Hideaki Onda

Molecular cloning of a novel brain-type Na(+)-dependent inorganic phosphate cotransporter.

Abstract: We have isolated a human cDNA encoding a protein, designated DNPI, that shows 82% amino acid identity and 92% similarity to the human brain‐specific Na+‐dependent inorganic phosphate (Na+/Pi) cotransporter (BNPI), which is localized exclusively to neuron‐rich regions. Expression of DNPI mRNA in Xenopus oocytes resulted in a significant increase in Na+‐dependent Pi transport, indicating that DNPI is a novel Na+/Pi cotransporter. Northern blot analysis shows that DNPI mRNA is expressed predominantly in brain, where the highest levels are observed in medulla, substantia nigra, subthalamic nucleus, and thalamus, all of which express BNPI mRNA at low levels. In contrast, DNPI mRNA is expressed at low levels in cerebellum and hippocampus, where BNPI mRNA is expressed at high levels. No hybridizing signal for DNPI mRNA is observed in the glia‐rich region of corpus callosum. In other regions examined, both mRNAs are moderately or highly expressed. These results indicate that BNPI and DNPI, which coordinate Na+‐dependent Pi transport in the neuron‐rich regions of the brain, may form a new class within the Na+/Pi cotransporter family.

Genomewide-Linkage and Haplotype-Association Studies Map Intracranial Aneurysm to Chromosome 7q11

Rupture of intracranial aneurysms (IAs) causes subarachnoid hemorrhage, a devastating condition with high morbidity and mortality. Angiographic and autopsy studies show that IA is a common disorder, with a prevalence of 3%-6%. Although IA has a substantial genetic component, little attention has been given to the genetic determinants. We report here a genomewide linkage study of IA in 104 Japanese affected sib pairs in which positive evidence of linkage on chromosomes 5q22-31 (maximum LOD score [MLS] 2.24), 7q11 (MLS 3.22), and 14q22 (MLS 2.31) were found. The best evidence of linkage is detected at D7S2472, in the vicinity of the elastin gene (ELN), a candidate gene for IA. Fourteen distinct single-nucleotide polymorphisms (SNPs) were identified in ELN, and no obvious allelic association between IA and each SNP was observed. The haplotype between the intron-20/intron-23 polymorphism of ELN is strongly associated with IA (P=3.81x10-6), and homozygous patients are at high risk (P=.002), with an odds ratio of 4.39. These findings suggest that a genetic locus for IA lies within or close to the ELN locus on chromosome 7.

Identification of genes differentially expressed in canine vasospastic cerebral arteries after subarachnoid hemorrhage.

To understand the molecular processes of continuous vasospasm of cerebral arteries after subarachnoid hemorrhage, mRNA differential display and screening of cDNA expression array were performed to identify genes that are differentially expressed in vasospastic arteries of canine two-hemorrhage models. The expression levels of 18 genes were found to be upregulated, and those of two genes to be down-regulated. Of these, 12 represent known genes or homologues of genes characterized previously, and the other eight genes are not related to any sequences in the databases. The known genes include five upregulated inflammation-related genes encoding monocyte chemotactic protein-1, cystatin B, inter-α-trypsin inhibitor family heavy chain-related protein, serum amyloid A protein, and glycoprotein 130, suggesting that inflammatory reaction may be involved in the development of cerebral vasospasm. The upregulation of three known genes encoding stress-related proteins of vascular endothelial growth factor, BiP protein, and growth-arrest and DNA-damage–inducible protein may be involved in possible cell survival in the damaged arteries. A full-length cDNA for the unknown clone DVS 27, whose expression was most highly upregulated, was isolated from the cerebral artery cDNA library by hybridization. Characterization of these genes should help to clarify the molecular mechanism of continuous cerebral vasospasm after subarachnoid hemorrhage.

Quantitative Analysis of Gene Expressions Related to Inflammation in Canine Spastic Artery After Subarachnoid Hemorrhage

Background and Purpose— The possible role of inflammatory reaction of the cerebral artery in the pathogenesis of cerebral vasospasm has been noted in recent studies. We quantitatively measured the levels of expression of genes related to inflammation in the spastic artery in a canine double-hemorrhage model. Methods— Twenty dogs were assigned to 4 groups: group D0, control; group D2, dogs killed 2 days after cisternal injection of blood; group D7, dogs given double cisternal injections of blood and killed 7 days after the first injection; and group D14. Angiography was performed twice: on the first day and before the animals were killed. Total RNA was extracted from the basilar artery. The expressions of interleukin (IL)-1&agr;, IL-6, IL-8, IL-10, tumor necrosis factor-&agr;, E-secretin, fibronectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule-1, transforming growth factor-&bgr;, basic fibroblast growth factor, and collagen types I, III, and IV were examined with TaqMan real-time quantitative reverse transcription–polymerase chain reaction. Results— Prolonged arterial narrowing peaking on 7 day was observed. There was a significant difference in vessel caliber between D0, D2, D7, and D14 groups (P <0.0001). There were significant differences in mRNA expression in the basilar artery for IL-1&agr;, IL-6, IL-8, ICAM-1, and collagen type I between D0, D2, D7, and D14 groups (P =0.0079, 0.0196, 0.0040, 0.0017, and <0.0001, respectively). The average level of mRNA was highest in D7 for IL-1&agr;, IL-6, IL-8, and ICAM-1 (17-, 16-, 131-, and 1.7-fold compared with those of D0, respectively) and in D14 for collagen type I (10.9-fold). Conclusions— Increased expression of genes related to inflammation in the spastic artery suggests that inflammatory reaction of the cerebral artery is associated with sustained contraction.

Application of Nicardipine Prolonged-release Implants: Analysis of 97 Consecutive Patients with Acute Subarachnoid Hemorrhage

To the Editor: We read with great interest the article by Kasuya et al. (2) on the application of nicardipine prolonged-release implants for subarachnoid hemorrhage. They analyzed 97 consecutive patients and compared 69 treated patients with 28 untreated patients. The authors concluded that application of local nicardipine prolonged-release implants leads to a reduced incidence of a delayed ischemic neurological deficit. Although the rate of clinical vasospasm could be roughly reduced by 50%, we would have expected a more marked effect for a therapy with the potential of adding a new dimension to the current strategy of prevention and treatment of cerebral vasospasm. However, some shortcomings of the currently reported methodology and the study design still leave hope that local intrathecal therapy of cerebral vasospasm can become a significant step forward. This prospective nonrandomized Phase II-type study resulted in a comparison between two inhomogeneous patient groups concerning the known risk factors of cerebral vasospasm. The control group without local pharmacological treatment consisted of 16 Fisher Grade 2 patients, nine Fisher Grade 3 patients, and three Fisher Grade 4 patients. Therefore, the natural risk for vasospasm differed between the pharmacologically treated group with a great majority of Fisher Grade 3 patients and the control group with predominant Fisher Grades of 2 and 4 and, therefore, a lower risk of vasospasm (1, 4). Besides, it is important to emphasize, that an incidence of clinical vasospasm of 11% in the untreated group and 6% in the medical group seems unusually low for a patient group dominated by Fisher Grade 3 hemorrhages. The current report leaves some open questions with regard to pharmacokinetics and effectiveness of the clinical set-up. The patients received from 2 to 12 pellets, resulting in different dosages. There was no correlation done between the quantity of nicardipine and the incidence of vasospasm. Only a positive local effect as documented by angiography was available as control. There was no monitoring of cerebrospinal fluid (CSF) levels of nicardipine. It seems unlikely that the released nicardipine could be sequestrated within the local cistern by the surrounding blood clots. In an animal study done on dogs, Pradilla et al. (3) documented an excellent diffusion of Evans blue dye from controlledrelease polymers throughout the subarachnoid space after experimental hemorrhage. In summary, we believe in the potential benefit of local therapy as a major step ahead in the treatment of vasospasm. Nicardipine may not be the optimum agent, or the dosing must be optimized. Perhaps a cocktail of several agents achieves a superior effect.

Clinical analysis of incidentally discovered unruptured aneurysms.

Background and Purpose We analyzed the risk factors for rupture of an intracranial aneurysm based on a retrospective angiographic study of ruptured and unruptured aneurysms. Methods The 44 cases of asymptomatic aneurysms were selected from 1612 patients whose lesions had been discovered fortuitously by angiography (2.7%) during the period from 1980 to 1989. All these patients were free from any sign of intracranial aneurysm. The variations in age, sex, and location of the aneurysms were analyzed compared with 638 ruptured aneurysms that had been treated in our institute during the same period. The size, shape, and arterial geometry of the unruptured aneurysms were examined angiographically. Results Unruptured aneurysm was discovered fortuitously in 44 (2.7%) of 1612 patients, with greater incidence in women aged older than 60 years. Unruptured aneurysms were less likely to occur in the anterior communicating artery (12.8%) and the middle cerebral artery (6.4%). However, they were frequently found in the internal carotid artery, with an incidence of 10.6% in the cavernous portion of the internal carotid artery, 19.1% in the internal cartoid-ophthalmic artery, 19.1% in the internal carotid-posterior communicating artery, and 12.8% in the internal carotid-anterior choroidal artery. Seven of the nine internal carotid-posterior communicating artery aneurysms showed a hypoplastic or aplastic posterior communicating artery. The mean diameter of the unruptured aneurysms was 4.8 mm, and 80% were smaller than 6 mm. Conclusions Intracranial aneurysms are formed not only at the bifurcation of an artery but also at its branching and bending points. However, an aneurysm located at the bifurcation, such as the anterior communicating artery and the middle cerebral artery, bleeds easily in contrast with lateral aneurysms such as those found at the branching and bending points on the internal carotid artery.

Efficacy and Safety of Nicardipine Prolonged-Release Implants for Preventing Vasospasm in Humans

Background and Purpose— Despite extensive investigative efforts, there are few treatments that can prevent vasospasm after subarachnoid hemorrhage. This study was conducted to examine the efficacy and safety of nicardipine prolonged-release implants (NPRI) for humans, which have already been proven in dogs. Methods— Twenty consecutive subarachnoid hemorrhage patients with thick subarachnoid clot were treated with NPRI (a pellet of diameter 2 mm, length 10 mm, containing 4 mg of nicardipine) during surgery after clipping of their aneurysm. The number and location of pellets depended on the amount and site of subarachnoid clot on preoperative CT and on craniotomy. Results— Two to 10 pellets were implanted in the cistern of the internal carotid, middle cerebral, and/or anterior cerebral artery, where thick clots existed and therefore vasospasm related to delayed ischemic neurological deficits was highly likely. Delayed ischemic neurological deficits and cerebral infarctions were seen in 1 patient. Angiography performed on days 7 to 12 revealed no vasospasm in any arteries near which NPRI were placed. No complications were experienced. Conclusions— Vasospasm was completely prevented for the arteries in thick clot cisterns, when NPRI were placed adjacent to the arteries during surgery. This drug-delivery system offers a promising approach for preventing vasospasm.

Collagen Type I α2 (COL1A2) Is the Susceptible Gene for Intracranial Aneurysms

Background and Purpose— The collagen &agr;2(I) gene (COL1A2) on chromosome 7q22.1, a positional and functional candidate for intracranial aneurysm (IA), was extensively screened for susceptibility in Japanese IA patients. Methods— Twenty-one single nucleotide polymorphisms (SNPs) of COL1A2 were genotyped in genomic DNA from 260 IA patients (including 115 familial cases) (mean age, 59.9 years) and 293 controls (mean age, 61.6 years). Differences in allelic and genotypic frequencies between the patients and controls were evaluated with the &khgr;2 test. Circular dichroism spectrometry was monitored with collagen-related peptides that mimic triple-helical models of type I collagen with Ala-459 and Pro-459 to estimate the conformation and stability of alterations. Results— Significant genotypic association in the dominant model was observed between an exonic SNP of COL1A2 and familial IA patients (&khgr;2=11.08; df =1; P =0.00087; odds ratio=3.19; 95% CI, 2.22 to 6.50). This SNP induces Ala to Pro substitution at amino acid 459, located on a triple-helical domain. Circular dichroism spectra showed that the Pro-459 peptide had a higher thermal stability than the Ala-459 peptide. Conclusions— The variant of COL1A2 could be a genetic risk factor for IA patients with family history.

Identification of mutations in the hepatocyte nuclear factor (HNF)-1 alpha gene in Japanese subjects with IDDM

One form of maturity-onset diabetes of the young, MODY3, is characterized by a severe insulin secretory defect, compared with MODY2, a glucokinase-deflcient diabetes. It has recently been shown that mutations of the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1α cause MODY3. Because of the rapid progress to overt diabetes and the high prevalence of required insulin treatment in patients with MODY3, we screened the HNF-1α gene for mutations in Japanese subjects with IDDM. Ten exons and flanking introns of the HNF-1α gene in these subjects were amplified by polymerase chain reaction and direct sequencing of the products. Mutations were identified in three (5.5%) of the 55 unrelated subjects with IDDM. A missense mutation of R272H (replacement of Arg by His in codon 272) in the DNA binding domain of HNF-1α was found in a subject who developed IDDM 1 year after diagnosis of NIDDM at 8 years of age. A frameshift mutation of P291fsinsC (insertion of a C in a polyC tract around codon 291 for Pro), which would generate a mutant truncated protein of 340 amino acids, was found in a subject who started insulin treatment when hyperglycemia and ketonuria were noticed at 13 years of age. A missense mutation of R583G (replacement of Arg by Gly in codon 583) in the transactivation domain of HNF-1α was found in a subject with sudden-onset IDDM at 20 years of age. None of these mutations were present in 100 nondiabetic subjects (200 normal chromosomes). These results indicate that the HNF-1α gene defects could lead to the development of not only early-onset NIDDM but also IDDM, implicating the importance of subclassification of HNF-1α-deficient IDDM from a classical type of autoimmune-based IDDM in Japanese.

Role of p38 Mitogen-Activated Protein Kinase on Cerebral Vasospasm After Subarachnoid Hemorrhage

Background and Purpose— Inflammatory cytokines are involved in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). This study was conducted to examine the role of p38 mitogen-activated protein kinase (MAPK) in the development of vasospasm and cytokine production. Methods— We measured the expression levels of genes and proteins related to inflammation in human vascular smooth muscle cells (hVSMCs) treated with hemolysate and FR167653 (FR) (1μmol/L), a selective p38MAPK inhibitor, for 48 hours by TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) and ELISA. Twenty-one dogs were assigned to 3 groups of 7 animals: control, placebo, and FR-treated (1 mg/kg/d) groups in a double-hemorrhage model. The effects were assessed through the caliber of the basilar artery, and the changes in gene expressions and the activation of p38MAPK were assessed by Western blot analysis. Results— Treatment of hVSMCs with hemolysate induced significant upregulation of interleukin (IL)-1α, IL-1β, and IL-8 gene and protein expressions, which was suppressed significantly with FR. The mean vessel caliber on day 7, as a percentage of that of day 0, was 49% in the placebo, and 74% in the FR group (P = 0.0001). The gene expression levels of IL-1α, IL-1β, and IL-8 in the arterial wall were extremely elevated in the placebo, and significantly suppressed in the FR group (P = 0.0027, 0.0002, and 0.0073). p38MAPK phosphorylation was stimulated in the placebo and hemolysate in vitro, and suppressed in the FR group. Conclusions— These results suggest that p38MAPK is activated in the arterial wall after SAH, leading to the development of vasospasm, possibly through the upregulation of inflammatory cytokines.