Mutations in the gene encoding c-Abl-binding protein SH3BP2 cause cherubism.

Cherubism (MIM 118400) is an autosomal dominant inherited syndrome characterized by excessive bone degradation of the upper and lower jaws followed by development of fibrous tissue masses, which causes a characteristic facial swelling. Here we describe seven mutations in the SH3-binding protein SH3BP2 (MIM 602104) on chromosome 4p16.3 that cause cherubism.

Increase in prostaglandin E(2) production by interleukin-1beta in arterial smooth muscle cells derived from patients with moyamoya disease.

Moyamoya disease is a progressive cerebrovascular occlusive disease that primarily affects children. The cause is unknown. We examined the production of prostanoids and the expression of cyclooxygenase-2 (COX-2) in cultured arterial smooth muscle cells (SMCs) derived from patients with moyamoya disease. Twelve moyamoya and 8 control cell strains were examined. The steady-state levels of prostanoids in the culture medium did not differ between moyamoya and control SMCs. When the cells were stimulated with interleukin-1beta (IL-1beta), prostaglandin E(2) (PGE(2)) release into the medium was significantly greater from moyamoya SMCs than from control SMCs, whereas the amounts of prostacyclin and thromboxane B(2) did not differ. IL-1beta-induced PGE(2) production by moyamoya SMCs was completely blocked by the addition of indomethacin or NS-398. IL-1beta significantly stimulated cell migration and DNA synthesis in control SMCs but had an inhibitory effect on moyamoya SMCs. The inhibitory effects on the growth and migration of moyamoya SMCs were caused by excessive secretion of PGE(2) and was reversed with indomethacin treatment. Immunofluorescence studies and Western blot analysis showed greater amounts of COX-2 protein expression in IL-1beta-stimulated moyamoya SMCs. These findings suggest that moyamoya SMCs respond to inflammatory stimuli to produce excess amounts of PGE(2) through the activation of COX-2, which increases vascular permeability and decreases vascular tone. This facilitates the exposure of vessels to blood constituents and promotes the development of intimal thickening in moyamoya disease.

Tissue specific regulation of VEGF expression during bone development requires Cbfa1/Runx2.

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis during development, but little is known about the factors that control its expression. We provide the first example of tissue specific loss of VEGF expression as a result of targeting a single gene, Cbfa1/Runx2. During endochondral bone formation, invasion of blood vessels into cartilage is associated with upregulation of VEGF in hypertrophic chondrocytes and increased expression of VEGF receptors in the perichondrium. This upregulation is lacking in Cbfa1 deficient mice, and cartilage angiogenesis does not occur. Finally, over-expression of Cbfa1 in fibroblasts induces an increase in their VEGF mRNA level and protein production by stimulating VEGF transcription. The results demonstrate that Cbfa1 is a necessary component of a tissue specific genetic program that regulates VEGF during endochondral bone formation.

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.

Matrilysin cleavage of corneal collagen type XVIII NC1 domain and generation of a 28-kDa fragment.

PURPOSE: To localize endostatin and collagen type XVIII in human corneas and to characterize the enzymatic action of matrix metalloproteinases (MMPs) in the cleavage of collagen type XVIII and generation of endostatin in the cornea. METHODS: Anti-endostatin and anti-hinge antibodies were generated using peptide fragments corresponding to the endostatin region and the adjacent nonendostatin hinge region of collagen XVIII noncollagenous (NC)1 domain, respectively. Confocal immunostaining was performed to localize collagen XVIII in human corneas. SV40-immortalized corneal epithelial cells were immunoprecipitated and incubated with active MMP-1, -2, -3, -7, or -9, and Western blot analysis was performed to study collagen XVIII cleavage. Incubation with MMP-7 was performed at various concentrations (0, 2, 4, and 6 microg/ml) and time intervals (0, 1, 5, and 12 hours). Purified recombinant NC1 fragment of collagen XVIII was also digested with MMP-7, and the cleavage product was sequenced. RESULTS: Collagen XVIII was immunolocalized to the human corneal epithelium, epithelial basement membrane, and Descemet membrane. Western blot analysis demonstrated a 180- to 200-kDa band corresponding to collagen XVIII. MMP-7 (but not MMP-1, -2, -3, and -9) cleaved corneal epithelium-derived collagen XVIII to generate a 28-kDa endostatin-spanning fragment in a time- and concentration-dependent fashion. MMP-7 cleaved purified recombinant 34-kDa NC1 fragment of collagen XVIII in the hinge region to generate a 28-kDa fragment. CONCLUSIONS: Collagen XVIII is present in human cornea. MMP-7 cleaves the collagen XVIII NC1 domain to generate a 28-kDa fragment in the cornea.

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.

A new surgical treatment of moyamoya disease in children: a preliminary report.

A new operative method, encephalo-duro-arterio-synangiosis, for the surgical treatment of pediatric moyamoya disease has been developed. The rationale of the operation is to help promote the natural tendency of this disease to develop cerebrovascular collaterals. The method is to transplant a scalp artery with a strip of galea, leaving the distal as well as the proximal arteries intact, to a narrow linear dural opening made under an osteoplastic craniotomy. A representative case is described and the operative procedure is outlined. Our new method is compared with other surgical treatments of this disease.

Very early expression of vascular endothelial growth factor in brain oedema tissue associated with brain contusion.

BACKGROUND: Brain oedema associated with cerebral contusion can be life-threatening. Mechanisms of the development of brain oedema are still unclear. METHOD: We investigated the expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (KDR/Flk-1) in the contusional brain tissue obtained during neurosurgery from 5 patients. FINDINGS: VEGF is expressed in some but not all the astrocytes, and KDR/Flk-1 is expressed in vascular endothelial cells in the con-tusional tissue as early as 3 hours after onset. CONCLUSION: The results suggested that the VEGF is induced in the contusional tissue in the very early period after onset, and that it increases capillary permeability via KDR/Flk-1 resulting in vasogenic type brain oedema.

[Bacterial meningitis in the elderly with neurosurgical procedures].

Bacterial meningitis is one of complications in the elderly with neurosurgical procedures. In an attempt to find the clinical features of this complication we analyzed 10 cases, which were found in 30 cases of the bacterial meningitis in Tokyo Metropolitan Geriatric Hospital from 1972 to 1989. The patients were 4 males and 6 females, 52-86 years old (the mean, 69). While 2 Enterococcus species were isolated after craniotomy, Staphylococci were common pathogens (4 S. aureus, 4 S. epidermidis and 1 P. aeruginosa) in patients with shunt infection. Most of these patients lacked typical manifestations of meningitis except the fever. Symptoms occurred long after surgery with little abnormality in the data of serum and cerebrospinal fluid. However, blood cultures were positive in 75% of the cases. Removal of the infected catheter was effective in the cases of shunt infection.

[The anatomy and pathology of the brain stem demonstrated by thin slice CT cisternography].

We demonstrated normal CT anatomy of the brain stem and adjacent structures using thin slice CT cisternography. Reducing the streak artifact caused by bony structures, thin slice CT cisternography can visualize the detailed features of the brain stem and its pathological changes. In addition, reconstructed images are also useful in diagnosis.

[A fragment of collagen XVIII inhibits angiogenesis]. / Et kollagen XVIII-fragment hemmer angiogenese.

BACKGROUND: Malignant tumours may produce substances with both stimulatory and inhibitory effect on angiogenesis. MATERIAL AND METHODS: A protein fragment with angiogenesis-inhibiting potential was recently identified in conditioned media from a murine endothelial tumour cell line. RESULTS: The angiogenesis inhibitor, endostatin, is a 20 kDa C-terminal fragment of collagen XVIII, a proteoglycan/collagen found in vessel walls and basement membranes. The generation of endostatin or endostatin-like collagen XVIII fragments is catalyzed by proteolytic enzymes, including cathepsin L and matrix metalloproteases, that cleave peptide bonds within the protease-sensitive hinge region of the C-terminal domain. INTERPRETATION: The physiological processing of collagen XVIII to endostatin may represent a local control mechanism for the regulation of angiogenesis. The outcome of ongoing clinical trials will determine the role of endostatin as a possible angiogenesis-inhibiting drug in the future.

Kinetics of 125I-PDGF binding and down-regulation of PDGF receptor in arterial smooth muscle cells derived from patients with moyamoya disease.

Progressive stenosis or occlusion of bilateral internal carotid arteries by fibrocellular intimal thickening results in cerebral ischemia in moyamoya disease. We recently found that cultured smooth muscle cells (SMC) derived from arteries of patients with moyamoya disease responded poorly to serum mitogens, especially to platelet-derived growth factor (PDGF). In the present study, we investigated further the binding and processing of 125I-PDGF, as well as down-regulation of the PDGF receptor in arterial SMC derived from patients with moyamoya disease. The specific binding sites of 125I-PDGF were reduced significantly at both 4 degrees C and 22 degrees C on SMC from moyamoya disease compared with those from control (4.78 vs. 11.92 x 10(4)/cell at 4 degrees C), though the apparent dissociation constant (Kd) were the same. Kinetics of 125I-PDGF binding at 37 degrees C in cells from moyamoya disease showed fewer binding sites (less than 1/3 of controls) and lower degradation per cell than in those from controls, though no difference was observed in either internalization or degradation of each receptor. When SMC were exposed to lower concentrations of nonlabeled PDGF at 37 degrees C, the percentage of remaining binding sites on cells from moyamoya disease was significantly less than that from controls. This excess down-regulation of PDGF receptor in SMC from moyamoya disease may be interpreted as insufficient recycling or a decreased intracellular pool of the PDGF receptor. These results are closely correlated with the diminished proliferation responses to PDGF in SMC from moyamoya disease and provide evidence that functional alterations in vascular cells are involved in the mechanism of development of intimal thickening in moyamoya disease.

Altered cellular responses to serum mitogens, including platelet-derived growth factor, in cultured smooth muscle cells derived from arteries of patients with moyamoya disease.

Progressive stenosis or occlusion of bilateral internal carotid arteries by fibrocellular intimal thickening results in cerebral ischemia in moyamoya disease. The etiology is unknown. We examined cultured arterial smooth muscle cells (SMC) from scalp arteries of five patients with moyamoya disease. In this study we investigated the responsiveness of the cells in culture to serum mitogens including platelet-derived growth factor (PDGF), a major mitogen of SMC, and compared the response to that of cells derived from age-matched control patients. SMC from patients with moyamoya disease proliferated less rapidly in a medium with 15% serum than did control SMC and responded poorly to the addition of PDGF to 5% serum. PDGF alone did not stimulate SMC in a quiescent state to initiate DNA synthesis in moyamoya disease, without serum factors other than bovine serum albumin, though it significantly stimulated the controls. Simultaneous additions of epidermal growth factor, insulin-like growth factor-I, and PDGF stimulated initiation of DNA synthesis in cells from moyamoya disease, but not as much as PDGF alone did in the controls. Although direct correlations with the pathogenesis of the disease remain to be clarified, the results indicate altered interrelations between serum factors and the cellular responses in vessels of moyamoya disease.

Increase in elastin gene expression and protein synthesis in arterial smooth muscle cells derived from patients with Moyamoya disease.

BACKGROUND AND PURPOSE: Moyamoya disease is a progressive cerebrovascular occlusive disease that is rare in all ages but frequently presents in children. The etiology of the disease is unknown. We examined elastin gene transcripts and elastin synthesis in cultured arterial smooth muscle cells (SMCs) derived from moyamoya patients and compared them with those in SMCs from age-matched control subjects. METHODS: We used six cell strains from moyamoya patients and four from controls. The expression of elastin protein was observed by Western blot analysis and metabolic labeling with 3H-valine. Elastin gene transcripts were identified by Northern blot analysis. RESULTS: Elastin mRNA and protein levels were elevated in all SMCs from moyamoya patients compared with control SMCs. Although transforming growth factor-beta 1 (TGF-beta 1), a potent enhancer of the expression of elastin in arterial SMCs, upregulated elastin mRNA and protein levels in SMCs from both moyamoya patients and control subjects, the maximum levels of elastin synthesis and elastin gene transcripts in response to exogenous TGF-beta 1 were significantly greater in moyamoya SMCs than control SMCs. In addition, quiescent moyamoya SMCs secreted significantly more TGF-beta 1 into the culture medium than quiescent control SMCs (P < .01). CONCLUSIONS: Our findings suggest that moyamoya disease may result, at least in part, from an abnormal regulation of extracellular matrix metabolism that leads to increased steady state levels of elastin mRNA and elastin accumulation in the intimal thickening and that increased elastin accumulation is a stable marker of SMCs from patients with moyamoya disease.

The matrix metalloproteinase-14 (MMP-14) gene is structurally distinct from other MMP genes and is co-expressed with the TIMP-2 gene during mouse embryogenesis.

The matrix metalloproteinases (MMPs) are a family of zinc-containing matrix degrading endopeptidases. A subfamily of membrane type (MT) -MMPs has been described recently. We have determined the structure of the gene (Mmp14) encoding the first MT-MMP to be described, MT1-MMP (MMP-14), and mapped it to mouse chromosome 14. The mouse MMP-14 protein is encoded by ten exons. The novel C-terminal peptide domains of MMP-14 are encoded by a single large exon that also encodes the 3'-untranslated region. The structure of the exons encoding the catalytic domain and pro-domain of MMP-14 is distinct from previously described MMP genes, whereas the exons encoding the hemopexin-like domains are similar to those of most other MMP genes. Mmp14 and the gene for tissue inhibitor of metalloproteinases-2 (Timp2) show a temporally and spatially co-regulated expression during mouse development. They are co-expressed during vascular and urogenital development and during the development of osteocartilaginous and musculotendinous structures. The stringent co-expression of these two genes suggests common regulatory pathways that may have important functional implications for the activation of pro-gelatinase A in health and disease.

Mouse clavicular development: analysis of wild-type and cleidocranial dysplasia mutant mice.

Cleidocranial dysplasia (CCD) is an autosomal dominant disease characterized by hypoplasia or aplasia of clavicles, open fontanelles, and other skeletal anomalies. A mouse mutant, shown by clinical and radiographic analysis to be strikingly similar to the human disorder and designated Ccd, was used as a model for the human disorder. Since malformation of the clavicle is the hallmark of CCD, we studied clavicular development in wild-type and Ccd mice. Histology and in situ hybridization experiments were performed to compare the temporal and spatial expression of several genes in wild-type and Ccd mutant mouse embryos. Bone and cartilage specific markers--type I, II, and X collagens, Sox9, aggrecan, and osteopontin were used as probes. The analyses covered the development of the clavicle from the initial mesenchymal condensation at embryonic day 13 (E13) to the late mineralization stage at embryonic day 15.5. At day 13.5, cells in the center of the condensation differentiate into characteristic precursor cells that were not observed in other bone anlagen. In the medial part of the anlage these cells express markers of the early cartilage lineage (type II collagen and Sox9), whereas cells of the lateral part express markers of the osteoblast lineage (type I collagen). With further development the medial cells differentiate into chondrocytes and start to express chondrocyte-specific markers such as aggrecan. Cells of the lateral part differentiate into osteoblasts as indicated by the production of bone matrix and the expression of osteopontin. At day 14.5 a regular growth plate has developed between the two parts where type X collagen expression can be demonstrated in hypertrophic chondrocytes. The data indicate that the medial part of the clavicle develops by endochondral bone formation while the lateral part ossifies as a membranous bone. The clavicle of Ccd mice showed a smaller band of mesenchymal cell condensation than in wild-type mice. Cells of the condensation failed to express type I and type II collagen at E13.5. In the lateral part of the clavicle type I collagen expression was not detected until E14.5 and osteopontin expression only appeared at E15.5. At E15.5, a small ossification center appears in the lateral part which is, in contrast to the wild-type clavicular bone, solid and without primary spongiosa as well as bone marrow. In the medial portion, type II collagen expression and endochondral ossification never occurs in Ccd mice; this portion of the clavicle is therefore missing in Ccd.

Collagen XVIII/endostatin structure and functional role in angiogenesis.

The angiogenesis inhibitor endostatin is a 20 kDA C-terminal fragment of collagen XVIII, a proteoglycan/collagen found in vessel walls and basement membranes. The endostatin fragment was originally identified in conditioned media from a murine endothelial tumor cell line. Endostatin inhibits endothelial cell migration in vitro and appears to be highly effective in murine in vivo studies. The molecular mechanisms behind the inhibition of angiogenesis have not yet been elucidated. Studies of the crystal structure of endostatin have shown a compact globular fold, with one face particularly rich in arginine residues acting as a heparin-binding epitope. It was initially suggested that zinc binding was essential for the antiangiogenic mechanism but later studies indicate that zinc has a structural rather than a functional role in endostatin. The generation of endostatin or endostatin-like collagen XVIII fragments is catalyzed by proteolytic enzymes, including cathepsin L and matrix metalloproteases, that cleave peptide bonds within the protease-sensitive hinge region of the C-terminal domain. The processing of collagen XVIII to endostatin may represent a local control mechanism for the regulation of angiogenesis.

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, Fisher's 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.