The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis

PTEN is an important tumor suppressor gene. Hereditary mutation of PTEN causes tumor-susceptibility diseases such as Cowden disease. We used the Cre-loxP system to generate an endothelial cell-specific mutation of Pten (Tie2CrePten) in mice. Tie2CrePten(flox/+) mice displayed enhanced tumorigenesis due to an increase in angiogenesis driven by vascular growth factors. This effect was partially dependent on the PI3K subunits p85alpha and p110gamma. In vitro, Tie2CrePten(flox/+) endothelial cells showed enhanced proliferation/migration. Tie2CrePten(flox/flox) mice died before embryonic day 11.5 (E11.5) due to bleeding and cardiac failure caused by impaired recruitment of pericytes and vascular smooth muscle cells to blood vessels, and of cardiomyocytes to the endocardium. These phenotypes depend strongly on p110gamma rather than on p85alpha and were associated with decreased expression of Ang-1, VCAM-1, connexin 40, and ephrinB2 but increased expression of Ang-2, VEGF-A, VEGFR1, and VEGFR2. Pten is thus indispensable for normal cardiovascular morphogenesis and post-natal angiogenesis, including tumor angiogenesis.     

Apoptotic signaling pathway activated by Helicobacter pylori infection and increase of apoptosis-inducing activity under serum-starved conditions

The enhanced gastric epithelial cell apoptosis observed during infection with Helicobacter pylori has been suggested to be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. To investigate the cell death signaling induced by H. pylori infection, human gastric epithelial cells were incubated with H. pylori for up to 72 h. H. pylori infection induced the activation of caspase -8, -9, and -3 and the expression of the proapoptotic Bcl-2 family proteins Bad and Bid. The peak of the activity of the caspases occurred at 24 h. At this time, the inhibition of caspase-8 or -9 almost completely suppressed H. pylori-induced apoptosis. Inhibition of caspase-8 suppressed the expression of Bad and Bid and the subsequent activation of caspase-9 and -3. These observations indicate that H. pylori induces apoptosis through a pathway involving the sequential induction of apical caspase-8 activity, the proapoptotic proteins Bad and Bid, caspase-9 activity, and effector caspase-3 activity. Activation of the pathway was independent of CagA or vacuolating toxin. A membrane fraction of H. pylori was sufficient to activate this pathway, and treatment with proteinase K eliminated the activity. Apoptotic activity of the membrane fraction was significantly increased by incubating the bacteria under serum-starved conditions for 24 h. These observations suggest that environmental conditions in the human stomach could induce H. pylori-mediated pathogenesis, leading to a variety of clinical outcomes.     

Neuroexcitatory actions of Tamiflu and its carboxylate metabolite

Oseltamivir (Tamiflu) is now being stockpiled by several governments as a first line treatment for an anticipated outbreak of avian influenza caused by H5N1. However, abnormal behaviors and death associated with the use of Tamiflu have developed into a major issue in Japan where Tamiflu is often prescribed for seasonal influenza. Thus, it is critical to determine neuropsychiatric effects of oseltamivir and to establish methods for safe administration. Using juvenile rats and rat hippocampal slices, we investigated whether oseltamivir has adverse effects on the central nervous system. Systemic injection of oseltamivir (50 mg/kg i.p.) produced no change in behavior within 2 h. However, prior injection of oseltamivir significantly altered the duration of loss of lightning reflex following ethanol injection (3.3 g/kg, i.p.). Ethanol injection in the presence of oseltamivir also resulted in enhanced hypothermia. In the CA1 region of hippocampal slices, oseltamivir (100 μM) induced paired-pulse facilitation in population spikes without changes in excitatory postsynaptic potentials. Similarly, 3 μM oseltamivir carboxylate, the active metabolite of oseltamivir, facilitated neuronal firing, though the facilitation did not involve GABAergic disinhibition. Moreover, oseltamivir carboxylate produced further facilitation following administration of 60 mM ethanol. These findings indicate that oseltamivir has effects on the central nervous system, especially when combined with other agents.     

Cats are protected against feline immunodeficiency virus infection following vaccination with a homologous AP-1 binding site-deleted mutant

Summary.  Following establishment, via the vaginal route, of infection with an AP-1 binding-site deleted mutant (ΔAP-1) of feline immunodeficiency virus (FIV), cats were challenged with a homologous intact strain (TM2) of FIV. The cats were observed for 23 weeks to evaluate the efficacy of the ΔAP-1 against the homologous TM2 strain challenge. These two viruses were differentiated by Southern blotting after amplification of proviral DNA by semi-nested polymerase chain reaction in DNAs of peripheral blood mononuclear cells and tissues. A TM2-specific band was detected in one cat exposed to but not infected with ΔAP-1, but not in two ΔAP-1-infected. These results indicate that ΔAP-1 could protect against subsequent challenge with homologous FIV TM2 strain. Received December 23, 1998 Accepted March 31, 1998     

Correlation of C3d fixing circulating immune complexes with disease activity and clinical parameters in patients with systemic lupus erythematosus

Using anti-C3d as a solid phase reagent, C3d fixing circulating immune complexes (CIC) were detected in sera from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis, membranous nephropathy and IgA nephropathy. Particularly, sera from SLE showed the highest CIC levels and highest incidence of positivity among these diseases. In the 51 serum samples from 48 patients with SLE we studied, the CIC detected by the anti-C3d assay correlated well (P less than 0.01) with the CIC detected by the solid phase C1q assay, but not with those detected by the conglutinin assay. In addition, the CIC detected by the anti-C3d assay correlated more significantly (P less than 0.001) with disease activity, as well as some clinical parameters (serum anti-dsDNA antibodies, CH50 and C3 levels) than CIC detected by the other two assays of SLE sera. The anti-C3d binding materials were found to be of intermediate (8-19S) and small (7S) sizes in a small number of SLE sera which we analysed.     

Significance of beta-catenin and pRB pathway components in malignant ovarian germ cell tumours: INK4A promoter CpG island methylation is associated with cell proliferation

To clarify the mechanisms underlying cell cycle promotion in malignant germ cell tumours of the ovary (MGCTOs), beta-catenin and components of the pRB pathway, cyclin D1 and p16, were analysed in relation to cell proliferation. Immunohistochemically, p16 protein was not expressed in a number of MGCTOs (9 of 42 tumours: 21.4%) and was associated with p16 gene (INK4A) promoter 5'-CpG islands methylation. Amplification of the cyclin D1 gene (CCND1) was detected in a small number of MGCTOs (5 of 42 tumours: 13.5%). Reduced expression of p16 due to promoter methylation correlated significantly with increased cell proliferation as evidenced by Ki-67 labelling index (p < 0.001) and mitotic index (p < 0.01). In some tumour types, nuclear localization of beta-catenin has been reported to be associated with beta-catenin gene (CTNNB1) mutation, cyclin D1 overexpression, and increased cell proliferation. Nuclear localization of beta-catenin, which was observed in MGCTOs other than dysgerminoma, was not associated with cyclin D1 expression and increased cell proliferation, but appeared to be related to tumour differentiation. Furthermore, CTNNB1 mutations were not detected in any of the MGCTOs examined. Our results suggest that reduced expression of p16 due to INK4A promoter methylation is one of the principal factors that promote cell proliferation in MGCTOs. Thus, p16 may be a novel target for gene therapies to treat MGCTOs.     

Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test

In order to evaluate the utility of the mouse lymphoma assay(MLA) for detecting in vitro clastogens and spindle poisonsand to compare it with the in vitro chromosomal aberration test(CA), we conducted an international collaborative study of theMLA that included 45 Japanese laboratories and seven overseaslaboratories under the cooperation of the Ministry of Healthand Welfare of Japan and the Japanese Pharmaceutical Manufacturer'sAssociation. We examined 40 chemicals; 33 were reportedly positivein the CA but negative in the bacterial reverse mutation assay,six were negative in both assays and one was positive in both.We assayed mutations of the thymidine kinase (TK) locus (tk)of L5178Y tk^+/– mouse lymphoma cells using the microwellmethod. According to our standard protocol, cells were exposedto the chemical for 3 h, cultured for 2 days and TK-deficientmutants were expressed in 96-well plates under trifluorothymidine.Each chemical was coded and tested by two or three laboratories.Among the 34 CA-positive chemicals, positive MLA results wereobtained for 20 and negative results were obtained for nine.The remaining five chemicals were inconclusive or equivocalbecause of discrepant inter-laboratory results or reproduceddiscrepant results, respectively. Among the six CA-negativechemicals, one was negative in the MLA, two were positive andthree were inconclusive. Thus, the MLA could detect only 59%(20/34) of CA-positive chemicals. We concluded that the MLAwas not as sensitive as the CA. Some MLA-negative chemicalsevoked positive responses in the CA only after long continuoustreatment. These might also be genotoxic in the MLA with longcontinuous treatment. Improvement of the MLA protocol, includingalteration of the duration of the treatment, might render theMLA as sensitive as the CA. ⁸ To whom correspondence should be addressed. Tel: +81 3 37009847; Fax: +81 3 3700 2348; Email: sofuni{at}nihs.go.jp     

Does gene therapy become pharmacotherapy?

Recent progress in molecular and cellular biology has led to the development of numerous effective cardiovascular drugs. However, there are still a number of diseases for which no known effective therapy exists, such as peripheral arterial disease, ischaemic heart disease, restenosis after angioplasty, and vascular bypass graft occlusion. Currently, gene therapy is emerging as a potential strategy for the treatment of cardiovascular disease despite its limitations. The first human trial in gene therapy for cardiovascular disease was started at 1994 to treat peripheral vascular disease using vascular endothelial growth factor (VEGF). Then, many different potent angiogenic growth factors were tested in clinical trials to treat peripheral arterial disease and ischaemic heart disease. Improvement of clinical symptoms in peripheral arterial disease and ischaemic heart disease has been reported. This review focuses on the future potential of gene therapy for the treatment of cardiovascular disease. In the future, gene therapy might become a real pharmacotherapy to treat cardiovascular disease.     

Two different roles of purified CD45+c-Kit+Sca-1+Lin- cells after transplantation in muscles

Recent studies have indicated that bone marrow cells can regenerate damaged muscles and that they can adopt phenotypes of other cells by cell fusion. Our direct visualization system gave evidence of massive muscle regeneration by green fluorescent protein (GFP)-labeled CD45+c-Kit+Sca-1+Lin- cells (KSL cells), and we investigated the role of KSL cells in muscle regeneration after transplantation with or without lethal irradiation. In the early phase, GFP signals were clearly observed in all the muscles of only irradiated mice. Transverse cryostat sections showed GFP+myosin+ muscle fibers, along with numerous GFP+ hematopoietic cells in damaged muscle. These phenomena were temporary, and GFP signals had dramatically reduced 30 days after transplantation. After 6 months, GFP+ fibers could hardly be detected, but GFP+c-Met+ mononuclear cells were located beneath the basal lamina where satellite cells usually exist in both conditioned mice. Immunostaining of isolated single fibers revealed GFP+PAX7+, GFP+MyoD+, and GFP+Myf5+ satellite-like cells on the fibers. Single-fiber cultures from these mice showed proliferation of GFP+ fibers. These results indicate two different roles of KSL cells: one leading to regeneration of damaged muscles in the early phase and the other to conversion into satellite cells in the late phase.     

A case of mixed connective tissue disease developed into overlap syndrome of progressive systemic sclerosis, systemic lupus erythematosus, polymyositis and Sj?gren's syndrome

We encountered a patient who developed an overlap syndrome of progressive systemic sclerosis (PSS), systemic lupus erythematosus (SLE), polymyositis (PM) and Sj?gren's syndrome (SjS) while we were treating her for mixed connective tissue disease (MCTD). This 42-year-old woman had been photosensitive since 18 years of age. In 1986, Raynaud's phenomenon, swollen hands and arthralgia appeared; therefore, we started to treat this patient based on a diagnosis of MCTD. At that time, her anti-RNP antibody titer was 82,920, but she was negative to anti-Sm antibody. In 1988, she was admitted to our hospital with chief complaints of aggravation of polyarthralgia and myalgia. On physical examination, she showed difficulty in opening her mouth, systemic dermal sclerosis, a decrease in muscular strength and rales. In laboratory tests, her myogenic enzyme level was increased, and she was found to be positive to LE cells, antinuclear antibody, anti-DNA antibody, anti-ENA antibody and anti-SSA antibody. Furthermore, histological features clearly corresponding to those of PSS were found by skin biopsy, myogenic changes by electromyography, evidence of chronic inflammation of the salivary glands by lip biopsy, and proliferative changes in the mesangium were detected by renal biopsy. The concept of MCTD, especially the differences from overlap syndrome, is vague. Therefore we need further study about many cases. Since there have been no reports on cases having sufficient evidence of the development of the overlap syndrome of PSS, SLE, PM and SjS during a course of MCTD, our patient would provide very useful data contributing to the study of MCTD.