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.

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.

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.

Single-Cell RNA sequencing investigation of female-male differences under PAD conditions.

Peripheral arterial disease (PAD) is an age-related medical condition affecting mostly muscular arteries of the limb. It is the 3rd leading cause of atherosclerotic morbidity. The mechanical environment of endothelial cells (ECs) in PAD is characterized by disturbed blood flow (d-flow) and stiff extracellular matrices. In PAD, the stiffness of arteries is due to decreased elastin function and increased collagen content. These flow and stiffness parameters are largely missing from current models of PAD. It has been previously proven that ECs exposed to d-flow or stiff substrates lead to proatherogenic pathways, but the effect of both, d-flow and stiffness, on EC phenotype has not been fully investigated. In this study, we sought to explore the effect of sex on proatherogenic pathways that could result from exposing endothelial cells to a d-flow and stiff environment. We utilized the scRNA-seq tool to analyze the gene expression of ECs exposed to the different mechanical conditions both in vitro and in vivo. We found that male ECs exposed to different mechanical stimuli presented higher expression of genes related to fibrosis and d-flow in vitro. We validated our findings in vivo by exposing murine carotid arteries to d-flow via partial carotid artery ligation. Since women have delayed onset of arterial stiffening and subsequent PAD, this work may provide a framework for some of the pathways in which biological sex interacts with sex-based differences in PAD.

HIF1A inhibitor PX-478 reduces pathological stretch-induced calcification and collagen turnover in aortic valve.

HIF1A is significantly upregulated in calcified human aortic valves (AVs). Furthermore, HIF1A inhibitor PX-478 was shown to inhibit AV calcification under static and disturbed flow conditions. Since elevated stretch is one of the major mechanical stimuli for AV calcification, we investigated the effect of PX-478 on AV calcification and collagen turnover under a pathophysiological cyclic stretch (15%) condition. Porcine aortic valve (PAV) leaflets were cyclically (1 Hz) stretched at 15% for 24 days in osteogenic medium with or without PX-478. In addition, PAV leaflets were cyclically stretched at a physiological (10%) and 15% for 3 days in regular medium to assess its effect of on HIF1A mRNA expression. It was found that 100 µM (high concentration) PX-478 could significantly inhibit PAV calcification under 15% stretch, whereas 50 µM (moderate concentration) PX-478 showed a modest inhibitory effect on PAV calcification. Nonetheless, 50 µM PX-478 significantly reduced PAV collagen turnover under 15% stretch. Surprisingly, it was observed that cyclic stretch (15% vs. 10%) did not have any significant effect on HIF1A mRNA expression in PAV leaflets. These results suggest that HIF1A inhibitor PX-478 may impart its anti-calcific and anti-matrix remodeling effect in a stretch-independent manner.

Hypoxia inducible factor 1α inhibitor PX-478 reduces atherosclerosis in mice.

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.

Sox13 is a novel flow-sensitive transcription factor that prevents inflammation by repressing chemokine expression in endothelial cells.

Atherosclerosis is a chronic inflammatory disease and occurs preferentially in arterial regions exposed to disturbed blood flow (d-flow) while the stable flow (s-flow) regions are spared. D-flow induces endothelial inflammation and atherosclerosis by regulating endothelial gene expression partly through the flow-sensitive transcription factors (FSTFs). Most FSTFs, including the well-known Kruppel-like factors KLF2 and KLF4, have been identified from in vitro studies using cultured endothelial cells (ECs). Since many flow-sensitive genes and pathways are lost or dysregulated in ECs during culture, we hypothesized that many important FSTFs in ECs in vivo have not been identified. We tested the hypothesis by analyzing our recent gene array and single-cell RNA sequencing (scRNAseq) and chromatin accessibility sequencing (scATACseq) datasets generated using the mouse partial carotid ligation model. From the analyses, we identified 30 FSTFs, including the expected KLF2/4 and novel FSTFs. They were further validated in mouse arteries in vivo and cultured human aortic ECs (HAECs). These results revealed 8 FSTFs, SOX4, SOX13, SIX2, ZBTB46, CEBPß, NFIL3, KLF2, and KLF4, that are conserved in mice and humans in vivo and in vitro. We selected SOX13 for further studies because of its robust flow-sensitive regulation, preferential expression in ECs, and unknown flow-dependent function. We found that siRNA-mediated knockdown of SOX13 increased endothelial inflammatory responses even under the unidirectional laminar shear stress (ULS, mimicking s-flow) condition. To understand the underlying mechanisms, we conducted an RNAseq study in HAECs treated with SOX13 siRNA under shear conditions (ULS vs. oscillatory shear mimicking d-flow). We found 94 downregulated and 40 upregulated genes that changed in a shear- and SOX13-dependent manner. Several cytokines, including CXCL10 and CCL5, were the most strongly upregulated genes in HAECs treated with SOX13 siRNA. The robust induction of CXCL10 and CCL5 was further validated by qPCR and ELISA in HAECs. Moreover, the treatment of HAECs with Met-CCL5, a specific CCL5 receptor antagonist, prevented the endothelial inflammation responses induced by siSOX13. In addition, SOX13 overexpression prevented the endothelial inflammation responses. In summary, SOX13 is a novel conserved FSTF, which represses the expression of pro-inflammatory chemokines in ECs under s-flow. Reduction of endothelial SOX13 triggers chemokine expression and inflammatory responses, a major proatherogenic pathway.

Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis.

Atherosclerosis 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 of d-flow and s-flow are mediated in part by the global changes in endothelial cell (EC) 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 mouse arterial ECs, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo mouse models and in vitro studies with cultured 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 against d-flow-induced permeability dysfunction and inflammation in human artery ECs, as determined by NFκB activation, expression of vascular cell adhesion molecule 1 and intracellular adhesion molecule 1, and monocyte adhesion. Furthermore, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

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

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.

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

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