Takahisa Kondo

Smoking Cessation Rapidly Increases Circulating Progenitor Cells in Peripheral Blood in Chronic Smokers

Objective—Circulating endothelial progenitor cells (EPCs) contribute to postnatal angiogenesis. The number of circulating EPCs has an inverse correlation with coronary risk scores. However, the effect of smoking on the number of circulating EPCs is not well-known. Methods and Results—We examined the effects of chronic smoking and of smoking cessation on EPC levels. Circulating EPCs were quantified by flow cytometry as CD45lowCD34+CD133+ (progenitor cells [PCs]) or CD45lowCD34+CD133+VEGFR2+ (EPCs) in 14 nonsmokers and in 15 smokers. All smokers quit smoking. Eight quit smoking with nicotine patch and 7 without nicotine patch. PC/EPC levels were inversely correlated with the number of cigarettes smoked. Circulating PCs/EPCs increased rapidly after cessation (P < 0.0001) and decreased again after resumption of smoking to the level similar to that before cessation (P = 0.0031). The magnitude of increase in EPCs was greater in light smokers than in heavy smokers. Conclusions—The number of circulating PCs/EPCs was reduced in chronic smokers. Smoking cessation led to a rapid restoration of PC/EPC levels. The recovery of EPC levels was greater in light smokers than in heavy smokers. The decreased number of circulating EPCs would make smokers susceptible to cardiovascular disease, and even short-time cessation of smoking may be an effective means to reduce cardiovascular risk.

Comparison of Circulating Adiponectin and Proinflammatory Markers Regarding Their Association With Metabolic Syndrome in Japanese Men

Background—Anti-inflammatory and proinflammatory molecules purportedly play an important role in developing metabolic syndrome (MetS). However, little is known as to the relative importance of these molecules in the association with MetS. Methods and Results—We studied 624 middle-aged Japanese men without medical history of cardiovascular disease or cancer and investigated the associations of circulating tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), and adiponectin with MetS. We used the respective definitions proposed by the National Cholesterol Education Program Adult Treatment Panel III (ATP-III), the International Diabetes Federation, and the Japanese Society of Internal Medicine. Decreased serum adiponectin was observed in those with any of the ATP-III–MetS components, whereas this was not the case with increased TNF-α, IL-6, or CRP. Adiponectin and CRP levels linearly deteriorated with an increasing number of ATP-III–MetS components (trend P<0.001, respectively). Significantly higher CRP and lower adiponectin levels were observed in those who met any MetS criteria, whereas increased TNF-α was observed in only those with ATP-III–MetS. Finally, odds ratios (ORs) for MetS prevalence of a 1-SD increase/decrease in log-transformed 4 markers were calculated with multivariate logistic regression analyses. Consequently, decreased adiponectin was associated most strongly with ATP-III–MetS (adiponectin: OR, 1.90 [95% CI, 1.44 to 2.51]; P<0.001; CRP: OR, 1.33 [95% CI, 1.01 to 1.74]; P=0.03; TNF-α: OR, 1.25 [95% CI, 0.94 to 1.67]; P=0.12; and IL-6: OR, 0.87 [95% CI, 0.63 to 1.19]; P=0.37). This result was not altered by using the other 2 criteria. Conclusions—The present results raise the possibility that decreased serum adiponectin might be fundamentally involved in the development of MetS.

Sinoatrial Node Dysfunction and Early Unexpected Death of Mice With a Defect of klotho Gene Expression

Background—Homozygous mutant mice with a defect of klotho gene expression (kl/kl) show multiple age-related disorders and premature death from unknown causes. Methods and Results—The kl/kl mice subjected to 20-hour restraint stress showed a high rate (20/30) of sudden death, which was associated with sinoatrial node dysfunction (conduction block or arrest). Heart rate and plasma norepinephrine of kl/kl mice, unlike those of wild-type (WT) mice, failed to increase during the stress. Intrinsic heart rate after pharmacological blockade of autonomic nerves in kl/kl mice was significantly lower than that in WT mice (380±33 versus 470±44 bpm; n=7). The sinus node recovery time after an overdrive pacing (600 bpm, 30 seconds) in kl/kl mice was significantly longer than in WT mice (392±37 versus 233±24 ms; n=6). In isolated sinoatrial node preparations, the positive chronotropic effect of isoproterenol was significantly less, whereas the negative chronotropic effect of acetylcholine was significantly greater in kl/kl than in WT mice. There was no degenerative structural change in the sinoatrial node of kl/kl mice. The precise localization of klotho was analyzed in newly prepared klotho-null mice with a reporter gene system (kl−geo). Homozygous kl− geo mice showed characteristic age-associated phenotypes that were almost identical to those of kl/kl mice. In the kl− geo mice, klotho expression was recognized exclusively in the sinoatrial node region in the heart in addition to parathyroid, kidney, and choroid plexus. Conclusions—In the heart, klotho is expressed solely at the sinoatrial node. klotho gene expression is essential for the sinoatrial node to function as a dependable pacemaker under conditions of stress.

Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin.

The serine/threonine protein kinase Akt is involved in a variety of cellular processes including cell proliferation, survival, metabolism and gene expression. It is essential in vascular endothelial growth factor (VEGF)-mediated angiogenesis; however, it is not known how Akt regulates the migration of endothelial cells, a crucial process for vessel sprouting, branching and the formation of networks during angiogenesis. Here we report that Akt-mediated phosphorylation of Girdin, an actin-binding protein, promotes VEGF-dependent migration of endothelial cells and tube formation by these cells. We found that exogenously delivered adenovirus harbouring Girdin short interfering RNA in Matrigel embedded in mice, markedly inhibited VEGF-mediated angiogenesis. Targeted disruption of the Girdin gene in mice impaired vessel remodelling in the retina and angiogenesis from aortic rings, whereas Girdin was dispensable for embryonic vasculogenesis. These findings demonstrate that the Akt/Girdin signalling pathway is essential in VEGF-mediated postneonatal angiogenesis.

The Impact of the Capability of Circulating Progenitor Cell to Differentiate on Myocardial Salvage in Patients With Primary Acute Myocardial Infarction

Background— Circulating endothelial progenitor cells (EPCs) are known to be involved in vasculogenesis and mobilized after acute myocardial infarction (AMI). To test the hypothesis that the angiogenic function of EPCs affects post-myocardial infarction (MI) myocardial salvage, we evaluated the number and potential differentiation of EPCs and compared these data with clinical parameters 6 months after MI. Methods and Results— Consecutive 51 patients (age, 61±8 years, mean±SD) with primary AMI who were successfully treated with stenting were enrolled. EPC identified as CD45low, CD34+, CD133+, and VEGFR2+ was quantified by a flow cytometry. The potential of EPCs to differentiate into endothelial cells (EPC differentiation) was also confirmed by the upregulation of CD31 and VEGFR2 after 7 days of culture. According to the proportion of EPC fraction, patients were divided into 2 groups (cut-off value=median). Although no difference was seen in myocardial damage shown by mean peak CK leakage and mean area at risk between the differentiated group (n=26) and nondifferentiated group (n=25), the number of attached cell was greater in differentiated group than in the nondifferentiated group (P=0.023). Left ventricular function and ischemic damaged area were assessed by scintigraphic images of 123I-BMIPP in the acute phase and 99mTc-tetrofosmin in the chronic phase. We found that a greater increase in myocardial salvage (P=0.0091), decrease in end-systolic volume (P=0.012), and recovery of ejection fraction (P=0.011) occurred in the group with differentiated EPCs than in the nondifferentiated group. Conclusions— In patients with primary AMI, the capability of EPCs to differentiate influences the functional improvement and infarct size reduction, indicating that manipulation of EPCs could be a novel therapeutic target to salvage ischemic damage.

Increased Expression of Plasminogen Activator Inhibitor-1 in Cardiomyocytes Contributes to Cardiac Fibrosis after Myocardial Infarction

Plasminogen activator inhibitor-1 (PAI-1) plays a critical role in tissue fibrosis by inactivating matrix metalloproteinases, which might effect on the progression of left ventricular dysfunction. However, little has been known about the expression of PAI-1 during cardiac remodeling. We used a mouse model of myocardial infarction (MI) by coronary ligation, in which the progression of left ventricular remodeling was confirmed by echocardiography. Histological examination showed that interstitial and perivascular fibrosis progressed in the post-MI (PMI) heart at 4 weeks after the procedure. We observed the dramatic induction of cardiac PAI-1 mRNA and PAI-1 antigen in plasma in the PMI mice, as compared with the sham-operated (sham) mice. In situ hybridization analysis demonstrated that strong signals for PAI-1 mRNA were localized to cardiomyocytes in the border of infarct area and around fibrous lesions, and to perivascular mononuclear cells, which seemed to be mast cells, only in hearts of the PMI mice. Importantly, less development of cardiac fibrosis after MI was observed in mice deficient in PAI-1 as compared to wild-type mice. The mRNA expression of cytokines, transforming growth factor-beta, and tumor necrosis factor-alpha, was also increased in hearts of the PMI mice, but not in the sham mice. These observations suggest that cardiomyocytes and mast cells contribute to the increased PAI-1 expression, resulting in the development of interstitial and perivascular fibrosis in the PMI heart, and that the regional induction of cytokines may be involved in this process.

Mechanisms Underlying the Impairment of Ischemia-Induced Neovascularization in Matrix Metalloproteinase 2–Deficient Mice

Matrix metalloproteinases (MMPs) have been implicated in the process of neovascularization. However, the exact roles of individual MMPs in vessel formation are poorly understood. To study the putative role of MMP-2 in ischemia-induced neovascularization, a hindlimb ischemia model was applied to MMP-2+/+ and MMP-2−/− mice. Serial laser Doppler blood-flow analysis revealed that the recovery of the ischemic/normal blood-flow ratio in MMP-2−/− young and old mice remained impaired throughout the follow-up period. At day 35, microangiography and anti–l-lectin immunohistochemical staining revealed lesser developed collateral vessels and capillary formation in both old and young MMP-2−/− mice compared with the age-matched MMP-2+/+ mice. An aortic-ring culture assay showed a markedly impaired angiogenic response in MMP-2−/− mice, which was partially recovered by supplementation of the culture medium with recombinant MMP-2. Aorta-derived endothelial cells or bone marrow–derived endothelial progenitor cell (EPC)-like c-Kit+ cells from MMP-2−/− showed marked impairment of invasive or/and proliferative abilities. At day 7, plasma and ischemic tissues of vascular endothelial growth factor protein were reduced in MMP-2−/−. Flow cytometry showed that the numbers of EPC-like CD31+c-Kit+ cells in peripheral blood markedly decreased in MMP-2–deficient mice. Transplantation of bone marrow–derived mononuclear cells from MMP-2+/+ mice restored neovascularization in MMP-2−/− young mice. These data suggest that MMP-2 deficiency impairs ischemia-induced neovascularization through a reduction of endothelial cell and EPC invasive and/or proliferative activities and EPC mobilization.

Molecular Evaluation of Endothelial Progenitor Cells in Patients With Ischemic Limbs Therapeutic Effect by Stem Cell Transplantation

Objective—Although some patients with limb ischemia have recently undergone therapeutic angiogenesis by cell transplantation, their angiogenic potential has not been well characterized. It is also important to evaluate endothelial progenitor cell (EPC) contents in different stem cell sources to choose the best material for therapeutic angiogenesis. Methods and Results—We quantitated the mRNA expression of EPC-specific molecules (eg, Flk-1, Flt-1, CD133, VE-cadherin, etc) in bone marrow-derived or peripheral blood-derived mononuclear cells obtained from patients with ischemic limbs, using real-time reverse-transcription polymerase chain reaction technique. The mRNA expression level of EPC markers was significantly lower in the patients than in healthy controls, which was consistent with results of flow cytometric analysis. However, the implantation of autologous bone marrow mononuclear cells increased the circulating EPCs in the peripheral blood of patients. We furthermore revealed the different expression pattern of EPC markers in possible sources for stem cell transplantation, including normal bone marrow, peripheral blood obtained from recombinant granulocyte colony–stimulating factor-treated donor, and umbilical cord blood. Conclusions—Patients with peripheral obstructive arterial diseases may have lower angiogenic potential because of decreased expression of EPC specific molecules in their marrow and blood. Therapeutic angiogenesis by transplantation of autologous marrow mononuclear cells increased circulating EPCs in the patients and improved ischemic symptoms.

Composite implantation of mesenchymal stem cells with endothelial progenitor cells enhances tissue-engineered bone formation

For successful tissue engineering, neovascularization of the implanted tissue is critical. Factors generated by endothelial cells are also considered crucial for the process of osteogenesis. The direct effects of supplementing tissue engineered constructs with cultured endothelial progenitor cells (EPCs) for enhancing bone regeneration have not been reported. In this study, we investigated the potential of EPCs to facilitate neovascularization in implants and evaluated their influence on bone regeneration. The influence of EPC soluble factors on osteogenic differentiation of mesenchymal stem cells (MSCs) was tested by adding EPC culture supernatant to MSC culture medium. To evaluate the influence of EPCs on MSC osteogenesis, canine MSCs-derived osteogenic cells and EPCs were seeded independently onto collagen fiber mesh scaffolds and co-transplanted to nude mice subcutaneously. Results from coimplant experiments were compared to implanted cells absent of EPCs 12 weeks after implantation. Factors from the culture supernatant of EPCs did not influence MSC differentiation. Coimplanted EPCs increased neovascularization and the capillary score was 1.6-fold higher as compared to the MSC only group (p < 0.05). Bone area was also greater in the MSC + EPC group (p < 0.05) and the bone thickness was 1.3-fold greater in the MSC + EPC group than the MSC only group (p < 0.05). These results suggest that soluble factors generated by EPCs may not facilitate the osteogenic differentiation of MSCs; however, newly formed vasculature may enhance regeneration of tissue-engineered bone.

Nifedipine Improves Endothelial Function: Role of Endothelial Progenitor Cells

Nifedipine has been shown to improve endothelial function. Recent studies have indicated that endothelial function is correlated with the number of circulating endothelial progenitor cells (EPCs), but it is unclear whether nifedipine affects the number and function of EPCs. The aims of this study were to determine the effects of nifedipine on the number and function of EPCs and to investigate the relationship between improvement of endothelial function and EPC numbers in patients with hypertension. Stage 1 hypertensive men (n=37) were randomly divided into the nifedipine group and the control untreated group. The nifedipine group was administered slow-release nifedipine (20 mg) once daily. At baseline and after 4 weeks, flow-mediated dilation, blood pressure, biochemical data, and number of circulating CD34+CD133+ progenitor cells and EPCs were measured. The direct effects of nifedipine on EPC number and function were assessed in vitro. In the nifedipine group, flow-mediated dilation and the numbers of circulating CD34+CD133+ progenitor cells and EPCs were increased, along with a decrease of serum malondialdehyde low-density lipoprotein. The improvement of flow-mediated dilation by nifedipine was correlated with the increase of circulating CD34+CD133+ progenitor cells. Nifedipine also improved angiogenesis-related functions of EPCs (differentiation, migration, and resistance to oxidative stress) in vitro. Thus, nifedipine improved endothelial function and EPC function in stage 1 hypertensive subjects. The latter action may be mediated by reduction of oxidative stress and suppression of EPC apoptosis. These results demonstrate that nifedipine preserves endothelial integrity in patients with hypertension, at least partly, by enhancing EPC numbers and activity.