Kimiyasu Egami

Role of host angiotensin II type 1 receptor in tumor angiogenesis and growth

Although the renin angiotensin system (RAS) is a major regulator of vascular homeostasis, the role of the RAS in tumor angiogenesis is little understood. Here we show that host angiotensin II (ATII) type 1 (AT1) receptor plays an important role in angiogenesis and growth of tumor cells engrafted in mice. Subcutaneous B16-F1 melanoma-induced angiogenesis as assessed by tissue capillary density and microangiography was prominent in WT mice but was reduced in AT1a receptor-deficient (AT1a-/-) mice. Consequently, tumor growth rate was significantly slower, and the mouse survival rate was greater, in AT1a-/- mice than in WT mice. Tumor growth was also reduced in WT mice treated with TCV-116, a selective blocker of AT1 receptor. Because the beta-galactosidase gene was inserted into the AT1a gene locus in AT1a-/- mice, the site of beta-galactosidase expression represents the AT1a receptor expression in these mutant mice. In tumor-implanted AT1a-/- mice, the major site of the beta-galactosidase expression was macrophages in tissues surrounding tumors. Moreover, the number of infiltrated macrophages was significantly lower in AT1a-/- mice than in WT mice, and double-immunofluorescence staining revealed that these macrophages expressed VEGF protein intensively. Therefore, the host ATII-AT1 receptor pathway supports tumor-associated macrophage infiltration, which results in enhanced tissue VEGF protein levels. The host ATII-AT1 receptor pathway thereby plays important roles in tumor-related angiogenesis and growth in vivo.

Angiogenesis and Vasculogenesis Are Impaired in the Precocious-Aging klotho Mouse

Background—The effects of aging on angiogenesis (vascular sprouting) and vasculogenesis (endothelial precursor cell [EPC] incorporation into vessels) are not well known. We examined whether ischemia-induced angiogenesis/vasculogenesis is altered in klotho (kl) mutant mice, an animal model of typical aging. Methods and Results—After unilateral hindlimb ischemia, laser Doppler blood-flow (LDBF) analysis revealed a decreased ischemic-normal LDBF ratio in kl mice. Tissue capillary density was also suppressed in kl mice (+/+>+/kl>kl/kl). Aortic-ring culture assay showed impaired angiogenesis in kl/kl mice, accompanied by reduced endothelium-derived nitric oxide release. Moreover, the rate of transplanted homologous bone marrow cells incorporated into capillaries in ischemic tissues (vasculogenesis) was lower in kl/kl mice than in wild-type (+/+) mice, which was associated with a decrease in the number of c-Kit+CD31+ EPC-like mononuclear cells in bone marrow and in peripheral blood. Finally, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor cerivastatin restored the impaired neovascularization in kl/kl mice, accompanied by an increase in c-Kit+CD31+ cells in bone marrow and peripheral blood, and enhanced angiogenesis in the aortic-ring culture. Conclusions—Angiogenesis and vasculogenesis are impaired in kl mutant mice, a model of typical aging. Moreover, the age-associated impairment of neovascularization might be a new target of statin therapy.

Hypoxic preconditioning augments efficacy of human endothelial progenitor cells for therapeutic neovascularization.

A subset of human peripheral blood mononuclear cells (PB-MNCs) differentiate into endothelial progenitor cells (EPCs) that participate in postnatal neovascularization. Although tissue ischemia can mobilize EPCs from bone marrow, the effects of hypoxia on differentiation and angiogenic function of EPCs are little known. We examined whether hypoxic conditioning would modulate differentiation and function of human PB-MNC-derived EPCs. A subset of PB-MNCs gave rise to EPC-like attaching (AT) cells under either normoxic or hypoxic conditions. However, hypoxia much enhanced the differentiation of AT cells from PB-MNCs compared with normoxia. AT cells released vascular endothelial growth factor (VEGF) protein and expressed CD31 and kinase insert domain receptor/VEGFR-2, endothelial lineage markers, on their surface, which were also enhanced by hypoxia. Both a neutralizing anti-VEGF mAb and a KDR-specific receptor tyrosine kinase inhibitor, SU1498, suppressed PB-MNC differentiation into EPC-like AT cells in a dose-dependent manner. Migration of AT cells in response to VEGF as examined by a modified Boyden chamber apparatus was also enhanced by hypoxia. Finally, in vivo neovascularization efficacy was significantly enhanced by in vitro hypoxic conditioning of AT cells when cells were transplanted into the ischemic hindlimb of immunodeficient nude rats. In conclusion, hypoxia directly stimulated differentiation of EPC-like AT cells from human PB-MNC culture. Moreover, hypoxic preconditioning of AT cells before in vivo transplantation is a useful means to enhance therapeutic vasculogenesis.

Ischemia-induced angiogenesis: role of inflammatory response mediated by P-selectin

P‐selectin is a 140‐kDa glycoprotein expressed on endothelial cells and platelets. P‐selectin mediates the tethering and rolling of leukocytes along the endothelium, an early step of leukocyte extravasation. Although inflammation is a requisite process for ischemia‐induced angiogenesis, little is known regarding the role of P‐selectin in angiogenesis in the setting of tissue ischemia. We examined whether ischemia‐induced angiogenesis is altered in P‐selectin knockout (P‐selectin−/−) mice. Angiogenesis was evaluated n a surgically induced hind‐limb ischemia model using laser Doppler blood flowmetry (LDBF) and histological capillary density (CD). After left hind‐limb ischemia, the ischemic/normal limb LDBF ratio was persistently lower in P‐selectin−/− mice compared with wild‐type (WT) mice. CD was also significantly lower in P‐selectin−/− mice than in WT mice on Postoperative Day 14. Fewer numbers of total CD45+ inflammatory leukocytes infiltrated into the ischemic tissues in P‐selectin−/− mice than in WT mice, and immunohistochemical analysis revealed the number of infiltrated leukocytes expressing vascular endothelial growth factor was also decreased in P‐selectin−/− mice. P‐selectin mRNA expression was augmented after hind‐limb ischemia in WT mice. In conclusion, P‐selectin may play an important role in ischemia‐induced angiogenesis by promoting early inflammatory mononuclear cell infiltration. P‐selectin would become one possible target molecule for modulating inflammatory angiogenesis.

Older Age Is a Risk Factor for the Development of Cardiovascular Sequelae in Kawasaki Disease

Objectives. To clarify the characteristics of Kawasaki disease (KD) in children 6 years and older and to determine whether age is a risk factor for cardiovascular abnormalities. Methods. Patients who had KD and were reported between 1999 and 2000 in the 16th nationwide survey of KD in Japan (n = 15 314) were analyzed. Patients who were aged 6 years or older (older group) were matched with patients who were aged 6 months to 3 years and were treated at the same hospital (younger groups). The total number of analyzed patients was 1498 (749 matched pairs). Results. The proportion of complete KD in the older group was similar to that in the younger group. Recurrent cases in the older group were significantly more common than those in the younger group (9% vs 2%). The proportion of patients who were treated with intravenous γ-globulin in the older group was significantly lower than that in the younger group (82% vs 87%). The proportion of older group patients who were treated with intravenous γ-globulin at or after 7 days of illness was significantly higher than that in the younger group (35% vs 14%). There was a higher prevalence of cardiovascular abnormalities in the older group than in the younger group (20% vs 15%). Multivariate logistic regression analysis showed that older age was an independent risk factor for cardiovascular sequelae (odds ratio: 1.58; 95% confidence interval: 1.01–2.46). Conclusions. In children older than 6 years, age is an independent risk factor for cardiovascular sequelae in KD.

Rescue of hypercholesterolemia-related impairment of angiogenesis by oral folate supplementation

OBJECTIVES We examined whether oral folate supplementation would rescue a hypercholesterolemia (HC)-related impairment of ischemia-induced angiogenesis. BACKGROUND Folate protects against endothelial dysfunction, but the effect of folate supplementation on angiogenesis is little known. METHODS Sprague-Dawley rats were divided into four groups. Control rats were fed a normal diet (n = 18); HC rats (n = 18) were fed 2% cholesterol diet; and HC + folate (HC+F) rats were fed an HC diet with oral folate (0.003% in water). The left femoral artery and vein were surgically excised, and angiogenesis in the ischemic limb was evaluated. We also examined the effects of Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, on angiogenesis in the HC+F state. RESULTS Laser Doppler blood flow (LDBF) analysis showed lower ischemic/normal LDBF ratio in the HC group than in the control group. Angiographic and histologic analyses on day 14 revealed a smaller angiographic score (p < 0.001) and capillary density (p < 0.001) in the HC group than in controls, which were associated with reduced tissue NOx and cyclic guanosine monophosphate (cGMP) levels. The LDBF ratio, angiographic score, and capillary density were significantly restored in the HC+F group (p < 0.01 vs. HC), which were associated with increased serum folate and tissue NOx and cGMP levels. Finally, L-NAME treatment abolished the beneficial action of folate on angiogenesis in the HC state. CONCLUSIONS Ischemia-induced angiogenesis was inhibited by HC, which was rescued by oral folate supplementation, at least in part, via an NO-dependent manner.

Soluble forms of the selectin family in children with Kawasaki disease: prediction for coronary artery lesions

Aim: To investigate the relationship between the plasma levels of soluble forms of the selectin family and the incidence of coronary artery lesions (CALs) in patients with Kawasaki disease (KD). Methods: Thirty‐three patients with KD, including group A patients (n= 22) who had no CALs and group B patients (n= 11) who had CALs, as well as age‐matched febrile (n= 10) and afebrile controls (n= 11), were studied. Results: Peak plasma E‐selectin levels (172.0 ± 58.6 ng ml‐1) occurred during the acute phase of KD, while peak plasma P‐selectin levels (260.3 ± 43.2 ng ml‐1) occurred during the subacute phase of the illness (p± 0.05). Plasma L‐selectin levels (1757.3 ± 244.3 ng ml‐1) during the convalescent phase tended to be higher than in either the acute or the subacute phase (not significant). Before intravenous immunoglobulin treatment, the plasma levels of E‐ (225.1 ± 46.8 ng ml‐1) and P‐selectin (259.4 ± 76.2 ng ml‐1) of patients with CALs (n= 11) were significantly higher than those of patients (n= 22) with no CALs (E‐selectin, 131.6 ± 36.9 ng ml‐1; P‐selectin, 184.9 ± 84.6 ng ml‐1; p± 0.05). When a plasma E‐selectin value before immunoglobulin treatment of ± 184.7 ng ml‐1 was used as the cut‐off point, the sensitivity and specificity for the incidence of CALs were 81.8% and 90.9%, respectively. These findings demonstrate the relationship between plasma levels of selectins and disease severity of Kawasaki vasculitis.

Assessment of the Ability of Myocardial Contrast Echocardiography with Harmonic Power Doppler Imaging to Identify Perfusion Abnormalities in Patients with Kawasaki Disease at Rest and During Dipyridamole Stress

The aim of our study was to assess the ability of myocardial contrast echocardiography (MCE) with harmonic power Doppler imaging (HPDI) to identify perfusion abnormalities in patients with Kawasaki disease at rest and during pharmacological stress imaging with dipyridamole. Results were compared with those of 99mTc-tetrofosmin single-photon emission computed tomography (SPECT) imaging as the clinical reference standard. MCE with HPDI was performed on 20 patients with a history of Kawasaki disease. Images were obtained at baseline and during dipyridamole infusion (0.56 mg kg?1) in the apical two- and four-chamber views. Myocardial opacification suitable for the analysis was obtained in all patients. Nine patients with stenotic lesions had a reversible defect after dipyridamole infusion detected by both MCE with HPDI and SPECT, and 3 patients with a history of myocardial infarction had a partially or completely irreversible defect detected by both methods. Three patients with coronary aneurysm without stenotic lesion, 4 patients with regressed coronary aneurysm, and 2 patients with normal coronary artery in acute phase also had normal perfusion at rest and after pharmacological stress by both methods. A 96% concordance (? = 0.87) was obtained when comparing the respective segmental perfusion scores using the two methods at baseline, and an 86% concordance (? = 0.81) was obtained at postdipyridamole infusion. After combining baseline and postdipyridamole images, each segment was labeled as having normal perfusion, irreversible defects, or reversible defects. Using these classifications, concordance for the two methods was 92% (? = 0.87). MCE with HPDI is a safe and feasible method by which to detect asymptomatic ischemia due to severe stenotic lesion, and it may be an important addition to the modalities used to identify patients at risk for myocardial infarction as a complication of Kawasaki disease.

Serum Sodium Levels in Patients with Kawasaki Disease

The purpose of this study was to assess the hypothesis that lower serum sodium levels are associated with cardiovascular sequelae in patients with Kawasaki disease (KD). We used the database of the 16th nationwide survey of KD in Japan. We investigated the distribution of serum sodium levels and the relationship between serum sodium levels and cardiovascular sequelae. Of the reported cases, serum sodium levels were reported in 13,569 patients (89%). The proportion of patients with serum sodium levels 130 mEq/L or less, was greater in complete cases than in incomplete cases. The proportion of patients with serum sodium levels 130 mEq/L or less was increased with age. The largest proportion of patients with serum sodium levels 130 mEq/L or less was found in the category of 3–5 days since onset of illness. A serum sodium of level 135 mEq/L or less was an independent risk factor for cardiovascular sequelae (odds ratio, 1.79, 95% confidence interval, 1.42–2.26). Among patients with KD, there are significant differences in serum sodium levels between diagnostic categories, age, and days since the onset of illness. The sodium level may be a simple predictor of cardiovascular sequelae.