HEG1 Protects Against Atherosclerosis by Regulating Stable Flow-Induced KLF2/4 Expression in Endothelial Cells.

BACKGROUND: Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow, and stable flow (s-flow) protects against atherosclerosis by incompletely understood mechanisms. METHODS: Our single-cell RNA-sequencing data using the mouse partial carotid ligation model was reanalyzed, which identified Heart-of-glass 1 (HEG1) as an s-flow-induced gene. HEG1 expression was studied by immunostaining, quantitive polymerase chain reaction, hybridization chain reaction, and Western blot in mouse arteries, human aortic endothelial cells (HAECs), and human coronary arteries. A small interfering RNA-mediated knockdown of HEG1 was used to study its function and signaling mechanisms in HAECs under various flow conditions using a cone-and-plate shear device. We generated endothelial-targeted, tamoxifen-inducible HEG1 knockout (HEG1iECKO) mice. To determine the role of HEG1 in atherosclerosis, HEG1iECKO and littermate-control mice were injected with an adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9] and fed a Western diet to induce hypercholesterolemia either for 2 weeks with partial carotid ligation or 2 months without the surgery. RESULTS: S-flow induced HEG1 expression at the mRNA and protein levels in vivo and in vitro. S-flow stimulated HEG1 protein translocation to the downstream side of HAECs and release into the media, followed by increased messenger RNA and protein expression. HEG1 knockdown prevented s-flow-induced endothelial responses, including monocyte adhesion, permeability, and migration. Mechanistically, HEG1 knockdown prevented s-flow-induced KLF2/4 (Kruppel-like factor 2/4) expression by regulating its intracellular binding partner KRIT1 (Krev interaction trapped protein 1) and the MEKK3-MEK5-ERK5-MEF2 pathway in HAECs. Compared with littermate controls, HEG1iECKO mice exposed to hypercholesterolemia for 2 weeks and partial carotid ligation developed advanced atherosclerotic plaques, featuring increased necrotic core area, thin-capped fibroatheroma, inflammation, and intraplaque hemorrhage. In a conventional Western diet model for 2 months, HEG1iECKO mice also showed an exacerbated atherosclerosis development in the arterial tree in both sexes and the aortic sinus in males but not in females. Moreover, endothelial HEG1 expression was reduced in human coronary arteries with advanced atherosclerotic plaques. CONCLUSIONS: Our findings indicate that HEG1 is a novel mediator of atheroprotective endothelial responses to flow and a potential therapeutic target.

Endothelial UCP2 Is a Mechanosensitive Suppressor of Atherosclerosis.

BACKGROUND: Inflamed endothelial cells (ECs) trigger atherogenesis, especially at arterial regions experiencing disturbed blood flow. UCP2 (Uncoupling protein 2), a key mitochondrial antioxidant protein, improves endothelium-dependent relaxation in obese mice. However, whether UCP2 can be regulated by shear flow is unknown, and the role of endothelial UCP2 in regulating inflammation and atherosclerosis remains unclear. This study aims to investigate the mechanoregulation of UCP2 expression in ECs and the effect of UCP2 on endothelial inflammation and atherogenesis. METHODS: In vitro shear stress simulation system was used to investigate the regulation of UCP2 expression by shear flow. EC-specific Ucp2 knockout mice were used to investigate the role of UCP2 in flow-associated atherosclerosis. RESULTS: Shear stress experiments showed that KLF2 (Krüppel-like factor 2) mediates fluid shear stress-dependent regulation of UCP2 expression in human aortic and human umbilical vein ECs. Unidirectional shear stress, statins, and resveratrol upregulate whereas oscillatory shear stress and proinflammatory stimuli inhibit UCP2 expression through altered KLF2 expression. KLF2 directly binds to UCP2 promoter to upregulate its transcription in human umbilical vein ECs. UCP2 knockdown induced expression of genes involved in proinflammatory and profibrotic signaling, resulting in a proatherogenic endothelial phenotype. EC-specific Ucp2 deletion promotes atherogenesis and collagen production. Additionally, we found endothelial Ucp2 deficiency aggravates whereas adeno-associated virus-mediated EC-Ucp2 overexpression inhibits carotid atherosclerotic plaque formation in disturbed flow-enhanced atherosclerosis mouse model. RNA-sequencing analysis revealed FoxO1 (forkhead box protein O1) as the major proinflammatory transcriptional regulator activated by UCP2 knockdown, and FoxO1 inhibition reduced vascular inflammation and disturbed flow-enhanced atherosclerosis. We showed further that UCP2 level is critical for phosphorylation of AMPK (AMP-activated protein kinase), which is required for UCP2-induced inhibition of FoxO1. CONCLUSIONS: Altogether, our studies uncover that UCP2 is novel mechanosensitive gene under the control of fluid shear stress and KLF2 in ECs. UCP2 expression is critical for endothelial proinflammatory response and atherogenesis. Therapeutic strategies enhancing UCP2 level may have therapeutic potential against atherosclerosis.

Constructing Lipoparticles Capable of Endothelial Cell-Derived Exosome-Mediated Delivery of Anti-miR-33a-5p to Cultured Macrophages.

Atherosclerosis is driven by intimal arterial macrophages accumulating cholesterol. Atherosclerosis also predominantly occurs in areas consisting of proinflammatory arterial endothelial cells. At time of writing, there are no available clinical treatments that precisely remove excess cholesterol from lipid-laden intimal arterial macrophages. Delivery of anti-miR-33a-5p to macrophages has been shown to increase apoAI-mediated cholesterol efflux via ABCA1 upregulation but delivering transgenes to intimal arterial macrophages is challenging due to endothelial cell barrier integrity. In this study, we aimed to test whether lipoparticles targeting proinflammatory endothelial cells can participate in endothelial cell-derived exosome exploitation to facilitate exosome-mediated transgene delivery to macrophages. We constructed lipoparticles that precisely target the proinflammatory endothelium and contain a plasmid that expresses XMOTIF-tagged anti-miR-33a-5p (LP-pXMoAntimiR33a5p), as XMOTIF-tagged small RNA demonstrates the capacity to be selectively shuttled into exosomes. The cultured cells used in our study were immortalized mouse aortic endothelial cells (iMAECs) and RAW 264.7 macrophages. From our results, we observed a significant decrease in miR-33a-5p expression in macrophages treated with exosomes released basolaterally by LPS-challenged iMAECs incubated with LP-pXMoAntimiR33a5p when compared to control macrophages. This decrease in miR-33a-5p expression in the treated macrophages caused ABCA1 upregulation as determined by a significant increase in ABCA1 protein expression in the treated macrophages when compared to the macrophage control group. The increase in ABCA1 protein also simulated ABCA1-dependent cholesterol efflux in treated macrophages-as we observed a significant increase in apoAI-mediated cholesterol efflux-when compared to the control group of macrophages. Based on these findings, strategies that involve combining proinflammatory-targeting lipoparticles and exploitation of endothelial cell-derived exosomes appear to be promising approaches for delivering atheroprotective transgenes to lipid-laden arterial intimal macrophages.

Myeloid CCN3 protects against aortic valve calcification.

BACKGROUND: Cellular communication network factor 3 (CCN3) has been implicated in the regulation of osteoblast differentiation. However, it is not known if CCN3 can regulate valvular calcification. While macrophages have been shown to regulate valvular calcification, the molecular and cellular mechanisms of this process remain poorly understood. In the present study, we investigated the role of macrophage-derived CCN3 in the progression of calcific aortic valve disease. METHODS: Myeloid-specific knockout of CCN3 (Mye-CCN3-KO) and control mice were subjected to a single tail intravenous injection of AAV encoding mutant mPCSK9 (rAAV8/D377Y-mPCSK9) to induce hyperlipidemia. AAV-injected mice were then fed a high fat diet for 40 weeks. At the conclusion of high fat diet feeding, tissues were harvested and subjected to histologic and pathologic analyses. In vitro, bone marrow-derived macrophages (BMDM) were obtained from Mye-CCN3-KO and control mice and the expression of bone morphogenic protein signaling related gene were verified via quantitative real-time PCR and Western blotting. The BMDM conditioned medium was cocultured with human valvular intersititial cells which was artificially induced calcification to test the effect of the conditioned medium via Western blotting and Alizarin red staining. RESULTS: Echocardiography revealed that both male and female Mye-CCN3-KO mice displayed compromised aortic valvular function accompanied by exacerbated valve thickness and cardiac dysfunction. Histologically, Alizarin-Red staining revealed a marked increase in aortic valve calcification in Mye-CCN3-KO mice when compared to the controls. In vitro, CCN3 deficiency augmented BMP2 production and secretion from bone marrow-derived macrophages. In addition, human valvular interstitial cells cultured with conditioned media from CCN3-deficient BMDMs resulted in exaggerated pro-calcifying gene expression and the consequent calcification. CONCLUSION: Our data uncovered a novel role of myeloid CCN3 in the regulation of aortic valve calcification. Modulation of BMP2 production and secretion in macrophages might serve as a key mechanism for macrophage-derived CCN3's anti-calcification function in the development of CAVD. Video Abstract.

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

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-ß2-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-ß 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.

Very late vasomotor responses and gene expression with bioresorbable scaffolds and metallic drug-eluting stents.

OBJECTIVES: To investigate the long-term vasomotor response and inflammatory changes in Absorb bioresorbable vascular scaffold (BVS) and metallic drug-eluting stent (DES) implanted artery. BACKGROUND: Clinical evidence has demonstrated that compared to DES, BVS is associated with higher rates of target lesion failure. However, it is not known whether the higher event rates observed with BVS are related to endothelial dysfunction or inflammation associated with polymer degradation. METHODS: Ten Absorb BVS and six Xience V DES were randomly implanted in the main coronaries of six nonatherosclerotic swine. At 4-years, vasomotor response was evaluated in vivo by quantitative coronary angiography response to intracoronary infusion of Ach and ex vivo by the biomechanical response to prostaglandin F2-α (PGF2-α), substance P and bradykinin and gene expression analysis. RESULTS: Absorb BVS implanted arteries showed significantly restored vasoconstrictive responses after Ach compared to in-stent Xience V. The contractility of Absorb BVS treated segments induced by PGF2-α was significantly greater compared to Xience V treated segments and endothelial-dependent vasorelaxation was greater with Absorb BVS compared to Xience V. Gene expression analyses indicated the pro-inflammatory lymphotoxin-beta receptor (LTßR) signaling pathway was significantly upregulated in arteries treated with a metallic stent compared to Absorb BVS treated arterial segments. CONCLUSIONS: At 4 years, arteries treated with Absorb BVS compared with Xience V, demonstrate significantly greater restoration of vasomotor responses. Genetic analysis suggests mechanobiologic reparation of Absorb BVS treated arteries at 4 years as opposed to Xience V treated vessels.

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

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.

Atorvastatin and blood flow regulate expression of distinctive sets of genes in mouse carotid artery endothelium.

Hypercholesterolemia is a well-known pro-atherogenic risk factor and statin is the most effective anti-atherogenic drug that lowers blood cholesterol levels. However, despite systemic hypercholesterolemia, atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while the stable flow (s-flow) regions are spared. Given their predominant effects on endothelial function and atherosclerosis, we tested whether (1) statin and flow regulate the same or independent sets of genes and (2) statin can rescue d-flow-regulated genes in mouse artery endothelial cells in vivo. To test the hypotheses, C57BL/6 J mice (8-week-old male, n=5 per group) were pre-treated with atorvastatin (10mg/kg/day, Orally) or vehicle for 5 days. Thereafter, partial carotid ligation (PCL) surgery to induce d-flow in the left carotid artery (LCA) was performed, and statin or vehicle treatment was continued. The contralateral right carotid artery (RCA) remained exposed to s-flow to be used as the control. Two days or 2 weeks post-PCL surgery, endothelial-enriched RNAs from the LCAs and RCAs were collected and subjected to microarray gene expression analysis. Statin treatment in the s-flow condition (RCA+statin versus RCA+vehicle) altered the expression of 667 genes at 2-day and 187 genes at 2-week timepoint, respectively (P<0.05, fold change (FC)≥±1.5). Interestingly, statin treatment in the d-flow condition (LCA+statin versus LCA+vehicle) affected a limited number of genes: 113 and 75 differentially expressed genes at 2-day and 2-week timepoint, respectively (P<0.05, FC≥±1.5). In contrast, d-flow in the vehicle groups (LCA+vehicle versus RCA+vehicle) differentially regulated 4061 genes at 2-day and 3169 genes at 2-week timepoint, respectively (P<0.05, FC≥±1.5). Moreover, statin treatment did not reduce the number of flow-sensitive genes (LCA+statin versus RCA+statin) compared to the vehicle groups: 1825 genes at 2-day and 3788 genes at 2-week, respectively, were differentially regulated (P<0.05, FC≥±1.5). These results revealed that both statin and d-flow regulate expression of hundreds or thousands of arterial endothelial genes, respectively, in vivo. Further, statin and d-flow regulate independent sets of endothelial genes. Importantly, statin treatment did not reverse d-flow-regulated genes except for a small number of genes. These results suggest that both statin and flow play important independent roles in atherosclerosis development and highlight the need to consider their therapeutic implications for both.

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.

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.

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

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.

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

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 G0/G1 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.

Vascular Semaphorin 7A Upregulation by Disturbed Flow Promotes Atherosclerosis Through Endothelial ß1 Integrin.

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 Sema7A-/-ApoE-/- mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of ApoE-/- 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 (N-[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 Sema7A+/+ApoE-/- mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in Sema7A-/-ApoE-/- mice. Further studies showed that blockage of ß1 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 ß1 integrin-dependent manner.

Disturbed Flow Promotes Arterial Stiffening Through Thrombospondin-1.

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-ß 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-ß. In vitro and in vivo testing under d-flow conditions identified a possible role for TSP-1 activation of transforming growth factor-ß 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-ß. 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.

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

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.

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

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-/y mice on C57 background, we found that the eNOS-/- mice developed more advanced lesions, while the NOX1-/y 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-/y_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.

Mechanosensitive PPAP2B Regulates Endothelial Responses to Atherorelevant Hemodynamic Forces.

RATIONALE: PhosPhatidic Acid Phosphatase type 2B (PPAP2B), an integral membrane protein known as lipid phosphate phosphatase (LPP3) that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide association studies. However, it is unclear whether genome-wide association studies-identified coronary artery disease genes, including PPAP2B, participate in mechanotransduction mechanisms by which vascular endothelia respond to local atherorelevant hemodynamics that contribute to the regional nature of atherosclerosis. OBJECTIVE: To establish the critical role of PPAP2B in endothelial responses to hemodynamics. METHODS AND RESULTS: Reduced PPAP2B was detected in vivo in mouse and swine aortic arch (AA) endothelia exposed to chronic disturbed flow, and in mouse carotid artery endothelia subjected to surgically induced acute disturbed flow. In humans, PPAP2B was reduced in the downstream part of carotid plaques where low shear stress prevails. In culture, reduced PPAP2B was measured in human aortic endothelial cells under atherosusceptible waveform mimicking flow in human carotid sinus. Flow-sensitive microRNA-92a and transcription factor KLF2 were identified as upstream inhibitor and activator of endothelial PPAP2B, respectively. PPAP2B suppression abrogated atheroprotection of unidirectional flow; inhibition of lysophosphatidic acid receptor 1 restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. PPAP2B inhibition resulted in myosin light-chain phosphorylation and intercellular gaps, which were abolished by lysophosphatidic acid receptor 1/2 inhibition. Expression quantitative trait locus mapping demonstrated PPAP2B coronary artery disease risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in human aortic endothelial cells. CONCLUSIONS: Atherorelevant flows dynamically modulate endothelial PPAP2B expression through miR-92a and KLF2. Mechanosensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under atheroprotective flow.

Deep transcriptomic profiling reveals the similarity between endothelial cells cultured under static and oscillatory shear stress conditions.

Atherosclerosis is a multifactorial disease that preferentially develops in specific regions in the arterial tree. This characteristic is mainly attributed to the unique pattern of hemodynamic shear stress in vivo. High laminar shear stress (LS) found in straight lumen exerts athero-protective effects. Low or oscillatory shear stress (OS) present in regions of lesser curvature and arterial bifurcations predisposes arterial intima to atherosclerosis. Shear stress-regulated endothelial function plays an important role in the process of atherosclerosis. Most in vitro research studies focusing on the molecular mechanisms of endothelial function are performed in endothelial cells (ECs) under cultured static (ST) condition. Some findings, however, are not recapitulated in subsequent translational studies, mostly likely due to the missing biomechanical milieu. Here, we profiled the whole transcriptome of primary human coronary arterial endothelial cells (HCAECs) under different shear stress conditions with RNA sequencing. Among 16,313 well-expressed genes, we detected 8,177 that were differentially expressed in OS vs. LS conditions and 9,369 in ST vs. LS conditions. Notably, only 1,618 were differentially expressed in OS vs. ST conditions. Hierarchical clustering of ECs demonstrated a strong similarity between ECs under OS and ST conditions at the transcriptome level. Subsequent pairwise heat mapping and principal component analysis gave further weight to the similarity. At the individual gene level, expressional analysis of representative well-known genes as well as novel genes showed a comparable amount at mRNA and protein levels in ECs under ST and OS conditions. In conclusion, the present work compared the whole transcriptome of HCAECs under different shear stress conditions at the transcriptome level as well as at the individual gene level. We found that cultured ECs are significantly different from those under LS conditions. Thus using cells under ST conditions is unlikely to elucidate endothelial physiology. Given the revealed high similarities of the endothelial transcriptome under OS and ST conditions, it may be helpful to understand the underlying mechanisms of OS-induced endothelial dysfunction from static cultured endothelial studies.

The role of endothelial mechanosensitive genes in atherosclerosis and omics approaches.

Atherosclerosis is the leading cause of morbidity and mortality in the U.S., and is a multifactorial disease that preferentially occurs in regions of the arterial tree exposed to disturbed blood flow. The detailed mechanisms by which d-flow induces atherosclerosis involve changes in the expression of genes, epigenetic patterns, and metabolites of multiple vascular cells, especially endothelial cells. This review presents an overview of endothelial mechanobiology and its relation to the pathogenesis of atherosclerosis with special reference to the anatomy of the artery and the underlying fluid mechanics, followed by a discussion of a variety of experimental models to study the role of fluid mechanics and atherosclerosis. Various in vitro and in vivo models to study the role of flow in endothelial biology and pathobiology are discussed in this review. Furthermore, strategies used for the global profiling of the genome, transcriptome, miR-nome, DNA methylome, and metabolome, as they are important to define the biological and pathophysiological mechanisms of atherosclerosis. These "omics" approaches, especially those which derive data based on a single animal model, provide unprecedented opportunities to not only better understand the pathophysiology of atherosclerosis development in a holistic and integrative manner, but also to identify novel molecular and diagnostic targets.