Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that induces a variety of biological responses in diverse cell types. Many, if not all, of these responses are mediated by members of the EDG (endothelial differentiation gene) family G protein-coupled receptors EDG1, EDG3, and EDG5 (AGR16). Among prominent activities of S1P is the regulation of cell motility; S1P stimulates or inhibits cell motility depending on cell types. In the present study, we provide evidence for EDG subtype-specific, contrasting regulation of cell motility and cellular Rac activity. In CHO cells expressing EDG1 or EDG3 (EDG1 cells or EDG3 cells, respectively) S1P as well as insulin-like growth factor I (IGF I) induced chemotaxis and membrane ruffling in phosphoinositide (PI) 3-kinase-and Rac-dependent manners. Both S1P and IGF I induced a biphasic increase in the amount of the GTP-bound active form of Rac. In CHO cells expressing EDG5 (EDG5 cells), IGF I similarly stimulated cell migration; however, in contrast to what was found for EDG1 and EDG3 cells, S1P did not stimulate migration but totally abolished IGF I-directed chemotaxis and membrane ruffling, in a manner dependent on a concentration gradient of S1P. In EDG5 cells, S1P stimulated PI 3-kinase activity as it did in EDG1 cells but inhibited the basal Rac activity and totally abolished IGF I-induced Rac activation, which involved stimulation of Rac-GTPase-activating protein activity rather than inhibition of Rac-guanine nucleotide exchange activity. S1P induced comparable increases in the amounts of GTP-RhoA in EDG3 and EDG5 cells. Neither S1P nor IGF I increased the amount of GTP-bound Cdc42. However, expression of N 17 -Cdc42, but not N 19 -RhoA, suppressed S1P-and IGF I-directed chemotaxis, suggesting a requirement for basal Cdc42 activity for chemotaxis. Taken together, the present results demonstrate that EDG5 is the first example of a hitherto-unrecognized type of receptors that negatively regulate Rac activity, thereby inhibiting cell migration and membrane ruffling.

Recent studies (1-3) provide increasing evidence of roles for lysosphingolipids as mediators to elicit a variety of physiological and pathophysiological responses. Thus, the lysosphingolipids SP 1 and SPC have been shown to evoke diverse cellular responses in various cell types, including mitogenesis (1, 2), inhibition of migration (4, 5), cell shape change (6), and microfilament reorganization (6, 7). Stimulation of cells with the lysosphingolipids triggers the activation of multiple intracellular signaling molecules, including phospholipase C (2, 5, 8, 9), phospholipase D (8), PKC (10), MAPK (5, 11), and K ϩ channel (muscarinic K ϩ current) (12). Many of the lysosphingolipidinduced responses are demonstrated to be inhibited by PTX pretreatment (5, 8 -13). In addition, either an increase or a decrease in cellular cAMP content in response to SP has been reported, depending on cell types used (5, 13). These observations suggest the existence of multiple G protein-coupled cell surface receptors for SP and SPC.Recently, the orphan G protein-coupled receptor EDG2 was identified as a functional receptor for LPA (14). Moreover, EDG4 was very recently identified to be the second LPA receptor (15). EDG2 and EDG4 are members of the EDG family of receptors comprising EDG1 (16), EDG3 (17), and AGR16 (18)/ H218 (19), which have 36 -58% homology in amino acid sequences with each other. SP is related in its structure to LPA, and in some cell types, LPA and SP have been suggested to share a cell surface receptor (20, 21). These observations prompted us to examine the possibility that members of the EDG family receptors could function as a receptor for the lysosphingolipids. Many of cell lines usually used for expression of exogenous genes, including COS, NIH3T3 and HEK293 cells, respond to SP (13), which hampered expression cloning of SP receptor gene and functional analysis of cloned SP receptor gene. In the present study, by using carefully selected mammalian cell expression systems, we found that EDG1 is a functional receptor with a high specificity and affinity for SP. We demonstrate that EDG1 is coupled via a G i/o protein to multiple effector pathways, including phospholipase C, adenylate cyclase, and Ras/MAPK. MATERIALS AND METHODS Cells-CHO-K1(CHO) and HEL cells, obtained from RIKEN CellBank and the Japanese Cancer Research Resources Bank (Tokyo, Japan), respectively, were grown in Ham's F-12 (CHO) and RPMI (HEL) media supplemented with 10% fetal calf serum (Equitech-Bio, Ingram, TX), 100 units/ml penicillin, and 100 g/ml streptomycin (Wako Pure Chemicals, Osaka, Japan). Before each experiment, cells were switched to the respective medium supplemented with 1% fetal calf serum.

Hepatocyte growth factor (HGF) is a potent angiogenic factor. The efficacy and safety of intramuscular injection of a naked plasmid encoding human HGF gene (beperminogene perplasmid, Collategene) was investigated in patients with critical limb ischemia (CLI) in a multicenter, randomized, double-blind, placebo-controlled trial. The randomization ratio for plasmid to placebo was 2:1. Injection sites were selected in each patient limb based on angiographic findings. Placebo or plasmid was injected on days 0 and 28. Evaluation of efficacy was carried out after 12 weeks. The primary end point was the improvement of rest pain in patients without ulcers (Rutherford 4) or the reduction of ulcer size in patients with ulcer(s) (Rutherford 5).Secondary end points were ankle-brachial pressure index, amputation, and quality of life (QOL). Forty-four patients were treated, and we performed interim analysis of efficacy in 40 patients. The overall improvement rate of the primary end point was 70.4% (19/27) in HGF group and 30.8% (4/13) in placebo group, showing a significant difference (P ¼ 0.014). In Rutherford 5 patients, HGF achieved a significantly higher improvement rate (100% [11/11]) than placebo (40% [2/5]; P ¼ 0.018). HGF plasmid also improved QOL. There were no major safety problems. HGF gene therapy is safe and effective for CLI.

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Background— Surgical interventions have been performed to ameliorate the complications of Takayasu’s arteritis. However, the efficacy of surgery to increase long-term survival has not been established. Methods and Results— A retrospective review was performed on the survival of 106 consecutive patients with Takayasu’s arteritis who underwent surgical treatment during the past 40 years. Their ages ranged from 5 to 69 years (mean±SEM, 31.7±1.3 years). Survival was compared with the reported results of medically treated patients according to Ishikawa’s prognostic classification. There were 12 hospital deaths, and the remaining 94 patients were followed up from 8 months to 41.8 years (mean, 19.8 years). A serious long-term complication was anastomotic aneurysm, with a cumulative incidence at 20 years of 13.8%. Thirty-one late deaths were observed, and the major cause was congestive heart failure. The overall cumulative survival rate at 20 years was 73.5%. The prognostic classification by Ishikawa had little influence on the survival of surgically treated patients. For stage 3 patients, surgery seemed to increase survival; however, surgery-related complications conversely decreased the survival of stage 1 patients. Conclusions— Surgery seems to increase the long-term survival of patients with stage 3 Takayasu’s arteritis, whereas conservative treatment is recommended for those with stage 1 or 2 disease. An anastomotic aneurysm may occur at any time after surgery, and regular follow-up using imaging modalities such as multi-detector CT, MRI, or ultrasonography at least once every several years for the rest of the patient’s life is mandatory for the early detection of anastomotic aneurysm.

Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor (bFGF), a new strategy for the treatment of chronic vascular occlusive disease, was examined in a rabbit model of hind limb ischemia. The left femoral artery was completely excised to induce an ischemic state in the hind limb of male rabbits. Simultaneously, a skin section was resected from the wound, and host fibroblasts were cultured. The cultured fibroblasts were infected with adenovirus vector containing modified human bFGF cDNA with the secretory signal sequence (AxCAMAssbFGF) or LacZ cDNA (AxCALacZ). At 21 days after femoral artery excision, the gene-transduced fibroblasts were administered through the left internal iliac artery. The fibroblasts significantly accumu-

These findings suggested that bFGF-induced collateral development exceeded VEGF-induced collateral development in the induction of arteriogenesis, and that combined gene delivery of VEGF and bFGF produced additive or synergistic effects of collateral development as compared with the effects induced by transfer of each gene alone.

Spontaneous dissection of the splanchnic arteries is rare and reportedly carries a high risk of mortality. Two cases with spontaneous dissection of the main trunk of the superior mesenteric artery followed by a self-limited clinical course are presented. Current management strategies, including bypass operation, patch angioplasty, and conservative treatments, are discussed. Emphasis is placed on the role of nonsurgical management with careful follow-up with the use of new technologies such as duplex and computed tomography scanning.