Mitsuo Kinugasa

Role of Afadin in Vascular Endothelial Growth Factor– and Sphingosine 1-Phosphate–Induced Angiogenesis

Rationale: Angiogenesis contributes to physiological and pathological conditions, including atherosclerosis. The Rap1 small G protein regulates vascular integrity and angiogenesis. However, little is known about the effectors of Rap1 involved in angiogenesis. It is not known whether afadin, an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1, plays a role in angiogenesis. Objective: We investigated the role of endothelial afadin in angiogenesis and attempted to clarify the underlying molecular mechanism. Methods and Results: Treatment of human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) induced the activation of Rap1. Activated Rap1 regulated intracellular localization of afadin. Knockdown of Rap1 or afadin by small interfering RNA inhibited the VEGF- and S1P-induced capillary-like network formation, migration, and proliferation, and increased the serum deprivation-induced apoptosis of HUVECs. Knockdown of Rap1 or afadin decreased the accumulation of adherens and tight junction proteins to the cell–cell contact sites. Rap1 regulated the interaction between afadin and phosphatidylinositol 3-kinase (PI3K), recruitment of the afadin–PI3K complex to the leading edge, and the activation of Akt, indicating the involvement of Rap1 and afadin in the PI3K–Akt signaling pathway. Binding of afadin to Rap1 regulated the activity of Rap1 in a positive-feedback manner. In vivo, conditional deletion of afadin in mouse vascular endothelium using a Cre-loxP system impaired the VEGF- and S1P-induced angiogenesis. Conclusions: These results demonstrate a novel molecular mechanism by which Rap1 and afadin regulate the VEGF- and S1P-induced angiogenesis.

Necl-5/Poliovirus Receptor Interacts With VEGFR2 and Regulates VEGF-Induced Angiogenesis

Rationale: Vascular endothelial growth factor (VEGF), a major proangiogenic agent, exerts its proangiogenic action by binding to VEGF receptor 2 (VEGFR2), the activity of which is regulated by direct interactions with other cell surface proteins, including integrin &agr;V&bgr;3. However, how the interaction between VEGFR2 and integrin &agr;V&bgr;3 is regulated is not clear. Objective: To investigate whether Necl-5/poliovirus receptor, an immunoglobulin-like molecule that is known to bind integrin &agr;V&bgr;3, regulates the interaction between VEGFR2 and integrin &agr;V&bgr;3, and to clarify the role of Necl-5 in the VEGF-induced angiogenesis. Methods and Results: Necl-5-knockout mice displayed no obvious defect in vascular development; however, recovery of blood flow after hindlimb ischemia and the VEGF-induced neovascularization in implanted Matrigel plugs were impaired in Necl-5-knockout mice. To clarify the mechanism of the regulation of angiogenesis by Necl-5, we investigated the roles of Necl-5 in the VEGF-induced angiogenic responses in vitro. Knockdown of Necl-5 by siRNAs in human umbilical vein endothelial cells (HUVECs) inhibited the VEGF-induced capillary-like network formation on Matrigel, migration, and proliferation, and conversely, enhanced apoptosis. Coimmunoprecipitation assays showed the interaction of Necl-5 with VEGFR2, and knockdown of Necl-5 prevented the VEGF-induced interaction of integrin &agr;V&bgr;3 with VEGFR2. Knockdown of Necl-5 suppressed the VEGFR2-mediated activation of downstream proangiogenic and survival signals, including Rap1, Akt, and endothelial nitric oxide synthase. Conclusions: These results demonstrate the critical role of Necl-5 in angiogenesis and suggest that Necl-5 may regulate the VEGF-induced angiogenesis by controlling the interaction of VEGFR2 with integrin &agr;v&bgr;3, and the VEGFR2-mediated Rap1-Akt signaling pathway.

Osteoblast-like Differentiation of Cultured Human Coronary Artery Smooth Muscle Cells by Bone Morphogenetic Protein Endothelial Cell Precursor-derived Regulator (BMPER)

Background: Osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) is a mechanism of vascular calcification. Results: Bone morphogenetic protein endothelial cell precursor-derived regulator (BMPER) was expressed in VSMCs and enhanced osteoblast-like differentiation of VSMCs via NF-κB activation. Conclusion: BMPER is a novel regulator of osteoblast-like differentiation of VSMCs. Significance: BMPER may be a potential target for prevention of vascular calcification. Differentiation of vascular smooth muscle cells (SMCs) into osteoblast-like cells is considered to be a mechanism of vascular calcification. However, regulators of osteoblast-like differentiation of vascular SMCs are not fully elucidated. Here, we investigated the expression of bone morphogenetic protein (BMP)-binding endothelial cell precursor-derived regulator (BMPER), a vertebrate homologue of Drosophila crossveinless-2, in vascular SMCs and the role and mode of action of BMPER in osteoblast-like differentiation of human coronary artery SMCs (HCASMCs). BMPER was expressed in cultured human vascular SMCs, including HCASMCs. Silencing of endogenous BMPER expression by an RNA interference technique inhibited osteoblast-like differentiation of HCASMCs, as evaluated by up-regulation of osteoblast markers such as alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2), by down-regulation of a SMC marker α-smooth muscle actin (αSMA), and by mineralization. Treatment with recombinant BMPER enhanced, whereas BMP-2 reduced osteoblast-like differentiation. BMPER antagonized BMP-2-induced phosphorylation of Smad 1/5/8, suggesting that the effect of BMPER was mediated by antagonizing the action of BMP. BMPER increased IκBα phosphorylation and NF-κB activity and specific NF-κB decoy oligonucleotides deteriorated osteoblast-like differentiation of HCASMCs by BMPER. In human coronary artery with atherosclerotic plaque containing calcification, the BMPER-positive signals were observed in the neointimal and medial SMCs in the vicinity of the plaque. These findings indicate that BMPER is a novel regulator of the osteoblast-like differentiation of HCASMCs.

Family with Sequence Similarity 5, Member C (FAM5C) Increases Leukocyte Adhesion Molecules in Vascular Endothelial Cells: Implication in Vascular Inflammation

Identification of the regulators of vascular inflammation is important if we are to understand the molecular mechanisms leading to atherosclerosis and consequent ischemic heart disease, including acute myocardial infarction. Gene polymorphisms in family with sequence similarity 5, member C (FAM5C) are associated with an increased risk of acute myocardial infarction, but little is known about the function of this gene product in blood vessels. Here, we report that the regulation of the expression and function of FAM5C in endothelial cells. We show here that FAM5C is expressed in endothelial cells in vitro and in vivo. Immunofluorescence microcopy showed localization of FAM5C in the Golgi in cultured human endothelial cells. Immunohistochemistry on serial sections of human coronary artery showed that FAM5C-positive endothelium expressed intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1). In cultured human endothelial cells, the overexpression of FAM5C increased the reactive oxygen species (ROS) production, nuclear factor-κB (NF-κB) activity and the expression of ICAM-1, VCAM-1 and E-selectin mRNAs, resulting in enhanced monocyte adhesion. FAM5C was upregulated in response to inflammatory stimuli, such as TNF-α, in an NF-κB- and JNK-dependent manner. Knockdown of FAM5C by small interfering RNA inhibited the increase in the TNF-α-induced production of ROS, NF-κB activity and expression of these leukocyte adhesion molecule mRNAs, resulting in reduced monocyte adhesion. These results suggest that in endothelial cells, when FAM5C is upregulated in response to inflammatory stimuli, it increases the expression of leukocyte adhesion molecules by increasing ROS production and NF-κB activity.

Optimal angulations for obtaining an en face view of each coronary aortic sinus and the interventricular septum: Correlative anatomy around the left ventricular outflow tract

An optimal image intensifier angulation used for obtaining an en face view of a target structure is important in electrophysiologic procedures performed around each coronary aortic sinus (CAS). However, few studies have revealed the fluoroscopic anatomy of the target area. This study investigated the optimal angulation for each CAS and the interventricular septum (IVS). The study included 102 consecutive patients who underwent computed tomography coronary angiography. The optimal angle for each CAS was determined by rotating the volume‐rendered image around the vertical axis. The angle formed between the anteroposterior axis and IVS was measured using the horizontal section. The frontal direction was defined as zero, positive, or negative if the en face view of the target CAS was obtained in the frontal view, left anterior oblique (LAO) direction, or right anterior oblique (RAO) direction, respectively. The optimal angles for the left, right, and non‐CASs were 120.3 ± 10.5°, 4.8 ± 16.3°, and −110.0 ± 13.8°, respectively. The IVS angle was 42.6 ± 8.5°. Accordingly, the optimal image intensifier angulations for the left, right, and non‐CASs and the IVS were estimated to be RAO 60°, LAO 5°, LAO 70°, and RAO 50°, respectively. The IVS angle was the most common independent predictor of the optimal angle for each CAS. Differences in the optimal angulations for each CAS and the IVS are demonstrated. The biplane angulation needs to be tailored according to the individual patients and target structures for electrophysiologic procedures. Clin. Anat. 28:494–505, 2015.

Association between the rotation and three-dimensional tortuosity of the proximal ascending aorta

Age‐related morphological changes of the aorta, including dilatation and elongation, have been reported. However, rotation has not been fully investigated. We focused on the rotation of the ascending aorta and investigated its relationship with tortuosity. One hundred and two consecutive patients who underwent computed tomography coronary angiography were studied. The angle at which the en face view of the volume‐rendered image of the right coronary aortic sinus (RCS) was obtained without foreshortening was defined as the rotation index. It was defined as zero if the RCS was squarely visible in the frontal view, positive if it rotated clockwise toward the left anterior oblique (LAO) direction, and negative if it rotated counter‐clockwise toward the right anterior oblique (RAO) direction. The tortuosity was evaluated by measuring the biplane tilt angles formed between the ascending aorta and the horizontal line. The mean rotation index, posterior tilt angle viewed from the RAO direction (αRAO), and anterior tilt angle viewed from the LAO direction (αLAO) were 4.8 ± 16.3, 60.7 ± 7.0°, and 63.6 ± 9.0°, respectively. Although no correlation was observed between the rotation index and the αLAO (β = −0.0761, P = 0.1651), there was a significant negative correlation between the rotation index and αRAO (β = −0.1810, P < 0.0001). In multivariate regression analysis, the rotation index was an independent predictor of the αRAO (β = −0.1274, P = 0.0008). Clockwise rotation of the proximal ascending aorta exacerbates the tortuosity by tilting the aorta toward the posterior direction. Clin. Anat. 27:1200–1211, 2014.

Three-dimensional quantification and visualization of aortic calcification by multidetector-row computed tomography: A simple approach using a volume-rendering method

OBJECTIVE Three-dimensional (3-D) visualization and quantification of vascular calcification (VC) are important to accelerate the multidisciplinary investigation of VC. Agatston scoring is the standard approach for evaluating coronary artery calcification. However, regarding aortic calcification (AC), quantification methods appear to vary among studies. The aim of this study was to introduce a simple technique of simultaneous quantification and 3-D visualization of AC and provide validation data. METHODS The main study comprised of 126 patients who underwent the thoracoabdominal plain computed tomography scan as preoperative general evaluation. AC was quantified using a volume-rendering (VR) method (VR AC volume) by extracting the volume with a density ≥130 HU within the total aorta. The concordance and reproducibility of the VR AC volume were validated in comparison with the conventional slice-by-slice voxel-based AC quantification (volumetric AC score) using the Agatston scoring software. RESULTS Excellent concordance between the VR AC volume and volumetric AC score was confirmed (Spearman correlation coefficient = 0.9997, mean difference = -0.05 ± 0.23 mL, p <0.0001). Excellent intraobserver and interobserver reliabilities were demonstrated using the Bland-Altman analysis as the mean intraobserver difference was 0.00 mL (p = 0.9863) and the mean interobserver difference was -0.01 mL (p = 0.6612). CONCLUSION The VR method was validated to be feasible. This simple approach could overcome the limitation of the current method based on slice-by-slice pixel or voxel summation, which lacks 3-D visual information. Accordingly, this approach would be promising for accelerating the investigation of VC.

Nectin-Like Molecule-5 Regulates Intimal Thickening After Carotid Artery Ligation in Mice

Objective—Intimal thickening is considered to result from the dedifferentiation of medial smooth muscle cells (SMCs) from a contractile to a synthetic phenotype, and their subsequent migration and proliferation. It is unknown whether nectin-like molecule (Necl)-5, which is overexpressed in cancer cells, is involved in intimal thickening. Approach and Results—Necl-5 was upregulated in mouse carotid artery after ligation. Compared with wild-type mice, intimal thickening after carotid artery ligation was milder in Necl-5 knockout mice. In vitro, the expression levels of SMC differentiation markers were higher, whereas the expression level of an SMC dedifferentiation marker was lower, in Necl-5 knockout mouse aortic SMCs (MASMCs) compared with wild-type MASMCs. The migration, proliferation, and extracellular signal–regulated kinase activity in response to serum were decreased in Necl-5 knockout MASMCs compared with wild-type MASMCs. In wild-type MASMCs, inhibition of extracellular signal–regulated kinase activity increased the expression levels of SMC differentiation markers and decreased their migration and proliferation in response to serum. Conclusions—The present findings indicate that Necl-5 plays a role in the formation of intimal thickening after carotid artery ligation by regulating dedifferentiation, migration, and proliferation of SMCs in an extracellular signal–regulated kinase-dependent manner. Our results suggest that Necl-5 may represent a potential therapeutic target to limit intimal thickening after vascular injury.

Serum phosphate is an independent predictor of the total aortic calcification volume in non-hemodialysis patients undergoing cardiovascular surgery.

BACKGROUND A high serum phosphate level is a well-known risk factor for vascular calcification (VC) in patients on hemodialysis (HD). However, the association between the serum phosphate level and VC in non-HD patients is unclear. Our aim was to assess the impact of serum phosphate level on aortic calcification (AC) volume in non-HD patients undergoing cardiovascular surgery. METHODS A total of 117 patients who underwent thoracoabdominal computed tomography as a preoperative general evaluation before cardiovascular surgery were enrolled. The total AC volume was quantified using the volume-rendering method by extracting the area ≥130HU within the entire aorta. The total AC volume index (AC-VI) was estimated as the total AC volume divided by the body surface area. RESULTS In the 117 patients (64.7±13.1 years, 39% women), the median total AC-VI was 1.23mL/m(2). The mean estimated glomerular filtration rate (eGFR), adjusted serum calcium levels, and serum phosphate levels were 63.8±19.9mL/min/1.73m(2), 9.1±0.4mg/dL, and 3.6±0.6mg/dL, respectively. When the patients were classified into four quartiles based on their total AC-VI value, the serum phosphate level showed a positive correlation with a probability of being in the highest AC-VI quartile (R(2)=0.0146, p=0.0383) whereas the adjusted serum calcium level did not show a significant correlation (R(2)=0.0040, p=0.2615). A similar relationship between the serum phosphate level, adjusted serum calcium level, and AC-VI was confirmed when the total AC-VI was divided into the thoracic AC-VI and abdominal AC-VI. Multivariate analysis indicated that the serum phosphate level was an independent positive predictor of higher total AC-VI quartiles (β=0.8013, p=0.0160). CONCLUSIONS An increase in serum phosphate level was associated with an increased AC burden in non-HD patients undergoing cardiovascular surgery.