Kiyotaka Yamamoto

Early Development of Intimal Thickening in Superficial Temporal Arteries in Patients With Moyamoya Disease

BACKGROUND AND PURPOSE Moyamoya disease is a progressive cerebrovascular occlusive disease that occurs in children. The etiology is unknown. We examined the superficial temporal arteries from patients with moyamoya disease, particularly children, to determine whether the extracranial arteries as well as the intracranial arteries are involved in this disease. METHODS Small branches of the superficial temporal arteries were obtained from 22 patients with moyamoya disease during indirect arterial bypass surgery. Histological examinations were performed, and the findings were compared with those of arteries from 12 control patients. RESULTS Intimal thickening was observed in 9 of 17 patients with moyamoya disease younger than 20 years but in none of 7 control patients under the age of 20 years (P < .02, Fishers exact test). Intimal thickening appeared from age 20 years in control patients. The arteries of moyamoya patients showed fibrocellular intimal thickening with a paucity of lipid. The arteries from moyamoya patients contained strongly stained multilayered elastic fibers in the thickened intima, while those from control patients showed only weakly stained elastic fibers in the intima. CONCLUSIONS Our findings suggest that moyamoya disease is a systemic vascular disease. The results indicate systemic etiologic factors that may promote the early development of intimal thickening in moyamoya disease.

Human Leukocyte Antigen in Patients With Moyamoya Disease

BACKGROUND AND PURPOSE Progressive stenosis or occlusion of bilateral internal carotid arteries by fibrocellular intimal thickening results in cerebral ischemia in moyamoya disease. In an attempt to elucidate the still-unknown etiologic factors in moyamoya disease, we assessed human leukocyte antigens in patients with this disease. METHODS We investigated 32 unrelated Japanese patients with moyamoya disease for typing of human leukocyte antigen A, B, C, and DR/DQ and compared the results with those from 178 unrelated control subjects. RESULTS We found a significant association of human leukocyte antigen B51 with moyamoya disease (corrected P < .05, chi 2 test). Although no significant associations were observed in DR/DQ typing, the frequency of the B51-DR4 combination was significantly higher in moyamoya patients than in control subjects (P < .002, Fishers exact test). CONCLUSIONS These findings suggest that there may be a genetic predisposition for moyamoya disease and that host factors may play a role in the development of intimal thickening in early childhood.

Changes in osteopontin mRNA expression during phenotypic transition of rabbit arterial smooth muscle cells

Abstract Transition from a contractile to a synthetic phenotype appears to be an early key event during the development of intimal thickening after arterial wall injury. We examined the expression of osteopontin mRNA, proliferation, and phenotypic properties of smooth muscle cells (SMCs) in rabbit neointima after balloon denudation and in primary culture. A strong osteopontin mRNA signal was detected in the thickened intima 1 week after balloon denudation and in the surface layer of the intima 2 weeks after balloon denudation. Ki-67 immunohistochemistry showed that osteopontin mRNA expression increased when SMCs entered the proliferating phase in the intima. Rabbit arterial SMCs on type I collagen after 1 day of primary culture with growth factors, as well as freshly isolated cells, were in the G0 phase (contractile phenotype) and did not express osteopontin mRNA. After 3 days of culture, most cells entered the G1B phase (synthetic phenotype) and expressed osteopontin mRNA. In the absence of growth factors, most cells transferred to the G1A phase (intermediate phenotype) after 3 and 7 days, but did not express osteopontin mRNA. Our findings indicate that the osteopontin gene provides a marker that can be used to distinguish the phenotypic properties of vascular SMCs.

Smooth muscle cell proliferation, elastin formation, and tropoelastin transcripts during the development of intimal thickening in rabbit carotid arteries after endothelial denudation

Abstract Extracellular matrix formation and smooth muscle cell proliferation are two major factors contributing to the development of intimal thickening after arterial injury. We investigated the elastin formation, tropoelastin transcripts, and proliferation of smooth muscle cells during the development of intimal thickening in rabbit carotid arteries after balloon endothelial denudation. Tropoelastin transcripts, identified by in situ hybridization using a digoxigenin-labeled probe, and elastin staining in the thickened intima were minimal 1 week after endothelial denudation when smooth muscle cell proliferation appeared throughout the thickened intima. A strong signal for tropoelastin transcripts was seen in the basal layer of the thickened intima 2 weeks after endothelial denudation, and then in the surface layer of the thickened intima 4 weeks after endothelial denudation. Immunohistochemistry for proliferating cell nuclear antigen and Ki-67, both markers for proliferating cell nuclei, showed that tropoelastin transcripts and elastin formation increased when smooth muscle cells enter quiescence after the end of the proliferative phase in the intima.Our findings strongly suggest that elastin synthesis and smooth muscle cell proliferation are tightly regulated during the repair of arterial wall injury.

Immunohistochemical Detection of Ki-67 in Replicative Smooth Muscle Cells of Rabbit Carotid Arteries After Balloon Denudation

BACKGROUND AND PURPOSE Ki-67 immunohistochemistry has been shown to be useful for paraffin sections from human species after wet heating. We applied Ki-67 immunohistochemistry to rabbit arteries after balloon denudation and compared the results with proliferating cell nuclear antigen (PCNA) immunohistochemistry. METHODS Monoclonal antibodies MIB-1 for Ki-67 and PC-10 for PCNA were used to detect replicative smooth muscle cells in rabbit carotid arteries during a period of 4 weeks after balloon endothelial denudation. RESULTS We demonstrated clear immunoreactivities for Ki-67 in paraffin sections of rabbit arteries after hydrated autoclaving. Ki-67-positive smooth muscle cells appeared throughout the thickened intima 1 week after endothelial denudation. At 2 weeks, Ki-67-positive cells were confined to the surface layer of the intima. PCNA-positive cells appeared in almost the same location by 2 weeks after endothelial denudation but were significantly greater in number than Ki-67-positive cells. PCNA-positive cells remained in the surface layer of the intima 4 weeks after endothelial denudation, while Ki-67-positive cells had almost disappeared from the intima. CONCLUSIONS Our results indicate that Ki-67 immunohistochemistry using MIB-1 monoclonal antibody provides a powerful tool, even in rabbit species, for the detection of replicative smooth muscle cells during the repair of arterial injury and that it detects replicative cells more accurately than PCNA immunohistochemistry.

Development of intimal thickening in superficial temporal arteries in patients with Moyamoya disease

To investigate the possible mechanism of neointimal formation in Moyamoya disease, we histologically examined the superficial temporal arteries and also investigated cultured smooth muscle cells (SMCs) from the arteries. Intimal thickening of the scalp arteries developed significantly at an early age in Moyamoya patients compared with control subjects. The histopathological findings of the neointima in scalp arteries were almost similar to those in intracranial arteries in Moyamoya patients. SMCs cultured from Moyamoya arteries responded significantly less to serum mitogens, especially to platelet derived growth factor (PDGF), than those of control patients, the finding of which was explained by the reduced number of PDGF receptor on Moyamoya SMCs. Our findings indicate the presence of systemic factors that promote migration and proliferation of SMCs from the media to the intima in Moyamoya disease. Our results suggest that alteration in vascular cells may contribute to the development of intimal thickening in Moyamoya disease.

Human arterial smooth muscle cell strains derived from patients with moyamoya disease: changes in biological characteristics and proliferative response during cellular aging in vitro

Moyamoya disease is a progressive cerebrovascular occlusive disease that occurs frequently in children. The etiology is unknown. We examined changes in biological characteristics and responsiveness to serum mitogens during the in vitro cellular aging of arterial smooth muscle cell strains derived from patients with moyamoya disease (HMSMC) and compared them with those of cells from age-matched control patients (HCSMC). HMSMC had a normal human diploid chromosome constitution. HMSMC and HCSMC had almost the same in vitro life span and the age-related patterns of biological parameters were essentially the same. However, the doubling time at the early passages was significantly longer in moyamoya SMC than control SMC, although there was no significant difference at the late passages. Furthermore, the poor responsiveness of moyamoya SMC to platelet-derived growth factor was retained throughout the life span in vitro. These results support the hypothesis that functional alterations in vascular cells are involved in the mechanism of development of intimal thickening in moyamoya disease.

Expression of p53 protein and p53 gene transcripts in rabbit carotid arteries after balloon denudation

Abstract Smooth muscle cell (SMC) proliferation may be positively or negatively regulated by various factors after arterial wall injury. We investigated the hypothesis that the p53 protein may play a role in regulating SMC proliferation. We examined p53 protein expression and p53 gene transcript distribution in rabbit carotid arteries over a period of 6 weeks after balloon denudation in relation to SMC proliferation. The number of p53-positive SMCs in the neointima reached a maximum 2 weeks after balloon denudation when proliferating SMCs decreased and were confined to the luminal surface of the intima. The p53-positive SMCs in the neointima almost paralleled the distribution of proliferating cell nuclear antigen (PCNA)-positive SMCs but not that of Ki-67-positive cells. Double immunofluorescence showed the simultaneous nuclear localization of both p53 protein and PCNA in intimal SMCs. Strong signals for p53 gene transcripts, identified by in situ hybridization, were observed in SMCs showing positive immunostaining for p53 protein. Our results indicate that p53 protein and p53 gene transcript levels increase, are closely linked to the proliferation of SMCs in the thickened intima, and play a key role in the regulation of cell proliferation during the repair process after arterial wall injury.

Elevated promotion of prostacyclin production by synthetic lipid A analogs in aged human endothelial cells in culture

We examined the effects of E. coli lipopolysaccharide (LPS) and synthetic analogs of lipid A, a bioactive moiety of LPS, on the prostacyclin (PGI2) production by young and old human endothelial cells in vitro. PGI2 production by endothelial cells has been shown to decrease during in vitro cellular senescence as well as in vivo. LPS and all the analogs tested in this study did not stimulate PGI2 production by young endothelial cells more than twofold. However, LPS and the majority of the lipid A analogs examined stimulated the PGI2 production by old cells more than twofold (approximately two- to sixfold). These results indicate that the responses to certain stimuli sometimes differ markedly between young and old cells, and this should be carefully considered when evaluating the biological effects of various compounds. Furthermore, these results suggest that certain synthetic lipid A analogs can be used as drugs to prevent some age-related vascular diseases.