Satoshi Shintani

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.

Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissue injury following myocardial infarction

Ischemia resulting from myocardial infarction (MI) promotes VEGF expression, leading to vascular permeability (VP) and edema, a process that we show here contributes to tissue injury throughout the ventricle. This permeability/edema can be assessed noninvasively by MRI and can be observed at the ultrastructural level as gaps between adjacent endothelial cells. Many of these gaps contain activated platelets adhering to exposed basement membrane, reducing vessel patency. Following MI, genetic or pharmacological blockade of Src preserves endothelial cell barrier function, suppressing VP and infarct volume, providing long-term improvement in cardiac function, fibrosis, and survival. To our surprise, an intravascular injection of VEGF into healthy animals, but not those deficient in Src, induced similar endothelial gaps, VP, platelet plugs, and some myocyte damage. Mechanistically, we show that quiescent blood vessels contain a complex involving Flk, VE-cadherin, and beta-catenin that is transiently disrupted by VEGF injection. Blockade of Src prevents disassociation of this complex with the same kinetics with which it prevents VEGF-mediated VP/edema. These findings define a molecular mechanism to account for the Src requirement in VEGF-mediated permeability and provide a basis for Src inhibition as a therapeutic option for patients with acute MI.

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.

Implantation of Adipose-Derived Regenerative Cells Enhances Ischemia-Induced Angiogenesis

Objective—Therapeutic angiogenesis using autologous stem/progenitor cells represents a novel strategy for severe ischemic diseases. Recent reports indicated that adipose tissues could supply adipose-derived regenerative cells (ADRCs). Accordingly, we examined whether implantation of ADRCs would augment ischemia-induced angiogenesis. Method and Results—Adipose tissue was obtained from C57BL/6J mice, and ADRCs were isolated using standard methods. ADRCs expressed stromal cell–derived factor 1 (SDF-1) mRNA and proteins. Hind limb ischemia was induced and culture-expanded ADRCs, PBS, or mature adipocytes (MAs) as control cells were injected into the ischemic muscles. At 3 weeks, the ADRC group had a greater laser Doppler blood perfusion index and a higher capillary density compared to the controls. Implantation of ADRCs increased circulating endothelial progenitor cells (EPCs). SDF-1 mRNA abundance at ischemic tissues and serum SDF-1 levels were greater in the ADRC group than in the control group. Finally, intraperitoneal injection of an anti–SDF-1 neutralizing antibody reduced the number of circulating EPCs and therapeutic efficacies of ADRCs. Conclusions—Adipose tissue would be a valuable source for cell-based therapeutic angiogenesis. Moreover, chemokine SDF-1 may play a pivotal role in the ADRCs-mediated angiogenesis at least in part by facilitating mobilization of EPCs.

Evidence for the importance of angiotensin II type 1 receptor in ischemia-induced angiogenesis.

The role of the renin-angiotensin system (RAS) in angiogenesis is little known. Here, we show that the angiotensin II (ATII) type 1 (AT1) receptor plays an important role in ischemia-induced angiogenesis. Well-developed collateral vessels and angiogenesis were observed in wild-type (WT) mice in response to hindlimb ischemia, whereas these responses were reduced in ATII type 1a receptor knockout (AT1a(-/-)) mice. Ischemia-induced angiogenesis was also impaired in WT mice treated with the AT1 receptor blocker TCV-116. These effects were not due to reduced systemic blood pressure (SBP), because hydralazine treatment preserved angiogenesis in WT mice although it reduced SBP to a level similar to that of AT1a(-/-) mice. Infiltration of inflammatory mononuclear cells (MNCs), including macrophages and T lymphocytes, was suppressed in the ischemic tissues of AT1a(-/-) mice compared with WT mice. Double immunofluorescence staining revealed that infiltrated macrophages and T lymphocytes expressed VEGF, and the expression of VEGF and monocyte chemoattractant protein-1 was also decreased in AT1a(-/-). Finally, the impaired angiogenesis in AT1a(-/-) mice was rescued by intramuscular transplantation of MNCs obtained from WT mice, further indicating the importance of MNC infiltration in ischemia-induced angiogenesis. Thus, the ATII--AT1 receptor pathway promotes early angiogenesis by supporting inflammatory cell infiltration and angiogenic cytokine expression.

Impact of a Single Intracoronary Administration of Adiponectin on Myocardial Ischemia/Reperfusion Injury in a Pig Model

Background—Adiponectin plays a protective role in the development of obesity-linked disorders. We demonstrated that adiponectin exerts beneficial actions on acute ischemic injury in mice hearts. However, the effects of adiponectin treatment in large animals and its feasibility in clinical practice have not been investigated. This study investigated the effects of intracoronary administration of adiponectin on myocardial ischemia-reperfusion (I/R) injury in pigs. Methods and Results—The left anterior descending coronary artery was occluded in pigs for 45 minutes and then reperfused for 24 hours. Recombinant adiponectin protein was given as a bolus intracoronary injection during ischemia. Cardiac functional parameters were measured by a manometer-tipped catheter. Apoptosis was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining. Tumor necrosis factor-α and interleukin-10 transcripts were analyzed by real-time polymerase chain reaction. Serum levels of derivatives of reactive oxygen metabolites and biological antioxidant potential were measured. Adiponectin protein was determined by immunohistochemical and Western blot analyses. Intracoronary administration of adiponectin protein led to a reduction in myocardial infarct size and improvement of left ventricular function in pigs after I/R. Injected adiponectin protein accumulated in the I/R-injured heart. Adiponectin treatment resulted in decreased tumor necrosis factor-α and increased interleukin-10 mRNA levels in the myocardium after I/R. Adiponectin-treated pigs had reduced apoptotic activity in the I/R-injured heart and showed increased biological antioxidant potential levels and decreased derivatives of reactive oxygen metabolite levels in the blood stream after I/R. Conclusions—These data suggest that adiponectin protects against I/R injury in a preclinical pig model through its ability to suppress inflammation, apoptosis, and oxidative stress. Administration of intracoronary adiponectin could be a useful adjunctive therapy for acute myocardial infarction.

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

AbstractAims/hypothesisResistin 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.MethodsThe 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, HbA1c 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.ResultsA 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/interpretationOur 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 Funding: This study was supported by Grants-in-Aids for Scientific Research from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Autologous Adipose-Derived Regenerative Cells for Therapeutic Angiogenesis

Therapeutic angiogenesis is an important means to salvage tissues against severe ischemic diseases in patients with no option for other vascular intervention. A number of recent studies implicated potentials of cell-based therapeutic angiogenesis using autologous bone marrow mononuclear cells, CD34(+) cells, peripheral blood mononuclear cells, and so on. Subcutaneous adipose tissues can be harvested by relatively easy methods. Recent studies indicated that adipose tissues contain progenitor cells or regenerative cells that can give rise to several mesenchymal lineages. Moreover, these progenitor cells can release multiple angiogenic growth factors and cytokines/chomokines including vascular endothelial growth factor (VEGF), hypatocyte growth factor (HGF) and chemokine stromal cell-derived factor-1 (SDF-1). The combination of these biological properties of adipose-derived regenerative cells (ADRCs) implicates that autologous adipose tissue will be a useful cell source for therapeutic angiogenesis in the next generation.

Comparison Between Valsartan and Amlodipine Regarding Cardiovascular Morbidity and Mortality in Hypertensive Patients With Glucose Intolerance: NAGOYA HEART Study

It has not been fully examined whether angiotensin II receptor blocker is superior to calcium channel blocker to reduce cardiovascular events in hypertensive patients with glucose intolerance. A prospective, open-labeled, randomized, controlled trial was conducted for Japanese hypertensive patients with type 2 diabetes mellitus or impaired glucose tolerance. A total of 1150 patients (women: 34%; mean age: 63 years; diabetes mellitus: 82%) were randomly assigned to receive either valsartan- or amlodipine-based antihypertensive treatment. Primary outcome was a composite of acute myocardial infarction, stroke, coronary revascularization, admission attributed to heart failure, or sudden cardiac death. Blood pressure was 145/82 and 144/81 mm Hg, and glycosylated hemoglobin was 7.0% and 6.9% at baseline in the valsartan group and the amlodipine group, respectively. Both of them were equally controlled between the 2 groups during the study. The median follow-up period was 3.2 years, and primary outcome had occurred in 54 patients in the valsartan group and 56 in the amlodipine group (hazard ratio: 0.97 [95% CI: 0.66–1.40]; P=0.85). Patients in the valsartan group had a significantly lower incidence of heart failure than in the amlodipine group (hazard ratio: 0.20 [95% CI: 0.06–0.69]; P=0.01). Other components and all-cause mortality were not significantly different between the 2 groups. Composite cardiovascular outcomes were comparable between the valsartan- and amlodipine-based treatments in Japanese hypertensive patients with glucose intolerance. Admission because of heart failure was significantly less in the valsartan group.

Metabolic syndrome and all-cause mortality, cardiac events, and cardiovascular events: a follow-up study in 25,471 young- and middle-aged Japanese men

Aim: The association between subjects with metabolic syndrome (MS) who were considered not to require medication by their attending physicians and all-cause mortality, ischemic heart disease (IHD) and cardiovascular disease (CVD) remains unknown and should be clarified. Methods and results: This is an observational longitudinal cohort study with a median follow-up of 7.5 years performed for 25,471 Japanese men aged 20–61 years who were not on medication. We used a modified definition of MS from the Japanese Society of Internal Medicine and the NCEP ATPIII, both of which employed body mass index instead of waist circumference. MS was associated with increased rates of all-cause death (adjusted hazard ratio (HR): 4.88 [95% confidence interval, 2.96–7.66]), IHD (3.17 [1.06–7.65]), and CVD (2.63 [1.32–4.72]). In contrast, overweight subjects with no component or one component had similar rates to subjects of normal weight. Any combination of the three MS components was associated with significantly greater rates of all-cause mortality (HR: 3.18–11.2) and IHD (HR: 3.17–8.24), whereas blood pressure elevation plus dyslipidaemia was associated with a significantly higher rate of CVD (HR: 3.27). In any endpoint, MS defined by Japanese criteria had higher HRs than defined by NCEP ATP III criteria. Conclusion: Young and middle-aged Japanese men with MS who had been viewed as not needing medication already showed increased rates of all-cause mortality, IHD and CVD. Additionally, the event rate depended on the specific combination of metabolic syndrome components.