Metabolic predictors of ischemic heart disease and cerebrovascular attack in elderly diabetic individuals: difference in risk by age.

BACKGROUND: High LDL-cholesterol (LDL-C) and glucose levels are risk factors for ischemic heart disease (IHD) in middle-aged diabetic individuals; however, the risk among the elderly, especially the very elderly, is not well known. The aim of this study was to identify factors that predict IHD and cerebrovascular attack (CVA) in the elderly and to investigate their differences by age. METHODS: We performed a prospective cohort study (Japan Cholesterol and Diabetes Mellitus Study) with 5.5 years of follow-up. A total of 4,014 patients with type 2 diabetes and without previous IHD or CVA (1,936 women; age 67.4 ± 9.5 years, median 70 years; <65 years old, n = 1,261; 65 to 74 years old, n = 1,731; and ≥ 75 years old, n = 1,016) were recruited on a consecutive outpatient basis from 40 hospitals throughout Japan. Lipids, glucose, and other factors related to IHD or CVA risk, such as blood pressure (BP), were investigated using the multivariate Cox hazard model. RESULTS: One hundred fifty-three cases of IHD and 104 CVAs (7.8 and 5.7/1,000 people per year, respectively) occurred over 5.5 years. Lower HDL-cholesterol (HDL-C) and female gender were correlated with IHD in patients ≥75 years old (hazard ratio (HR):0.629, P < 0.01 and 1.132, P < 0.05, respectively). In contrast, systolic BP (SBP), HbA1C, LDL-C and non-HDL-C were correlated with IHD in subjects <65 years old (P < 0.05), and the LDL-C/HDL-C ratio was correlated with IHD in all subjects. HDL-C was correlated with CVA in patients ≥75 years old (HR: 0.536, P < 0.01). Kaplan-Meier estimator curves showed that IHD occurred more frequently in patients <65 years old in the highest quartile of the LDL-C/HDL-C ratio. In patients ≥75 years old, IHD and CVA were both the most frequent among those with the lowest HDL-C levels. CONCLUSIONS: IHD and CVA in late elderly diabetic patients were predicted by HDL-C. LDL-C, HbA1C, SBP and non-HDL-C are risk factors for IHD in the non-elderly. The LDL-C/HDL-C ratio may represent the effects of both LDL-C and HDL-C. These age-dependent differences in risk are important for developing individualized strategies to prevent atherosclerotic disease. TRIAL REGISTRATION: UMIN-CTR, UMIN00000516.

Age, gender, insulin and blood glucose control status alter the risk of ischemic heart disease and stroke among elderly diabetic patients.

BACKGROUND: We analyzed the effects of insulin therapy, age and gender on the risk of ischemic heart disease (IHD) and cerebrovascular accident (CVA) according to glycemic control. METHODS AND RESULTS: We performed a prospective cohort study (Japan Cholesterol and Diabetes Mellitus Study) of type 2 diabetes patients (n = 4014) for 2 years. The primary endpoint was the onset of fatal/non-fatal IHD and/or CVA, which occurred at rates of 7.9 and 7.2 per 1000 person-years, respectively. We divided diabetic patients into four groups based on age (≤ 70 and > 70) and hemoglobin A1C levels (≤ 7.0 and > 7.0%). Multiple regression analysis revealed that IHD was associated with high systolic blood pressure and low HDL-C in patients under 70 years of age with fair glycemic control and was associated with low diastolic blood pressure in the older/fair group. Interestingly, insulin use was associated with IHD in the older/poor group (OR = 2.27, 95% CI = 1.11-5.89; p = 0.026) and was associated with CVA in the older/fair group (OR = 2.09, 95% CI = 1.06-4.25; p = 0.028). CVA was associated with lower HDL-C and longer duration of diabetes in younger/poor glycemic control group. Results by stepwise analysis were similar. Next, patients were divided into four groups based on gender and diabetic control(hemoglobinA1C < or > 7.0%). Multiple regression analysis revealed that IHD was associated with high systolic blood pressure in male/fair glycemic control group, age in male/poor control group, and short duration of diabetic history in females in both glycemic control groups. Interestingly, insulin use was associated with IHD in the male/poor group(OR = 4.11, 95% CI = 1.22-8.12; p = 0.018) and with CVA in the female/poor group(OR = 3.26, 95% CI = 1.12-6.24; p = 0.02). CVA was associated with short duration of diabetes in both female groups. CONCLUSIONS: IHD and CVA risks are affected by specific factors in diabetics, such as treatment, gender and age. Specifically, insulin use has a potential role in preventing IHD but may also be a risk factor for CVA among the diabetic elderly, thus revealing a need to develop improved treatment strategies for diabetes in elderly patients. The Japan Cholesterol and Diabetes Mellitus Study was formulated to evaluate them(Umin Clinical Trials Registry, clinical trial reg. no. UMIN00000516; http://www.umin.ac.jp/ctr/index.htm).

Augmentation of vascular remodeling by uncoupled endothelial nitric oxide synthase in a mouse model of diabetes mellitus.

OBJECTIVE: Diabetes mellitus is associated with increased oxidative stress, which induces oxidation of tetrahydrobiopterin (BH4) in vessel wall. Without enough BH4, eNOS is uncoupled to L-arginine and produces superoxide rather than NO. We examined the role of uncoupled eNOS in vascular remodeling in diabetes. METHODS AND RESULTS: Diabetes mellitus was produced by streptozotocin in C57BL/6J mice. Under stable hyperglycemia, the common carotid artery was ligated, and neointimal formation was examined 4 weeks later. In diabetic mice, the neointimal area was dramatically augmented. This augmentation was associated with increased aortic superoxide formation, reduced aortic BH4/dihydrobiopterin (BH2) ratio, and decreased plasma nitrite and nitrate (NOx) levels compared with nondiabetic mice. Chronic BH4 treatment (10 mg/kg/d) reduced the neointimal area in association with suppressed superoxide production and inflammatory changes in vessels. BH4/BH2 ratio in vessel wall was preserved, and plasma NOx levels increased. Furthermore, in the presence of diabetes, overexpression of bovine eNOS resulted in augmentation of neointimal area, accompanied by increased superoxide production in the endothelium. CONCLUSIONS: In diabetes, increased oxidative stress by uncoupled NOSs, particularly eNOS, causes augmentation of vascular remodeling. These findings indicate restoration of eNOS coupling has an atheroprotective benefit in diabetes.

Central role of calcium-dependent tyrosine kinase PYK2 in endothelial nitric oxide synthase-mediated angiogenic response and vascular function.

BACKGROUND: The involvement of Ca2+-dependent tyrosine kinase PYK2 in the Akt/endothelial NO synthase pathway remains to be determined. METHODS AND RESULTS: Blood flow recovery and neovessel formation after hind-limb ischemia were impaired in PYK2-/- mice with reduced mobilization of endothelial progenitors. Vascular endothelial growth factor (VEGF)-mediated cytoplasmic Ca2+ mobilization and Ca2+-independent Akt activation were markedly decreased in the PYK2-deficient aortic endothelial cells, whereas the Ca2+-independent AMP-activated protein kinase/protein kinase-A pathway that phosphorylates endothelial NO synthase was not impaired. Acetylcholine-mediated aortic vasorelaxation and cGMP production were significantly decreased. Vascular endothelial growth factor-dependent migration, tube formation, and actin cytoskeletal reorganization associated with Rac1 activation were inhibited in PYK2-deficient endothelial cells. PI3-kinase is associated with vascular endothelial growth factor-induced PYK2/Src complex, and inhibition of Src blocked Akt activation. The vascular endothelial growth factor-mediated Src association with PLCgamma1 and phosphorylation of 783Tyr-PLCgamma1 also were abolished by PYK2 deficiency. CONCLUSION: These findings demonstrate that PYK2 is closely involved in receptor- or ischemia-activated signaling events via Src/PLCgamma1 and Src/PI3-kinase/Akt pathways, leading to endothelial NO synthase phosphorylation, and thus modulates endothelial NO synthase-mediated vasoactive function and angiogenic response.

Local overexpression of toll-like receptors at the vessel wall induces atherosclerotic lesion formation: synergism of TLR2 and TLR4.

OBJECTIVE: Atherosclerosis is now considered as a chronic inflammatory disease, and inflammation is closely related to immune systems, which consist of innate-immunity and adaptive-immunity. Recently, toll-like receptors (TLRs) have been identified as key components of innate-immunity. We examined the role of local expressions of TLRs at the vessel wall in atherosclerosis. METHODS AND RESULTS: We transfected cDNA encoding human TLR2 and TLR4 into the carotid arterial vessel wall of rabbits fed high-cholesterol diets with the use of HVJ-liposome. The rabbits were transfected with (1) pCMV-beta-gal, (2) empty vector, (3) TLR2, (4) TLR4, (5) TLR2+4. X-gal staining and immunohistochemical analysis showed that the transfected plasmids were mainly expressed in the media. Neither TLR2 nor TLR4 transfection induced significant augmentation of atherosclerosis. Transfection of TLR2- and TLR4-containing HVJ synergistically accelerated atherosclerosis and increased expressions of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and MCP-1. Moreover, transfection of TLR2 and TLR4 resulted in synergistic activation of NF-kappaB at the vessel wall in vivo, and in vascular smooth muscle cells in vitro. CONCLUSIONS: Expressions of both TLR2 and TLR4 at the vessel wall synergistically accelerated atherosclerosis. The present study revealed the role of TLRs expressed locally at the vessel wall in the early stage of atherosclerosis.

A specific role for eNOS-derived reactive oxygen species in atherosclerosis progression.

OBJECTIVE: When the availability of tetrahydrobiopterin (BH4) is deficient, endothelial nitric oxide synthase (eNOS) produces superoxide rather than NO (uncoupled eNOS). We have shown that the atherosclerotic lesion size was augmented in apolipoprotein E-deficient (ApoE-KO) mice overexpressing eNOS because of the enhanced superoxide production. In this study, we addressed the specific importance of uncoupled eNOS in atherosclerosis, and the potential mechanistic role for specific versus nonspecific antioxidant strategies in restoring eNOS coupling. METHODS AND RESULTS: We crossed mice overexpressing eNOS in the endothelium (eNOS-Tg) with mice overexpressing GTP-cyclohydrolase I (GCH), the rate-limiting enzyme in BH4 synthesis, to generate ApoE-KO/eNOS-Tg/GCH-Tg mice. As a comparison, ApoE-KO/eNOS-Tg mice were treated with vitamin C. Atherosclerotic lesion formation was increased in ApoE-KO/eNOS-Tg mice compared with ApoE-KO mice. GCH overexpression in ApoE-KO/eNOS-Tg/GCH-Tg mice increased vascular BH4 levels and reduced plaque area. This reduction was associated with decreased superoxide production from uncoupled eNOS. Vitamin C treatment failed to reduce atherosclerotic lesion size in ApoE-KO/eNOS-Tg mice, despite reducing overall vascular superoxide production. CONCLUSION: In contrast to vitamin C treatment, augmenting BH4 levels in the endothelium by GCH overexpression reduced the accelerated atherosclerotic lesion formation in ApoE-KO/eNOS-Tg mice, associated with a reduction of superoxide production from uncoupled eNOS.

Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice.

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is regarded as a protective factor against atherosclerosis. Therefore, augmentation of eNOS expression or NO production by pharmacological intervention is postulated to inhibit atherosclerosis. We crossed eNOS-overexpressing (eNOS-Tg) mice with atherogenic apoE-deficient (apoE-KO) mice to determine whether eNOS overexpression in the endothelium could inhibit the development of atherosclerosis. After 8 weeks on a high-cholesterol diet, the atherosclerotic lesion areas in the aortic sinus were unexpectedly increased by more than twofold in apoE-KO/eNOS-Tg mice compared with apoE-KO mice. Also, aortic tree lesion areas were approximately 50% larger in apoE-KO/eNOS-Tg mice after 12 weeks on a high-cholesterol diet. Expression of eNOS and NO production in aortas from apoE-KO/eNOS-Tg mice were significantly higher than those in apoE-KO mice. However, eNOS dysfunction, demonstrated by lower NO production relative to eNOS expression and enhanced superoxide production in the endothelium, was observed in apoE-KO/eNOS-Tg mice. Supplementation with tetrahydrobiopterin, an NOS cofactor, reduced the atherosclerotic lesion size in apoE-KO/eNOS-Tg mice to the level comparable to apoE-KO mice, possibly through the improvement of eNOS dysfunction. These data demonstrate that chronic overexpression of eNOS does not inhibit, but accelerates, atherosclerosis under hypercholesterolemia and that eNOS dysfunction appears to play important roles in the progression of atherosclerosis in apoE-KO/eNOS-Tg mice.

Stoichiometric relationships between endothelial tetrahydrobiopterin, endothelial NO synthase (eNOS) activity, and eNOS coupling in vivo: insights from transgenic mice with endothelial-targeted GTP cyclohydrolase 1 and eNOS overexpression.

Endothelial dysfunction in vascular disease states is associated with reduced NO bioactivity and increased superoxide (O2*-) production. Some data suggest that an important mechanism underlying endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O2*- rather than NO, possibly because of a mismatch between eNOS protein and its cofactor tetrahydrobiopterin (BH4). However, the mechanistic relationship between BH4 availability and eNOS coupling in vivo remains undefined because no studies have investigated the regulation of eNOS by BH4 in the absence of vascular disease states that cause pathological oxidative stress through multiple mechanisms. We investigated the stoichiometry of BH4-eNOS interactions in vivo by crossing endothelial-targeted eNOS transgenic (eNOS-Tg) mice with mice overexpressing endothelial GTP cyclohydrolase 1 (GCH-Tg), the rate-limiting enzyme in BH4 synthesis. eNOS protein was increased 8-fold in eNOS-Tg and eNOS/GCH-Tg mice compared with wild type. The ratio of eNOS dimer:monomer was significantly reduced in aortas from eNOS-Tg mice compared with wild-type mice but restored to normal in eNOS/GCH-Tg mice. NO synthesis was elevated by 2-fold in GCH-Tg and eNOS-Tg mice but by 4-fold in eNOS/GCH-Tg mice compared with wild type. Aortic BH4 levels were elevated in GCH-Tg and maintained in eNOS/GCH-Tg mice but depleted in eNOS-Tg mice compared with wild type. Aortic and cardiac O2*- production was significantly increased in eNOS-Tg mice compared with wild type but was normalized after NOS inhibition with Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME), suggesting O2*- production by uncoupled eNOS. In contrast, in eNOS/GCH-Tg mice, O2*- production was similar to wild type, and L-NAME had no effect, indicating preserved eNOS coupling. These data indicate that eNOS coupling is directly related to eNOS-BH4 stoichiometry even in the absence of a vascular disease state. Endothelial BH4 availability is a pivotal regulator of eNOS activity and enzymatic coupling in vivo.

Possible role of brain-derived neurotrophic factor in the pathogenesis of coronary artery disease.

BACKGROUND: The neurotrophin (NT) family, including nerve growth factor NT-3 and brain-derived neurotrophic factor (BDNF), has a critical role in the survival, growth, maintenance, and death of central and peripheral neurons. NTs and their receptors are expressed in atherosclerotic lesions; however, their significance in cardiovascular disease remains unclear. METHODS AND RESULTS: To clarify the role of NTs in the pathogenesis of coronary artery disease, NT plasma levels in the aorta, coronary sinus, and peripheral veins of patients with unstable angina (n=38), stable effort angina (n=45), and non-coronary artery disease (n=24) were examined. In addition, regional expression of BDNF in coronary arteries was examined in autopsy cases and patients with angina pectoris by directional coronary atherectomy. The difference in BDNF levels, but not NT-3, between the coronary sinus and aorta was significantly greater in the unstable angina group compared with the stable effort angina and non-coronary artery disease groups. Immunohistochemical investigations demonstrated BDNF expression in the atheromatous intima and adventitia in atherosclerotic coronary arteries. BDNF expression was enhanced in macrophages and smooth muscle cells in atherosclerotic coronary arteries. Stimulation with recombinant BDNF significantly enhanced NAD(P)H oxidase activity and the generation of reactive oxygen species in cultured human coronary artery smooth muscle cells. CONCLUSIONS: BDNF has an important role in atherogenesis and plaque instability via the activation of NAD(P)H oxidase.

Angiotensin II type 1 receptor blocker telmisartan suppresses superoxide production and reduces atherosclerotic lesion formation in apolipoprotein E-deficient mice.

Angiotensin II is involved in the process of atherosclerosis and stimulates superoxide production from cardiovascular cells. We examined the effect of telmisartan, an angiotensin II type 1 receptor blocker, on atherosclerosis. We chronically treated apolipoprotein E-deficient mice with two different doses of telmisartan dissolved in drinking water (0.3 and 3 mg/kg) starting from 4 weeks of age for 12 weeks. Lipid contents were not different in both telmisartan-treated groups compared with control group. Systolic blood pressure was significantly reduced with 3 mg/kg, but unchanged with 0.3 mg/kg. The total atherosclerotic lesion size at the aortic sinus was reduced with 0.3 mg/kg compared with control, and additional reduction was proved with 3 mg/kg. The fibrotic change was not different among three groups, but MOMA-2-, malondialdehyde-, 4-hydroxy-2-nonenal-immunostained areas were reduced by telmisartan. As the mechanism, we revealed that both doses of telmisartan markedly reduced superoxide production from in situ vessels assessed by lucigenin-enhanced chemiluminescence and dihydroethidium staining. And NAD(P)H dependent oxidase activity in vessels was reduced by telmisartan. Further, 8-iso-prostaglandin F2alpha level, a systemic oxidative stress marker, obtained from urine and plasma samples were significantly reduced by telmisartan. Telmisartan reduced atherosclerosis in apolipoprotein E-deficient mice at least partly via the suppression of oxidative stress.

Increased eNOS accounts for changes in connexin expression in renal arterioles during diabetes.

Previous studies have shown that connexin (Cx) expression is considerably higher in the preglomerular compared to postglomerular vasculature and that these differences are accentuated during diabetes. Since nitric oxide (NO) has been reported to alter Cx expression in endothelial cells and muscle cells and NO bioavailability is altered in diabetes, we hypothesized that NO may be responsible for the changes during diabetes. Cx expression was studied using immunohistochemistry in mice in which eNOS expression was either upregulated (eNOS transgenic) or downregulated (eNOS knockout). Diabetes was induced intraperitoneally with a single dose of alloxan or multiple low doses of streptozotocin. Expression of Cx40 in smooth muscle cells of afferent arterioles was increased, while expression of Cx43 in endothelial cells of efferent arterioles was absent in eNOS transgenic mice, similar to the changes occurring in wild-type mice during diabetes. Expression of Cx40 and Cx43 in eNOS knockout mice was not different from control; however, induction of diabetes in eNOS knockout mice failed to produce any changes in Cx40 or Cx43 in either afferent or efferent arterioles. Immunohistochemistry showed that eNOS expression was increased in the endothelium of renal arterioles in wild-type diabetic and eNOS transgenic mice, but absent from arterioles of eNOS knockout mice. We conclude that changes occurring in Cx expression in afferent and efferent arterioles during diabetes may result from increased eNOS.

Toll-like receptor 4 expressions on peripheral blood monocytes were enhanced in coronary artery disease even in patients with low C-reactive protein.

Toll-like receptors (TLRs) play important roles in the pathogenesis of atherosclerosis. On the other hand, serum high sensitivity C-reactive protein (hsCRP) is known as an independent coronary risk factor, but cardiovascular events do occur even in low hsCRP levels. We investigated whether the TLR4 expression levels on human peripheral blood monocytes were associated with serum hsCRP levels or the occurrence of coronary artery diseases (CAD). One hundred CAD patients and 100 non-CAD subjects were enrolled. There were 72 non-CAD subjects and 53 CAD patients with low serum hsCRP levels. Among the low-hsCRP subjects, the TLR4 expression levels were higher in CAD patients than in non-CAD subjects (P < 0.05, after being adjusted for other risk factors). Moreover, TLR4 expression levels in stable angina pectoris (SAP) patients were elevated compared with those in non-CAD subjects (P < 0.05), and those in acute coronary syndrome patients were higher than SAP patients even in low-hsCRP subjects (P < 0.01). In conclusion, the TLR4 expression levels on peripheral blood monocytes in CAD patients were higher than those in non-CAD subjects and correlated with disease activity, even in low-hsCRP subjects. The combined measurement of serum hsCRP and the TLR4 expression on peripheral blood monocytes, especially among low-hsCRP subjects, may become a new coronary risk marker.

Soluble CD40 ligand and interleukin-6 in the coronary circulation after acute myocardial infarction.

BACKGROUND: Inflammation, operated by blood, vascular and immune cells interaction, is implicated in plaque disruption and CD40 ligand (CD40L) was identified on activated T cells and platelets. We sought to investigate the roles of local inflammation in acute myocardial infarction (AMI). METHODS: Coronary sinus (CS) and arterial (A) levels of interleukin (IL)-6 and soluble CD40L (sCD40L) and matrix metalloproteinase (MMP)-9 activity in serial blood samples obtained until 48 h after percutaneous coronary intervention (PCI) were determined. In tissue specimens obtained by aspirating thrombectomy and directional coronary atherectomy, CD40L was immunohistochemically stained. RESULTS: Trans-cardiac gradient (CS-A) of IL-6, indicating cardiac release into the coronary circulation, significantly increased at 24 h after PCI in patients with AMI (group MI, n=17) in contrast with angina pectoris (n=10). Soluble CD40L levels in CS showed earlier peak, yielding trans-cardiac gradient, at 9 h in both groups. The maximum (max) release of IL-6 in MI, but not sCD40L, positively correlated with end-diastolic volume index (R=0.84) and negatively with ejection fraction (R=-0.66) by contrast ventriculography at 6-month follow up. Immunohistological study revealed the expression of CD40L in intra-coronary occlusive and mural thrombi. Aspirating thrombectomy significantly reduced the increase in both sCD40L levels and MMP-9 activity, but not max IL-6 release in MI. CONCLUSIONS: In contrast with myocardial injury represented by IL-6 release, acute rise in sCD40L levels with the MMP-9 activation in the coronary circulation may possibly reflect local inflammation with platelet activation and be a novel marker of plaque damage by PCI.

Xenogenic smooth muscle cell immunization reduces neointimal formation in balloon-injured rabbit carotid arteries.

OBJECTIVE: Intimal hyperplasia plays an important role in a variety of types of vascular remodeling, particularly luminal narrowing after vascular injury. The vascular smooth muscle cells (VSMCs) in the neointimal area are a synthetic phenotype and have different epitopes from VSMCs in the normal media. The synthetic VSMCs in the neointima contain various possible antigens that can be targeted by the immune system. In this study, we tried to develop a new immunotherapy, which targets the synthetic VSMCs, for prevention of neointimal formation after angioplasty. METHOD AND RESULTS: Rabbits were repeatedly immunized with fixed xenogenic rat cultured VSMCs suspended in adjuvant as immunogens or injected with adjuvant and phosphate-buffered saline (PBS) or rat hepatocytes as controls every 2 weeks for 3 times. One week after the last immunization/injection, balloon injury of the left common carotid artery was performed. Four weeks after the injury, rabbits were euthanized and the neointimal lesion formation was assessed. The mean neointimal area of the PBS-injected, non-immunized group and the rat hepatocyte-immunized, control group was not statistically different (0.339 +/- 0.036 and 0.350 +/- 0.041 mm(2), P = NS). However, immunization with rat VSMCs significantly reduced the intimal lesion area (0.219 +/- 0.0286 mm(2); P < 0.05 vs. PBS-injected, non-immunized group and rat hepatocyte-immunized group.) PCNA-immunopositive proliferating VSMCs in the neointima were suppressed by the rat VSMC immunization (1.34 +/- 0.49% vs. 5.78 +/- 0.47%; P < 0.05 vs. PBS-injected, non-immunized group). Rat VSMC immunization induced antibodies which had strong cross-reactivity against rabbit synthetic VSMCs. In experiments in vitro, proliferation and migration of rabbit VSMCs that were stimulated by serum, angiotensin (AT) II, platelet-derived growth factor (PDGF)-BB, fibroblast growth factor (FGF), and the phorbol ester PMA were significantly suppressed by treatment with immunoglobulin extracted from the VSMC-immunized rabbit plasma, implying that the immunoglobulin had some global effects on VSMCs. The rat VSMC-immunized rabbit immunoglobulin bound the rabbit AT1a receptor protein, which was expressed in COS7 cells by transfection of rabbit AT1a receptor pcDNA3. This binding to AT1a receptor may be one of mechanisms of the effects of VSMC-immunized immunoglobulin. CONCLUSION: Xenogenic, synthetic rat VSMC immunization in rabbits induced auto-antibodies against synthetic rabbit VSMCs in a cross-reaction. The induced auto-antibodies against synthetic VSMCs may provide a possibility of new immunotherapy for vascular remodeling that forms neointimal lesions.

Optical coherence tomography study of chronic-phase vessel healing after implantation of bare metal and paclitaxel-eluting self-expanding nitinol stents in the superficial femoral artery.

BACKGROUND: This study aimed to assess chronic-phase suppression of neointimal proliferation and arterial healing following paclitaxel-coated (PTX) and bare metal stent (BMS) implantation in the superficial femoral artery using optical coherence tomography (OCT). METHODS: Twenty-five patients with 68 stents underwent an 8-month OCT follow-up. Besides standard OCT variables, neointimal characterization and frequencies of peri-strut low-intensity area (PLIA), macrophage accumulation, and in-stent thrombi were evaluated. RESULTS: The mean neointimal thickness was significantly less with PTX stents (544.9±202.2 µm vs. 865.0±230.6 µm, p<0.0001). The covered and uncovered strut frequencies were significantly smaller and larger, respectively, in the PTX stent group vs. the BMS group (93.7% vs. 99.4%; p<0.0001, 4.0% vs. 0.4%; p<0.0001, respectively). Heterogeneous neointima was only observed in the PTX stent group (12.5% vs. 0%, p=0.017). The frequencies of PLIA and macrophage accumulation were significantly greater in the PTX stent group (87.2% vs. 67.6%, p=0.001 and 46% vs. 9.1%, p=0.003, respectively). CONCLUSION: After 8 months, reduced neointimal proliferation was observed with PTX stent implantation. On the other hand, delayed arterial healing was observed compared with BMS.

Transplantation of cardiotrophin-1-expressing myoblasts to the left ventricular wall alleviates the transition from compensatory hypertrophy to congestive heart failure in Dahl salt-sensitive hypertensive rats.

OBJECTIVES: We investigated whether autologous transplantation of skeletal myoblasts (MB) transferred with cardiotrophin-1 (CT-1) gene could retard the transition to heart failure (HF) in Dahl salt-sensitive (DS) hypertensive rats. BACKGROUND: Although MB is a therapeutic candidate for chronic HF, little is known about the efficiency of this strategy when applied in nonischemic HF. Cardiotrophin-1 has potent hypertrophic and survival effects on cardiac myocytes. We hypothesized that transplantation of CT-1-expressing myoblasts could provide cardioprotective effects against ventricular remodeling in DS hypertensive rats. METHODS: The DS rats were fed a high salt diet for 6 weeks and developed left ventricular (LV) hypertrophy at 11 weeks. At this stage, animals underwent MB to the myocardium with skeletal myoblasts transferred with CT-1 gene using retrovirus (transplantation of CT-1-expressing myoblasts [MB + CT], n = 31) or myoblasts alone (MB, n = 31). The sham group rats were injected with phosphate-buffered saline (n = 24). RESULTS: At 17 weeks, MB and MB + CT groups showed a significant alleviation of LV dilation and contractile dysfunction compared with the sham group. The degree of alleviation was significantly greater in the MB + CT group than the MB group (LV end-diastolic dimension: sham 7.06 +/- 0.14 mm, MB 6.51 +/- 0.16 mm, MB + CT 6.24 +/- 0.07 mm; fractional shortening: sham 32.1 +/- 1.4%, MB 38.5 +/- 1.5%, MB + CT 43.2 +/- 0.8%). Histological examination revealed that the myocyte size was 20% larger in the MB + CT group at 17 weeks than in the age-matched sham group. Upregulation of renin-angiotensin and endothelin systems during the transition to HF was attenuated by myoblast transplantation, and this effect was enhanced in the MB + CT group. CONCLUSIONS: Transplantation of skeletal myoblasts combined with CT-1-gene transfer could be a useful therapeutic strategy for HF.

Dysfunction of endothelial nitric oxide synthase and atherosclerosis.

Atherosclerosis is associated with an impairment of endothelium-dependent relaxations, which represents the reduced bioavailability of nitric oxide (NO) produced from endothelial NO synthase (eNOS). Among various mechanisms implicated in the impaired EDR in atherosclerosis, superoxide generated from dysfunctional eNOS has attracted attention. Under conditions in which vascular tissue levels of tetrahydrobiopterin (BH4), a cofactor for NOS, are deficient or lacking, eNOS becomes dysfunctional and produces superoxide rather than NO. Experimental studies in vitro have revealed that NO from eNOS constitutes an anti-atherogenic molecule. A deficiency of eNOS was demonstrated to accelerate atherosclerotic lesion formation in eNOS knockout mice. In contrast, eNOS overexpression with hypercholesterolemia may promote atherogenesis via increased superoxide generation from dysfunctional eNOS. Thus, eNOS may have 2 faces in the pathophysiology of atherosclerosis depending on tissue BH4 metabolisms. An improved understanding of tissue BH4 metabolisms in atherosclerotic vessels is needed, which would help in developing new strategies for the inhibition and treatment of atherosclerosis.

Development of an animal model for spontaneous myocardial infarction (WHHLMI rabbit).

OBJECTIVE: Coronary heart disease is the most common cause of death in developed countries. However, there are no suitable animal models that mimic spontaneous myocardial infarction in humans. In this study, we attempted to obtain a rabbit strain with spontaneous myocardial infarction by selective breeding of coronary atherosclerosis-prone Watanabe heritable hyperlipidemic (WHHL) rabbits, designated as WHHLMI rabbits. METHODS AND RESULTS: WHHLMI rabbits were characterized by the high incidence of fatal myocardial infarction at ages 11 to 35 months, being increased from 23% to 97% after the selective breeding. The ECG on WHHLMI rabbits showed a typical feature of myocardial infarction. Histological examination of hearts from suddenly deceased WHHLMI rabbits revealed old myocardial infarction accompanied by fresh myocardial lesions. The culprit coronary arteries exhibited severe atheromatous plaques (>90% lumen area stenosis), suggesting that coronary atherosclerosis is responsible for myocardial infarction observed in WHHLMI rabbits. In addition, the coronary plaques showed vulnerable features including macrophage-rich thin cap and large necrotic core. CONCLUSIONS: To the best of our knowledge, this is the first report of spontaneous myocardial infarction in rabbits, and it is suggested that this WHHLMI rabbit strain will be a useful animal model to study human myocardial infarction.

Involvement of endothelial nitric oxide in sphingosine-1-phosphate-induced angiogenesis.

Nitric oxide (NO) has been implicated as a critical signaling molecule of angiogenesis. Recently, sphingosine-1-phosphate (S1P) has emerged as a mediator of angiogenesis, and S1P-induced NO synthesis in endothelial cells (ECs) has been reported. To analyze the signaling pathways involved in S1P-induced angiogenesis and clarify the role of NO in this process, we performed in vivo and in vitro angiogenesis assays. S1P activated the phosphatidylinositol-3-kinase (PI3K)/Akt/endothelial NO synthase (eNOS) pathway in ECs, since S1P-stimulated eNOS phosphorylation and NO production were blocked by inhibition of activities of PI3K and Akt. S1P increased capillary ingrowth into subcutaneously implanted Matrigel plugs in mice, and the effect of S1P was significantly reduced in mice that received N(G)-nitro- L-arginine methyl ester (L-NAME), an inhibitor of NOS. S1P stimulated EC migration and tube formation on Matrigel, which processes were significantly decreased by inhibition of activities of PI3K, Akt, or eNOS, whereas treatment with LY294002, a PI3K inhibitor, but not L-NAME, inhibited EC viability and proliferation. Thus, our results demonstrate the crucial role of NO in S1P-induced angiogenesis in vivo and in vitro and suggest the divergent roles of NO in the S1P-induced angiogenic response.

Propagermanium reduces atherosclerosis in apolipoprotein E knockout mice via inhibition of macrophage infiltration.

Monocyte chemoattractant protein-1 (MCP-1), which binds to C-C chemokine receptor 2, has been implicated as the primary source of monocyte chemoattractant function in the early stages of atherosclerosis. Recently, propagermanium, a drug used clinically for the treatment of chronic hepatitis in Japan, has been shown to inhibit C-C chemokine receptor 2 function and suppress monocyte/macrophage infiltration in vitro and in vivo. Given the importance of monocyte infiltration in atherogenesis, the inhibition of it by propagermanium might prevent atherosclerosis. Apolipoprotein E knockout (apoE-KO) mice were fed an atherogenic high cholesterol diet with or without 0.005% propagermanium for 8 or 12 weeks. Although the plasma lipid levels were unchanged by the drug treatment, atherosclerotic lesion area in the aortic root was reduced by 50% in the drug-treated apoE-KO mice compared with the nontreated apoE-KO mice after 8 weeks of cholesterol feeding (0.62+/-0.12 versus 1.27+/-0.07 mm2, respectively; P<0.01). Moreover, the accumulation of macrophages in the lesions was markedly reduced in the drug-treated group (macrophage positive area, 0.23+/-0.06 mm2 [drug-treated group] versus 0.67+/-0.07 mm2 [control group]; P<0.01). After 12 weeks of cholesterol feeding, atherosclerotic lesion formation in the aortic root and in the descending thoracic aorta was significantly reduced in the drug-treated group. Inhibition of macrophage infiltration by propagermanium prevented the formation of atherosclerotic lesions in apoE-KO mice. This drug may serve as a therapeutic tool for the treatment of atherosclerosis.