Masayoshi Iwasaki

MicroRNA-92a Controls Angiogenesis and Functional Recovery of Ischemic Tissues in Mice

Of Life, Limb, and a Small RNA Gene expression in mammals is controlled not only by proteins but by small noncoding RNAs called microRNAs. The involvement of these RNAs provides powerful clues about the molecular origins of human diseases and how they might be treated. Ischemic diseases arise from an inadequate blood supply. Bonauer et al. (p. 1710, published online 21 May) find that a specific microRNA that is expressed in the cells lining blood vessels (called miR-92a) functions to repress the growth of new blood vessels. MiR-92a probably acts through effects on expression of integrins, proteins involved in cell adhesion and migration. In mouse models in which an inadequate blood supply had caused damage either to heart or limb muscle, therapeutic inhibition of miR-92a led to an increase in blood vessel density in the damaged tissues and enhanced functional recovery. Inhibition of a microRNA that represses blood vessel growth enhances the recovery of tissue damaged by an inadequate blood supply. MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by binding to target messenger RNAs (mRNAs), leading to translational repression or degradation. Here, we show that the miR-17~92 cluster is highly expressed in human endothelial cells and that miR-92a, a component of this cluster, controls the growth of new blood vessels (angiogenesis). Forced overexpression of miR-92a in endothelial cells blocked angiogenesis in vitro and in vivo. In mouse models of limb ischemia and myocardial infarction, systemic administration of an antagomir designed to inhibit miR-92a led to enhanced blood vessel growth and functional recovery of damaged tissue. MiR-92a appears to target mRNAs corresponding to several proangiogenic proteins, including the integrin subunit alpha5. Thus, miR-92a may serve as a valuable therapeutic target in the setting of ischemic disease.

Interleukin-10 From Transplanted Bone Marrow Mononuclear Cells Contributes to Cardiac Protection After Myocardial Infarction

Bone marrow mononuclear cells (BM-MNCs) have successfully been used as a therapy for the improvement of left ventricular (LV) function after myocardial infarction (MI). It has been suggested that paracrine factors from BM-MNCs may be a key mechanism mediating cardiac protection. We previously performed microarray analysis and found that the pleiotropic cytokine interleukin (IL)-10 was highly upregulated in human progenitor cells in comparison with adult endothelial cells and CD14+ cells. Moreover, BM-MNCs secrete significant amounts of IL-10, and IL-10 could be detected from progenitor cells transplanted in infarcted mouse hearts. Specifically, intramyocardial injection of wild-type BM-MNCs led to a significant decrease in LV end-diastolic pressure (LVEDP) and LV end-systolic volume (LVESV) compared to hearts injected with either diluent or IL-10 knock-out BM-MNCs. Furthermore, intramyocardial injection of wild-type BM-MNCs led to a significant increase in stroke volume (SV) and rate of the development of pressure over time (+dP/dt) compared to hearts injected with either diluent or IL-10 knock-out BM-MNCs. The IL-10–dependent improvement provided by transplanted cells was not caused by reduced infarct size, neutrophil infiltration, or capillary density, but rather was associated with decreased T lymphocyte accumulation, reactive hypertrophy, and myocardial collagen deposition. These results suggest that BM-MNCs mediate cardiac protection after myocardial infarction and this is, at least in part, dependent on IL-10.

Mobilization of Bone Marrow Cells by G-CSF Rescues Mice from Cisplatin-Induced Renal Failure, and M-CSF Enhances the Effects of G-CSF

Cisplatin, which is a broadly used anticancer drug, is widely known to induce acute renal failure as a result of renal tubular injury. This article examines whether G-CSF and/or M-CSF rescues mice from renal failure induced by cisplatin. BALB/c mice received intraperitoneal injections with or without G-CSF and/or M-CSF for 5 d (from day -5 to day -1). The day after the last injection of G-CSF and/or M-CSF (day 0), the mice received an intraperitoneal injection of cisplatin. When pretreated with G-CSF or G-CSF + M-CSF, the mice showed longer survival and lower serum creatinine and blood urea nitrogen levels than mice that had been received injections of M-CSF or saline. Histologically, pretreatment with G-CSF or G-CSF + M-CSF attenuated the damage to renal tubules induced by cisplatin. BALB/c mice that had received a transplant of bone marrow cells of enhanced green fluorescent protein (EGFP)-transgenic mice ([EGFP-->BALB/c] mice) were treated with or without G-CSF and/or M-CSF, followed by injection of cisplatin as well as above. [EGFP-->BALB/c] mice that were treated with G-CSF or G-CSF + M-CSF showed a significantly higher number of EGFP(+) tubular epithelial cells in the kidney than mice that were treated with only M-CSF or saline. These results suggest that bone marrow cells mobilized by G-CSF accelerate the improvement in renal functions and prevent the renal tubular injury induced by cisplatin and that M-CSF enhances the effects of G-CSF.

Modified resveratrol Longevinex improves endothelial function in adults with metabolic syndrome receiving standard treatment

Resveratrol is known to improve endothelial function in animals, but little is known about its effect on human subjects. Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors underlying endothelial dysfunction. We hypothesized that the modified resveratrol, Longevinex, improves endothelial function in patients with MetS. Thirty-four patients who had been treated for MetS and lifestyle-related disease were randomly assigned to group A, in which Longevinex was administered for 3 months and then discontinued for 3 months, whereas in the time-matched group B, Longevinex was administered between 3 and 6 months. These 2 groups of patients received similar drugs at baseline for diabetes mellitus, dyslipidemia, or hypertension. Flow-mediated dilatation significantly increased during the administration of Longevinex but decreased to baseline 3 months after the discontinuation of Longevinex in the group A patients. Conversely, in the group B patients, flow-mediated dilatation remained unchanged for the first 3 months without Longevinex but was significantly increased 3 months after the treatment with Longevinex. Longevinex did not significantly affect blood pressure, insulin resistance, the lipid profile or inflammatory markers during 6-month follow-up. These results demonstrate that Longevinex specifically improves endothelial function in subjects with MetS who were receiving standard therapy for lifestyle-related disease.

Notch Signaling Contributes to the Expression of Cardiac Markers in Human Circulating Progenitor Cells

It has been demonstrated that adult human circulating endothelial progenitor cells (EPCs) can differentiate to a cardiomyogenic phenotype. Notch signaling promotes epithelial-to-mesenchymal transformation and plays a role in heart and vessel development. Here, we investigated the role of Notch activation for cardiac differentiation of EPCs in a coculture system with neonatal cardiomyocyte. After coculture, Notch activation was transiently detected in EPCs, as determined by immunhistochemical detection of NICD (the intracellular cleavage fragment of Notch-1) and expression of human Notch target genes. Inhibition of &ggr;-secretase blocked Notch cleavage and NICD translocation. Furthermore, the expression of the cardiac marker protein &agr;-sarcomeric actinin and troponin T was significantly suppressed by &ggr;-secretase inhibition or addition of soluble recombinant Jagged-1, indicating that Notch activation facilitates cardiac marker gene expression. Because noncanonical Wnts have previously been shown to promote cardiac differentiation, we additionally determined the influence of Notch activation on the expression of Wnt5a and Wnt11. Wnt5a and Wnt11 expression in the human cells was induced by the coculture and was blocked by &ggr;-secretase inhibition. Likewise, stimulation of Notch signaling by immobilized Jagged-1 promoted Wnt5a expression in EPCs. These data suggest that Notch is activated upon coculture of EPCs with neonatal rat cardiac myocytes. &ggr;-Secretase–dependent Notch activation is required for cardiac gene expression in human cells and induces the expression of noncanonical Wnt proteins, which may act in a paracrine manner to further amplify cardiac differentiation.

Macrophage colony-stimulating factor (M-CSF), as well as granulocyte colony-stimulating factor (G-CSF), accelerates neovascularization

It has been reported that bone marrow cells (BMCs) differentiate into endothelial cells of blood vessels, and that granulocyte colony‐stimulating factor (G‐CSF) mobilizes progenitors in the BMCs to the peripheral blood, while macrophage colony‐stimulating factor (M‐CSF) augments the production of monocytes. We examined whether M‐CSF augments the differentiation of BMCs into endothelial cells of blood vessels using a hindlimb‐ischemic model. Either G‐CSF or M‐CSF, or both, was administered to the hindlimb‐ischemic mice for 3 days. Both M‐CSF and G‐CSF augmented the differentiation of BMCs into endothelial cells of blood vessels through vascular endothelial cell growth factor (VEGF), resulting in early recovery of blood flow in the ischemic limbs.

Acute myocardial infarction activates progenitor cells and increases Wnt signalling in the bone marrow

AIMS We aimed to characterize the influence of acute myocardial infarction (AMI) on the metabolic activity of the bone marrow (BM) and on the composition and functional activity of BM-derived mononuclear cells (BMC). Acute ischaemia or other stressors induce the mobilization of progenitor cells from the BM stem cell niche. The effect of AMI on the numbers and functional activity of cells within the BM is unknown. METHODS AND RESULTS In patients of the REPAIR-AMI trial as well as in mice, the number and functionality of BMC was compared with respect to the time interval from AMI. Activation of Wnt signalling was assessed after AMI induction in TOP-GAL transgenic reporter mice, carrying a β-galactosidase gene driven by an LEF/TCF/β-catenin responsive promoter. The metabolic activity of the BM, as determined by F-18-fluorodeoxyglucose-positron emission tomography, was significantly higher in patients with AMI compared with patients with chronic post-ischaemic heart failure. Moreover, the number of haematopoietic CD34(+) (P < 0.05) and CD133(+) (P < 0.05) cells in the BM aspirates was significantly increased in patients within 7 days after AMI. In order to confirm these clinical data, we induced AMI in mice, which time-dependently increased the number of c-kit + Sca-1 + lin- cells and colony-forming units in the BM. Activation of the BM by AMI induced a significant increase in Wnt signalling, which is known to induce proliferation of haematopoietic stem cells, and demonstrated increased levels of the Wnt target Axin-2 in BM-derived cells on Day 7 (P < 0.01 vs. control). CONCLUSION Acute myocardial infarction is associated with an increased metabolic activity and increased levels of progenitor cells within days after AMI. These findings document an activation of the stem cell niche within the BM following AMI, which may have important implications for the optimal timing of cell aspirations used for therapeutic application in patients with AMI.

Treatment and transfer of emphysema by a new bone marrow transplantation method from normal mice to Tsk mice and vice versa.

We have recently established a new bone marrow transplantation (BMT) method in which bone marrow cells are injected into the intrabone marrow (IBM). In the present study, we used an animal model for emphysema (tight‐skin [Tsk] mouse) to examine whether IBM‐BMT could be used to treat emphysema in Tsk mice. IBM‐BMT was carried out from C3H mice into Tsk mice (8–10 weeks old) that had already shown emphysema. Six months after transplantation, the lungs of all the Tsk mice treated with IBM‐BMT [C3H→Tsk] showed similar structures to those of normal mice, whereas the [Tsk→Tsk] mice showed emphysema, as seen in age‐matched Tsk mice. Next, we attempted to transfer emphysema from Tsk mice to C3H mice by IBM‐BMT. Six months after IBM‐BMT, the [Tsk→C3H] mice showed emphysema. These results strongly suggest that emphysema in Tsk mice originates from defects of stem cells in the bone marrow.

Hepatocyte Growth Factor Delivered by Ultrasound-Mediated Destruction of Microbubbles Induces Proliferation of Cardiomyocytes and Amelioration of Left Ventricular Contractile Function in Doxorubicin-Induced Cardiomyopathy

At present, there is no curative strategy for advanced cardiomyopathy except for cardiac transplantation, which is not easily performed, mainly due to a shortage of donors. It has been reported that myocardial progenitor cells exist even in the postnatal heart, suggesting that myocardial progenitor cells could proliferate under some situations and might improve cardiac function in cardiomyopathy‐induced hearts. In this study, recombinant human hepatocyte growth factor (rhHGF) was delivered using ultrasound‐mediated destruction of microbubbles (UMDM) into the cardiomyopathy‐induced heart by doxorubicin (20 mg/kg). Intravenous injection of rhHGF (IV‐rhHGF) alone or UMDM alone failed to improve the morphology or the function of the cardiomyopathy‐induced heart, but (IV‐rhHGF + UMDM) treatment significantly improved the heart morphologically and functionally, and repetitive treatments of (IV‐rhHGF + UMDM) enhanced the effects. The number of bromodeoxy‐uridine‐positive cardiomyocytes significantly increased in the (IV‐rhHGF + UMDM)–treated hearts compared with the untreated hearts. Moreover, Sca‐1+ myocardial progenitor cells express c‐Met, a receptor for HGF. These results suggest that (IV‐rhHGF + UMDM) treatment could morphologically and functionally improve the heart in the case of doxorubicin‐induced cardiomyopathy through the proliferation of the myocardial progenitor cells.

The Effect of Residential Exercise Training on Baroreflex Control of Heart Rate and Sympathetic Nerve Activity in Patients With Acute Myocardial Infarction

STUDY OBJECTIVES Exercise training has been shown to favorably affect the prognosis after acute myocardial infarction (AMI), but the mechanisms of such favorable effects remain speculative. The aim of this study was to determine whether exercise training improves baroreflex control of heart rate and muscle sympathetic nerve activity (MSNA) in patients with AMI. DESIGN Prospective randomized clinical study. PARTICIPANTS Thirty patients with an uncomplicated AMI were randomized into trained or untrained groups. Arterial BP, heart rate, and MSNA were measured at rest, and during baroreceptor stimulation (phenylephrine infusion) and baroreceptor deactivation (nitroprusside infusion). These measurements were performed at baseline and after 4 weeks of exercise training. MEASUREMENTS AND RESULTS Peak oxygen uptake increased significantly (12.3 +/- 10.7% [mean +/- SD]) with exercise training. Resting MSNA reduced from 34 +/- 12 to 27 +/- 8 bursts/min in the trained group but not in the untrained group. Arterial baroreflex sensitivity (BRS) [from 8.9 +/- 3.0 to 10.3 +/- 3.0 ms/mm Hg, p < 0.05] and MSNA response to baroreceptor stimulation (change of integrated MSNA from - 47 +/- 23 to - 70 +/- 21%, p < 0.01) improved significantly in the trained group, but not in the untrained group. Despite baroreceptor deactivation improving MSNA response in both groups, there was no significant difference between the two groups. CONCLUSIONS Exercise training increased arterial BRS and decreased sympathetic nerve traffic after AMI, which indicate that the sympathoinhibitory effect of exercise training may, at least in part, contribute to the beneficial effect of exercise training in patients with AMI.