Koichi Tabayashi

A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Cre‐mediated recombination

The success of Cre‐mediated conditional gene targeting depends on the specificity of Cre recombinase expression in Cre‐transgenic mouse lines. As a tool to evaluate the specificity of Cre expression, we developed a reporter transgenic mouse strain that expresses enhanced green fluorescent protein (EGFP) upon Cre‐mediated recombination. We demonstrate that the progeny resulting from a cross between this reporter strain and a transgenic strain expressing Cre in zygotes show ubiquitous EGFP fluorescence. This reporter strain should be useful to monitor the Cre expression directed by various promoters in transgenic mice, including mice in which Cre is expressed transiently during embryogenesis under a developmentally regulated promoter.

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.

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.

Increased von Willebrand Factor in the Endocardium as a Local Predisposing Factor for Thrombogenesis in Overloaded Human Atrial Appendage

OBJECTIVES We investigated immunoreactive von Willebrand factor (vWF), a platelet adhesion molecule, in the endocardial endothelium and its relationship to thrombogenesis in the human atrial appendage. BACKGROUND Intra-atrial thrombogenesis is generally thought to be induced by blood stasis in the atrial appendage involved with atrial fibrillation (AF). Little attention has been paid to alterations of the endocardial endothelium on which the thrombus develops. METHODS Atrial appendage tissue was obtained at heart surgery or at autopsy from AF and non-AF cardiac patients and from noncardiac patients. Immunohistochemistry for endothelial cell markers including vWF, CD31, CD34 and endothelial nitric oxide synthase (eNOS) and platelet glycoprotein Ib/IX or IIb/IIIa was performed and semiquantitatively graded. RESULTS In contrast to the apparent immunostaining for CD31, CD34 and eNOS, only focal or little immunoreactive vWF was seen in the endocardium of noncardiac patients. Immunoreactive vWF in the endocardial endothelium was increased in most cardiac patients, particularly in the left, but not in the right, atrial appendage of patients with mitral valvular disease, irrespective of whether AF was present. Platelet adhesion/thrombus formation in the endocardium was found in limited sites in which the overlying endothelium was deficient in eNOS and CD34. When warfarin-treated cases were excluded, there was a significant correlation between the immunohistochemical grade for vWF and the degree of platelet adhesion/thrombus formation in the endocardium. CONCLUSIONS Immunoreactive vWF in the endocardial endothelium was increased in overloaded human atrial appendage, which may be a local predisposing factor for intraatrial thrombogenesis.

Apoptosis of Motor Neurons With Induction of Caspases in the Spinal Cord After Ischemia

BACKGROUND AND PURPOSE Some neuronal subpopulations are especially vulnerable to ischemic injury. In the spinal cord, large motor neurons are vulnerable to ischemia and are selectively lost after transient ischemia. However, the mechanisms of the neuronal loss have been uncertain. We hypothesized that spinal motor neurons might be lost by apoptosis and investigated a possible mechanism of neuronal death by detection of double-strand breaks in genomic DNA and immunohistochemical analysis for caspases, ie, interleukin-1beta converting enzyme (ICE), Nedd-2, and CPP32. METHODS We used a rabbit spinal cord ischemia model created with a balloon catheter. The spinal cord was removed at 8 hours, 1, 2, or 7 days after 15 minutes of transient ischemia, and histological changes were studied with hematoxylin-eosin staining. To detect double-strand breaks in DNA, a staining with terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed. Furthermore, expression of ICE, Nedd-2, and CPP32 was investigated by Western blotting and immunohistochemical analysis. RESULTS Motor neurons were selectively lost at 7 days after transient ischemia. TUNEL study demonstrated that no cells were positively labeled until 1 day after ischemia, but nuclei of some motor neurons were positively labeled at 2 days. Western blot analysis revealed no immunoreactivity for ICE and slight immunoreactivities for Nedd-2 and CPP32 in the sham-operated spinal cords. However, immunoreactivity became apparent at 8 hours after transient ischemia, decreased at 1 day, and returned to baseline level at 2 days. Immunohistochemical analysis demonstrated that motor neurons were responsible for induction of those caspases. CONCLUSIONS Double-strand breaks in genomic DNA and induction of three caspases were demonstrated. These results indicate that motor neuron death in the spinal cord after transient ischemia is profoundly associated with activation of apoptotic processes.

Enhancement of heat shock protein expression after transient ischemia in the preconditioned spinal cord of rabbits.

PURPOSE This investigation was designed to evaluate the mechanism used to acquire a tolerance to spinal ischemia. We investigated inductions of the heat shock protein (HSP) 70 gene and protein in rabbit spinal cord with or without preconditioning. METHODS Neurologic function, morphologic changes, and inductions of HSP70 messenger RNA (mRNA) and protein were compared in the cases of a 15-minute ischemia 2 days after sham treatment and a 15-minute ischemia 2 days after 10-minute preconditioning. RESULT HSP70 mRNA was induced at 8 hours of reperfusion after a 15-minute ischemia 2 days after sham treatment. HSP70 protein was induced slightly in selective motor neuron cells at 8 hours of reperfusion, and about 70% of motor neuron cells showed selective cell death after 7 days of reperfusion (p < 0.01). On the other hand, large populations of the motor neuron cells survived at 7 days after the 15-minute ischemia that was applied at 2 days after preconditioning (p < 0.01). HSP70 mRNA was induced persistently as compared with the case of a 15-minute ischemia 2 days after sham treatment. The motor neuron cells strongly produced immunoreactive HSP70 from 8 hours to 2 days. CONCLUSION Preconditioning with 10-minute ischemia enhanced and prolonged the HSP70 gene expression at both mRNA and protein levels and saved the motor neuron cells from subsequent lethal ischemia. These changes of HSP70 gene expression may play an important role in the acquisition of ischemic tolerance of motor neuron cells in rabbit spinal cord.

Isolation and Characterization of Vasohibin-2 as a Homologue of VEGF-Inducible Endothelium-Derived Angiogenesis Inhibitor Vasohibin

Objective—We recently isolated vasohibin, a novel vascular endothelial growth factor (VEGF)-inducible endothelium-derived angiogenesis inhibitor. Our aim is to find DNA sequences homologous to vasohibin and determine their expression profile. Methods and Results—By the search of DNA sequences in the database, we found one homologous gene and designated it vasohibin-2. Overall amino acid sequence homology between the prototype vasohibin (vasohibin-1) and vasohibin-2 was >50%. Vasohibin-2 exhibited antiangiogenic activity. Vasohibin-2 expression in cultured endothelial cells was low and not inducible by the stimulation that induced vasohibin-1. However, the immunohistochemical analysis revealed that vasohibin-1 and -2 were diffusely expressed in endothelial cells in embryonic organs during mid-gestation. After that time point, vasohibin-1 and -2 became faint, but persisted to a certain extent in arterial endothelial cells from late gestation to neonate. Expression of vasohibin-1 and -2 could be augmented in vivo by local transfection with the VEGF gene in the embryonic brain or by cutaneous wounding in adult mice. Conclusion—These results suggest that vasohibin-2, in combination with vasohibin-1, forms a novel family of angiogenesis inhibitors.

Selective motor neuron death and heat shock protein induction after spinal cord ischemia in rabbits

Paraplegia is a serious complication that sometimes results from operation on the thoracic aorta. The mechanism of spinal cord injury has been thought to involve tissue ischemia, and spinal motor neurons are suggested to be vulnerable to ischemia. The exact mechanism, however, is not fully understood. To evaluate the mechanism of such vulnerability of motor neurons, we attempted to make a reproducible model for spinal cord ischemia and statistically analyzed cell damage. With this model, induction of heat shock protein 70 (HSP70) and heat shock cognate protein (HSC70) messenger ribonucleic acid molecules were investigated with Northern blot analysis for up to 7 days of reperfusion after 5 or 15 minutes of ischemia. Immunohistochemical studies of their proteins were also done. (heat shock proteins are a set of markers of neuronal injury after ischemia.) After 5 minutes of ischemia, there was no induction of HSP70 and HSC70 messenger ribonucleic acid molecules or their proteins, and all cells remained intact. In contrast, after 15 minutes of ischemia, HSP70 messenger ribonucleic acid was induced at 8 hours of reperfusion, and HSC70 messenger ribonucleic acid was expressed continuously at the control level. Immunoreactivity of HSP70 protein was slightly induced at 8 hours of reperfusion selectively in motor neurons, and about 70% of motor neuron cells showed selective cell death after 7 days of reperfusion. This study demonstrated induction of HSP70 messenger ribonucleic acid and its protein in motor neuron cells after transient ischemia in the spinal cord. This phenomenon was not accompanied by HSC70 induction.

Delayed and selective motor neuron death after transient spinal cord ischemia: A role of apoptosis?

OBJECTIVE The mechanism of spinal cord injury has been thought to be related to tissue ischemia, and spinal motor neuron cells are suggested to be vulnerable to ischemia. We hypothesized that delayed and selective motor neuron death is apoptosis. METHODS Thirty-seven Japanese domesticated white rabbits weighing 2 to 3 kg were used in this study and were divided into two subgroups: a 15-minute ischemia group and a sham control group. Animals were allowed to recover at ambient temperature and were killed at 8 hours, and 1, 2, 4, and 7 days after reperfusion (n = 3 at each time point). By means of this model, cell damage was histologically analyzed. Detection of ladders of oligonucleosomal DNA fragment was investigated with gel electrophoresis up to 7 days of the reperfusion. Immunocytochemistry, in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining was also performed. RESULTS After 15 minutes of ischemia, most of the motor neurons showed selective cell death at 7 days of reperfusion. Typical ladders of oligonucleosomal DNA fragments were detected at 2 days of reperfusion. Immunocytochemistry showed in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end staining was detected at 2 days of reperfusion selectively in the nuclei of motor neurons. CONCLUSION These results suggest that delayed and selective death of the motor neuron cells after transient ischemia may not be necrotic but rather predominantly apoptotic.

Delayed selective motor neuron death and fas antigen induction after spinal cord ischemia in rabbits.

The mechanism of spinal cord injury has been thought to be related with tissue ischemia, and spinal motor neuron cells are suggested to be vulnerable to ischemia. To evaluate the mechanism of such vulnerability of motor neurons, we attempted to make a reproducible model for spinal cord ischemia. Using this model, cell damage was histologically analyzed. Detection of ladders of oligonucleosomal DNA fragment was investigated with gel electrophoresis up to 7 days of the reperfusion. Time course expression of Fas antigen, identified as a apoptosis-regulating molecules, was also assessed in rabbit spinal cord following transient ischemia. Spinal cord sections from animals sacrificed at 8 h, 1 day, 2 days, and 7 days following 15-min ischemia were immunohistochemically evaluated using monoclonal antibodies for Fas antigen. Following 15-min ischemia, the majority of motor neuron showed selective cell death at 7 days of reperfusion. Typical ladders of oligonucleosomal DNA fragments were detected at 2 days of reperfusion. Immunoreactivity of Fas antigen were induced at 8 h to 1 day of reperfusion selectively in motor neuron cells. The expression of Fas antigen may be related to the activation of apoptosis signal in motor neuron cells after spinal cord ischemia in rabbits.