Impact of diabetes and glycaemic control on peripheral artery disease in Japanese patients with end-stage renal disease: long-term follow-up study from the beginning of haemodialysis.

AIMS/HYPOTHESIS: End-stage renal disease (ESRD) patients with diabetes have been regarded as being at the highest risk of cardiovascular disease. We therefore investigated the relationship between diabetes and the incidence of peripheral artery disease (PAD) in new haemodialysis patients. METHODS: We enrolled 1,513 ESRD patients who had just begun haemodialysis therapy. They were divided into two groups: those with (n = 739) and those without diabetes (n = 774). The endpoint was the development of PAD, defined as ankle brachial pressure index ≤ 0.9 or toe brachial pressure index <0.7 in patients with an ankle brachial pressure index >0.9. RESULTS: According to the Kaplan-Meier method, the 10 year event-free rate for development of PAD and lower limb amputation was significantly lower in the diabetes group than in the non-diabetes group (60.3% vs 82.8%, HR 2.99, 95% CI 2.27, 3.92, p<0.0001 and 93.9% vs 98.9%, HR 5.59, 95% CI 2.14, 14.7, p = .0005 for PAD and lower limb amputation, respectively). In patients with diabetes, quartile analysis of HbA1c levels showed that the highest quartile group (≥ 6.8% [51 mmol/mol]) had significant development of PAD and lower limb amputation compared with lower quartile groups (PAD HR 1.63, 95% CI 1.17, 2.28, p = .0038; lower limb amputation HR 2.99, 95% CI 1.17, 7.70, p = .023). CONCLUSIONS/INTERPRETATION: Diabetes was a strong predictor of PAD after initiation of haemodialysis therapy in patients with ESRD. In addition, higher HbA1c levels were associated with increased risk of developing PAD and requiring limb amputation in such diabetic populations.

Prospective evaluation of corrected QT intervals and arrhythmias after exposure to epirubicin, cyclophosphamide, and 5-fluorouracil in women with breast cancer.

BACKGROUND: Corrected QT (QTc) interval prolongation can induce fatal arrhythmias such as torsade de pointes. PATIENTS AND METHODS: To assess the characteristics of QTc intervals and arrhythmias in women with early breast cancer who received FEC100 adjuvant chemotherapy, electrocardiograms (ECGs) were recorded before and after each chemotherapy. Associations between QTc interval prolongation and single nucleotide polymorphisms (SNPs) of potassium channel genes were also investigated. RESULTS: A total of 131 ECG records were obtained in 34 patients who received 153 cycles of FEC100. QTc intervals could be measured in 127 records. There was a significant trend toward QTc interval prolongation after each treatment, persisting through four cycles of chemotherapy (P < 0.001). Median QTc interval prolongations were 13, 11, 18, and 14 ms in the first through fourth cycles of chemotherapy, respectively. QTc intervals differed significantly between cycles 1 and 4 before treatment as well as after treatment (P < 0.05). A single supraventricular premature contraction was noted in 3 (2.3%) of the 131 cycles in 2 (5.9%) of the 34 patients. There was no significant association between QTc interval prolongation and SNPs of potassium channel genes. CONCLUSION: This prospective study confirmed that FEC100 is associated with significant QTc interval prolongation in women with early breast cancer.

Cilostazol stimulates revascularisation in response to ischaemia via an eNOS-dependent mechanism.

OBJECTIVES: Cilostazol is known to be a selective inhibitor of phosphodiesterase 3 and is generally used to treat intermittent claudication caused by peripheral arterial disease. However, there is little information concerning the effect of cilostazol on angiogenesis. Here, we investigated whether cilostazol modulates the angiogenic process in vivo employing a hindlimb model of ischaemia-induced angiogenesis. DESIGN: This was an experimental study. MATERIALS AND METHODS: Wild-type (WT) mice were randomly divided into two groups and were treated with or without cilostazol. One week later, the mice were subjected to unilateral hindlimb ischaemia. Angiogenesis was determined by laser Doppler analysis and capillary density stained with CD31. The expression of endothelial nitric oxide synthase (eNOS) was assessed by immunoblotting. RESULTS: WT mice treated with cilostazol showed accelerated neo-vascularisation following hindlimb ischaemic surgery on post-operative day 14 based upon laser Doppler measurements of blood flow (cilostazol-treated group, 0.54 ± 0.13 vs. control group, 0.38 ± 0.11; P-<-0.05). The capillary density in the ischaemic hindlimb was also significantly greater in WT mice treated with cilostazol than in non-treated WT mice (cilostazol-treated group, 1.63 ± 0.10 vs. control group, 1.15 ± 0.12; P-<-0.01). Cilostazol stimulated an ischaemia-induced increase in the phosphorylation of eNOS in the ischaemic limbs. Administration of NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) abolished cilostazol-induced increase in limb perfusion. CONCLUSIONS: Our observations indicate that cilostazol can promote neo-vascularisation in response to tissue ischaemia via an eNOS-dependent mechanism. Cilostazol could be useful for treatment of ischaemic limb diseases.

Association of a polymorphism of BTN2A1 with type 2 diabetes mellitus in Japanese individuals.

AIMS: We previously showed that the C→T polymorphism (rs6929846) of BTN2A1 was significantly associated with myocardial infarction in Japanese individuals by a genome-wide association study. Given that diabetes mellitus is an important risk factor for myocardial infarction, the association of rs6929846 of BTN2A1 with myocardial infarction might be attributable, at least in part, to its effect on susceptibility to diabetes. The purpose of this study was to examine the relation of rs6929846 of BTN2A1 to Type 2 diabetes mellitus. METHODS: A total of 8650 Japanese individuals from two independent subject panels were examined: Panel A comprised 1141 individuals with Type 2 diabetes and 3161 control subjects and panel B comprised 1664 individuals with Type 2 diabetes and 2684 control subjects. RESULTS: The chi-square test revealed that rs6929846 of BTN2A1 was significantly related to the prevalence of Type 2 diabetes in subject panel A (P = 0.0002) and subject panel B (P=0.006). Multivariable logistic regression analysis with adjustment for age, sex, body mass index and smoking status revealed that rs6929846 was significantly associated with Type 2 diabetes (P = 0.0006; odds ratio 1.25) in all individuals, with the T allele representing a risk factor for this condition. Multiple regression analysis with adjustment for age, sex and body mass index revealed that rs6929846 was significantly (P=0.04) related to blood glycosylated haemoglobin content in control subjects. CONCLUSIONS: BTN2A1 may be a susceptibility gene for Type 2 diabetes in Japanese individuals.

Reciprocal relation between VEGF and NO in the regulation of endothelial integrity.

Balloon angioplasty disrupts the protective endothelial lining of the arterial wall, rendering arteries susceptible to thrombosis and intimal thickening. We show here that vascular endothelial growth factor (VEGF), an endothelial cell mitogen, is upregulated in medial smooth muscle cells of the arterial wall in response to balloon injury. Both protein kinase C (PKC) and tyrosine kinase pp60src mediate augmented VEGF expression. In contrast, nitric oxide (NO) donors inhibit PKC-induced VEGF upregulation by interfering with binding of the transcription factor activator protein-1 (AP-1) to the VEGF promoter. Inhibition of VEGF promoter activation suggests that NO secreted by a restored endothelium functions as the negative feedback mechanism that downregulates VEGF expression to basal levels. Administration of a neutralizing VEGF antibody impaired reendothelialization following balloon injury performed in vivo. These findings establish a reciprocal relation between VEGF and NO in the endogenous regulation of endothelial integrity following arterial injury.

Plasma resistin concentration determined by common variants in the resistin gene and associated with metabolic traits in an aged Japanese population.

AIMS/HYPOTHESIS: Resistin is a cytokine derived from adipose tissue and is implicated in obesity-related insulin resistance and type 2 diabetes mellitus. Polymorphisms of the resistin gene (RETN) have been shown to affect the plasma resistin concentration. The aims of this study were to identify polymorphisms of RETN that influence plasma resistin concentration and to clarify the relation between plasma resistin level and metabolic disorders in an aged Japanese cohort. METHODS: The study participants comprised 3133 individuals recruited to a population-based prospective cohort study (KING study). Plasma resistin concentration, BMI, abdominal circumference, blood pressure, fasting plasma glucose and serum insulin concentrations, HbA(1c) content and serum lipid profile were measured in all participants. The HOMA index of insulin resistance (HOMA-IR) was also calculated. Eleven polymorphisms of RETN were genotyped. RESULTS: A combination of ANOVA and multiple linear regression analysis in screening and large-scale subsets of the study population revealed that plasma resistin concentration was significantly associated with rs34861192 and rs3745368 polymorphisms of RETN. Multiple linear regression analysis with adjustment for age and sex also showed that the plasma resistin level was significantly associated with serum concentrations of HDL-cholesterol, triacylglycerol and insulin, as well as with BMI. CONCLUSIONS/INTERPRETATION: Our results implicate the rs34861192 and rs3745368 polymorphisms of RETN as robust and independent determinants of plasma resistin concentration in the study population. In addition, plasma resistin level was associated with dyslipidaemia, serum insulin concentration and obesity. TRIAL REGISTRATION: ClinicalTrials.gov NCT00262691.

Isolation of putative progenitor endothelial cells for angiogenesis.

Putative endothelial cell (EC) progenitors or angioblasts were isolated from human peripheral blood by magnetic bead selection on the basis of cell surface antigen expression. In vitro, these cells differentiated into ECs. In animal models of ischemia, heterologous, homologous, and autologous EC progenitors incorporated into sites of active angiogenesis. These findings suggest that EC progenitors may be useful for augmenting collateral vessel growth to ischemic tissues (therapeutic angiogenesis) and for delivering anti- or pro-angiogenic agents, respectively, to sites of pathologic or utilitarian angiogenesis.

Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization.

Endothelial precursor cells (EPCs) have been identified in adult peripheral blood. We examined whether EPCs could be isolated from umbilical cord blood, a rich source for hematopoietic progenitors, and whether in vivo transplantation of EPCs could modulate postnatal neovascularization. Numerous cell clusters, spindle-shaped and attaching (AT) cells, and cord-like structures developed from culture of cord blood mononuclear cells (MNCs). Fluorescence-trace experiments revealed that cell clusters, AT cells, and cord-like structures predominantly were derived from CD34-positive MNCs (MNC(CD34+)). AT cells and cell clusters could be generated more efficiently from cord blood MNCs than from adult peripheral blood MNCs. AT cells incorporated acetylated-LDL, released nitric oxide, and expressed KDR, VE-cadherin, CD31, and von Willebrand factor but not CD45. Locally transplanted AT cells survived and participated in capillary networks in the ischemic tissues of immunodeficient nude rats in vivo. AT cells thus had multiple endothelial phenotypes and were defined as a major population of EPCs. Furthermore, laser Doppler and immunohistochemical analyses revealed that EPC transplantation quantitatively augmented neovascularization and blood flow in the ischemic hindlimb. In conclusion, umbilical cord blood is a valuable source of EPCs, and transplantation of cord blood-derived EPCs represents a promising strategy for modulating postnatal neovascularization.

Nitric oxide synthase modulates angiogenesis in response to tissue ischemia.

We tested the hypothesis that endothelial nitric oxide synthase (eNOS) modulates angiogenesis in two animal models in which therapeutic angiogenesis has been characterized as a compensatory response to tissue ischemia. We first administered L-arginine, previously shown to augment endogenous production of NO, to normal rabbits with operatively induced hindlimb ischemia. Angiogenesis in the ischemic hindlimb was significantly improved by dietary supplementation with L-arginine, compared to placebo-treated controls; angiographically evident vascularity in the ischemic limb, hemodynamic indices of limb perfusion, capillary density, and vasomotor reactivity in the collateral vessel-dependent ischemic limb were all improved by oral L-arginine supplementation. A murine model of operatively induced hindlimb ischemia was used to investigate the impact of targeted disruption of the gene encoding for ENOS on angiogenesis. Angiogenesis in the ischemic hindlimb was significantly impaired in eNOS-/- mice versus wild-type controls evaluated by either laser Doppler flow analysis or capillary density measurement. Impaired angiogenesis in eNOS-/- mice was not improved by administration of vascular endothelial growth factor (VEGF), suggesting that eNOS acts downstream from VEGF. Thus, (a) eNOS is a downstream mediator for in vivo angiogenesis, and (b) promoting eNOS activity by L-arginine supplementation accelerates in vivo angiogenesis. These findings suggest that defective endothelial NO synthesis may limit angiogenesis in patients with endothelial dysfunction related to atherosclerosis, and that oral L-arginine supplementation constitutes a potential therapeutic strategy for accelerating angiogenesis in patients with advanced vascular obstruction.

Fatal thrombosis of antithrombin-deficient mice is rescued differently in the heart and liver by intercrossing with low tissue factor mice.

BACKGROUND: We previously reported that the targeted disruption of murine antithrombin (AT) gene resulted in embryonic lethality before 16.5 gestational days (gd) because of severe cardiac and hepatic thrombosis. OBJECTIVE AND METHODS: To investigate the influences of lowered tissue factor (TF) activity upon hypercoagulation of AT-/- embryos, we crossed AT+/- with low TF (mTF-/- hTF+) mice to yield homozygous AT-deficient mice with the extremely low TF activity, that is expressed from the inserted human TF mini gene. RESULTS: AT-/- embryos either with 50% TF (AT-/- mTF+/- hTF+) or with low (approximately 1% TF, AT-/- mTF-/- hTF+) were not born, although the survival was prolonged until 18.5 gd. In both genotypes, histological examination showed disseminated thrombosis in hepatic sinusoidal space or in the portal veins, suggesting that the thrombogenesis caused loss of hepatic blood flow. As in original AT-/-, AT-/- mTF+/- hTF+ showed subcutaneous (s.c.) bleeding and also suffered from the myocardial degeneration apparently because of coronary thrombus formation. However, AT-/- mTF-/- hTF+ had no skin hemorrhage and the thrombosis and degeneration were completely abolished in the heart. Myocardium of adult low TF mice had exhibited fibrosis secondary to hemorrhage; however, it was significantly decreased in low TF mice with AT+/-. CONCLUSIONS: Our current model suggests that, in the heart, TF plays an important role in the thrombogenesis and it counterbalances AT-dependent anticoagulation. AT may be a potent anticoagulant during mice development and the activation and subsequent regulation of TF-procoagulant activity take place differently between the liver and the heart. These differences appear to point to local regulatory mechanisms in murine hemostasis.

Highly purified eicosapentaenoic acid ameliorates cardiac injury and adipose tissue inflammation in a rat model of metabolic syndrome.

INTRODUCTION: n-3 Polyunsaturated fatty acids such as eicosapentaenoic acid (EPA), which are abundant in fish oil, have been shown to delay the onset of cardiovascular events. We previously established DahlS.Z-Leprfa/Leprfa (DS/obese) rats, which are derived from a cross between Dahl salt-sensitive and Zucker rats, as a model of metabolic syndrome. This study has now explored the influence of highly purified EPA on cardiac and adipose tissue pathophysiology in this animal model. MATERIALS AND METHODS: DS/obese rats were administered EPA (300 or 1,000 mg kg-1 d-1, per os) or vehicle from age 9 to 13 weeks. Homozygous lean (DahlS.Z-Lepr+/Lepr+, or DS/lean) littermates were studied as controls. RESULTS: Whereas EPA had no effect on body weight, food intake or systolic blood pressure in DS/obese rats, it attenuated cardiac fibrosis, diastolic dysfunction, oxidative stress and inflammation in these animals. In addition, EPA did not affect insulin resistance but reduced adipocyte hypertrophy and inflammation in visceral fat of DS/obese rats. Moreover, EPA increased circulating levels of adiponectin as well as attenuated both the down-regulation of AMP-activated protein kinase phosphorylation and the up-regulation of phosphorylation of the p65 subunit of nuclear factor-kB in the heart of DS/obese rats. CONCLUSIONS: Treatment of DS/obese rats with EPA did not affect hypertension but reduced cardiac fibrosis and diastolic dysfunction, with the latter effects being accompanied by AMP-activated protein kinase activation and inactivation of nuclear factor-kB signalling in the heart, possibly as a result of an increase in adiponectin secretion. EPA may be suitable for the treatment of cardiac injury associated with metabolic syndrome.

Attenuation of cold stress-induced exacerbation of cardiac and adipose tissue pathology and metabolic disorders in a rat model of metabolic syndrome by the glucocorticoid receptor antagonist RU486.

OBJECTIVES: Chronic stress affects the central nervous system as well as endocrine, metabolic and immune systems. However, the effects of cold stress on cardiovascular and metabolic disorders in metabolic syndrome (MetS) have remained unclear. We recently characterized DahlS.Z-Lepr(fa)/Lepr(fa) (DS/obese) rats, derived from a cross between Dahl salt-sensitive and Zucker rats, as a new animal model of MetS. We have now investigated the effects of chronic cold stress and glucocorticoid receptor (GR) blockade on cardiac and adipose tissue pathology as well as on metabolic parameters in this model. METHODS: DS/obese rats were exposed to cold stress (immersion in ice-cold water to a depth of 1-2 cm for 2 h per day) with or without subcutaneous injection of the GR antagonist RU486 (2 mg kg(-1)day(-1)) for 4 weeks beginning at 9 weeks of age. Age-matched homozygous lean (DahlS.Z-Lepr(+)/Lepr(+)) littermates served as a control. RESULTS: Chronic cold stress exacerbated hypertension as well as left ventricular (LV) hypertrophy, fibrosis and diastolic dysfunction in DS/obese rats in a manner sensitive to RU486 treatment. Cold stress with or without RU486 did not affect body weight or fat mass. In contrast, cold stress further increased cardiac oxidative stress as well as macrophage infiltration and proinflammatory gene expression in LV and visceral fat tissue, with all of these effects being attenuated by RU486. Cold stress also further increased GR and 11ß-hydroxysteroid dehydrogenase type 1 mRNA and protein abundance in LV and visceral adipose tissue, and these effects were again inhibited by RU486. In addition, RU486 ameliorated the stress-induced aggravation of dyslipidemia, glucose intolerance and insulin resistance in DS/obese rats. CONCLUSIONS: Our results implicate GR signaling in cold stress-induced exacerbation of cardiac and adipose tissue pathology as well as of abnormal glucose and lipid metabolism in a rat model of MetS.

Intraplatelet tetrahydrobiopterin plays an important role in regulating canine coronary arterial thrombosis by modulating intraplatelet nitric oxide and superoxide generation.

BACKGROUND: Platelet-derived nitric oxide inhibits platelet aggregation via constitutive NO synthase (NOS). Tetrahydrobiopterin (BH(4)), a cofactor of NOS, augments NO formation, whereas its deficiency decreases NO bioactivity and increases superoxide generation by NOS. The roles of intraplatelet BH(4) in platelet aggregation and thrombus formation, however, are unknown. Accordingly, we investigated whether intraplatelet BH(4) is involved in regulating cyclic flow variations (CFVs) and platelet aggregation in a canine model with stenosed and endothelium-injured coronary arteries that mimics acute coronary syndromes in humans. METHODS AND RESULTS: After developing CFVs, dogs received saline or BH(4) (10 or 30 mg/kg) intravenously. Intraplatelet BH(4) and cGMP levels were decreased and intraplatelet nitrotyrosine production was increased during CFVs. ADP- and U46619-induced ex vivo platelet aggregation and platelet P-selectin expression were augmented during CFVs. BH(4) administration restored intraplatelet BH(4) and cGMP levels and decreased intraplatelet nitrotyrosine production, resulting in reduced CFVs and inhibited ex vivo platelet aggregation and platelet P-selectin expression. CFVs again developed after N(G)-monomethyl-L-arginine, an inhibitor of NOS, in BH(4)-treated dogs. Ex vivo platelet NOS activity at baseline, during CFVs, and after BH(4) administration did not differ. CONCLUSIONS: Intraplatelet BH(4) may play an important role in regulating thrombus formation by modulating platelet-derived nitric oxide and superoxide generation by platelet NOS.

Augmentation of postnatal neovascularization with autologous bone marrow transplantation.

BACKGROUND: Endothelial progenitor cells (EPCs) have been identified in adult human peripheral blood. Because circulating EPCs should originate from bone marrow (BM), we examined whether BM mononuclear cells (BM-MNCs) can give rise to functional EPCs and whether transplantation of autologous BM-MNCs might augment angiogenesis and collateral vessel formation in a rabbit model of hindlimb ischemia. METHODS AND RESULTS: Rabbit BM-MNCs were isolated by centrifugation through a Histopaque density gradient and cultured on fibronectin. EPCs developed from BM-MNCs in vitro, as assessed by acetylated LDL incorporation, nitric oxide (NO) release, and expression of von Willebrand factor and lectin binding. Unilateral hindlimb ischemia was surgically induced in rabbits (n=8), and fluorescence-labeled autologous BM-MNCs were transplanted into the ischemic tissues. Two weeks after transplantation, fluorescence microscopy revealed that transplanted cells were incorporated into the capillary network among preserved skeletal myocytes. In contrast, transplanted autologous BM-fibroblasts did not participate in EC capillary network formation (n=5). Then, in an additional 27 rabbits, saline (control; n=8), autologous BM-MNCs (n=13; 6.9+/-2.2x10(6) cells/animal), or BM-fibroblasts (n=6; 6.5+/-1.5x10(6) cells/animal) were injected into the ischemic tissues at postoperative day 7. Four weeks after transplantation, the BM-MNC-transplanted group had more angiographically detectable collateral vessels (angiographic score: 1.5+/-0.34 versus 0.94+/-0.26 and 1.1+/-0.14; P:<0.05), a higher capillary density (23+/-5.8 versus 10+/-1.9 and 11+/-0.8 per field; P:<0.001), and a greater laser Doppler blood perfusion index (505+/-155 versus 361+/-35 and 358+/-22 U; P:<0.05) than the control and BM-fibroblast-transplanted groups. CONCLUSIONS: Direct local transplantation of autologous BM-MNCs seems to be a useful strategy for therapeutic neovascularization in ischemic tissues in adults, consistent with "therapeutic vasculogenesis."

Hypercholesterolemia inhibits angiogenesis in response to hindlimb ischemia: nitric oxide-dependent mechanism.

BACKGROUND: Endothelium-derived nitric oxide (EDNO) plays an important role in the regulation of angiogenesis, whereas hypercholesterolemia (HC) impairs EDNO release. We examined the hypothesis that HC may inhibit ischemia-induced angiogenesis by inhibition of EDNO in a rat model of unilateral hindlimb ischemia and that oral L-arginine supplementation, a substrate for NO synthase, may prevent HC-related impairment of angiogenesis. METHODS AND RESULTS: Male Sprague-Dawley rats were fed (A) standard diet (control), (B) 2% high-cholesterol diet (HC group), or (C) high-cholesterol diet with oral L-arginine (2.25% in drinking water) (HC+L-arg group). At 2 weeks of the dietary intervention, unilateral limb ischemia was surgically induced in all animals. Dietary HC groups (B and C) revealed elevated total and LDL cholesterol levels compared with control animals. Laser Doppler blood flow analyses showed significant decreases in the ischemic/normal limb blood flow ratio in the HC group compared with controls (P:<0.05) when followed up until 4 weeks after surgery. Selective angiography and immunohistochemical analyses in the ischemic limb at postoperative day 14 revealed significantly lower angiographic scores (P:<0.01) and capillary densities (P:<0.01) in the HC group than controls, which were associated with decreased tissue contents of NO(x) and cGMP. Oral L-arginine supplementation (HC+L-arg) significantly improved all parameters of the laser Doppler blood perfusion ratio, angiographic scores, and capillary densities (P:<0.01 versus HC group), which were accompanied by significant elevations in serum L-arginine levels and tissue NO(x) and cGMP contents. CONCLUSIONS: Collateral vessel formation and angiogenesis in response to hindlimb ischemia were significantly attenuated in rats with dietary HC. The mechanism may be related to the reduced NO bioactivity in the ischemic tissues. Augmentation of the tissue NO activity by oral L-arginine supplementation restored the impaired angiogenesis in HC.

Mobilization of endothelial progenitor cells in patients with acute myocardial infarction.

BACKGROUND: Endothelial progenitor cells (EPCs) circulate in adult peripheral blood (PB) and contribute to neovascularization. However, little is known regarding whether EPCs and their putative precursor, CD34-positive mononuclear cells (MNC(CD34+)), are mobilized into PB in acute ischemic events in humans. METHODS AND RESULTS: Flow cytometry revealed that circulating MNC(CD34+) counts significantly increased in patients with acute myocardial infarction (n=16), peaking on day 7 after onset, whereas they were unchanged in control subjects (n=8) who had no evidence of cardiac ischemia. During culture, PB-MNCs formed multiple cell clusters, and EPC-like attaching cells with endothelial cell lineage markers (CD31, vascular endothelial cadherin, and kinase insert domain receptor) sprouted from clusters. In patients with acute myocardial infarction, more cell clusters and EPCs developed from cultured PB-MNCs obtained on day 7 than those on day 1. Plasma levels of vascular endothelial growth factor significantly increased, peaking on day 7, and they positively correlated with circulating MNC(CD34+) counts (r=0.35, P=0.01). CONCLUSIONS: This is the first clinical demonstration showing that lineage-committed EPCs and MNC(CD34+), their putative precursors, are mobilized during an acute ischemic event in humans.

Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines.

BACKGROUND: Bone marrow implantation (BMI) was shown to enhance angiogenesis in a rat ischemic heart model. This preclinical study using a swine model was designed to test the safety and therapeutic effectiveness of BMI. METHODS AND RESULTS: BM-derived mononuclear cells (BM-MNCs) were injected into a zone made ischemic by coronary artery ligation. Three weeks after BMI, regional blood flow and capillary densities were significantly higher (4.6- and 2.8-fold, respectively), and cardiac function was improved. Angiography revealed that there was a marked increase (5.7-fold) in number of visible collateral vessels. Implantation of porcine coronary microvascular endothelial cells (CMECs) did not cause any significant increase in capillary densities. Labeled BM-MNCs were incorporated into approximately 31% of neocapillaries and corresponded to approximately 8.7% of macrophages but did not actively survive as myoblasts or fibroblasts. There was no bone formation by osteoblasts or malignant ventricular arrhythmia. Time-dependent changes in plasma levels for cardiac enzymes (troponin I and creatine kinase-MB) did not differ between the BMI, CMEC, and medium-alone implantation groups. BM-MNCs contained 16% of endothelial-lineage cells and expressed basic fibroblast growth factor>>vascular endothelial growth factor>angiopoietin 1 mRNAs, and their cardiac levels were significantly upregulated by BMI. Cardiac interleukin-1beta and tumor necrosis factor-alpha mRNA expression were also induced by BMI but not by CMEC implantation. BM-MNCs were actively differentiated to endothelial cells in vitro and formed network structure with human umbilical vein endothelial cells. CONCLUSIONS: BMI may constitute a novel safety strategy for achieving optimal therapeutic angiogenesis by the natural ability of the BM cells to secrete potent angiogenic ligands and cytokines as well as to be incorporated into foci of neovascularization.

Augmented oxidative stress of platelets in chronic smokers. Mechanisms of impaired platelet-derived nitric oxide bioactivity and augmented platelet aggregability.

OBJECTIVES: We investigated whether impaired platelet-derived nitric oxide (PDNO) bioactivity and augmented platelet aggregability in chronic smokers are related to the imbalance of the intraplatelet redox state through increased oxidative stress. BACKGROUND: Chronic smoking impairs PDNO release and augments platelet aggregability. However, their mechanisms are unknown. METHODS: Collagen-induced PDNO release, platelet aggregation, plasma and intraplatelet vitamin C and reduced glutathione (GSH), intraplatelet cyclic guanosine 3',5'-monophosphate (cGMP) and intraplatelet nitrotyrosine production, which is a marker of the peroxynitrite formation, were measured in 11 chronic smokers and 10 age-matched nonsmokers. RESULTS: Release of PDNO and levels of intraplatelet cGMP were lower, and platelet aggregation was greater, in smokers than in nonsmokers. Intraplatelet vitamin C and GSH levels were lower in smokers than in nonsmokers. Intraplatelet nitrotyrosine production was greater in smokers than in nonsmokers. Next, we investigated the effects of oral vitamin C administration (2 g). After vitamin C administration, intraplatelet vitamin C levels were increased and not different at 2 h between the two groups. Then, PDNO release, intraplatelet cGMP levels and platelet aggregation in smokers were restored to the levels of nonsmokers. In smokers, PDNO release and consumption of GSH during platelet aggregation were inversely correlated, and consumption was much less after vitamin C administration. Vitamin C administration decreased intraplatelet nitrotyrosine production in smokers. CONCLUSIONS: Impaired PDNO bioactivity and augmented platelet aggregability may be caused by an imbalance of the intraplatelet redox state through increased oxidative stress in smokers.

Enhanced external counterpulsation improves exercise tolerance, reduces exercise-induced myocardial ischemia and improves left ventricular diastolic filling in patients with coronary artery disease.

OBJECTIVES: We examined whether enhanced external counterpulsation (EECP) improves myocardial ischemia, exercise tolerance and cardiac function in patients with coronary artery disease (CAD). BACKGROUND: Enhanced external counterpulsation reduces angina and improves exercise tolerance in patients with CAD. Some objective improvements of ischemia by EECP have been reported, but they should be confirmed further. Detailed hemodynamic effects of EECP have been less well documented. METHODS: Enhanced external counterpulsation was performed for a total of 35 h in patients with stable CAD (n = 12) who showed evidence of exercise-induced myocardial ischemia despite conventional medical or surgical therapies. All patients had significant stenotic lesions in major coronary arteries. RESULTS: Enhanced external counterpulsation improved all exercise test parameters (p < 0.05): exercise duration, time to 1-mm ST segment depression, rate-pressure product at peak exercise and rate-pressure product at 1-mm ST segment depression. Moreover, the prevalence of exercise-induced reversible perfusion defects by thallium scintigraphy decreased after treatment (p < 0.01). Enhanced external counterpulsation did not alter systolic function but improved diastolic filling, left ventricular (LV) end-diastolic pressure (p < 0.05) by cardiac catheterization and LV peak filling rate end-diastolic volume/s (p < 0.01) and time to peak filling rate (p < 0.05) by radionuclide scintigraphy. These hemodynamic improvements were associated with decreased plasma brain natriuretic peptides levels after EECP (p < 0.05). CONCLUSIONS: Thus, EECP treatment improves exercise tolerance and reduced myocardial ischemia by thallium scintigraphy in association with improved LV diastolic filling in patients with stable CAD.

Hyperhomocysteinemia impairs angiogenesis in response to hindlimb ischemia.

Hyperhomocysteinemia (HH) is an independent risk factor for atherosclerosis, including peripheral arterial occlusive disease (PAOD). Because angiogenesis and collateral vessel formation are important self-salvage mechanisms for ischemic PAOD, we examined whether HH modulates angiogenesis in vivo in a rat model of hindlimb ischemia. Rats were divided into 3 groups: the control group was given tap water, the HH group was given water containing L-methionine (1 g x kg(-1) x d(-1)), and the HH+L-arg group was given water containing methionine (1 g x kg(-1) x d(-1)) and l-arginine (2.25 vol%). At day 14 of the dietary modifications, the left femoral artery and vein were excised, and the extent of angiogenesis and collateral vessels in the ischemic limb were examined for 4 weeks. Plasma homocysteine levels significantly increased (P:<0.001), and plasma and tissue contents of nitrite+nitrate as well as tissue cGMP levels significantly decreased in the HH group compared with the control group (P:<0.01). Laser Doppler blood flowmetry (LDBF) revealed a significant decrease in the ischemic/normal limb LDBF ratio in the HH group at days 7, 14, 21, and 28 (P:<0.01 versus control). Angiography revealed a significant decrease in the angiographic score in the HH group at day 14 (P:<0.001 versus control). Immunohistochemistry of ischemic tissue sections showed a significant reduction in the capillary density in the HH group (P:<0. 001 versus control). Oral l-arginine supplementation in rats with HH (HH+L-arg) restored the decreased plasma and tissue nitrite+nitrate and cGMP contents (P:<0.05) as well as angiogenesis, as assessed by LDBF (P:<0.05 versus HH), angiographic score (P:<0.01 versus HH), and capillary density (P:<0.001 versus HH). In summary, HH impaired ischemia-induced angiogenesis and collateral vessel formation in a rat model of hindlimb ischemia in vivo. The mechanism of the HH-induced impairment of angiogenesis might be mediated in part by a reduced bioactivity of endogenous NO in the HH state.