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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 (P0.05, 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 (P0.05, 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 (P0.05, 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 (P0.05, 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.

Antisense oligonucleotides (ASOs) are single-stranded short nucleic acids that silence the expression of target mRNAs and show increasing therapeutic potential. Since ASOs are internalized by many cell types, both normal and diseased cells, gene silencing in unwanted cells is a significant challenge for their therapeutic use. To address this challenge, we created conditional ASOs that become active only upon detecting transcripts unique to the target cell. As a proof-of-concept, we modified an HIF1α ASO (EZN2968) to generate miRNA-specific conditional ASOs, which can inhibit HIF1α in the presence of a hepatocyte-specific miRNA, miR-122, via a toehold exchange reaction. We characterized a library of nucleic acids, testing how the conformation, thermostability, and chemical composition of the conditional ASO impact the specificity and efficacy in response to miR-122 as a trigger signal. Optimally designed conditional ASOs demonstrated knockdown of HIF1α in cells transfected with exogenous miR-122 and in hepatocytes expressing endogenous miR-122. We confirmed that conditional ASO activity was mediated by toehold exchange between miR-122 and the conditional ASO duplex, and the magnitude of the knockdown depended on the toehold length and miR-122 levels. Using the same concept, we further generated another conditional ASO that can be triggered by miR-21. Our results suggest that conditional ASOs can be custom-designed with any miRNA to control ASO activation in targeted cells while reducing unwanted effects in nontargeted cells.

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

Atherosclerosis is the primary cause of hardening and narrowing arteries, leading to cardiovascular disease accounting for the high mortality in the United States. For developing effective treatments for atherosclerosis, considerable efforts have been devoted to developing

Objective: CCN3, an inhibitor in osteoblast differentiation, can modulate calcium signaling, yet, whether CCN3 can regulate valvular calcification is unknown. While it has been suggested that macrophages are important in the regulation of valvular calcification, the molecular and cellular mechanisms of this process remain poorly understood. In the present study, we investigated the specific role that macrophage-derived CCN3 plays during the progression of calcific aortic valve disease (CAVD). Methods and Results: Myeloid-specific knockout of CCN3 (Mye-CCN3-KO) and control mice were subjected to a single injection of AAV encoding mutant mPCSK9 (rAAV8/D377Y-mPCSK9) to induce hyperlipidemia. AAV-injected mice were then fed a high fat diet (HFD) for 40 weeks. At the conclusion of HFD feeding, tissues were harvested for subsequent histological and pathological analysis. Echocardiography revealed that both male and female mye-CCN3-KO animals displayed compromised aortic valvular function accompanied by exacerbated valve thickness and cardiac dysfunction. Histologically, alizarin red and OsteoSense staining revealed a marked increase of aortic valve calcification in mye-CCN3-KO animals when compared to controls. In vitro, CCN3 deficiency augmented bmp2 production and secretion from bone marrow derived macrophages (BMDMs). Further, human valvular interstitial cells (VICs) cocultured with conditional media from CCN3-deficient BMDMs resulted in exaggerated pro-calcifying gene expression and the attendant calcification as revealed by alizarin red staining. Conclusions: 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 CCN3’s anti-calcification function in the development of CAVD.

IntroductionAtherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects ofd-flowands-floware mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (KLK10, a secreted serine protease) as a flow-sensitive gene in arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown.Methods and ResultsHere, we show that KLK10 is upregulated unders-flowconditions and downregulated underd-flowconditions usingin vivomouse models andin vitrostudies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated KLK10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected againstd-flow-induced inflammation and permeability dysfunction in human artery ECs (HAECs). Further, treatment of micein vivowith rKLK10 decreased arterial endothelial inflammation ind-flowregions. Additionally, rKLK10 injection or ultrasound-mediated transfection of KLK10-expressing plasmids inhibited atherosclerosis inApoE-/-mice. Studies using pharmacological inhibitors and siRNAs revealed that the anti-inflammatory effects of KLK10 were mediated by a Protease Activated Receptors (PAR1/2)-dependent manner. However, unexpectedly, KLK10 did not cleave the PARs. Through a proteomics study, we identified HTRA1 (High-temperature requirement A serine peptidase 1), which bound and cleaved KLK10. Further, siRNA knockdown of HTRA1 prevented KLK10’s anti-inflammatory and barrier protective function in HAECs, suggesting that HTRA1 regulates KLK10 function. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques.ConclusionKLK10 is a flow-sensitive endothelial protein and, in collaboration with HTRA1, serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

BACKGROUND AND AIMS: Hypoxia inducible factor 1α (HIF1α) plays a critical role in atherosclerosis as demonstrated in endothelial-targeted HIF1α -deficient mice. However, it has not been shown if specific pharmacological inhibitors of HIF1α can be used as potential drugs for atherosclerosis. PX-478 is a selective inhibitor of HIF1α, which was used to reduce cancer and obesity in animal models. Here, we tested whether PX-478 can be used to inhibit atherosclerosis. METHODS: We first tested PX-478 in human aortic endothelial cells (HAEC) and found that it significantly inhibited expression of HIF1α and its targets, including Collagen I. Next, two independent atherosclerosis models, C57BL/6 mice treated with AAV-PCSK9 and ApoE(−/−) mice, were used to test the efficacy of PX-478. Both mouse models were fed a Western diet for 3 months with bi-weekly treatment with PX-478 (40 mg/kg) or saline. RESULTS: PX-478 treatment reduced atherosclerotic plaque burden in the aortic trees in both mouse models, while plaque burden in the aortic sinus was reduced in the AAV-PCSK9 mouse model, but not in the ApoE(−/−) mice. Russell-Movat’s Pentachrome and Picrosirius Red staining showed a significant reduction in extracellular matrix remodeling and collagen maturation, respectively, in the PX-478-treated mice. As expected, PX-478 treatment reduced diet-induced weight-gain and abdominal adipocyte hypertrophy. Interestingly, PX-478 reduced plasma LDL cholesterol by 69% and 30% in AAV-PCSK9 and ApoE(−/−) mice, respectively. To explore the cholesterol-lowering mechanisms, we carried out an RNA sequencing study using the liver tissues from the ApoE(−/−) mouse study. We found 450 genes upregulated and 381 genes downregulated by PX-478 treatment in the liver. Further, gene ontology analysis showed that PX-478 treatment upregulated fatty acid and lipid catabolic pathways, while downregulating lipid biosynthesis and plasma lipoprotein particle remodeling processes. Of interest, Cfd, Elovl3, and Insig2 were some of the most downregulated genes by PX-478, and have been implicated in fat storage, fatty acid elongation, and cholesterol metabolism. The downregulation of Cfd, Elovl3, and Insig2 was further validated by qPCR in the liver tissues of ApoE(−/−) mice treated with PX-478. CONCLUSIONS: These results suggest that PX-478 is a potential anti-atherogenic drug, which targets vascular endothelium and hepatic cholesterol pathways.

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 (LTβR) 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.

Supplemental Digital Content is available in the text., Rationale: Understanding the mechanisms that regulate arterial flow recovery is important to design treatment options for peripheral artery disease patients ineligible for invasive revascularization. Transcriptional orchestrators of this recovery process represent an appealing target for treatment design. We previously identified Prdm (positive regulatory domain-containing protein) 16 as an arterial-specific endothelial transcription factor but its in vivo role in arteries remains completely unknown. Objective: To unravel the role of Prdm16 in arteries under physiological and pathological conditions, more specifically during peripheral artery disease. Methods and Results: Within the vasculature, Prdm16 expression was strictly confined to arterial endothelial and smooth muscle cells. Heterozygous loss of Prdm16 caused a modest reduction of the inner arterial diameter and smooth muscle cell coating without compromising vasomotor function. Upon femoral artery ligation, Prdm16+/− mice featured significantly impaired flow recovery to ischemic limbs. This impairment was recapitulated in mice with a Prdm16 deletion specifically in endothelial cells (EC-Prdm16−/−) but not smooth muscle cells. Structural collateral remodeling was normal in both Prdm16+/− and EC-Prdm16−/− mice, but significant endothelial dysfunction postligation was present in EC-Prdm16−/− mice as evidenced by impaired endothelial-dependent relaxation. Upon ligation, endothelial Prdm16 deficiency altered the expression of genes encoding endothelial cell function regulators, many related to nitric oxide bioavailability and Ca2+ homeostasis. Accordingly, Prdm16 overexpression in cultured endothelial cells affected both total cellular Ca2+ levels and store-operated Ca2+ entry. Conclusions: We showed that Prdm16 is indispensable for arterial flow recovery under pathological challenge not because it affects structural remodeling but due to its role in maintaining endothelial function. It, therefore, represents an appealing target for designing novel therapeutic strategies for no-option patients with peripheral artery disease.

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 hours in regular medium and for one 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 stretchinduced calcific AV disease.

Cancer and cardiovascular diseases are the leading causes of death globally. Given the high percentage of cancer patients with co-existing atherosclerosis due to many shared risk factors, the development of novel cancer therapeutic agents with strong anti-tumor efficacy and therapeutic benefit on atherosclerosis can significantly improve the outcome of cancer therapy. Immune checkpoint inhibition (ICI) therapy using therapeutic antibodies has shown promises in the treatment of several types of human cancers. However, many cancer patients showed a poor response due to low delivery efficiency, lack of effector T cells, an immunosuppressive tumor microenvironment, and intrinsic resistance in solid tumors. Increasing numbers of patients developed immunotherapy related adverse effects (irAEs). It is well known that macrophages and effector T cells drive the progression of atherosclerosis. Recent clinical studies revealed that cancer patients received ICI therapy have 3-fold higher risk of atherosclerosis and 3-fold increases in plaque progression detected by PET imaging. To enhance therapeutic efficacy of tumor immunotherapy and decrease irAEs, we have developed a hyaluronic acid nanoparticle (HANP) conjugated with PD1 mimetic peptides that target and block PD-L1 function, and encapsulated with Avasimibe (PD1Y-HANP/Ava). Avasimibe is a multifunctional agent that decreases cholesterol accumulation, inhibits tumor growth, and enhances immune response by activating cytotoxic T cells. We found that systemic administrations of PD1Y-HANP/Ava led to targeted delivery into tumors and atherosclerotic plaques in a dual colon cancer and atherosclerosis mouse model, established by injecting mouse colon tumor cells into Apoe knockout mice on a high fat diet. PD1Y-HANP/Ava treatments resulted in 78% of tumor growth inhibition. Following surgical resection of residual tumors, 80% of PD1Y-HANP/Ava treated mice had disease-free survival of >120 days and were protected from tumor growth after tumor cell re-challenging. Histological analysis of the major arteries revealed that the volume of atherosclerotic plaques decreased 70% in the mice treated with PD1Y-HANP/Ava compared the no-treated mice. Our results showed that PD1Y-HANP/Ava treatment increased infiltration of CD8+ T effector and dendritic cells, and activated cytotoxic T cells in mouse colon tumors. Colon tumor specific antibodies were detected in the mouse serum following PD1Y-HANP/Ava treatment. However, the levels of CD8+ T cells, dendritic cells, and macrophages were decreased in the atherosclerotic plaques, which could prevent ICI-induced cardiovascular irAEs. Results of this study should provide us with preclinical evidence for translational development of targeted immunotherapy for colon cancer patients with comorbid atherosclerosis. Citation Format: Lei Zhu, Weiping Qian, Minglong Chen, Tongrui Liu, Charles A. Staley, Bassel El-Rayes, Hanjoong Jo, Lily Yang. Development of an immune modulating and tumor inhibiting hyaluronic acid nanoparticle encapsulated with Avasimibe for the treatment of cancer patients with comorbid atherosclerosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4152.

POLDIP2 is a multifunctional protein whose roles are only partially understood. Our laboratory previously reported physiological studies performed using a mouse gene trap model, which suffered from two limitations: perinatal lethality in homozygotes and constitutivePoldip2inactivation. To overcome these limitations, we developed a new conditional floxedPoldip2model. The first part of the present study shows that our initial floxed mice were affected by an unexpected mutation, which was not readily detected by Southern blotting and traditional PCR. It consisted of a 305 kb duplication aroundPoldip2with retention of the wild type allele and could be traced back to the original targeted ES cell clone. We offer simple suggestions to rapidly detect similar accidents, which may affect genome editing using both traditional and CRISPR-based methods. In the second part of the present study, correctly targeted floxedPoldip2mice were generated and used to produce a new constitutive knockout line by crossing with a Cre deleter. In contrast to the gene trap model, many homozygous knockout mice were viable, in spite of having no POLDIP2 expression. To further characterize the effects ofPoldip2ablation in the vasculature, an RNA-seq experiment was performed in constitutive knockout carotid arteries. Results support the involvement of POLDIP2 in multiple cellular processes and provide new opportunities for future in-depth study of its functions.

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.

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 (10 mg/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

Introduction: Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow ( d-flow ) while the straight regions exposed to stable flow ( s-flow ) are protected. The proatherogenic and atheroprotective effects of flow are mediated in large part by the global changes in endothelial cell gene expression, which regulate endothelial dysfunction and inflammation. Previously, we identified Kallikrein-Related Peptidase 10 (KLK10) as one of the most flow-sensitive genes in arterial endothelial cells using the partial carotid ligation model of d-flow -induced atherosclerosis. KLK10 is a secreted serine protease, but its role in endothelial function and atherosclerosis is unknown. Methods/Results: Here, we validated that KLK10 was upregulated under s-flow conditions and downregulated under d-flow conditions using the in vivo mouse models and in vitro studies using endothelial cells (ECs). Through in vitro functional studies using ECs, we found that KLK10 produced by s-flow protected against endothelial inflammation and permeability dysfunction. Furthermore, treatment with rKLK10 or overexpression of KLK10 plasmids in vivo decreased endothelial inflammation the mouse model. Further, rKLK10 injection or ultrasound-mediated transfection of KLK10 plasmids in the hind leg muscles led to inhibition of atherosclerosis in ApoE-/- mice with the partial carotid ligation surgery. Studies using the pharmacological inhibitors and siRNAs showed that the anti-inflammatory effects of KLK10 was mediated by the Protease Activated Receptors 1 and 2, but without directly cleaving them. Further studies show that KLK10’s anti-inflammatory effect was mediated by the NFκB and VCAM1 and ICAM1 expression pathway. In addition, immunostaining showed that KLK10 expression is significantly reduced in human coronary arterial sections with atherosclerotic plaques compared to the non-diseased controls. Conclusions: We found that KLK10 is a potent flow-sensitive secreted protein, which serve as a novel anti-inflammatory and anti-atherogenic factor. KLK10 may be a potential anti-atherogenic therapeutic.

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., Graphical abstract Image 1, Highlights • Reactive oxygen species (ROS) are thought to promote atherosclerosis. • NoxO1 is a factor required for the activation of the ROS-producing Nox1. • Deletion of NoxO1 attenuated atherosclerosis development in female mice. • NoxO1 knockout suppressed inflammatory signatures in the female murine plasma.

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.

SUMMARY 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., Graphical Abstract, In Brief To determine the effect of proatherogenic disturbed flow on transcriptomic and epigenomic chromatin accessibility profiles in endothelial cells at single-cell resolution, Andueza et al. perform scRNA-seq and scATAC-seq analyses using mouse carotid arteries following the partial carotid ligation. Disturbed flow reprograms endothelial cells to proatherogenic phenotypes, including EndMT and endothelial-to-immune cell-like transition.

Significance Here we show that the histone demethylase JMJD2B is induced in endothelial cells by EndMT provoking stimuli and thereby contributes to the acquirement of a mesenchymal/smooth muscle phenotype. Silencing of JMJD2B inhibited EndMT in vitro and reduced the induction of EndMT after myocardial infarction in vivo. Inhibition of JMJD2B prevents the demethylation of repressive trimethylated histone H3 at lysine 9 (H3K9me3) at promoters of mesenchymal and EndMT-controlling genes, thereby reducing EndMT. Together, our study reports a crucial role for JMJD2B in controlling histone modifications during the transition of endothelial cells toward a mesenchymal phenotype., 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.

Calcific aortic valve disease (CAVD) is a leading cause of death. Due to insufficient understanding of the molecular mechanisms that govern the disease, the current therapeutic options lack medical therapies and are limited to aortic valve (AV) replacement. AV calcification occurs preferentially in the fibrosa side of the valve, which is exposed to unstable hemodynamic conditions. In contrast, the ventricularis side is exposed to more stable hemodynamic conditions and is relatively protected from the disease, suggesting the role of biomechanical forces in CAVD. Recent studies have shown the role and mechanisms of biomechanical forces on regulation of AV biology and CAVD pathophysiology. Here, we review the molecular mechanisms by which biomechanical forces regulate mechanosensitive genes, especially microRNAs, and their roles in CAVD. Expression of these microRNAs can be easily manipulated, leading to effective inhibition of CAVD. These mechanosensitive microRNAs could be used for detection and treatment of CAVD.

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 down regulated 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 up-regulation 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.

Original article has been updated to include names and affiliations of authors.

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.

PURPOSE: Calcific aortic valve disease (CAVD) is a major cause of morbidity in the aging population, but 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). METHODS AND RESULTS: 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. CONCLUSIONS: These studies suggest that targeting this shear-dependent miRNA may provide a novel non-invasive treatment for CAVD.

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.

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.

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.

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 have been developed and shown to deliver RNAi to ECs, but few of them have been 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 (VCAM-1) protein, overexpressed on inflamed EC membranes. To test this approach, the novel NPs were 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 reduced 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 were 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.

Atherosclerosis is a chronic, inflammatory disease of the vascular wall, which preferentially develops in regions under disturbed flow (d-flow). Once inflamed, the endothelial cells initiate a signaling cascade that culminates in the expression of pro-atherogenic genes such as mRNAs and miRNAs. Here we demonstrate that vascular cell adhesion molecule 1 (VCAM1), which is highly expressed on the surface of endothelial cells under d-flow, can serve as a marker for the targeted delivery of drug to the inflamed endothelium. We could selectively deliver anti-miR-712, an inhibitor of a key pro-atherogenic miRNA, to VCAM1-expressing immortalized mouse aortic endothelial cells (iMAECs) by hybridizing the inhibitor with a carrier DNA that has a complementary sequence and is conjugated to the surface of Au nanospheres. Our results suggest that the combination of VCAM1-binding peptide and Au nanosphere could provide an effective strategy for the selective delivery of anti-athero-miRNAs or other drugs into inflamed endothelium for the purpose of inhibiting the formation of atherosclerotic plaques.

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.

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.

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.

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 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.

Aims Oxidative stress is thought to be a risk for cardiovascular disease and NADPH oxidases of the Nox family are important producers of reactive oxygen species. Within the Nox family, the NADPH oxidase Nox4 has a unique position as it is constitutively active and produces H2O2 rather than O-2(-). Nox4 is therefore incapable of scavenging NO and its low constitutive H2O2 production might even be beneficial. We hypothesized that Nox4 acts as an endogenous anti-atherosclerotic enzyme.Methods and results Tamoxifen-induced Nox4-knockout mice were crossed with ApoE(-/-) mice and spontaneous atherosclerosis under regular chow as well as accelerated atherosclerosis in response to partial carotid artery ligation under high-fat diet were determined. Deletion of Nox4 resulted in increased atherosclerosis formation in both models. Mechanistically, pro-atherosclerotic and pro-inflammatory changes in gene expression were observed prior to plaque development. Moreover, inhibition of Nox4 or deletion of the enzyme in the endothelium but not in macrophages resulted in increased adhesion of macrophages to the endothelial surface.Conclusions The H2O2-producing NADPH oxidase Nox4 is an endogenous anti- atherosclerotic enzyme. Nox4 inhibitors, currently under clinical evaluation, should be carefully monitored for cardiovascular side-effects.

Atherosclerosis remains a significant cause of mortality and morbidity in the world. It is now well established that atherosclerosis is an inflammatory disease preferentially occurring in curved or branched arterial regions, whereas straight parts of the arteries are protected, suggesting a close relationship between flow and atherosclerosis. Animal models of atherosclerosis are an important tool to understand the pathophysiology of atherosclerosis and identify new therapies. Various animal models have been used throughout the years, each contributing to scientific progress in the field, including different species such as pigeon, mice, rabbit, pig and nonhuman primates. In many cases, one or a combination of multiple interventions, including genetic manipulation, hypercholesterolemia inducing diets, or surgical interventions are employed to induce atherosclerosis or to accelerate or vary the severity and complexity of the lesions. In recent years, new animal models have been developed to address specific needs in the field of atherosclerosis research. Some of these models use surgical interventions to create a disturbed flow profile, and a few models have used larger animals such as minipigs. While PCSK9 inhibition is being used as a new therapy for lowering cholesterol in hyperlipidemic patients, over-expression of PCSK9 has recently been exploited to induce atherosclerosis in animal models. No animal model can perfectly mimic the process in human disease, however each model may be successfully used to elucidate a particular aspect of the pathophysiology. It is essential to understand the strengths and weaknesses in each model in order to interpret the results as accurate as possible.

Epigenetic mechanisms that regulate endothelial cell gene expression are now emerging. DNA methylation is the most stable epigenetic mark that confers persisting changes in gene expression. Not only is DNA methylation important in rendering cell identity by regulating cell type–specific gene expression throughout differentiation, but it is becoming clear that DNA methylation also plays a key role in maintaining endothelial cell homeostasis and in vascular disease development. Disturbed blood flow causes atherosclerosis, whereas stable flow protects against it by differentially regulating gene expression in endothelial cells. Recently, we and others have shown that flow-dependent gene expression and atherosclerosis development are regulated by mechanisms dependent on DNA methyltransferases (1 and 3A). Disturbed blood flow upregulates DNA methyltransferase expression both in vitro and in vivo, which leads to genome-wide DNA methylation alterations and global gene expression changes in a DNA methyltransferase–dependent manner. These studies revealed several mechanosensitive genes, such as HoxA5, Klf3, and Klf4, whose promoters were hypermethylated by disturbed blood flow, but rescued by DNA methyltransferases inhibitors such as 5Aza-2-deoxycytidine. These findings provide new insight into the mechanism by which flow controls epigenomic DNA methylation patterns, which in turn alters endothelial gene expression, regulates vascular biology, and modulates atherosclerosis development.

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 in vivo 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 in vivo 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.

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

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.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.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.

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−/− 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 approximately 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.

Atherosclerosis preferentially occurs in arterial regions exposed to disturbed flow, in part, due to alterations in gene expression. MicroRNAs (miRNAs) are small, noncoding genes that post-transcriptionally regulate gene expression by targeting messenger RNA transcripts. Emerging evidence indicates that alteration of flow conditions regulate expression of miRNAs in endothelial cells both in vitro and in vivo. These flow-sensitive miRNAs, known as mechano-miRs, regulate endothelial gene expression and can regulate endothelial dysfunction and atherosclerosis. MiRNAs such as, miR-10a, miR-19a, miR-23b, miR-17–92, miR-21, miR-663, miR-92a, miR-143/145, miR-101, miR-126, miR-712, miR-205, and miR-155, have been identified as mechano-miRs. Many of these miRNAs were initially identified as flow sensitive in vitro and were later found to play a critical role in endothelial function and atherosclerosis in vivo through either gain-of-function or loss-of-function approaches. The key signaling pathways that are targeted by these mechano-miRs include the endothelial cell cycle, inflammation, apoptosis, and nitric oxide signaling. Furthermore, we have recently shown that the miR-712/205 family, which is upregulated by disturbed flow, contributes to endothelial inflammation and vascular hyperpermeability by targeting tissue inhibitor of metalloproteinase-3, which regulates metalloproteinases and a disintegrin and metalloproteinases. The mechano-miRs that are implicated in atherosclerosis are termed as mechanosensitive athero-miRs and are potential therapeutic targets to prevent or treat atherosclerosis. This review summarizes the current knowledge of mechanosensitive athero-miRs and their role in vascular biology and atherosclerosis.