CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer.

In atherosclerosis and Alzheimer's disease, deposition of the altered self components oxidized low-density lipoprotein (LDL) and amyloid-beta triggers a protracted sterile inflammatory response. Although chronic stimulation of the innate immune system is believed to underlie the pathology of these diseases, the molecular mechanisms of activation remain unclear. Here we show that oxidized LDL and amyloid-beta trigger inflammatory signaling through a heterodimer of Toll-like receptors 4 and 6. Assembly of this newly identified heterodimer is regulated by signals from the scavenger receptor CD36, a common receptor for these disparate ligands. Our results identify CD36-TLR4-TLR6 activation as a common molecular mechanism by which atherogenic lipids and amyloid-beta stimulate sterile inflammation and suggest a new model of TLR heterodimerization triggered by coreceptor signaling events.

Erythropoietic properties of human induced pluripotent stem cells-derived red blood cells in immunodeficient mice.

Transfusion of red blood cells (RBCs) is a life-saving intervention for anemic patients. Human induced pluripotent stem cells (iPSC) have the capability to expand and differentiate into RBCs (iPSC-RBCs). Here we developed a murine model to investigate the in vivo properties of human iPSC-RBCs. iPSC lines were produced from human peripheral blood mononuclear cells by transient expression of plasmids containing OCT4, SOX2, MYC, KLF4, and BCL-XL genes. Human iPSC-RBCs were generated in culture supplemented with human platelet lysate, and were CD34- CD235a+ CD233+ CD49dlow CD71low ; about 13% of iPSC-RBCs were enucleated before transfusion. Systemic administration of clodronate liposomes (CL) and cobra venom factor (CVF) to NOD scid gamma (NSG) mice markedly promoted the circulatory survival of human iPSC-RBCs following transfusion. While iPSC-RBCs progressively decreased with time, 90% of circulating iPSC-RBCs were enucleated 1 day after transfusion (CD235a+ CD233+ CD49d- CD71- ). Surprisingly, human iPSC-RBCs reappeared in the peripheral circulation at 3 weeks after transfusion at levels more than 8-fold higher than at 1 h after transfusion. Moreover, a substantial portion of the transfused nucleated iPSC-RBCs preferentially homed to the bone marrow, and were detectable at 24 days after transfusion. These results suggest that nucleated human iPSC-derived cells that homed to the bone marrow of NSG mice retained the capability to complete differentiation into enucleated erythrocytes and egress the bone marrow into peripheral blood. The results offer a new model using human peripheral blood-derived iPSC and CL/CVF-treated NSG mice to investigate the development and circulation of human erythroid cells in vivo.

BMI1 enables extensive expansion of functional erythroblasts from human peripheral blood mononuclear cells.

Transfusion of red blood cells (RBCs) from ABO-matched but genetically unrelated donors is commonly used for treating anemia and acute blood loss. Increasing demand and insufficient supply for donor RBCs, especially those of universal blood types or free of known and unknown pathogens, has called for ex vivo generation of functional RBCs by large-scale cell culture. However, generating physiological numbers of transfusable cultured RBCs (cRBCs) ex vivo remains challenging, due to our inability to either extensively expand primary RBC precursors (erythroblasts) or achieve efficient enucleation once erythroblasts have been expanded and induced to differentiation and maturation. Here, we report that ectopic expression of the human BMI1 gene confers extensive expansion of human erythroblasts, which can be derived readily from adult peripheral blood mononuclear cells of either healthy donors or sickle cell patients. These extensively expanded erythroblasts (E3s) are able to proliferate exponentially (>1 trillion-fold in 2 months) in a defined culture medium. Expanded E3 cells are karyotypically normal and capable of terminal maturation with approximately 50% enucleation. Additionally, E3-derived cRBCs can circulate in a mouse model following transfusion similar to primary human RBCs. Therefore, we provide a facile approach of generating physiological numbers of human functional erythroblasts ex vivo.

Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans.

A major challenge in vaccinology is to prospectively determine vaccine efficacy. Here we have used a systems biology approach to identify early gene 'signatures' that predicted immune responses in humans vaccinated with yellow fever vaccine YF-17D. Vaccination induced genes that regulate virus innate sensing and type I interferon production. Computational analyses identified a gene signature, including complement protein C1qB and eukaryotic translation initiation factor 2 alpha kinase 4-an orchestrator of the integrated stress response-that correlated with and predicted YF-17D CD8(+) T cell responses with up to 90% accuracy in an independent, blinded trial. A distinct signature, including B cell growth factor TNFRS17, predicted the neutralizing antibody response with up to 100% accuracy. These data highlight the utility of systems biology approaches in predicting vaccine efficacy.

GM-CSF and IL-4 Fusion Cytokine Induces B Cell-Dependent Hematopoietic Regeneration.

Hematopoietic stem cells (HSCs) have the capacity to self-renew and differentiate into hematopoietic cells and have been utilized to replace diseased bone marrow for patients with cancers and blood disorders. Although remarkable progress has been made in developing new tools to manipulate HSCs for clinic use, there is still no effective method to expand HSCs in vivo for quick repopulation of hematopoietic cells following sublethal irradiation. We have recently described a novel synthetic cytokine that is derived from the fusion of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4; named as GIFT4), and we have now discovered that GIFT4 fusokine promotes long-term hematopoietic regeneration in a B cell-dependent manner. We found that GIFT4 treatment triggered a robust expansion of endogenous bone marrow HSCs and multipotent progenitors in vivo. Delivery of GIFT4 protein together with B cells rescued lethally irradiated mice. Moreover, adoptive transfer of autologous or allogeneic GIFT4-treated B cells (GIFT4-B cells) enhanced long-term hematopoietic recovery in radiated mice and prevented the mice from irradiation-induced death. Our data suggest that GIFT4 as well as GIFT4-B cells could serve as means to augment HSC engraftment in the setting of bone marrow transplantation for patients with hematological malignancy.

Combination of serum lipids and cancer antigens as a novel marker for colon cancer diagnosis.

BACKGROUND: Colon cancer is a malignancy of the large intestine with high mortality and economic burden. Recent studies reveal a new relationship between blood lipids and the risk of cancer. The presents study aims to investigate the combination of serum lipids with cancer antigens as a novel diagnostic marker for colon cancer. METHODS: Two hundred of colon cancer patients or healthy subjects were recruited. Serum lipids and cancer antigens such as total cholesterol (TC), high-density lipoprotein (HDL), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were measured. RESULTS: There were significantly lower level of serum TC or HDL, and significantly higher level of serum CEA or CA19-9 in patients than in healthy subjects. Serum TC or HDL in patients with advanced colon cancer was significantly lower than the ones with early stage disease. The level of serum TC or HDL in patients after surgical removal of colon cancer was significantly higher compared to the ones before surgery, but serum CEA or CA19-9 after surgery was significantly reduced in comparison with the ones before surgery. Combined TC, HDL, CEA and CA19-9 as a diagnostic marker for colon cancer had the highest positive predictive rate in comparison with individual, two or three of the parameters. CONCLUSIONS: The combination of serum TC, HDL, CEA and CA19-9 can be used as an effective marker for colon cancer, and offers a novel strategy for clinical diagnosis and monitoring the disease.

Regulatory B Cells Induce Formation of IL-10-Expressing T Cells in Mice with Autoimmune Neuroinflammation.

Although B cells are traditionally known for their role in propagating proinflammatory immune responses, their immunosuppressive effects have only recently begun to be appreciated. How these regulatory B cells (Bregs) suppress the immune response remains to be worked out in detail. In this article, we show that Bregs can induce the formation of conventional FoxP3+ regulatory T cells (Tregs), as well as a more recently described CD49b+CD223+ regulatory T-cell subset, known as type 1 regulatory T cells (Tr1s). When Bregs are transferred into mice with experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, they home to the spleen and mesenteric lymph nodes, leading to an expansion of Tregs and Tr1 in vivo Tregs and Tr1s are also found in greater proportions in the CNS of mice with EAE treated with Bregs and are correlated with the remission of symptoms. The discovery that Bregs induce the formation of regulatory T-cell subsets in vivo may herald their use as immunosuppressive agents in adoptive cellular therapies for autoimmune pathologies. SIGNIFICANCE STATEMENT: Although B cells are traditionally known for their role in propagating proinflammatory immune responses, their immunosuppressive effects have only recently begun to be appreciated. How regulatory B cells (Bregs) suppress the immune response remains to be fully understood. In this article, we show that Bregs can induce the formation of conventional regulatory T cells (Tregs) as well as type 1 regulatory T cells (Tr1s). When Bregs are transferred into mice with experimental autoimmune encephalomyelitis (EAE), they home to secondary lymphoid organs, leading to an expansion of Tregs and Tr1s in vivo Tregs and Tr1s are also found in greater proportions in the CNS of mice with EAE treated with Bregs and are correlated with the remission of symptoms.

Expression of Death Receptor 4 Is Positively Regulated by MEK/ERK/AP-1 Signaling and Suppressed upon MEK Inhibition.

Death receptor 4 (DR4) is a cell surface receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and triggers apoptosis upon ligation with TRAIL or aggregation. MEK/ERK signaling is a well known and the best-studied effector pathway downstream of Ras and Raf. This study focuses on determining the impact of pharmacological MEK inhibition on DR4 expression and elucidating the underlying mechanism. We found that several MEK inhibitors including MEK162, AZD6244, and PD0325901 effectively decreased DR4 protein levels including cell surface DR4 in different cancer cell lines. Accordingly, pre-treatment of TRAIL-sensitive cancer cell lines with a MEK inhibitor desensitized them to TRAIL-induced apoptosis. These results indicate that MEK inhibition negatively regulates DR4 expression and cell response to TRAIL-induced apoptosis. MEK inhibitors did not alter DR4 protein stability, rather decreased its mRNA levels, suggesting a transcriptional regulation. In contrast, enforced activation of MEK/ERK signaling by expressing ectopic B-Raf (V600E) or constitutively activated MEK1 (MEK1-CA) or MEK2 (MEK2-CA) activated ERK and increased DR4 expression; these effects were inhibited when a MEK inhibitor was present. Promoter analysis through deletion and mutation identified the AP-1 binding site as an essential response element for enhancing DR4 transactivation by MEK1-CA. Furthermore, inhibition of AP-1 by c-Jun knockdown abrogated the ability of MEK1-CA to increase DR4 promoter activity and DR4 expression. These results suggest an essential role of AP-1 in mediating MEK/ERK activation-induced DR4 expression. Our findings together highlight a previously undiscovered mechanism that positively regulates DR4 expression through activation of the MEK/ERK/AP-1 signaling pathway.

GIFT4 fusokine converts leukemic B cells into immune helper cells.

BACKGROUND: Chronic lymphocytic leukemia (CLL) remains incurable with standard therapy, and is characterized by excessive expansion of monoclonal abnormal mature B cells and more regulatory immune properties of T cell compartment. Thus, developing novel strategies to enhance immune function merits further investigation as a possible therapy for CLL. METHODS: We generated a fusion cytokine (fusokine) arising from the combination of human GM-CSF and IL-4 (named GIFT4). Primary CLL cells were treated with GIFT4 or GM-CSG and IL-4 in vitro. GIFT4-triggered STAT5 signaling in CLL cells was examined by Western blot. The phenotype and secretome of GIFT4-treated CLL cells (GIFT4-CLL cells), and the immune stimulatory function of GIFT4-CLL cells on autologous T cells were analyzed by flow cytometry and luminex assay. RESULTS: GIFT4-CLL up-regulated the expression of co-stimulatory molecules CD40, CD80 and CD86 and adhesion molecule CD54. GIFT4-CLL cells secreted IL-1ß, IL-6, ICAM-1 and substantial IL-2 relative to unstimulated CLL cells. GIFT4 treatment led to JAK1, JAK2 and JAK3-mediated hyper-phosphorylation of STAT5 in primary CLL cells, which is essential for GIFT4-triggered conversion of CLL cells. GIFT4-CLL cells directly propelled the expansion of autologous IFN-γ-producing CD314(+) cytotoxic T cells in vitro, and that these could lyse autologous CLL cells. Furthermore, administration of GIFT4 protein promoted the expansion of human T cells in NOD-scid IL2Rγ(null) immune deficient mice adoptively pre-transferred with peripheral blood mononuclear cells from subjects with CLL. CONCLUSION: GIFT4 has potent capability to converts primary CLL cells into APC-like immune helper cells that initiate a T cell driven anti-CLL immune response.

Blocking the NOTCH pathway inhibits vascular inflammation in large-vessel vasculitis.

BACKGROUND: Giant cell arteritis is a granulomatous vasculitis of the aorta and its branches that causes blindness, stroke, and aortic aneurysm. CD4 T cells are key pathogenic regulators, instructed by vessel wall dendritic cells to differentiate into vasculitic T cells. The unique pathways driving this dendritic cell-T-cell interaction are incompletely understood, but may provide novel therapeutic targets for a disease in which the only established therapy is long-term treatment with high doses of corticosteroids. METHODS AND RESULTS: Immunohistochemical and gene expression analyses of giant cell arteritis-affected temporal arteries revealed abundant expression of the NOTCH receptor and its ligands, Jagged1 and Delta1. Cleavage of the NOTCH intracellular domain in wall-infiltrating T cells indicated ongoing NOTCH pathway activation in large-vessel vasculitis. NOTCH activation did not occur in small-vessel vasculitis affecting branches of the vasa vasorum tree. We devised 2 strategies to block NOTCH pathway activation: γ-secretase inhibitor treatment, preventing nuclear translocation of the NOTCH intracellular domain, and competing for receptor-ligand interactions through excess soluble ligand, Jagged1-Fc. In a humanized mouse model, NOTCH pathway disruption had strong immunosuppressive effects, inhibiting T-cell activation in the early and established phases of vascular inflammation. NOTCH inhibition was particularly effective in downregulating Th17 responses, but also markedly suppressed Th1 responses. CONCLUSIONS: Blocking NOTCH signaling depleted T cells from the vascular infiltrates, implicating NOTCH- NOTCH ligand interactions in regulating T-cell retention and survival in vessel wall inflammation. Modulating the NOTCH signaling cascade emerges as a promising new strategy for immunosuppressive therapy of large-vessel vasculitis.

Silence of FIZZ1 by Short Hairpin RNA Inhibits Atherosclerosis.

OBJECTIVE: Found in Inflammatory Zone 1 (FIZZ1) protein plays an important enhancive role in inflammation and angiogenesis. This study aims to explore the effects of FIZZ1 on murine atherosclerosis. METHODS: The murine aortic endothelial cells were treated with an adenoviral vector encoding FIZZ1 short hairpin RNA (Ad-shFIZZ1). Murine atherosclerosis model was established with ApoE-/- mice. The effects of FIZZ1 were studied in vitro and in vivo. RESULTS: Ad-shFIZZ1 treatment suppressed the expression of FIZZ1 and the formation of capillary tube formation in vitro. Administration of Ad-shFIZZ1 suppressed FIZZ1-mediated signaling, the growth of endothelial cells and the production of inflammatory molecules in murine aorta in vivo, and inhibited the development of atherosclerosis in vivo. CONCLUSION: FIZZ1 played a potent promotive role in atherosclerosis, and could serve as a novel therapeutic target for the treatment of the disease.

Th17 and Th1 T-cell responses in giant cell arteritis.

BACKGROUND: In giant cell arteritis (GCA), vasculitic damage of the aorta and its branches is combined with a syndrome of intense systemic inflammation. Therapeutically, glucocorticoids remain the gold standard because they promptly and effectively suppress acute manifestations; however, they fail to eradicate vessel wall infiltrates. The effects of glucocorticoids on the systemic and vascular components of GCA are not understood. METHODS AND RESULTS: The immunoprofile of untreated and glucocorticoid-treated GCA was examined in peripheral blood and temporal artery biopsies with protein quantification assays, flow cytometry, quantitative real-time polymerase chain reaction, and immunohistochemistry. Plasma interferon-gamma and interleukin (IL)-17 and frequencies of interferon-gamma-producing and IL-17-producing T cells were markedly elevated before therapy. Glucocorticoid treatment suppressed the Th17 but not the Th1 arm in the blood and the vascular lesions. Analysis of monocytes/macrophages in the circulation and in temporal arteries revealed glucocorticoid-mediated suppression of Th17-promoting cytokines (IL-1beta, IL-6, and IL-23) but sparing of Th1-promoting cytokines (IL-12). In human artery-severe combined immunodeficiency mouse chimeras, in which patient-derived T cells cause inflammation of engrafted human temporal arteries, glucocorticoids were similarly selective in inhibiting Th17 cells and leaving Th1 cells unaffected. CONCLUSIONS: Two pathogenic pathways mediated by Th17 and Th1 cells contribute to the systemic and vascular manifestations of GCA. IL-17-producing Th17 cells are sensitive to glucocorticoid-mediated suppression, but interferon-gamma-producing Th1 responses persist in treated patients. Targeting steroid-resistant Th1 responses will be necessary to resolve chronic smoldering vasculitis. Monitoring Th17 and Th1 frequencies can aid in assessing disease activity in GCA.

A MCP1 fusokine with CCR2-specific tumoricidal activity.

BACKGROUND: The CCL2 chemokine is involved in promoting cancer angiogenesis, proliferation and metastasis by malignancies that express CCR2 receptor. Thus the CCL2/CCR2 axis is an attractive molecular target for anticancer drug development. METHODS: We have generated a novel fusion protein using GMCSF and an N-terminal truncated version of MCP1/CCL2 (6-76) [hereafter GMME1] and investigated its utility as a CCR2-specific tumoricidal agent. RESULTS: We found that distinct to full length CCL2 or its N-truncated derivative (CCL2 5-76), GMME1 bound to CCR2 on mouse lymphoma EG7, human multiple myeloma cell line U266, or murine and human medulloblastoma cell lines, and led to their death by apoptosis. We demonstrated that GMME1 specifically blocked CCR2-associated STAT3 phosphorylation and up-regulated pro-apoptotic BAX. Furthermore, GMME1 significantly inhibited EG7 tumor growth in C57BL/6 mice, and induced apoptosis of primary myeloma cells from patients. CONCLUSION: Our data demonstrate that GMME1 is a fusokine with a potent, CCR2 receptor-mediated pro-apoptotic effect on tumor cells and could be exploited as a novel biological therapy for CCR2+ malignancies including lymphoid and central nervous system malignancies.

Toll-like receptors 4 and 5 induce distinct types of vasculitis.

Large vessel vasculitides, such as Takayasu arteritis and giant cell arteritis, affect vital arteries and cause clinical complications by either luminal occlusion or vessel wall destruction. Inflammatory infiltrates, often with granulomatous arrangements, are distributed as a panarteritis throughout all of the artery's wall layers or cluster in the adventitia as a perivasculitis. Factors determining the architecture and compartmentalization of vasculitis are unknown. Human macrovessels are populated by indigenous dendritic cells (DCs) positioned in the adventitia. Herein, we report that these vascular DCs sense bacterial pathogens and regulate the patterning of the emerging arteritis. In human temporal artery-SCID chimeras, lipopolysaccharides stimulating Toll-like receptor (TLR)4 and flagellin stimulating TLR5 trigger vascular DCs and induce T-cell recruitment and activation. However, the architecture of the evolving inflammation is ligand-specific; TLR4 ligands cause transmural panarteritis and TLR5 ligands promote adventitial perivasculitis. Underlying mechanisms involve selective recruitment of functional T cell subsets. Specifically, TLR4-mediated DC stimulation markedly enhances production of the chemokine CCL20, biasing recruitment toward CCL20-responsive CCR6(+) T cells. In adoptive transfer experiments, CCR6(+) T cells produce an arteritis pattern with media-invasive T cells damaging vascular smooth muscle cells. Also, CCR6(+) T cells dominate the vasculitic infiltrates in patients with panarteritic giant cell arteritis. Thus, depending on the original danger signal, vascular DCs edit the emerging immune response by differentially recruiting specialized T effector cells and direct the disease process toward distinct types of vasculitis.

Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain.

Phagocyte recognition and clearance of bacteria play essential roles in the host response to infection. In an on-going forward genetic screen, we identify the Drosophila melanogaster scavenger receptor Croquemort as a receptor for Staphylococcus aureus, implicating for the first time the CD36 family as phagocytic receptors for bacteria. In transfection assays, the mammalian Croquemort paralogue CD36 confers binding and internalization of Gram-positive and, to a lesser extent, Gram-negative bacteria. By mutational analysis, we show that internalization of S. aureus and its component lipoteichoic acid requires the COOH-terminal cytoplasmic portion of CD36, specifically Y463 and C464, which activates Toll-like receptor (TLR) 2/6 signaling. Macrophages lacking CD36 demonstrate reduced internalization of S. aureus and its component lipoteichoic acid, accompanied by a marked defect in tumor necrosis factor-alpha and IL-12 production. As a result, Cd36-/- mice fail to efficiently clear S. aureus in vivo resulting in profound bacteraemia. Thus, response to S. aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain, which initiates TLR2/6 signaling.

Next-generation sequencing specifies Angiostrongylus eosinophilic meningoencephalitis in infants: Two case reports.

RATIONALE: Angiostrongylus cantonensis-induced eosinophilic meningoencephalitis (AEM) in infants is a very rare but fatal disease. Utilization of genetic assay to detect the cerebral parasite plays an important role for the treatment of the infection. PATIENT CONCERNS: Two infants (<2 years) presented with cough, intermittent fever, mental fatigue, and poor diet. DIAGNOSIS: The patients were under clinical examination and laboratory test including cardiac ultrasound, chest X-ray, blood or cerebrospinal fluid (CSF) cell counting, serum enzyme-linked immunosorbent assay (ELISA), head magnetic resonance imaging (MRI) and next-generation sequencing (NGS) on DNA from CSF. Due to hypereosinophils in patients' peripheral blood and CSF, and abundant DNA sequences from A cantonensis in CSF, the patients were diagnosed with Angiostrongylus eosinophilic meningoencephalitis. INTERVENTIONS: The patients were treated with albendazole to deworm, and methylprednisolone to reduce inflammation. OUTCOME: The patients were completely recovered from AEM without relapse after 10-day treatment. LESSONS: ELISA and MRI are not sufficiently accurate for the diagnosis of AEM in infants. NGS can specify the infection by the cerebral parasite and offers a new effective approach for the early and precise diagnosis of AEM in infants.

Erratum: Crim1 suppresses left ventricular hypertrophy.

[This corrects the article DOI: 10.3892/br.2019.1214.].

Crim1 suppresses left ventricular hypertrophy.

Left ventricular hypertrophy is a leading cause of heart failure and sudden death. Cysteine-rich transmembrane bone morphogenetic protein regulator 1 (Crim1) is expressed at a high level in the heart and has a regulatory role in heart development. The present study aimed to test the hypothesis that Crim1 can have an inhibitory function on ventricular hypertrophy. Rat primary ventricular myocytes were stretched to induce myocyte hypertrophy, and treated with telmisartan or infected with Crim1-expressing recombinant adenovirus (Ad-Crim1). Rat ventricular hypertrophy was induced by abdominal aortic coarctation (AAC), and treated either with telmisartan or myocardial injection of Ad-Crim1 or empty adenovirus vector. The results showed that the expression of Crim1 decreased in the hypertrophic ventricle. The inhibition of angiotensin receptor type 1 (AT1R) by telmisartan in vitro and in vivo significantly increased the expression of Crim1 in the left ventricle. The overexpression of Crim1 by infection with Ad-Crim1 significantly inhibited stretch-induced ventricular myocyte hypertrophy in vitro. The overexpression of Crim1 by gavage with AT1R inhibitor telmisartan or myocardial injection of Ad-Crim1 markedly suppressed AAC-induced left ventricular hypertrophy in vivo. These results suggest that Crim1 has a suppressive function on ventricular hypertrophy and provides a novel therapeutic target for the treatment of cardiac hypertrophy.

Silencing of long non-coding RNA H19 downregulates CTCF to protect against atherosclerosis by upregulating PKD1 expression in ApoE knockout mice.

This study aimed to explore the interactions among long non-coding RNA H19, transcriptional factor CCCTC-binding factor (CTCF) and polycystic kidney disease 1 (PKD1), and to investigate its potentially regulatory effect on vulnerable plaque formation and angiogenesis of atherosclerosis. We established an atherosclerosis mouse model in ApoE knockout mice, followed by gain- and loss-of-function approaches. H19 was upregulated in aortic tissues of atherosclerosis mice, but silencing of H19 significantly inhibited atherosclerotic vulnerable plaque formation and intraplaque angiogenesis, accompanied by a downregulated expression of MMP-2, VEGF, and p53 and an upregulated expression of TIMP-1. Moreover, opposite results were found in the aortic tissues of atherosclerosis mice treated with H19 or CTCF overexpression. H19 was capable of recruiting CTCF to suppress PKD1, thus promoting atherosclerotic vulnerable plaque formation and intraplaque angiogenesis in atherosclerosis mice. The present study provides evidence that H19 recruits CTCF to downregulate the expression of PKD1, thereby promoting vulnerable plaque formation and intraplaque angiogenesis in mice with atherosclerosis.