Shinichi Kanno

Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells.

Vascular endothelial growth factor (VEGF) is a principal regulator of vasculogenesis and angiogenesis. VEGF expresses its effects by binding to two VEGF receptors, Flt-1 and KDR. However, properties of Flt-1 and KDR in the signal transduction of VEGF-mediated effects in endothelial cells (ECs) were not entirely clarified. We investigated this issue by using two newly developed blocking monoclonal antibodies (mAbs) against Flt-1 and KDR. VEGF elicits DNA synthesis and cell migration of human umbilical vein endothelial cells (HUVECs). The pattern of inhibition of these effects by two mAbs indicates that DNA synthesis is preferentially mediated by KDR. In contrast, the regulation of cell migration by VEGF appears to be more complicated. Flt-1 regulates cell migration through modulating actin reorganization, which is essential for cell motility. A distinct signal is generated by KDR, which influences cell migration by regulating cell adhesion via the assembly of vinculin in focal adhesion plaque and tyrosine-phosphorylation of focal adhesion kinase (FAK) and paxillin.

Protection against ischemia/reperfusion injury in cardiac and renal transplantation with carbon monoxide, biliverdin and both

Both carbon monoxide (CO) and biliverdin, products of heme degradation by heme oxygenase, have been shown to attenuate ischemia/reperfusion (I/R) injury. We hypothesized in this study that dual‐treatment with CO and biliverdin would induce enhanced protective effects against cold I/R injury. Heterotopic heart and orthotopic kidney transplantation were performed in syngeneic Lewis rats after 24‐h cold preservation in UW solution. While monotherapy with CO (20 ppm) or biliverdin (50 mg/kg, ip) did not alter the survival of heart grafts, dual‐treatment increased survival to 80% from 0% in untreated recipients, with a significant decrease of myocardial injury and improved cardiac function. Similarly, dual‐treatment significantly improved glomerular filtration rates of renal grafts and prolonged recipient survival compared to untreated controls. I/R injury‐induced up‐regulation of pro‐inflammatory mediators (e.g. TNF‐α, iNOS) and extravasation of inflammatory infiltrates were significantly less with dual‐treatment than untreated controls. In addition, dual‐treatment was effective in decreasing lipid peroxidation and improving graft blood flow through the distinctive action of biliverdin and CO, respectively. The study shows that the addition of byproducts of heme degradation with different mechanisms of action provides enhanced protection against transplant‐associated cold I/R injury of heart and kidney grafts.

Establishment of a Simple and Practical Procedure Applicable to Therapeutic Angiogenesis

BACKGROUND Therapeutic angiogenesis is thought to be beneficial for serious ischemic diseases. This investigation was designed to establish a simple and practical procedure applicable to therapeutic angiogenesis. METHODS AND RESULTS When cultured skeletal muscle cells were electrically stimulated at a voltage that did not cause their contraction, vascular endothelial growth factor (VEGF) mRNA was augmented at an optimal-frequency stimulation. This increase of VEGF mRNA was derived primarily from transcriptional activation. Electrical stimulation increased the secretion of VEGF protein into the medium. This conditioned medium then augmented the growth of endothelial cells. The effect of electrical stimulation was further confirmed in a rat model of hindlimb ischemia. The tibialis anterior muscle in the ischemic limb was electrically stimulated. The frequency of stimulation was 50 Hz and strength was 0.1 V, which was far below the threshold for muscle contraction. After a 5-day stimulation, there was a significant increase in blood flow within the muscle. Immunohistochemical analysis revealed that VEGF protein was synthesized and capillary density was significantly increased in the stimulated muscle. Rats tolerated this procedure very well, and there was no muscle contraction, muscle injury, or restriction in movement. CONCLUSIONS We propose this procedure as a simple and practical method of therapeutic angiogenesis.

Properties of Two VEGF Receptors, Flt-1 and KDR, in Signal Transductiona

Abstract: The properties of two VEGF receptors, Flt‐1 and KDR, in the signal transduction of VEGF in human umbilical vein endothelial cells (HUVECs) were investigated by using two newly developed blocking monoclonal antibodies (mAbs) against Flt‐1 and KDR. VEGF stimulated the expression of transcription factor Ets‐1 as well as matrix metalloproteinase‐1 (MMP‐1) and Flt‐1 in HUVECs. The KDR/Flt‐1 heterodimer and the KDR homodimer mediate the expression of Ets‐1, MMP‐1, and Flt‐1. VEGF also stimulated DNA synthesis and migration of HUVECs. DNA synthesis is mediated by the same signaling system as the expression of Ets‐1. In contrast, cell migration is regulated by two distinct signaling systems. The Flt‐1 homodimer is required for actin reorganization. The KDR/Flt‐1 heterodimer and the KDR homodimer are required for the assembly of vinculin in focal adhesion plaque by regulating the phosphorylation of focal adhesion kinase (FAK) and paxillin.

Signal transduction and transcriptional regulation of angiogenesis.

When quiescent endothelial cells (ECs) are exposed to angiogenic factor such as VEGF; ECs express proteases to degrade extracellular matrices, migrate, proliferate and form new vessels. However, the molecular mechanism of these events is not fully characterized yet. We are studying the signal transduction and transcriptional regulation of angiogenesis. We investigated the properties of two VEGF receptors, Flt-1 and KDR, by using two newly developed blocking monoclonal antibodies (mAbs), i.e., anti-human Flt-1 mAb and anti-human KDR mAb. VEGF elicited induction of transcription factor Ets-1 in human umbilical vein endothelial cells (HUVECs). This induction was mediated by the KDR/Flt-1 heterodimer and the KDR homodimer. The role of transcription factor Ets-1 in angiogenesis was further clarified. We established both high and low Ets-1 expressing EC lines, and compared angiogenic properties of these cell lines with a parental murine EC line, MSS31. The growth rate was almost identical among three cell lines. It appeared that gene expressions of matrix metalloproteinases (MMP-1, MMP-3, and MMP-9) as well as integrin beta 3 were correlated with the level of Ets-1 expression. As a result, the invasiveness was enhanced in high Ets-1 expressing cells and reduced in low Ets-1 expressing cells compared with parental cells, and high Ets-1 expressing cells made more tube-like structures in type 1 collagen gel. These results indicate that Ets-1 is a principle transcription factor converting ECs to the angiogeneic phenotype.

Effect of low-voltage electrical stimulation on angiogenic growth factors in ischaemic rat skeletal muscle.

1 Low‐voltage electrical stimulation (LVES) in skeletal muscle at a level far below the threshold of muscle contraction has been reported to promote local angiogenesis. However, the mechanism underlying the promotion of local angiogenesis by LVES has not been fully elucidated. In the present study, we evaluated whether angiogenic factors, such as vascular endotherial growth factor (VEGF), hepatocyte growth factor (HGF) and fibroblast growth factor (FGF), as well as other disadvantageous factors, such as inflammation (interleukin (IL)‐6) and hypoxia (hypoxia‐inducible factor (HIF)‐1α), contribute to the local angiogenesis produced by LVES. 2 We completely excised bilateral femoral arteries of male Sprague‐Dawley rats. After the operation, electrodes were implanted onto the centre of the fascia of the bilateral tibialis anterior (TA) muscles, tunnelled subcutaneously and exteriorized at the level of the scapulae. The right TA muscles of rats were stimulated continuously at a stimulus frequency of 50 Hz, with a 0.1 V stimulus strength and no interval, for 5 days. The left TA muscles served as controls. 3 We found that both VEGF and HGF protein were significantly increased by LVES in stimulated muscles compared with control. The VEGF level of the LVES group was 89.10 ± 17.19 ng/g, whereas that of the control group was 65.07 ± 12.88 ng/g, as determined by ELISA (P < 0.05). The HGF level of the LVES and control groups was 8.52 ± 1.96 and 5.80 ± 2.14 ng/g, respectively (P < 0.05). In contrast, there was no difference in FGF, IL‐6 and HIF‐1α between the LVES and control groups. 4 These results suggest that LVES in a hindlimb ischaemia model of rats increases not only VEGF, but also HGF, production, which may be the main mechanism responsible for the angiogenesis produced by LVES.

Galantamine and carbon monoxide protect brain microvascular endothelial cells by heme oxygenase-1 induction

Galantamine, a reversible inhibitor of acetylcholine esterase (AChE), is a novel drug treatment for mild to moderate Alzheimers disease and vascular dementia. Interestingly, it has been suggested that galantamine treatment is associated with more clinical benefit in patients with mild-to-moderate Alzheimer disease compared to other AChE inhibitors. We hypothesized that the protective effects of galantamine would involve induction of the protective gene, heme oxygenase-1 (HO-1), in addition to enhancement of the cholinergic system. Brain microvascular endothelial cells (mvECs) were isolated from spontaneous hypertensive rats. Galantamine significantly reduced H(2)O(2)-induced cell death of mvECs in association with HO-1 induction. These protective effects were completely reversed by nuclear factor-kappaB (NF-kappaB) inhibition or HO inhibition. Furthermore, galantamine failed to induce HO-1 in mvECs which lack inducible nitric oxide synthase (iNOS), supplementation of a nitric oxide (NO) donor or iNOS gene transfection on iNOS-deficient mvECs resulted in HO-1 induction with galantamine. These data suggest that the protective effects of galantamine require NF-kappaB activation and iNOS expression, in addition to HO-1. Likewise, carbon monoxide (CO), one of the byproducts of HO, up-regulated HO-1 and protected mvECs from oxidative stress in a similar manner. Our data demonstrate that galantamine mediates cytoprotective effects on mvECs through induction HO-1. This pharmacological action of galantamine may, at least in part, account for the superior clinical efficacy of galantamine in vascular dementia and Alzheimer disease.

Stochastic active contour for cardiac MR image segmentation

We develop an energy based automatic image segmentation algorithm using a novel active contour scheme. The algorithm overcomes some unique challenges arising in cardiac MR images. Two features are particularly relevant. The first is that it uses region-based information captured by a stochastic model. As a result, our method is robust to assumed initial conditions and can be applied to a large range of images, particularly when the contrast is low. The second feature is the incorporation of prior knowledge on the shape of the organ to be segmented. For cardiac image segmentation, it is sufficient to assume that the shape resembles an ellipse.

Nitric Oxide, Oxygen Radicals

Nitric oxide (NO) is one of the first gases discovered by Joseph Priestley in 1772. For more than 200 years, it was considered mainly as an environmental pollutant and product of bacterial metabolism. In 1980, Furchgott and Zawadzki reported a substance released from endothelial cells which is responsible for the relaxation of arterial smooth muscle by acetylcholine’. They named this substance endothelium-derived relaxing factor (EDRF). EDRF was proven to be NO in the late 1980s2-4. Since NO is known to be generated in many different mammalian cells, a deluge of articles has implicated this ubiquitous molecule in a wide variety of regulatory mechanisms ranging from vascular tone to neurotransmissions. NO is also involved in nonspecific immunity and participates in the complex mechanism of tissue injury, acting as a major mediator of inflammation-related processes6,7

Hearts transfected with inducible nitric oxide synthase have accelerated restoration of cardiac function following ischemia/reperfusion associated with cardiac transplantation

Abstract Purpose: Our laboratory has demonstrated the novel effect of iNOS gene therapy in preventing the development of cardiac allograft vasculopathy. The effect of this therapeutic strategy on graft function needs to be evaluated prior to initiating clinical trials. This study documents the effect that iNOS transfection has on graft function following a period of ischemia/reperfusion similar to what would be incurred during cardiac transplantation. Procedure: Isografts were transfected via continuous antegrade coronary perfusion with either AdiNOS or the reporter gene AdEGFP (5X 10 8 pfu)prior to heterotopic transplantation. Grafts were harvested 4 days later and placed on a Langendorf apparatus. Grafts were then subjected to 20 minutes of warm ischemia and then reperfused. Coronary blood flow (CBF), contractility (dP/dT), diastolic relaxation (-dP/dT), and left ventricular pressures (LVP) were recorded at 20 and 120 minute intervals after reperfusion. Results: As shown in the table below, grafts transfected with AdiNOS had a significant restoration in contractility, diastolic relaxation and coronary blood flow at 120 minutes when compared to their respective AdEGFP controls. ∗ . Pre EGFP Post EGFP 20 minutes Post EGFP 120 minutes Pre iNOS Post iNOS 20 minutes Post iNOS 120 minutes CF 12.03 ± 0.61 6.15 ± 0.44 4.28 ± 0.35 12.180 ± 0.74 5.75 ± 0.21 9.08 ± 0.61 ∗ dP/dT 2503 ± 822 1202 ± 129 1156 ± 470 2075 ± 511 960 ± 46.5 1826 ± 479 ∗ −dP/dT −1959 ± 591 −792 ± 102 −691 ± 201 −1475 ± 432 −630 ± 107 −1320.2 ± 624 ∗ LVP 107.7 ± 11.4 41.5 ± 5.3 42.3 ± 7.1 87.0 ± 10.7 34.8 ± 3.0 69.5 ± 10.1 ∗ ∗ p Conclusions: AdiNOS gene therapy accelerates the restoration of baseline cardiac function following ischemia/reperfusion. The mechanism by which nitric oxide ameliorates the toxic effect of ischemia/reperfusion needs to be further studied. These data suggest further evaluation of this therapeutic strategy to prevent allograft vasculopathy is warranted and support the initiation of clinical trials.