Deficiency of smooth muscle cell ILF3 alleviates intimal hyperplasia via HMGB1 mRNA degradation-mediated regulation of the STAT3/DUSP16 axis.

Intimal hyperplasia is a complicated pathophysiological phenomenon attributable to in-stent restenosis, and the underlying mechanism remains unclear. Interleukin enhancer-binding factor 3 (ILF3), a double-stranded RNA-binding protein involved in regulating mRNA stability, has been recently demonstrated to assume a crucial role in cardiovascular disease; nevertheless, its impact on intimal hyperplasia remains unknown. In current study, we used samples of human restenotic arteries and rodent models of intimal hyperplasia, we found that vascular smooth muscle cell (VSMC) ILF3 expression was markedly elevated in human restenotic arteries and murine ligated carotid arteries. SMC-specific ILF3 knockout mice significantly suppressed injury induced neointimal formation. In vitro, platelet-derived growth factor type BB (PDGF-BB) treatment elevated the level of VSMC ILF3 in a dose- and time-dependent manner. ILF3 silencing markedly inhibited PDGF-BB-induced phenotype switching, proliferation, and migration in VSMCs. Transcriptome sequencing and RNA immunoprecipitation sequencing depicted that ILF3 maintained its stability upon binding to the mRNA of the high-mobility group box 1 protein (HMGB1), thereby exerting an inhibitory effect on the transcription of dual specificity phosphatase 16 (DUSP16) through enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Therefore, the results both in vitro and in vivo indicated that the loss of ILF3 in VSMC ameliorated neointimal hyperplasia by regulating the STAT3/DUSP16 axis through the degradation of HMGB1 mRNA. Our findings revealed that vascular injury activates VSMC ILF3, which in turn promotes intima formation. Consequently, targeting specific VSMC ILF3 may present a potential therapeutic strategy for ameliorating cardiovascular restenosis.

Improved therapeutic effects on vascular intimal hyperplasia by mesenchymal stem cells expressing MIR155HG that function as a ceRNA for microRNA-205.

Coronary artery bypass grafting (CABG) is an effective treatment for coronary heart disease, with vascular transplantation as the key procedure. Intimal hyperplasia (IH) gradually leads to vascular stenosis, seriously affecting the curative effect of CABG. Mesenchymal stem cells (MSCs) were used to alleviate IH, but the effect was not satisfactory. This work aimed to investigate whether lncRNA MIR155HG could improve the efficacy of MSCs in the treatment of IH and to elucidate the role of the competing endogenous RNA (ceRNA). The effect of MIR155HG on MSCs function was investigated, while the proteins involved were assessed. IH was detected by HE and Van Gieson staining. miRNAs as the target of lncRNA were selected by bioinformatics analysis. qRT-PCR and dual-luciferase reporter assay were performed to verify the binding sites of lncRNA-miRNA. The apoptosis, Elisa and tube formation assay revealed the effect of ceRNA on the endothelial protection of MIR155HG-MSCs. We observed that MIR155HG improved the effect of MSCs on IH by promoting viability and migration. MIR155HG worked as a sponge for miR-205. MIR155HG/miR-205 significantly improved the function of MSCs, avoiding apoptosis and inducing angiogenesis. The improved therapeutic effects of MSCs on IH might be due to the ceRNA role of MIR155HG/miR-205.

Myocardial Bridge and Atherosclerosis, an Intimal Relationship.

PURPOSE OF REVIEW: This review investigates the relationship between myocardial bridges (MBs), intimal thickening in coronary arteries, and Atherosclerotic cardiovascular disease. It focuses on the role of mechanical forces, such as circumferential strain, in arterial wall remodeling and aims to clarify how MBs affect coronary artery pathology. REVIEW FINDINGS: MBs have been identified as influential in modulating coronary artery intimal thickness, demonstrating a protective effect against thickening within the MB segment and an increase in thickness proximal to the MB. This is attributed to changes in mechanical stress and hemodynamics. Research involving arterial hypertension models and vein graft disease has underscored the importance of circumferential strain in vascular remodeling and intimal hyperplasia. Understanding the complex dynamics between MBs, mechanical strain, and vascular remodeling is crucial for advancing our knowledge of coronary artery disease mechanisms. This could lead to improved management strategies for cardiovascular diseases, highlighting the need for further research into MB-related vascular changes.

Deletion of the P2Y2 receptor aggravates internal elastic lamina calcification in chronic kidney disease mice through upregulation of alkaline phosphatase and lipocalin-2.

Calcification of the medial layer, inducing arterial stiffness, contributes significantly to cardiovascular mortality in patients with chronic kidney disease (CKD). Extracellular nucleotides block the mineralization of arteries by binding to purinergic receptors including the P2Y2 receptor. This study investigates whether deletion of the P2Y2 receptor influences the development of arterial media calcification in CKD mice. Animals were divided into: (i) wild type mice with normal renal function (control diet) (n = 8), (ii) P2Y2 R-/- mice with normal renal function (n = 8), (iii) wild type mice with CKD (n = 27), and (iv) P2Y2 R-/- mice with CKD (n = 22). To induce CKD, animals received an alternating (0.2-0.3%) adenine diet for 7 weeks. All CKD groups developed a similar degree of chronic renal failure as reflected by high serum creatinine and phosphorus levels. Also, the presence of CKD induced calcification in the heart and medial layer of the aortic wall. However, deletion of the P2Y2 receptor makes CKD mice more susceptible to the development of calcification in the heart and aorta (aortic calcium scores (median ± IQR), CKD-wild type: 0.34 ± 4.3 mg calcium/g wet tissue and CKD-P2Y2 R-/- : 4.0 ± 13.2 mg calcium/g wet tissue). As indicated by serum and aortic mRNA markers, this P2Y2 R-/- mediated increase in CKD-related arterial media calcification was associated with an elevation of calcification stimulators, including alkaline phosphatase and inflammatory molecules interleukin-6 and lipocalin 2. The P2Y2 receptor should be considered as an interesting therapeutic target for tackling CKD-related arterial media calcification.

Tumor necrosis factor receptor-associated factor 5 protects against intimal hyperplasia by regulation of macrophage polarization via directly targeting PPARγ.

OBJECTIVES: Intimal hyperplasia is a serious clinical problem associated with the failure of therapeutic methods in multiple atherosclerosis-related coronary heart diseases, which are initiated and aggravated by the polarization of infiltrating macrophages. The present study aimed to determine the effect and underlying mechanism by which tumor necrosis factor receptor-associated factor 5 (TRAF5) regulates macrophage polarization during intimal hyperplasia. METHODS: TRAF5 expression was detected in mouse carotid arteries subjected to wire injury. Bone marrow-derived macrophages, mouse peritoneal macrophages and human myeloid leukemia mononuclear cells were also used to test the expression of TRAF5 in vitro. Bone marrow-derived macrophages upon to LPS or IL-4 stimulation were performed to examine the effect of TRAF5 on macrophage polarization. TRAF5-knockout mice were used to evaluate the effect of TRAF5 on intimal hyperplasia. RESULTS: TRAF5 expression gradually decreased during neointima formation in carotid arteries in a time-dependent manner. In addition, the results showed that TRAF5 expression was reduced in classically polarized macrophages (M1) subjected to LPS stimulation but was increased in alternatively polarized macrophages (M2) in response to IL-4 administration, and these changes were demonstrated in three different types of macrophages. An in vitro loss-of-function study with TRAF5 knockdown plasmids or TRAF5-knockout mice revealed high expression of markers associated with M1 macrophages and reduced expression of genes related to M2 macrophages. Subsequently, we incubated vascular smooth muscle cells with conditioned medium of polarized macrophages in which TRAF5 expression had been downregulated or ablated, which promoted the proliferation, migration and dedifferentiation of VSMCs. Mechanistically, TRAF5 knockdown inhibited the activation of anti-inflammatory M2 macrophages by directly inhibiting PPARγ expression. More importantly, TRAF5-deficient mice showed significantly aggressive intimal hyperplasia. CONCLUSIONS: Collectively, this evidence reveals an important role of TRAF5 in the development of intimal hyperplasia through the regulation of macrophage polarization, which provides a promising target for arterial restenosis-related disease management.

Effect of F11 Receptor/Junctional Adhesion Molecule-A-derived Peptide on Neointimal Hyperplasia in a Murine Model.

PURPOSE: To determine whether inhibition of the F11 receptor/JAM-A (F11R) using F11R-specific antagonist peptide 4D results in inhibition of smooth muscle cell (SMC) proliferation and migration in vivo, known as neointimal hyperplasia (NIH), using a mouse focal carotid artery stenosis model (FCASM). MATERIALS AND METHODS: The mouse FCASM was chosen to test the hypothesis because the dominant cell type at the site of stenosis is SMC, similar to that in vascular access stenosis. Fourteen C57BL/6 mice underwent left carotid artery (LCA) partial ligation to induce stenosis, followed by daily injection of peptide 4D in 7 mice and saline in the remaining 7 mice, and these mice were observed for 21 days and then euthanized. Bilateral carotid arteries were excised for histologic analysis of the intima and media areas. RESULTS: The mean intimal area was significantly larger in control mice compared with peptide 4D-treated mice (0.031 mm2 [SD ± 0.024] vs 0.0082 mm2 [SD ± 0.0103]; P = .011). The mean intima-to-intima + media area ratio was significantly larger in control mice compared with peptide 4D-treated mice (0.27 [SD ± 0.13] vs 0.089 [SD ± 0.081]; P = .0079). NIH was not observed in the right carotid arteries in both groups. CONCLUSIONS: Peptide 4D, an F11R antagonist, significantly inhibited NIH in C57BL/6 mice in a FCASM.

Inhibition of Intimal Thickening By PRH (Proline-Rich Homeodomain) in Mice.

BACKGROUND: Late vein graft failure is caused by intimal thickening resulting from endothelial cell (EC) damage and inflammation which promotes vascular smooth muscle cell (VSMC) dedifferentiation, migration, and proliferation. Nonphosphorylatable PRH (proline-rich homeodomain) S163C:S177C offers enhanced stability and sustained antimitotic effect. Therefore, we investigated whether adenovirus-delivered PRH S163C:S177C protein attenuates intimal thickening via VSMC phenotype modification without detrimental effects on ECs. METHODS: PRH S163C:S177C was expressed in vitro (human saphenous vein-VSMCs and human saphenous vein-ECs) and in vivo (ligated mouse carotid arteries) by adenoviruses. Proliferation, migration, and apoptosis were quantified and phenotype was assessed using Western blotting for contractile filament proteins and collagen gel contraction. EC inflammation was quantified using VCAM (vascular cell adhesion protein)-1, ICAM (intercellular adhesion molecule)-1, interleukin-6, and monocyte chemotactic factor-1 measurement and monocyte adhesion. Next Generation Sequencing was utilized to identify novel downstream mediators of PRH action and these and intimal thickening were investigated in vivo. RESULTS: PRH S163C:S177C inhibited proliferation, migration, and apoptosis and promoted contractile phenotype (enhanced contractile filament proteins and collagen gel contraction) compared with virus control in human saphenous vein-VSMCs. PRH S163C:S177C expression in human saphenous vein-ECs significantly reduced apoptosis, without affecting cell proliferation and migration, while reducing TNF (tumor necrosis factor)-α-induced VCAM-1 and ICAM-1 and monocyte adhesion and suppressing interleukin-6 and monocyte chemotactic factor-1 protein levels. PRH S163C:S177C expression in ligated murine carotid arteries significantly impaired carotid artery ligation-induced neointimal proliferation and thickening without reducing endothelial coverage. Next Generation Sequencing revealed STAT-1 (signal transducer and activator of transcription 1) and HDAC-9 (histone deacetylase 9) as mediators of PRH action and was supported by in vitro and in vivo analyses. CONCLUSIONS: We observed PRH S163C:S177C attenuated VSMC proliferation, and migration and enhanced VSMC differentiation at least in part via STAT-1 and HDAC-9 signaling while promoting endothelial repair and anti-inflammatory properties. These findings highlight the potential for PRH S163C:S177C to preserve endothelial function whilst suppressing intimal thickening, and reducing late vein graft failure.

Thrombus or tumor? A case report of a rare sarcoma entity: intimal sarcoma of the pulmonary arteries.

BACKGROUND: Tumor embolism is a very rare primary manifestation of cancers and the diagnosis is challenging, especially if located in the pulmonary arteries, where it can mimic nonmalignant pulmonary embolism. Intimal sarcoma is one of the least commonly reported primary tumors of vessels with only a few cases reported worldwide. A typical location of this malignancy is the pulmonary artery. Herein, we present a case report of an intimal sarcoma with primary manifestation in the pulmonary arteries. A 53-year-old male initially presented with dyspnea. On imaging, a pulmonary artery embolism was detected and was followed by thrombectomy of the right ventricular outflow tract, main pulmonary artery trunk, and right pulmonary artery after ineffective lysis therapy. Complementary imaging of the chest and abdomen including a PET-CT scan demonstrated no evidence of a primary tumor. Subsequent pathology assessment suggested an intimal sarcoma further confirmed by DNA methylation based molecular analysis. We initiated adjuvant chemotherapy with doxorubicin. Four months after the completion of adjuvant therapy a follow-up scan revealed a local recurrence without distant metastases. DISCUSSION: Primary pulmonary artery intimal sarcoma (PAS) is an exceedingly rare entity and pathological diagnosis remains challenging. Therefore, the detection of entity-specific molecular alterations is a supporting argument in the diagnostic spectrum. Complete surgical resection is the prognostically most important treatment for intimal cardiac sarcomas. Despite adjuvant chemotherapy, the prognosis of cardiac sarcomas remains very poor. This case of a PAS highlights the difficulty in establishing a diagnosis and the aggressive natural course of the disease. CONCLUSION: In case of atypical presentation of a pulmonary embolism, a tumor originating from the great vessels should be considered. Molecular pathology techniques support in establishing a reliable diagnosis.

Paricalcitol Has a Potent Anti-Inflammatory Effect in Rat Endothelial Denudation-Induced Intimal Hyperplasia.

Neointimal hyperplasia is the main cause of vascular graft failure in the medium term. Vitamin D receptor activation modulates the biology of vascular smooth muscle cells and has been reported to protect from neointimal hyperplasia following endothelial injury. However, the molecular mechanisms are poorly understood. We have now explored the impact of the selective vitamin D receptor activator, paricalcitol, on neointimal hyperplasia, following guidewire-induced endothelial cell injury in rats, and we have assessed the impact of paricalcitol or vehicle on the expression of key cell stress factors. Guidewire-induced endothelial cell injury caused neointimal hyperplasia and luminal stenosis and upregulated the expression of the growth factor growth/differentiation factor-15 (GDF-15), the cytokine receptor CD74, NFκB-inducing kinase (NIK, an upstream regulator of the proinflammatory transcription factor NFκB) and the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Immunohistochemistry confirmed the increased expression of the cellular proteins CD74 and NIK. Paricalcitol (administered in doses of 750 ng/kg of body weight, every other day) had a non-significant impact on neointimal hyperplasia and luminal stenosis. However, it significantly decreased GDF-15, CD74, NIK and MCP-1/CCL2 mRNA expression, which in paricalcitol-injured arteries remained within the levels found in control vehicle sham arteries. In conclusion, paricalcitol had a dramatic effect, suppressing the stress response to guidewire-induced endothelial cell injury, despite a limited impact on neointimal hyperplasia and luminal stenosis. This observation identifies novel molecular targets of paricalcitol in the vascular system, whose differential expression cannot be justified as a consequence of improved tissue injury.

New strategy of using double-network hydrogel extravascular stent for preventing venous graft restenosis after coronary artery bypass grafting.

OBJECTIVE: Effective therapies for the prevention of vein graft failure, which frequently occurs in coronary artery bypass grafting (CABG) due to intimal hyperplasia (IH), are still lacking. Here, we investigated the effects of the perivenous application of double-network hydrogel on vein grafts in carotid artery bypass grafting in a rabbit model. METHODS: Healthy New Zealand white rabbits were randomized into the following groups: no graft, graft, or graft + Double-network hydrogel external stent (DNHES). The rabbits' carotid artery was bypassed via the jugular vein. Double-network hydrogel external stent was wrapped around the jugular graft after the anastomoses were completed. Blood flow parameters and tissue histology of the vein grafts were evaluated. RESULTS: Compared with the untreated vein grafts at 12 weeks after the surgery, the DNHES significantly improved graft flow, attenuated intimal and medial thickening, reduced the anti-proliferating cell nuclear antigen proliferation index of the vein grafts, decreased the mRNA and protein expression of Mitogen-Activated Protein Kinase (MAPK) and Transforming Growth Factor-ß (TGF-ß), and increased the mRNA and protein expression of endothelial Nitric Oxide Synthase (eNOS). CONCLUSION: The perivenous application of DNHES exerts beneficial effects on vein grafts, reduces the inflammatory response in carotid artery bypass grafting in a rabbit model, and appears to be a safe and promising strategy to prevent vein graft failure.

TET2 Protects Against Vascular Smooth Muscle Cell Apoptosis and Intimal Thickening in Transplant Vasculopathy.

BACKGROUND: Coronary allograft vasculopathy (CAV) is a devastating sequela of heart transplant in which arterial intimal thickening limits coronary blood flow. There are currently no targeted therapies to prevent or reduce this pathology that leads to transplant failure. Vascular smooth muscle cell (VSMC) phenotypic plasticity is critical in CAV neointima formation. TET2 (TET methylcytosine dioxygenase 2) is an important epigenetic regulator of VSMC phenotype, but the role of TET2 in the progression of CAV is unknown. METHODS: We assessed TET2 expression and activity in human CAV and renal transplant samples. We also used the sex-mismatched murine aortic graft model of graft arteriopathy (GA) in wild-type and inducible smooth muscle-specific Tet2 knockout mice; and in vitro studies in murine and human VSMCs using knockdown, overexpression, and transcriptomic approaches to assess the role of TET2 in VSMC responses to IFNγ (interferon γ), a cytokine elaborated by T cells that drives CAV progression. RESULTS: In the present study, we found that TET2 expression and activity are negatively regulated in human CAV and renal transplant samples and in the murine aortic graft model of GA. IFNγ was sufficient to repress TET2 and induce an activated VSMC phenotype in vitro. TET2 depletion mimicked the effects of IFNγ, and TET2 overexpression rescued IFNγ-induced dedifferentiation. VSMC-specific TET2 depletion in aortic grafts, and in the femoral wire restenosis model, resulted in increased VSMC apoptosis and medial thinning. In GA, this apoptosis was tightly correlated with proliferation. In vitro, TET2-deficient VSMCs undergo apoptosis more readily in response to IFNγ and expressed a signature of increased susceptibility to extrinsic apoptotic signaling. Enhancing TET2 enzymatic activity with high-dose ascorbic acid rescued the effect of GA-induced VSMC apoptosis and intimal thickening in a TET2-dependent manner. CONCLUSIONS: TET2 is repressed in CAV and GA, likely mediated by IFNγ. TET2 serves to protect VSMCs from apoptosis in the context of transplant vasculopathy or IFNγ stimulation. Promoting TET2 activity in vivo with systemic ascorbic acid reduces VSMC apoptosis and intimal thickening. These data suggest that promoting TET2 activity in CAV may be an effective strategy for limiting CAV progression.

Circular RNA CircMAP3K5 Acts as a MicroRNA-22-3p Sponge to Promote Resolution of Intimal Hyperplasia Via TET2-Mediated Smooth Muscle Cell Differentiation.

BACKGROUND: Aberrant expression of circular RNA contributes to human diseases. Circular RNAs regulate gene expression by sequestering specific microRNAs. In this study, we investigated whether circMAP3K5 (circular mitogen-activated protein kinase 5) could act as a competing endogenous microRNA-22-3p (miR-22-3p) sponge and regulate neointimal hyperplasia. METHODS: Circular RNA profiling from genome-wide RNA sequencing data was compared between human coronary artery smooth muscle cells (SMCs) treated with or without platelet-derived growth factor. Expression levels of circMAP3K5 were assessed in human coronary arteries from autopsies on patients with dilated cardiomyopathy or coronary heart disease. The role of circMAP3K5 in intimal hyperplasia was further investigated in mice with adeno-associated virus 9-mediated circMAP3K5 transfection. SMC-specific Tet2 (ten-eleven translocation-2) knockout mice and global miR-22-3p knockout mice were used to delineate the mechanism by which circMAP3K5 attenuated neointimal hyperplasia using the femoral arterial wire injury model. RESULTS: RNA sequencing demonstrated that treatment with platelet-derived growth factor-BB significantly reduced expression of circMAP3K5 in human coronary artery SMCs. Wire-injured mouse femoral arteries and diseased arteries from patients with coronary heart disease (where platelet-derived growth factor-BB is increased) confirmed in vivo downregulation of circMAP3K5 associated with injury and disease. Lentivirus-mediated overexpression of circMAP3K5 inhibited the proliferation of human coronary artery SMCs. In vivo adeno-associated virus 9-mediated transfection of circMap3k5 (mouse circular Map3k5) specifically inhibited SMC proliferation in the wire-injured mouse arteries, resulting in reduced neointima formation. Using a luciferase reporter assay and RNA pull-down, circMAP3K5 (human circular MAP3K5) was found to sequester miR-22-3p, which, in turn, inhibited the expression of TET2. Both in vitro and in vivo results demonstrate that the loss of miR-22-3p recapitulated the antiproliferative effect of circMap3k5 on vascular SMCs. In SMC-specific Tet2 knockout mice, loss of Tet2 abolished the circMap3k5-mediated antiproliferative effect on vascular SMCs. CONCLUSIONS: We identify circMAP3K5 as a master regulator of TET2-mediated vascular SMC differentiation. Targeting the circMAP3K5/miR-22-3p/TET2 axis may provide a potential therapeutic strategy for diseases associated with intimal hyperplasia, including restenosis and atherosclerosis.

Increased frequency of proangiogenic tunica intima endothelial kinase 2 (Tie2) expressing monocytes in individuals with type 2 diabetes mellitus.

BACKGROUND: Individuals with type 2 diabetes mellitus (T2DM) have an increased risk for developing macrovascular disease (MVD) manifested by atherosclerosis. Phenotypically and functionally different monocyte subsets (classical; CD14++CD16-, non-classical; CD14+CD16++, and intermediate; CD14++CD16+) including pro-angiogenic monocytes expressing Tie2 (TEMs) can be identified. Here we investigated monocyte heterogeneity and its association with T2DM and MVD. METHODS: Individuals with (N = 51) and without (N = 56) T2DM were recruited and allocated to "non-MVD" or "with MVD" (i.e., peripheral or coronary artery disease) subgroups. Blood monocyte subsets were quantified based on CD14, CD16 and Tie2 expression levels. Plasma levels of Tie2-ligands angiopoietin-1 and angiopoietin-2 were determined using ELISA. Carotid endarterectomy samples from individuals with (N = 24) and without (N = 22) T2DM were stained for intraplaque CD68+ macrophages (inflammation) and CD34+ (angiogenesis), as plaque vulnerability markers. RESULTS: Monocyte counts were similar between individuals with T2DM and healthy controls (non-diabetic, non-MVD). Non-classical monocytes were reduced (p < 0.05) in T2DM, whereas the percentage of TEMs within the intermediate subset was increased (p < 0.05). T2DM was associated with increased angiopoietin-1 (p < 0.05) and angiopoietin-2 (p = 0.0001) levels. Angiopoietin-2 levels were higher in T2DM individuals with MVD compared with non-MVD (p < 0.01). Endarterectomized plaques showed no differences in macrophage influx and microvessel number between individuals with and without T2DM. CONCLUSIONS: Monocyte subset distribution is altered in T2DM with reduced non-classical monocytes and increased TEM percentage in the intermediate monocyte subset. Increased angiopoietin-2 levels together with increased frequency of TEMs might promote plaque vulnerability in T2DM which could however not be confirmed at tissue level in advanced atherosclerotic lesions.

Hydrogen Sulphide Release via the Angiotensin Converting Enzyme Inhibitor Zofenopril Prevents Intimal Hyperplasia in Human Vein Segments and in a Mouse Model of Carotid Artery Stenosis.

OBJECTIVE: Hypertension is a major risk factor for intimal hyperplasia (IH) and re-stenosis following vascular and endovascular interventions. Preclinical studies suggest that hydrogen sulphide (H2S), an endogenous gasotransmitter, limits re-stenosis. While there is no clinically available pure H2S releasing compound, the sulfhydryl containing angiotensin converting enzyme inhibitor zofenopril is a source of H2S. Here, it was hypothesised that zofenopril, due to H2S release, would be superior to other non-sulfhydryl containing angiotensin converting enzyme inhibitors (ACEi) in reducing intimal hyperplasia. METHODS: Spontaneously hypertensive male Cx40 deleted mice (Cx40-/-) or wild type (WT) littermates were randomly treated with enalapril 20 mg or zofenopril 30 mg. Discarded human vein segments and primary human smooth muscle cells (SMCs) were treated with the active compound enalaprilat or zofenoprilat. IH was evaluated in mice 28 days after focal carotid artery stenosis surgery and in human vein segments cultured for seven days ex vivo. Human primary smooth muscle cell (SMC) proliferation and migration were studied in vitro. RESULTS: Compared with control animals (intima/media thickness 2.3 ± 0.33 µm), enalapril reduced IH in Cx40-/- hypertensive mice by 30% (1.7 ± 0.35 µm; p = .037), while zofenopril abrogated IH (0.4 ± 0.16 µm; p < .002 vs. control and p > .99 vs. sham operated Cx40-/- mice). In WT normotensive mice, enalapril had no effect (0.9665 ± 0.2 µm in control vs. 1.140 ± 0.27 µm; p > .99), while zofenopril also abrogated IH (0.1623 ± 0.07 µm; p < .008 vs. control and p > .99 vs. sham operated WT mice). Zofenoprilat, but not enalaprilat, also prevented IH in human vein segments ex vivo. The effect of zofenopril on carotid and SMCs correlated with reduced SMC proliferation and migration. Zofenoprilat inhibited the mitogen activated protein kinase and mammalian target of rapamycin pathways in SMCs and human vein segments. CONCLUSION: Zofenopril provides extra beneficial effects compared with non-sulfhydryl ACEi in reducing SMC proliferation and re-stenosis, even in normotensive animals. These findings may hold broad clinical implications for patients suffering from vascular occlusive diseases and hypertension.

Intimal Hyperplasia of Arteriovenous Fistula.

Intimal hyperplasia (IH), a crucial histopathological injury, forms the basis of vascular stenosis and thrombogenesis. In addition, it is common in maladies such as stenosis at the anastomosis of arteriovenous fistula and restenosis after angioplasty. Various cellular and noncellular components play critical parts in the advancement of IH. This article reviews the distinctive components of IH, such as endothelial dysfunction, multiplication, and movement of vascular smooth muscle cells. Finally, in addition to synthesis of large amounts of extracellular matrix and inflammatory responses, which have frequently been studied in recent years, we offer a premise for clinical treatment with vascular smooth muscle cells.

Pericyte-specific deletion of ninjurin-1 induces fragile vasa vasorum formation and enhances intimal hyperplasia of injured vasculature.

Adventitial abnormalities including enhanced vasa vasorum malformation are associated with development and vulnerability of atherosclerotic plaque. However, the mechanisms of vasa vasorum malformation and its role in vascular remodeling have not been fully clarified. We recently reported that ninjurin-1 (Ninj1) is a crucial adhesion molecule for pericytes to form matured neovessels. The purpose is to examine if Ninj1 regulates adventitial angiogenesis and affects the vascular remodeling of injured vessels using pericyte-specific Ninj1 deletion mouse model. Mouse femoral arteries were injured by insertion of coiled wire. Four weeks after vascular injury, fixed arteries were decolorized. Vascular remodeling, including intimal hyperplasia and adventitial microvessel formation were estimated in a three-dimensional view. Vascular fragility, including blood leakiness was estimated by extravasation of fluorescein isothiocyanate (FITC)-lectin or FITC-dextran from microvessels. Ninj1 expression was increased in pericytes in response to vascular injury. NG2-CreER/Ninj1loxp mice were treated with tamoxifen (Tam) to induce deletion of Ninj1 in pericyte (Ninj1 KO). Tam-treated NG2-CreER or Tam-nontreated NG2-CreER/Ninj1loxp mice were used as controls. Intimal hyperplasia was significantly enhanced in Ninj1 KO compared with controls. Vascular leakiness was significantly enhanced in Ninj1 KO. In Ninj1 KO, the number of infiltrated macrophages in adventitia was increased, along with the expression of inflammatory cytokines. In conclusion, deletion of Ninj1 in pericytes induces the immature vasa vasorum formation of injured vasculature and exacerbates adventitial inflammation and intimal hyperplasia. Thus, Ninj1 contributes to the vasa vasorum maturation in response to vascular injury and to reduction of vascular remodeling.NEW & NOTEWORTHY Although abnormalities of adventitial vasa vasorum are associated with vascular remodeling such as atherosclerosis, the mechanisms of vasa vasorum malformation and its role in vascular remodeling have not been fully clarified. The present study provides a line of novel evidence that ninjurin-1 contributes to adventitial microvascular maturation during vascular injury and regulates vascular remodeling.

Focal Intimal Disruption Size at Multidetector CT and Disease Progression in Type B Aortic Intramural Hematoma.

Background Recent imaging techniques show that patients with aortic intramural hematoma (IMH) have various sizes of focal intimal disruptions (FIDs) that may affect clinical outcomes. Purpose To evaluate the relationship between size of FIDs detected at multidetector CT and disease progression in patients with type B IMH. Materials and Methods This retrospective study analyzed consecutive patients with type B IMH who underwent multidetector CT at admission within 24 hours after symptom onset from January 2011 to March 2017. FID was defined as a focal contrast material-filled pouch projecting outside of the aorta lumen with a communicating orifice greater than 3 mm. Large FID was defined as an FID of at least 10 mm in length and width and at least 5 mm in depth. Aorta-related events were defined as a composite of aortic rupture, surgical or endovascular aortic repair, progressive aortic enlargement, and development of aortic dissection. Results Seventy-six patients (mean age, 72 years ± 11 [standard deviation]; 54 men) were evaluated. Fifty patients (66%; 50 of 76) had 91 FIDs. Twenty-eight patients had large FIDs and 22 patients had small FIDs. Aorta-related events occurred in 15 of 28 patients with large FIDs, two of 22 patients with small FIDs, and four of 26 patients with no FID. Patients with large FIDs showed lower aorta-related event-free survival rates than did patients with small FIDs or no FID (small FIDS, 63% ± 10 vs 90% ± 7 at 3 years, respectively [P = .001]; and no FIDS, 63% ± 10 vs 84% ± 7 at 3 years, respectively [P = .005]). Multivariable analysis showed that a maximum aortic diameter of at least 40 mm (hazard ratio, 4.8; 95% CI: 1.8, 12.6; P = .001) and large FID (hazard ratio, 3.2; 95% CI: 1.1, 8.9; P = .03) were the independent predictors of aorta-related events. Conclusion A large portion of patients with B-intramural hematoma (IMH) had focal intimal disruption (FID) detected at CT at admission. Large FIDs were associated with disease progression of IMH. © RSNA, 2021 See also the editorial by Raptis and Braverman in this issue. Online supplemental material is available for this article.

Intimal sarcomas and undifferentiated cardiac sarcomas carry mutually exclusive MDM2, MDM4, and CDK6 amplifications and share a common DNA methylation signature.

Undifferentiated mesenchymal tumors arising from the inner lining (intima) of large arteries are classified as intimal sarcomas (ISA) with MDM2 amplification as their molecular hallmark. Interestingly, undifferentiated pleomorphic sarcomas (UPS) of the heart have recently been suggested to represent the cardiac analog of ISA due to morphological overlap and high prevalence of MDM2 amplifications in both neoplasms. However, little is known about ISAs and cardiac UPS without MDM2 amplifications and molecular data supporting their common classification is sparse. Here, we report a series of 35 cases comprising 25 ISAs of the pulmonary artery, one ISA of the renal artery and 9 UPS of the left atrium. Tumors were analyzed utilizing the Illumina Infinium MethylationEPIC BeadChip array, enabling copy number profile generation and unsupervised DNA methylation analysis. DNA methylation patterns were investigated using t-distributed stochastic neighbor embedding (t-SNE) analysis. Histologically, all ISAs and UPS of the left atrium resembled extra-cardiac UPS. All cases exhibited highly complex karyotypes with overlapping patterns between ISA and UPS. 29/35 cases showed mutually exclusive amplifications in the cell-cycle associated oncogenes MDM2 (25/35), MDM4 (2/35), and CDK6 (2/35). We further observed recurrent co-amplifications in PDGFRA (21/35), CDK4 (15/35), TERT (11/35), HDAC9 (9/35), and CCND1 (4/35). Sporadic co-amplifications occurred in MYC, MYCN, and MET (each 1/35). The tumor suppressor CDKN2A/B was frequently deleted (10/35). Interestingly, DNA methylation profiling (t-SNE) revealed an overlap of ISA and cardiac UPS. This "ISA" methylation signature was distinct from potential histologic and molecular mimics. In conclusion, our data reveal MDM4 and CDK6 amplifications in ISAs and UPS of the left atrium, lacking MDM2 amplification. We further report novel co-amplifications of various oncogenes, which may have therapeutic implications. Finally, the genetic and epigenetic concordance of ISAs and UPS of the left atrium further supports a shared pathogenesis and common classification.

Reversal of pulmonary arterial hypertension and neointimal formation by kinin B1 receptor blockade.

BACKGROUND: This study examined whether BI113823, a novel selective kinin B1 receptor antagonist can reverse established pulmonary arterial hypertension (PAH), prevent right heart failure and death, which is critical for clinical translation. METHODS: Left pneumonectomized male Wistar rats were injected with monocrotaline to induce PAH. Three weeks later, when PAH was well established, the rats received daily treatment of BI113823 or vehicle for 3 weeks. RESULTS: Treatment with BI113823 from day 21 to day 42 after monocrotaline injection reversed established PAH as shown by normalized values of mean pulmonary arterial pressure (mPAP). BI113823 therapy reversed pulmonary vascular remodeling, pulmonary arterial neointimal formation, and heart and lung fibrosis, reduced right ventricular pressure, right heart hypertrophy, improved cardiac output, and prevented right heart failure and death. Treatment with BI113823 reduced TNF-α and IL-1ß, and macrophages recruitment in bronchoalveolar lavage, reduced CD-68 positive macrophages and expression of proliferating cell nuclear antigen (PCNA) in the perivascular areas, and reduced expression of iNOS, B1 receptors, matrix metalloproteinase (MMP)-2 and MMP-9 proteins, and the phosphorylation of ERK1/2 and AKT in lung. Treatment with BI113823 reduced mRNA expression of ANP, BNP, ßMHC, CGTF, collange-I and IV in right heart, compared to vehicle treated controls. In human monocytes cultures, BI113823 reduced LPS-induced TNF-α production, MMP-2 and MMP-9 expression, and reduced TNF-α-induced monocyte migration. CONCLUSIONS: We conclude that BI113823 reverses preexisting severe experimental pulmonary hypertension via inhibition of macrophage infiltration, cytokine production, as well as down regulation of matrix metalloproteinase proteins.

Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-Mediated Cyclin D1 Transcription.

RATIONALE: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders, such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via FAK (focal adhesion kinase). OBJECTIVE: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. METHODS AND RESULTS: Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (Myh11-Cre-ERT2) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4- (GATA-binding protein 4) cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter, and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitant increase in GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control mice, but not in VS-4718-treated and SMC-specific FAK kinase-dead mice. Finally, lentiviral shGATA4 knockdown in the wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared with control mice. CONCLUSIONS: Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.