Intramuscular gene transfer of FGF-2 attenuates endothelial dysfunction and inhibits intimal hyperplasia of vein grafts in poor-runoff limbs of rabbit

We previously demonstrated that sustained disturbance of endothelium-dependent vasorelaxation and poor distal runoff in ischemic limbs were critical factors affecting the neointimal development of autologous vein grafts (VGs). Also, we recently showed the superior therapeutic potential of basic fibroblast growth factor (bFGF/FGF-2) boosted by the recombinant Sendai virus (SeV) for severe limb ischemia compared with that of vascular endothelial growth factor. Here, the effect of FGF-2 on neointimal hyperplasia of VGs was examined in a rabbit model of poor-runoff limbs. Two weeks after initial surgery for the induction of poor-runoff, SeV-expressing human FGF-2 (SeV-hFGF2) or that encoding firefly luciferase (109 plaque-forming units/head) was injected into the thigh and calf muscle. At that time, the femoral vein was implanted in the femoral artery in an end-to-end manner in some groups. FGF-2 gene-transferred limbs demonstrated significantly increased blood flow assessed not only by laser Doppler flow image but also by ultrasonic transit-time flowmeter (USTF). USTF also showed a significant increase in the blood flow ratio of the deep femoral artery to external iliac artery, indicating that collateral flow was significantly restored in the thigh muscles (P < 0.01). Reduction of neointimal hyperplasia was also observed in the VGs treated by SeV-hFGF2; these grafts demonstrated significant restoration of endothelium-dependent vasorelaxation. These findings thus extend the indications of therapeutic angiogenesis using SeV-hFGF2 to include not only limb salvage but also prevention of late graft failure.     

Identification of a stable chymase inhibitor using a pharmacophore-Based database search

In general, serine protease chymase inhibitors readily decompose in plasma. We previously found that thiazolidine-2,4-dione and thiadiazole derivatives are also unstable. Using a pharmacophore-based database search, we identified a benzo[b]thiophen-2-sulfonamide derivative as a stable chymase inhibitor. Finding a lead compound with adequate activity and stability by a pharmacophore-based approach is more efficient than modifying an unstable compound to reduce its instability without simultaneously decreasing its inhibitory activity. Our pharmacophore model of chymase inhibitors suggests that the two hydrophobic interactions in the S1 and S1' regions and the two H-bonding interactions between them play important roles in chymase inhibitors.     

Interisland Mutation of a Novel Phospholipase A<Subscript>2</Subscript> from <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom and Evolution of Crotalinae Group II Phospholipases A<Subscript>2</Subscript>

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A₂ (PLA₂). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA₂, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD₅₀ for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA₂s based on the amino acid sequences revealed that neurotoxic PLA₂s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA₂ groups such as PLA₂ type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA₂s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA₂s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA₂s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation. The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB102728 and AB102729.     

Two carboxyl-terminal activation regions of Epstein-Barr virus latent membrane protein 1 activate NF-kappaB through distinct signaling pathways in fibroblast cell lines

Latent membrane protein 1 (LMP1), an Epstein-Barr virus transforming protein, is able to activate NF-kappaB through its carboxyl-terminal activation region 1 (CTAR1) and 2 (CTAR2), but the exact role of each domain is not fully understood. Here we show that LMP1 activates NF-kappaB in different NF-kappaB essential modulator (NEMO)-defective cell lines, but not in cells lacking both IkappaB kinase 1 (IKK1) and 2 (IKK2). Mutational studies reveal that CTAR1, but not CTAR2, mediates NEMO-independent NF-kappaB activation and that this process largely depends on IKK1. Retroviral expression of LMP1 mutants in cells lacking either functional NF-kappaB inducing kinase (NIK), NEMO, IKK1, or IKK2 further illustrates distinct signals from the two activation regions of LMP1 for persistent NF-kappaB activation. One originates in CTAR2, operates through the canonical NEMO-dependent pathway, and induces NFKB2 p100 production; the second signal originates in CTAR1, utilizes NIK and IKK1, and induces the processing of p100. Our results thus help clarify how two functional domains of LMP1 persistently activate NF-kappaB through distinct signaling pathways.     

Capillary malformation-arteriovenous malformation, a new clinical and genetic disorder caused by RASA1 mutations

Capillary malformation (CM), or "port-wine stain," is a common cutaneous vascular anomaly that initially appears as a red macular stain that darkens over years. CM also occurs in several combined vascular anomalies that exhibit hypertrophy, such as Sturge-Weber syndrome, Klippel-Trenaunay syndrome, and Parkes Weber syndrome. Occasional familial segregation of CM suggests that there is genetic susceptibility, underscored by the identification of a large locus, CMC1, on chromosome 5q. We used genetic fine mapping with polymorphic markers to reduce the size of the CMC1 locus. A positional candidate gene, RASA1, encoding p120-RasGAP, was screened for mutations in 17 families. Heterozygous inactivating RASA1 mutations were detected in six families manifesting atypical CMs that were multiple, small, round to oval in shape, and pinkish red in color. In addition to CM, either arteriovenous malformation, arteriovenous fistula, or Parkes Weber syndrome was documented in all the families with a mutation. We named this newly identified association caused by RASA1 mutations "CM-AVM," for capillary malformation-arteriovenous malformation. The phenotypic variability can be explained by the involvement of p120-RasGAP in signaling for various growth factor receptors that control proliferation, migration, and survival of several cell types, including vascular endothelial cells.     

Cloning of rat ABCA7 and its preferential expression in platelets

We cloned the full-length cDNA of a rat orthologue of ABCA7 (rABCA7) from rat platelets. The cDNA of rABCA7 is 6510bp in length and encodes a protein of 2170 amino acids. The amino acid sequence of rABCA7 exhibits homology to those of mouse ABCA7 (92.5% identical in amino acid sequence) and human ABCA7 (76.6%). We obtained two clones of monoclonal antibodies against rABCA7 recognizing different epitopes. Analysis of CHO cells stably expressing rABCA7 by confocal laser-scanning microscopy indicated that rABCA7 is mainly located in the plasma membrane. Western blot analysis of rat tissues revealed that rABCA7 was preferentially expressed in platelets and that its apparent molecular mass was 250kDa. This is the first report of the tissue distribution of rABCA7 at the protein level and is the first reported case of ABC transporters being expressed in platelets, suggesting their important role in platelet function.     

A G protein-coupled receptor responsive to bile acids

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.     

The Alzheimer-related gene presenilin-1 facilitates sonic hedgehog expression in Xenopus primary neurogenesis

We analyzed the influence of presenilins on the genetic cascades that control neuronal differentiation in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduced the number of N-tubulin+ primary neurons and modulated Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increased shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduced shh expression. Both shh and presenilin promoted cell proliferation and apoptosis, but the effects of shh were widely distributed, while those resulting from presenilin injection coincided with the range of shh signaling. We suggest that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling.     

ecNOS gene polymorphism is associated with endothelium-dependent vasodilation in Type 2 diabetes

The association between endothelial constitutive nitric oxide synthase (ecNOS) gene polymorphism and vascular endothelial function has not been clarified. We investigated the impact of ecNOS gene polymorphism on endothelial function in 95 patients with Type 2 diabetes (ecNOS genotype: 4b/b, n = 62; 4b/a, n = 30; 4a/a, n = 3). Flow-mediated (endothelium dependent, FMD) and nitroglycerin-induced (endothelium independent, NTG) vasodilations of the right brachial artery were studied using a phase-locked echotracking system. There were no significant differences in clinical characteristics among the ecNOS genotypes. The FMD was significantly lower in the patients with ecNOS4a allele than in those without ecNOS4a allele (P < 0.05). Multiple regression analysis showed that ecNOS4a allele and mean blood pressure were significant independent determinants for reduced FMD in all patients (R(2) = 0.122, P = 0.0025). The ecNOS4a allele was an independent determinant for reduced FMD in smokers but not in nonsmokers. These results suggest that ecNOS4a allele is a genetic risk factor for endothelial dysfunction in diabetic patients, especially in smokers.