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Biomedical subjects

M Aoki

Publications and source records attributed to M Aoki.

At least 343 records · Page 19Linked to original sources
PubMed1997

In vivo transfer efficiency of antisense oligonucleotides into the myocardium using HVJ-liposome method.

Although antisense strategy has been at the center of interest in gene therapy, little is known about application of this strategy to cardiac diseases because of the lack of a suitable delivery method into the heart. Therefore, we compared the transfection efficiency of antisense oligodeoxynucleotides (ODN) using HVJ-liposome method and direct transfer in in vivo transfer into heart. To investigate the cellular fate and localization of ODN, transfer of "naked" FITC-labeled antisense ODN or ODN enwrapped in HVJ-liposome complex were examined. Following in vivo transfer using direct injection as well as in vitro transfer, fluorescence rapidly disappeared within 1 day, whereas transfer by HVJ-liposome method resulted in sustained fluorescence localized in the nucleus for at least 1 week. Measurement of fluorescence also demonstrated a significantly higher level in myocardium transfected by HVJ-liposome method than direct transfer. The present study demonstrated that HVJ-liposome method is more efficacious for ODN delivery by prolongation of half-life of ODN, suggesting its usefulness for gene therapy in cardiac diseases.

Animals↗
PubMed1997

Chick osteocyte-derived protein inhibits osteoclastic bone resorption.

In order to investigate the role of osteocytes in bone resorption, we examined the homogenate and conditioned medium from purified chick calvarial osteocytes in a pit-formation assay using unfractionated bone cells from mice. The osteocyte homogenate markedly inhibited pit formation, whereas the conditioned medium of osteocytes had no effect. This inhibitory activity was not the result of cytotoxicity of the homogenate. A novel bone-resorption-inhibitory protein was purified from collagenase-digested chick calvarial fragments enriched in osteocytes. The inhibitory protein, of molecular mass 18.5 kDa, showed significant dose-dependent inhibition of pit formation by unfractionated bone cells from mice and rabbits, and by human giant tumour cells. This protein also inhibited the bone-resorbing activity of purified osteoclasts in the pit-formation assay in the absence of other effector cells. Microinjection of the protein into osteoclasts caused disruption of the podosomes in the cells. The N-terminal 25-amino-acid sequence of the protein showed 68% identity to a part of Rho-GTP-dissociation inhibitor. Thus chick calvarial osteocytes may be involved in the regulation of bone resorption by osteoclasts.

Amino Acid Sequence↗
PubMed1997

Histologic changes in the intervertebral disc after intradiscal injections of methylprednisolone acetate in rabbits.

STUDY DESIGN: Histologic changes in the intervertebral disc were evaluated by light and electron microscopy after an intradiscal injection of methylprednisolone acetate. OBJECTIVE: To evaluate the histologic changes that occur in the disc after an intradiscal injection of cortico steroids. SUMMARY OF BACKGROUND DATA: Several orthopedic surgeons have managed lumbar disc herniation with intradiscal steroid injection. Few studies have addressed the histologic changes after this treatment. METHODS: Thirty rabbits were surgically treated. Rabbits were divided into two groups. Group A: 25 rabbits underwent intradiscal steroid injections. Methylprednisolone acetate was injected into the L3-L4 disc, whereas methylprednisolone sodium succinate was injected into the L4-L5 disc. Physiologic saline was injected into the L5-L6 disc as a control. Groups of five rabbits were killed 1 day, 1, 4, 12, and 24 weeks after the injection and subjected to microscopic evaluation. Group B: Another five rabbits underwent an injection of polyethylene glycol 4000, a vehicle for methylprednisolone acetate, into the L3-L4 and L4-L5 discs. All five rabbits were killed 24 weeks after the injection and subjected to microscopic evaluation. RESULTS: Group A: Light microscopic evaluation showed tissue in the nucleus pulposus, and the inner layer of the anulus fibrosus had degenerated in the methylprednisolone acetate group killed 24 weeks after the injection. Matrix vesicles, an indication of primary tissue calcification, were observed by electron microscopy. In the methylprednisolone sodium succinate group and saline group, however, no histologic changes were observed. Group B: Degeneration of the nucleus pulposus and primary tissue calcification were observed in the discs of all rabbits. CONCLUSIONS: Methylprednisolone acetate and its vehicle polyethylene glycol cause degeneration and primary calcification in discs.

Animals↗
PubMed1997

Ocular changes in patients with spinocerebellar degeneration and repeated trinucleotide expansion of spinocerebellar ataxia type 1 gene.

OBJECTIVE: To examine ocular changes in patients with spinocerebellar degeneration who have repeated trinucleotide expansion in the spinocerebellar ataxia type 1 (SCA1) gene. DESIGN: Ophthalmic findings in 6 patients from 3 families whose DNA analysis revealed that they had an expanded allele of the trinucleotide repeated in the SCA1 gene were compared with those of normal control subjects and other healthy family members. The DNA was extracted from peripheral blood lymphocytes of the neurodegenerative family and normal control subjects. SETTING: University medical center. RESULTS: Visual acuity gradually decreased in successive follow-up visits. Color vision and visual fields were gradually affected. Electroretinograms showed mild attenuation of oscillatory potentials. Corneal endothelial cell density was severely decreased from 600 to 1300 cells/mm2. These findings were not observed in the normal control subjects, other healthy family members, or other patients with spinocerebellar degeneration who had repeated trinucleotide expansion of other genes. CONCLUSION: To the best of our knowledge, this is the first report describing the association between ocular changes in patients with spinocerebellar degeneration and gene mutation. These ocular changes were considered specific to patients who had the expanded allele of the repeated trinucleotide in the SCA1 gene.

Adult↗
PubMed1997

Efficient in vivo gene transfer into the heart in the rat myocardial infarction model using the HVJ (Hemagglutinating Virus of Japan)--liposome method.

The lack of efficient treatment for myocardial infarction remains an unresolved problem in the field of cardiovascular disease. Gene therapy may be a potential therapeutic strategy for the treatment of myocardial infarction. However, current methods of in vivo gene transfer into the heart are limited by their low efficiency and/or potential toxicity. In the present study, we developed an efficient technique of gene transfer into the intact heart in vivo using the Sendai virus (HVJ: Hemagglutinating Virus of Japan)--liposome method. We used the beta-galactosidase gene, luciferase gene and human angiotensin converting enzyme (ACE) gene as markers. In vivo gene transfer into the rat heart was performed as follows: (1) direct injection into the rat heart, (2) incubation within the pericardium, and (3) infusion into a coronary artery. Direct injection of the HVJ-liposome complex containing the beta-galactosidase vector into the rat heart resulted in limited staining of beta-galactosidase 3 days after transfection. To compare transfection efficiency between "naked" plasmid DNA transfection and the HVJ-liposome method, we also transfected the luciferase reporter gene into the heart. Luciferase activity was significantly higher in hearts transfected by the HVJ-liposome method than that in hearts transfected by direct "naked" plasmid transfection (P < 0.01). To confirm the successful gene in the protein level, we measured ACE activity in the hearts. Cardiac ACE activity was significantly increased in hearts transfected with human ACE gene as compared to hearts transfected with control vector (P < 0.01). On the other hand, incubation of HVJ-liposome complex, containing beta-galactosidase vector, within the pericardium resulted in widespread staining of cardiac myocytes and fibroblasts, mainly located in several surface layers beneath the pericardium. More importantly, widespread stained areas of beta-galactosidase were also observed in the middle of the myocardium around the vasa vasorum. We also examined the efficiency of gene transfer by the HVJ-liposome method in a rat myocardial infarction model. In the infarction model, using the pericardium incubation approach, staining for beta-galactosidase was observed in the viable cells around the infarction area. Finally, direct infusion of the HVJ complex, containing the beta-galactosidase vector, into coronary artery also resulted in widespread staining of beta-galactosidase in cardiac myocytes around the microvasculature. Using direct injection, we found significant injury to the myocardium and severe fibrosis at the injection site, whereas no apparent injury was observed using pericardium incubation and coronary infusion. There was no evidence of cytotoxicity or inflammation caused by the HVJ-liposome complex itself. Overall, we have established an efficient in vivo gene transfer method into the heart using the HVJ-liposome method. Direct infusion into the coronary artery resulted in widespread transfection without damaging the myocytes; incubation within the pericardium demonstrated the usefulness of the HVJ-liposome method for studying cardiac function and as a means of gene therapy for cardiovascular diseases.

Animals↗
PubMed1997

Role of hepatocyte growth factor in endothelial regulation: prevention of high D-glucose-induced endothelial cell death by prostaglandins and phosphodiesterase type 3 inhibitor.

Injury of endothelial cells (EC) has been postulated as the initial trigger of the progression of atherosclerosis in patients with diabetes mellitus. We previously reported that decrease in a novel endothelium-specific growth factor, hepatocyte growth factor (HGF), by high D-glucose might be a trigger of endothelial injury. However, the physiological role of the local vascular HGF system has not yet been clarified. To investigate the role of HGF in endothelial injury, we initially examined the effects of HGF on endothelial injury induced by serum deprivation. Decrease in EC number by serum deprivation was significantly attenuated by addition of HGF as well as recombinant basic fibroblast growth factor, whereas vascular endothelial growth factor showed no effect. Apoptotic changes in EC induced by serum deprivation were also significantly attenuated by addition of HGF (p < 0.01). Given the protective action of HGF, we next studied the physiological role of local HGF production in endothelial regulation. We focused on the protective actions of prostaglandin (PG) I2, PGE and a phosphodiesterase type 3 inhibitor (cilostazol) on endothelial injury by high glucose, since these agents are widely used in the treatment of peripheral arterial disease which is frequently observed in diabetic patients. Treatment of human aortic EC with PGE1, PGE2, and a PGI2 analogue (beraprost sodium) as well as cilostazol stimulated EC growth. HGF concentration in conditioned medium from EC treated with PGE1, PGE2 or PGI2 analogue as well as cilostazol was significantly higher than that with vehicle (p < 0.01). Interestingly, treatment with PGI2 analogue or cilostazol attenuated high D-glucose-induced EC death, which was abolished by neutralizing anti-HGF antibody. Moreover, decreased local HGF production by high D-glucose was also significantly attenuated by PGI2 analogue or cilostazol. Finally, we tested the effects of PGE, PGI2 analogue and cilostazol on local HGF production in human aortic vascular smooth muscle cells (VSMC). Although high D-glucose treatment resulted in a significant increase in VSMC number, PGI2 analogue and/or cilostazol treatment had no effects on VSMC growth. However, the decrease in local HGF production by high D-glucose was significantly attenuated by addition of PGI2 analogue or cilostazol. Overall, this study demonstrated that treatment with PGE, PGI2 analogue or cilostazol prevented aortic EC death induced by high D-glucose, probably through the activation of local HGF production. Increased local vascular HGF production by prostaglandins and cilostazol may prevent endothelial injury, potentially resulting in the improvement of peripheral arterial disease.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Cytokine responses in mice infected with Angiostrongylus cantonensis.

For determination of the kinetics of cytokine production and its possible role in host resistance to Angiostrongylus cantonensis in the mouse, Th1 [interleukin 2 (IL-2) and interferon gamma (IFN-gamma] and Th2 (IL-5 and IL-4) cytokine production in the cerebrospinal fluid (CSF), sera, and culture supernatants of spleen cells (SC) or cervical lymph-node cells (CLNC) of infected BALB/c and C57BL/6 mice was assessed by a sandwich enzyme-linked immunosorbent assay (ELISA). IL-5 and IL-4 were detected in CSF of both strains, with a peak response occurring at around days 12-15 and 20 postinfection (p.i.), respectively. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay also revealed prominent IL-5 and IL-4 mRNA expression in T-cells but not in eosinophils in CSF. SC and CLNC stimulated with A. cantonensis young adult-worm antigen released IL-5 in vitro at and after day 20 p.i. Contrarily, IFN-gamma production in CSF and SC or CLNC culture supernatants was almost negligible before day 30 p.i. IL-5, IL-4, and IL-2 production in culture supernatants was rather prominent in resistant C57BL/6 mice as opposed to susceptible BALB/c mice as assessed by the magnitude of increase over preinfection levels. Antigen-specific IgG1 (but not IgG2a) responses were more prominent in C57BL/6 mice than in BALB/c mice. These data suggest that systemic and local Th2 cytokine responses, especially those involving IL-5, are predominant in A. cantonensis-infected mice and that IL-5 is an important cytokine underlying the innate resistance of the mouse against A. cantonensis.

Angiostrongylus cantonensis↗
PubMed1997

Eosinophilia and intracranial worm recovery in interleukin-5 transgenic and interleukin-5 receptor alpha chain-knockout mice infected with Angiostrongylus cantonensis.

We infected interleukin-5 (IL-5)-transgenic (IL-5-Tg) and IL-5 receptor alpha knockout (IL-5R alpha -/-) mice with Angiostrongylus cantonensis to determine the possible roles of IL-5 and eosinophils in A. cantonensis infection in mice. IL-5-Tg mice demonstrated significantly higher eosinophilia in bone marrow, blood and cerebrospinal fluid (CSF), lower intracranial worm recovery and smaller female worms than naive C3H/HeN mice. Both IL-5-Tg and C3H/HeN mice evoked antigen-specific serum and CSF IgA antibody responses as early as days 5 and 7 postinfection, respectively. Prominent eosinophil infiltration was noted around intracranial worms in the subarachnoid spaces of the mouse brains; eosinophils adhering to the worm surface were degranulated. In contrast, IL-5R alpha -/- mice yielded a higher worm recovery than wild-type or heterozygous mice at day 20 postinfection and failed to provoke CSF eosinophilia. These findings indicate that A. cantonensis infection in the mouse causes IL-5 production and subsequent CSF eosinophilia, the latter probably being involved in the killing of intracranial worms.

Angiostrongylus cantonensis↗
PubMed1997

Ventricular hypertrophy as a risk factor in ventricular septation for double-inlet left ventricle.

BACKGROUND: Ventricular septation is an option for surgical correlation of double-inlet or common-inlet left ventricle. However, the surgical risk factors of ventricular septation remain unknown. METHODS: Twenty-three patients with double-inlet or common-inlet left ventricle underwent ventricular septation. Preoperative data were compared between the survivors (n = 18) and the nonsurvivors (n = 5) to assess surgical risk factors. RESULTS: There were two early deaths (9.5%) and three late deaths (14.3%). Nonsurvivors of ventricular septation were significantly older at the time of operation (14.0 +/- 6.0 versus 7.0 +/- 5.4 years; p < 0.05) and had greater left ventricular mass (383% +/- 100% versus 206% +/- 57% of normal predicted value; p < 0.005) and greater left ventricular mass to left ventricular end-diastolic volume ratio (1.84% +/- 1.18% versus 0.77% +/- 0.17%/% of normal predicted value; p < 0.005). Univariate logistic regression analysis also revealed age at operation (p < 0.05) and mass/end-diastolic volume ratio (p < 0.05) as significant risk factors for death after operation. Multivariate regression analysis revealed that age at operation positively influenced increased mass/end-diastolic volume ratio (p < 0.001). These findings indicated that ventricular hypertrophy was one of the risk factors for ventricular septation, which had a tendency to progress with age. CONCLUSIONS: Early operation before progression of ventricular hypertrophy is recommended in patients with double-inlet or common-inlet left ventricle who have suitable anatomy for the ventricular septation procedure.

Adolescent↗
PubMed1997

Modified Fontan procedure in ninety-nine cases of atrioventricular valve regurgitation.

Between January 1985 and August 1995, among 242 patients who underwent a modified Fontan procedure, 99 had atrioventricular valve regurgitation ranging in degree from 1 to 4, for which concomitant repair of the atrioventricular valve regurgitation was done in the majority of cases. In all but 4 cases the atrioventricular valve was repaired mainly by circular annuloplasty and valve replacement was not done in any case. Although the hospital mortality rate was significantly higher in cases with atrioventricular valve regurgitation (12/99, 12%) than in cases without (4/143, 3%; p < 0.0037, chi 2 test), actuarial survival in atrioventricular valve regurgitation was 84% for years 5 through 10. The degree of atrioventricular valve regurgitation before operation was 1.6 +/- 0.7 on average: in 49 cases with higher than grade 2 regurgitation before operation there was a significant decrease to 0.4 +/- 0.49 (p < 0.0001) after operation in short-term survivors. Patients with atrioventricular valve regurgitation can be treated with reasonable risk, provided proper repair of the valve is done. Circular annuloplasty is a simple and uniformly effective method to control regurgitation even in cases of common atrioventricular valve.

Adolescent↗
PubMed1997

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.

Animals↗
PubMed1997

Biomechanical and histologic characteristics of canine flexor tendon repair using early postoperative mobilization.

The purpose of this experimental study was to evaluate the mechanical and histologic healing of flexor tendon repairs using an early active motion protocol. Three different flexor tendon repair techniques in zone II were used. Forty-seven lacerated canine flexor profundus tendons from 25 dogs were repaired and evaluated at 5, 10, and 21 days after surgery. Eight of 9 Kessler repairs ruptured at days 5 and 10. None of the 19 Savage repairs or the 19 dorsal tendon splint repairs ruptured; 3 of 19 dorsal tendon splint repairs failed owing to adhesions. Smooth tendon gliding was obtained in all specimens in which repair was successful. The gap strength values for both the Savage and dorsal tendon splint repairs improved significantly for day-21 specimens compared to day-5 or day-10 specimens. The ultimate tensile strength showed no reduction during the 3-week period of tendon healing for both repairs. Histologically, there was evidence of progressive healing without surrounding adhesions. The improved suture techniques have the potential to withstand the stress produced by active digital motion protocols.

Animals↗
PubMed1997

Effect of immobilization, immediate mobilization, and delayed mobilization on the resistance to digital flexion using a tendon injury model.

This study employed a tendon injury model in chickens to determine the effect of immobilization, immediate mobilization, and delayed mobilization on the energy required to fully flex the digit following surgical trauma to the tendon, as determined by measurement of the work of flexion (WOF). The immobilized group showed no increased WOF at 3 days compared to the zero time control values, followed by significant increased WOF at 1 week (36%), 2 weeks (41%), and 3 weeks (63%). The immediate mobilization tendons showed an initial 36% rise in the work of flexion at 3 days, which increased to 40% at 1 week, and then decreased to baseline control values by 3 weeks. Delaying the period of mobilization until 5 days was not shown to significantly lower the peak WOF value at 1 week, but delaying the period of mobilization until 3 days lowered the peak WOF value at 1 week dramatically to 13%; both the 3- and 5-day delayed-mobilization groups returned to baseline WOF values by 3 weeks. From these data, it can be assumed that there is an early increase in the work necessary for flexion of the injured operated digit and tendon that is present in the immediately mobilized tendons at 3 days and persists to 1 week and that does not appear until 1 week in the immobilized tendons; this rise in WOF can be blunted by delaying the institution of mobilization.

Animals↗
PubMed1997

In vivo transfection of cis element "decoy" against nuclear factor-kappaB binding site prevents myocardial infarction.

The transcriptional factor nuclear factor-kappaB (NFkappaB) plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes that might be involved in myocardial damage after ischemia and reperfusion. Therefore, we hypothesized that synthetic double-stranded DNA with high affinity for NFkappaB could be introduced in vivo as "decoy" cis elements to bind the transcriptional factor and to block the activation of genes mediating myocardial infarction, thus providing effective therapy for myocardial infarction. Treatment before and after infarction by transfection of NFkappaB decoy, but not scrambled decoy, oligodeoxynucleotides before coronary artery occlusion or immediately after reperfusion had a significant inhibitory effect on the area of infarction. Here, we report the first successful in vivo transfer of NFkappaB decoy oligodeoxynucleotides to reduce the extent of myocardial infarction following reperfusion, providing a new therapeutic strategy for myocardial infarction.

Animals↗
PubMed1997

Prevention of graft coronary arteriosclerosis by antisense cdk2 kinase oligonucleotide.

Graft coronary arteriosclerosis, which limits the long-term survival of allograft recipients, is characterized by diffuse intimal thickening composed of proliferative smooth muscle cells. We observed that messenger RNA of the cell cycle regulatory enzyme cyclin-dependent kinase (cdk) 2 kinase, which mediates smooth muscle cell proliferation, was elevated in the thickened intima of coronary arteries of murine heterotopic cardiac allografts. We studied the effects of antisense phosphorothioate oligodeoxynucleotide (ODN) against this enzyme using gene transfer mediated by a hemagglutinating virus of Japan (HVJ)-liposome complex intraluminally delivered to inhibit the intimal hyperplasia. At 30 days after transplantation, antisense cdk2 kinase ODN treatment had dramatically inhibited neointimal formation in the allografts. Expression of vascular cell adhesion molecule-1 was also suppressed by antisense cdk2 kinase. However, these effects were not observed in the sense or scrambled ODN-treated allografts. Thus, an intraluminal administration of antisense ODN directed to a specific cell cycle regulatory gene can inhibit neointimal formation after cardiac transplantation.

Animals↗
PubMed1997

Survival of grafts of genetically modified cardiac myocytes transfected with FITC-labeled oligodeoxynucleotides and the beta-galactosidase gene in the noninfarcted area, but not the myocardial infarcted area.

Since one of the attractions of gene therapy in the heart is the implantation of genetically modified cultured cells, we employed genetically modified myocytes transfected with FITC-labeled oligodeoxynucleotides (ODN) and beta-galactosidase gene in this study, to investigate the cellular localization and fate of grafted myocytes in the heart. In addition, we examined the feasibility of myocytes grafting into a myocardial infarction model. Delivery of FITC-labeled ODN with the HVJ-liposome method resulted in sustained fluorescence as compared to direct transfer of 'naked' ODN. Interestingly, implantation of cardiac myocytes transfected with FITC-labeled ODN ex vivo by the HVJ-liposome method resulted in sustained fluorescence for at least 1 week in the noninfarcted area, whereas little fluorescence was detected in the area of infarction. This observation was confirmed by measurement of fluorescence, which showed significantly higher levels in the noninfarcted area than the infarcted area. Positive staining for beta-galactosidase protein was also clearly observed 7 days after grafting of transfection of the beta-galactosidase gene, while no staining was detected in grafted myocytes in control rats. Survival of implanted genetically modified cardiac myocytes in the noninfarcted, but not infarcted area, provides new information for the local delivery of recombinant molecules to the heart using gene therapy.

Animals↗