDI and RhoGDI families, which likely maintained PDI/RhoGDI microsynteny along > 800-million years of evolution.
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http://dx.doi.org/10.1038/s41598-017-16947-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5722932PMC
December 2017

Mechanical Activation of Hypoxia-Inducible Factor 1α Drives Endothelial Dysfunction at Atheroprone Sites.

Arterioscler Thromb Vasc Biol 2017 11 7;37(11):2087-2101. Epub 2017 Sep 7.

From the Department of Infection, Immunity, and Cardiovascular Disease, INSIGNEO Institute for In Silico Medicine, and the Bateson Centre (S.F., N.B., M.F., C.S., H.S., M.M., D.P., B.T.A., A.A.R.T., V.R., P.C.E.) and Sheffield Institute for Translational Neuroscience (S.A.), University of Sheffield, United Kingdom; Institute for Cardiovascular Prevention, Ludwig-Maximilians University of Munich and DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Germany (S.A., C.W., A.S.); and Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta (H.J.).

Objective: Atherosclerosis develops near branches and bends of arteries that are exposed to low shear stress (mechanical drag). These sites are characterized by excessive endothelial cell (EC) proliferation and inflammation that promote lesion initiation. The transcription factor HIF1α (hypoxia-inducible factor 1α) is canonically activated by hypoxia and has a role in plaque neovascularization. We studied the influence of shear stress on HIF1α activation and the contribution of this noncanonical pathway to lesion initiation.

Approach And Results: Quantitative polymerase chain reaction and en face staining revealed that HIF1α was expressed preferentially at low shear stress regions of porcine and murine arteries. Low shear stress induced HIF1α in cultured EC in the presence of atmospheric oxygen. The mechanism involves the transcription factor nuclear factor-κB that induced HIF1α transcripts and induction of the deubiquitinating enzyme Cezanne that stabilized HIF1α protein. Gene silencing revealed that HIF1α enhanced proliferation and inflammatory activation in EC exposed to low shear stress via induction of glycolysis enzymes. We validated this observation by imposing low shear stress in murine carotid arteries (partial ligation) that upregulated the expression of HIF1α, glycolysis enzymes, and inflammatory genes and enhanced EC proliferation. EC-specific genetic deletion of HIF1α in hypercholesterolemic apolipoprotein E-defecient mice reduced inflammation and endothelial proliferation in partially ligated arteries, indicating that HIF1α drives inflammation and vascular dysfunction at low shear stress regions.

Conclusions: Mechanical low shear stress activates HIF1α at atheroprone regions of arteries via nuclear factor-κB and Cezanne. HIF1α promotes atherosclerosis initiation at these sites by inducing excessive EC proliferation and inflammation via the induction of glycolysis enzymes.
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http://dx.doi.org/10.1161/ATVBAHA.117.309249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659306PMC
November 2017

Disturbed Flow Promotes Arterial Stiffening Through Thrombospondin-1.

Circulation 2017 Sep 4;136(13):1217-1232. Epub 2017 Aug 4.

From Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta (C.W.K., A.P.-P., S.K., D.-W.K., J.R., R.L.G., H.J., L.P.B.); Department of Microbiology, College of Medicine, Inha University, Incheon, Republic of Korea (C.W.K.); Department of Surgery, Emory University, Atlanta, GA (A.P.-P., A.D.M., T.C., K.M.K., H.L., L.P.B.); Department of Radiology and Imaging Sciences, Emory University, Atlanta, GA (L.H.T.); Department of Bioengineering, University of Utah, Salt Lake City (L.H.T.); Mercer University School of Medicine, Macon, GA (S.D.); Life Science Center, Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan (H.Y.); George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta (R.L.G.); Surgical and Research Services, Atlanta VA Medical Center, Decatur, GA (L.P.B.); and Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta (L.P.B.).

Background: 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.

Methods: D-flow was induced in the carotid arteries of young and old mice of both sexes. Arterial stiffness was quantified ex vivo with cylindrical biaxial mechanical testing and in vivo from duplex ultrasound and compared with unmanipulated carotid arteries from 80-week-old mice. Gene expression and pathway analysis was performed on endothelial cell-enriched RNA and validated by immunohistochemistry. In vitro testing of signaling pathways was performed under oscillatory and laminar wall shear stress conditions. Human arteries from regions of d-flow and stable flow were tested ex vivo to validate critical results from the animal model.

Results: D-flow induced arterial stiffening through collagen deposition after partial carotid ligation, and the degree of stiffening was similar to that of unmanipulated carotid arteries from 80-week-old mice. Intimal gene pathway analyses identified transforming growth factor-β pathways as having a prominent role in this stiffened arterial response, but this was attributable to thrombospondin-1 (TSP-1) stimulation of profibrotic genes and not changes to transforming growth factor-β. In vitro and in vivo testing under d-flow conditions identified a possible role for TSP-1 activation of transforming growth factor-β in the upregulation of these genes. TSP-1 knockout animals had significantly less arterial stiffening in response to d-flow than wild-type carotid arteries. Human arteries exposed to d-flow had similar increases TSP-1 and collagen gene expression as seen in our model.

Conclusions: TSP-1 has a critical role in shear-mediated arterial stiffening that is mediated in part through TSP-1's activation of the profibrotic signaling pathways of transforming growth factor-β. Molecular targets in this pathway may lead to novel therapies to limit arterial stiffening and the progression of disease in arteries exposed to d-flow.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.116.026361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614852PMC
September 2017

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

Lab Invest 2017 08 15;97(8):935-945. Epub 2017 May 15.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.

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.
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http://dx.doi.org/10.1038/labinvest.2017.47DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563968PMC
August 2017

KLF2 and KLF4 control endothelial identity and vascular integrity.

JCI Insight 2017 02 23;2(4):e91700. Epub 2017 Feb 23.

Cardiovascular Research Institute, Department of Medicine, and.

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 and/or 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.
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http://dx.doi.org/10.1172/jci.insight.91700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313061PMC
February 2017

Mechanosensitive microRNA-181b Regulates Aortic Valve Endothelial Matrix Degradation by Targeting TIMP3.

Cardiovasc Eng Technol 2018 06 24;9(2):141-150. Epub 2017 Feb 24.

Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, GA, USA.

Calcific aortic valve disease (CAVD) is a major cause of morbidity in the aging population, but the underlying mechanisms of its progression remain poorly understood. Aortic valve calcification preferentially occurs on the fibrosa, which is subjected to disturbed flow. The side-specific progression of the disease is characterized by inflammation, calcific lesions, and extracellular matrix (ECM) degradation. Here, we explored the role of mechanosensitive microRNA-181b and its downstream targets in human aortic valve endothelial cells (HAVECs). Mechanistically, miR-181b is upregulated in OS and fibrosa, and it targets TIMP3, SIRT1, and GATA6, correlated with increased gelatinase/MMP activity. Overexpression of miR-181b led to decreased TIMP3 and exacerbated MMP activity as shown by gelatinase assay, and miR-181b inhibition decreased gelatinase activity through the repression of TIMP3 levels. Luciferase assay showed specific binding of miR-181b to the TIMP3 gene. Overexpression of miR-181b in HAVECs subjected to either LS or OS increased MMP activity, and miR-181b inhibition abrogated shear-sensitive MMP activity. These studies suggest that targeting this shear-dependent miRNA may provide a novel noninvasive treatment for CAVD.
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http://dx.doi.org/10.1007/s13239-017-0296-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568995PMC
June 2018