Differential expression of proteoglycans biglycan and decorin during neointima formation after stent implantation in normal and atherosclerotic rabbit aortas.

Proteoglycans decorin and biglycan, which bind to TGF-beta, are thought to participate in regulation of extracellular matrix accumulation in arterial intimal hyperplasia. To investigate the correlation of these proteoglycans with the cellular localization and phenotypic modulation of smooth muscle cells (SMCs), we analyzed the spatial and chronological distribution of these proteoglycans and two cytokines, TGF-beta and IL-1beta, in the process of neointima formation after stent implantation in the aortas of rabbits fed a high-cholesterol diet (atherosclerotic group) or a regular diet (control group). We implanted metallic stents in the rabbit aortas and harvested the aortas 4-56 days later for immunohistochemical and mRNA in situ hybridization analyses. In the control group, TGF-beta and biglycan expression was in correspondence with the chronology and localization of embryonic SMCs. In the atherosclerotic group, TGF-beta and biglycan expression was sustained throughout the experimental period, which was in accord with the prolonged expression of embryonic SMCs. Decorin, which did not occur in neointima in the control group, appeared in the atherosclerotic aortas in the confined area of vascular SMCs surrounding the macrophages around the stent wire. These results indicate that biglycan and decorin kinetics during neointima formation after arterial injury are distinct, despite their similar construction; biglycan synthesis correlates with embryonic SMCs.

Regulation of vascular smooth muscle cell apoptosis. Modulation of bad by a phosphatidylinositol 3-kinase-dependent pathway.

Our objective was to define the signaling mechanisms by which mitogens such as insulin-like growth factor-I (IGF-I) regulate vascular smooth muscle cell (VSMC) apoptosis. We confirmed that IGF-I inhibits serum withdrawal-induced apoptosis of cultured VSMCs in a dose-dependent and time-dependent fashion. To test the hypothesis that the phosphatidylinositol (PI) 3-kinase signaling pathway regulates VSMC survival, we examined the relationship between PI 3-kinase activity and cell fate. PI 3-kinase was elevated in viable VSMCs maintained in serum-containing medium, declined significantly in response to serum withdrawal, and increased in response to IGF-I-induced survival. Moreover, blockade of PI 3-kinase with 2 structurally dissimilar inhibitors (wortmannin or LY294002) abolished the capacity of IGF-I to maintain VSMC viability. Similarly, transient transfection of a dominant-negative Deltap85 PI 3-kinase mutant construct abrogated the capacity of IGF-I to prevent VSMC death. Thus, PI 3-kinase is a critical antiapoptotic signal in VSMCs. To define the distal element of the antiapoptotic cascade, we tested the hypothesis that IGF-I inhibits the influence of the proapoptotic gene Bad. Indeed, IGF-I stimulates increased expression of the inactive, phosphorylated form of Bad by a PI 3-kinase-dependent pathway. Moreover, the proapoptotic effect of Bad was attenuated by the stimulation of IGF-I. Thus, growth factors appear to prevent VSMC death by activating signal transduction pathways linked to apoptotic regulatory genes.

Association of smooth muscle cell phenotypic modulation with extracellular matrix alterations during neointima formation in rabbit vein grafts.

PURPOSE: To clarify the mechanisms of structural changes underlying vein graft stenosis that limits efficacy of bypass grafting operation, we examined the accumulation and distribution of various extracellular matrix (ECM) components during neointima formation in rabbit vein grafts and analyzed their correlation with proliferation and phenotypic modulation of smooth muscle cells (SMCs). METHODS AND RESULTS: An autologous external jugular vein graft was transplanted into the carotid artery in 25 rabbits. After the restoration of blood flow, the graft was markedly dilated. Medial SMCs in the graft appeared to be injured, and they began to proliferate at day 4 and subsequently migrated and formed the neointima at day 7. The neointima observed at days 7 and 14 contained ECM components, including type I collagen, heparan sulfate, and chondroitin sulfate, and the intimal SMCs were phenotypically modulated from the differentiated-type (SM2-positive and SM embryonic-negative) to the dedifferentiated-type (SM2-negative and SM embryonic-positive) as determined with immunostainings for myosin heavy chain isoforms. The intimal SMC proliferation was maximal at 2 weeks and then decreased rapidly. However, the neointima continued to thicken thereafter throughout the 6-month period of the experiment, and ECM accumulation, such as type I collagen and decorin, a small dermatan sulfate proteoglycan, was a prominent feature observed in the hypocellular region of the deep intima from 2 months after the transplantation. The phenotype of the intimal SMCs gradually returned to the differentiated-type from the deep intima after 2 months, but a small number of the intimal SMCs remained in the dedifferentiated phenotype even at 6 months after the operation. CONCLUSION: The neointima in the vein graft was formed initially by means of migration and proliferation of the phenotypically modulated, dedifferentiated-type SMCs and continued to thicken by means of sustained ECM accumulation, including type I collagen and decorin, in association with the prolonged presence of the dedifferentiated-type SMCs. These chronologic features in cell kinetics and ECM accumulation may contribute to the frequent occurrence of graft wall thickening that occurs in the vein grafts.

Gene transfer to vein graft wall by HVJ-liposome method: time course and localization of gene expression.

BACKGROUND: A novel gene transfer method using liposomes with a viral envelope of hemagglutinating virus of Japan (HVJ) has been reported to be very effective for gene transfection into somatic cells and might be applicable to improve the patency of vein grafts. The present study examined the time course and localization of gene expression to assess the feasibility of ex vivo gene transfer into the vein graft by the HVJ-liposome method. METHODS: The HVJ-liposome complex containing either beta-galactosidase plasmid DNA (deoxyribonucleic acid) or no genes (controls) (experiment 1) or fluorescein isothiocyanate-labeled oligonucleotides either with or without HVJ-liposomes (experiment 2) was infused into rabbit vein grafts and allowed to incubate before autologous transplantation to carotid arteries. RESULTS: In experiment 1, all grafts incubated with beta-galactosidase plasmid with HVJ-liposomes showed the blue staining of X-gal 7 days after operation, whereas the controls did not. The blue granules were present in the medial and adventitial tissue and were still present after 14 days. In experiment 2, many fluorescein isothiocyanate-labeled nuclei were observed in the graft wall 2 and 4 days after operation and remained present mainly in the media of HVJ-liposome-treated grafts after 7 and 14 days, when no fluorescein isothiocyanate activity was observed without HVJ-liposome treatment. CONCLUSIONS: These results demonstrated the feasibility of ex vivo transfection to the medial and adventitial tissue of the vein graft by the HVJ-liposome method and suggest the possibility of its clinical application to prevent vein graft failure.

Efficient transfer of oligonucleotides and plasmid DNA into the whole heart through the coronary artery.

Several of the current techniques for transfer of both oligonucleotide and plasmid DNA into the myocardium are impaired by low efficiency and toxicity. To improve gene transfer techniques, especially into the whole heart, a gene transfer method involving liposome in conjunction with a viral envelope (HVJ-liposome) was essayed as an alternative. FITC-labeled oligonucleotide (F-ODN) and the cDNA of beta-galactosidase (beta-gal) were introduced into the myocardium by coronary infusion of HVJ-liposome during cardioplegic arrest of adult Sprague-Dawley rat hearts. Then, transfected heart was ectopically transplanted into another rat abdomen of the same strain to maintain the transfected heart long enough to allow for protein synthesis. After 3 days of transfection, transfected heart was excised and the efficiency of gene transfection was evaluated. FITC was detected in the nuclei of more than 70% of the myocytes and endothelial cells both in the epicardium and endocardium. beta-Gal was expressed in the cytosol of more than 50% of the myocytes. beta-Gal expression was demonstrated by Western blotting analysis at day 3 after transfection and continued for at least 14 days. No significant histological damage of the myocardium or leakage of CPK were detected in the rats transfected by the HVJ-liposome method. These results clearly demonstrate that the hearts were efficiently transfected both by oligonucleotide and plasmid DNA as a result of coronary infusion of HVJ-liposome during cardioplegic arrest. This thus appears to be an efficient method for gene transfer into the whole heart, providing a new tool for research and therapy for heart diseases.

Efficient gene transfer method into the whole heart through the coronary artery with hemagglutinating virus of Japan liposome.

OBJECTIVE: To confirm gene transfer techniques especially into the whole heart, we tried out a gene transfer method involving liposome with the viral envelope hemagglutinating virus of Japan liposome as an alternative to existing techniques such as cationic lipofection or other viral vectors. METHOD: For this study, hemagglutinating virus of Japan liposome (H group) or cationic liposome (L group) was used to compare the efficacy of gene transfection of oligonucleotide labeled with fluorescein isothiocyanate and cDNA of beta-galactosidase and human manganese-superoxide dismutase. Fluorescein-labeled oligonucleotide, cDNA of beta-galactosidase, or manganese-superoxide dismutase was complexed with liposomes, DNA-binding nuclear protein, and the viral protein coat of hemagglutinating virus of Japan. After donor rat hearts arrested by cardioplegia had been harvested, the coronary artery during cardioplegic arrest was infused via an aortic cannula with the liposome-gene complex. Next, the hearts were transplanted into the abdomen of recipient rats of the same strain, and all recipients were put to death after 3 days of transfection. RESULTS: Fluorescein isothiocyanate was detected in the nuclei of more than 70% of the myocytes (75% +/- 14%, n = 5) in the H group compared with fewer than 10% in the L group (7% +/- 5%, n = 5). The intensity of fluorescein isothiocyanate was significantly higher in the H group (979 +/- 112 FI) than in the L group (116 +/- 68 FI). beta-Galactosidase was expressed in the cytosol of more than 50% of the myocytes in the H group (61% +/- 7%, n = 5) compared with none in the L group (0%, n = 5). After 3 days of gene transfection, and when exposed to ischemia (30 minutes, 37 degrees C) and reperfusion (30 minutes, 37 degrees C) with Langendorff apparatus, the hearts transfected with manganese-superoxide dismutase (S group, n = 5) showed a significantly higher percentage of recovery of left ventricular end-diastolic pressure (S vs C, 86% +/- 3% vs 54% +/- 12%) and coronary flow (98% +/- 2% vs 66% +/- 12%) than did the control hearts (C group, n = 5). Western blotting analysis showed an apparent increased expression of manganese-superoxide dismutase in the hearts transfected with manganese-superoxide dismutase compared with the control hearts. These results clearly demonstrated that the donor hearts were transfected with fluorescein-labeled oligonucleotide and the beta-galactosidase gene as a result of coronary infusion of the hemagglutinating virus of Japan liposome during cardioplegic arrest at the time of harvest. Furthermore, the hearts transfected with manganese-superoxide dismutase showed significant improvement in tolerance against ischemia reperfusion injury. CONCLUSION: We believe that this method represents a novel in vivo gene transfer technique for the heart and thus may provide a new tool for research and therapy of heart transplantation.

Efficiency of in vivo gene transfection into transplanted rat heart by coronary infusion of HVJ liposome.

BACKGROUND: Current methods of in vivo gene transfer into myocardium are limited by low efficiency. To improve in vivo gene transfer, a gene transfer method using hemagglutinating virus of Japan (HVJ) as a viral vector can be an alternative. METHODS AND RESULTS: In vivo gene transfection of FITC-labeled oligonucleotide (F-ODN) and cDNA of beta-galactosidase (beta-gal) was examined with use of the HVJ liposome (H group) or without it (C group). In the H group, F-ODN or cDNA of beta-gal were complexed with liposomes, DNA binding nuclear protein (HMG1), and the viral protein coat of HVJ. After the harvest of donor rat hearts arrested by cardioplegia, the coronary artery was infused with the liposome gene complex. The hearts were transplanted into the abdomens of recipient rats and harvested 3 days after transplantation. Regarding F-ODN, the H group clearly showed FITC staining in the nuclei of the myocytes and endothelial cells in almost all layers of the myocardium as compared with the C group. Regarding the expression of beta-gal, the H group showed a clear expression of beta-gal on myocytes, whereas very low expression of beta-gal was seen in the C group. CONCLUSIONS: The donor hearts were transfected with F-ODN and beta-gal gene in almost all layers of the myocardium as a result of coronary infusion of the HVJ liposome during cardioplegic arrest. Our method is seen as a novel in vivo gene transfer technique for the heart and may provide a new tool for both research and therapy of heart transplantation.

[Nearly total aortic replacement for DeBakey type-I aortic dissection in a patient with Marfan's syndrome].

A successful nearly total aortic replacement for DeBakey type-I aortic dissection in a patient with Marfan's syndrome is reported. A 36-year-old woman developed a DeBakey type-I aortic dissection in October 1987. We replaced the ascending aorta and resuspended the aortic valve urgently, when she had not been diagnosed with Marfan's syndrome. In August 1989, we replaced the distal portion of the descending aorta and the infra-renal abdominal aorta because of dilatation in the diameter of these regions. In September 1990, she had sudden severe chest pain. Computed tomography and Doppler ultrasonography revealed that a new aortic dissection developed in the aortic arch and the descending aorta (three-channeled dissection) and that the new false lumen compressed the true and the old false lumens obstructing the blood flow to the abdominal aorta. She was immediately placed an axilo-femoral bypass, and in January 1991 she underwent the arch and descending aorta replacement using permanent bypass to the supra-aortic branches and partial extracorporeal circulation (first reported by Larmi et al). Postoperative course was satisfactory, and she has been well at 3 years after the operation. Because progressive dilatation of an untreated dissecting aorta is common in patients with Marfan's syndrome, aggressive replacement of the diseased aorta is indicated in these patients.

[Surgical treatment of descending thoracic aortic aneurysm with simple aortic cross-clamping versus left heart bypass using centrifugal pump].

Surgical treatment has been employed in 52 patients (pts) with descending thoracic aortic aneurysm (DS-TAA). Based on the adjuncts during aneurysmal repair, the series is divided into 2 groups; simple aortic cross-clamping was utilized to manage the lesion in group SC (n = 42), while left heart bypass using a centrifugal pump was employed during the period of aortic occlusion in LHB group (n = 10). Of these 52 pts, 4 died in hospital (group SC:2, group LHB:2). The most common complication was the respiratory failure following the renal failure. No paraplegia occurred in both groups. Biochemistric measurements of alanine aminotransferase, creatinine (CRN) and amylase (AMY) showed no difference between group SC and group LHB. In pts of SC group with normal renal function, post-operative maximum (post Max) CRN during the first month had a logarithmic correlation with total aortic cross-clamp time (TAXT). The post Max CRN of LHB group with normal renal function remained less than 3.0 mg/dl even in the case with TAXT over 60 minutes. There is also a linear correlation of post Max AMY in pts of SC group. Late survival at 4 years, including hospital death, were 83% in SC group and 63% in LHB group. We conclude that DS-TAA cases with TAXT of less than 30 min with good distal organ function can be managed with simple aortic cross-clamping; otherwise usage of LHB was recommended to support distal circulation.

[Aortic valve re-replacement with a SJM valve by aortic ring re-enlargement with Nick's method--report of a successful case].

An 11-year-old girl had undergone an aortic valve replacement for congenital aortic stenosis with #17 Björk-Shiley valve and aortic ring enlargement by Nick's method eight years later, the pressure gradient across the prosthetic valve increased up to 100 mmHg. Re-aortic valve replacement was successfully performed with #21 St. Jude Medical valve, when aortic valve ring increased to accommodate #21 SJM valve.

[Influence of emotional stress and behavior during acute myocardial infarction on prognosis].

Seventeen cases of acute myocardial infarction (AMI) with emotional stress (group A) and 54 cases with basically stable emotion (group B) were compared with respect to three major complications (arrhythmia, cardiac insufficiency of grade 3 and 4 and cardiogenic shock) and therapeutic effect. The results showed that the incidences of the three complications in group A were significantly higher than those in group B (P less than 0.05), and the clinical condition was more serious in the former group. Response to narcotics showed that number of patients requiring more than 3 injections of either dolantine 50 mg or morphine 5 mg was significantly greater in group A than in group B (P less than 0.001). Sigma ST was not found to be significantly different between the two groups. However, the duration of elevation of ST segment was significantly longer in group A than in group B (P less than 0.01). It is suggested that relief of myocardial ischemia is slow in group A. There were five patients in group A with extended infarct size and died during the acute stage. None in the group B showed extension of infarction. The mortality rate in group A significantly higher than that in group B (53% VS 3.7%, P less than 0.001) and it bears no relation with sex, age and the site of infarction on admission. The greater the emotional upheaval the more unfavorable the prognosis. It is shown that excessive emotional stress is an important risk factor of AMI and aggressive measures are required to prevent worsening of the condition.