Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization

Retinal vasculogenesis and ischemic retinopathies provide good model systems for study of vascular development and neovascularization (NV), respectively. Vascular endothelial cell growth factor (VEGF) has been implicated in the pathogenesis of retinal vasculogenesis and in the development of retinal NV in ischemic retinopathies. However, insulin-like growth factor-I and possibly other growth factors also participate in the development of retinal NV and intraocular injections of VEGF antagonists only partially inhibit retinal NV. One possible conclusion from these studies is that it is necessary to block other growth factors in addition to VEGF to achieve complete inhibition of retinal NV. We recently demonstrated that a partially selective kinase inhibitor, PKC412, that blocks phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors and several isoforms of protein kinase C (PKC), completely inhibits retinal NV. In this study, we have used three additional selective kinase inhibitors with different selectivity profiles to explore the signaling pathways involved in retinal NV. PTK787, a drug that blocks phosphorylation by VEGF and PDGF receptors, but not PKC, completely inhibited retinal NV in murine oxygen-induced ischemic retinopathy and partially inhibited retinal vascularization during development. CGP 57148 and CGP 53716, two drugs that block phosphorylation by PDGF receptors, but not VEGF receptors, had no significant effect on retinal NV. These data and our previously published study suggest that regardless of contributions by other growth factors, VEGF signaling plays a critical role in the pathogenesis of retinal NV. Inhibition of VEGF receptor kinase activity completely blocks retinal NV and is an excellent target for treatment of proliferative diabetic retinopathy and other ischemic retinopathies.     

Cleavage of integrin by mu-calpain during hypoxia in human endometrial cells

PROBLEM: The distribution and activation of mu-calpain and possible cleavage of integrin in human endometrial cells under hypoxic condition were investigated. METHOD OF STUDY: Human endometrial epithelial and stromal cells were subjected to hypoxia, and subsequently used for immunostaining and western blot analysis. RESULTS: The proform of mu-calpain was detected in the cytoplasm of normal cells, and displayed a substantial decrease after hypoxia. Conversely, the active form of mu-calpain was not detected in normal cells, but was abundant after hypoxia. The cytoplasmic domain of integrin beta3 was also detected in the cytoplasm of endometrial cells. Western blot analysis confirmed that both the proform of mu-calpain and the integrin beta3 cytoplasmic domain decreased during hypoxia. CONCLUSIONS: Mu-calpain is activated in human endometrial cells during hypoxia and that subsequent cleavage of the integrin beta3 cytoplasmic domain may give some adverse effects to the function of human endometrium.     

Variable region sequences of pathogenic anti-mouse red blood cell autoantibodies from autoimmune NZB mice

New Zealand Black (NZB) mice spontaneously develop a severe autoimmune hemolytic anemia due to the production of anti-mouse red blood cell (MRBC) autoantibodies. The contribution of variable region genes and somatic mutations in the pathogenicity of anti-MRBC autoantibodies was investigated by mRNA sequencing of eight NZB anti-MRBC monoclonal autoantibodies, among which five are capable of inducing anemia in BALB/c mice. Here we report that at least three VH gene families (J558, J606 and 3609) and five Vchi subgroups (V chi 8, 9, 19, 21 and 28), in combination with several D, JH and Jchi gene segments, encode anti-MRBC autoantibodies. Thus, the NZB anti-MRBC autoantibodies, whether pathogenic or not, are encoded by a large number of immunoglobulin gene elements and by members of known VH and Vchi gene families with preferential usage of VH gene families most distal to the D regions. The presence of several mutations in the JH gene segments of both IgM and IgG anti-MRBC autoantibodies, whether pathogenic or not, strongly suggests that their VH regions may be highly mutated and that the mechanism of somatic diversification might be important in the generation of anti-MRBC autoantibodies. Our results support the idea that anti-MRBC autoimmune responses are likely to be generated by an antigen-driven mechanism.     

Specific atrial natriuretic peptide binding sites in rat cerebral capillaries

We examined the specific rat 125I-alpha-rat atrial natriuretic peptide(1-28)[ANF-(99-126)] (125I-rANP) binding sites in the cerebral capillaries from the cerebral cortex of male adult Wistar rats. The binding of 125I-rANP at 37 degrees C was saturable and of high affinity with a Kd of approximately 100 pM and Bmax of 152 fmol/mg protein. Divalent cations, Mn2+ (2.2 mM) and Ca2+ (1.8 mM) potently inhibited the binding. The rank order for inhibition of the binding was rANP, alpha-human ANP and ANF-(101-126) greater than ANF-(103-126) and ANF-(103-125)"ANF-(103-123). These data on specific binding sites of ANP in cerebral capillaries suggest a possible role for ANP in the blood-brain permeability of water and electrolytes.     

Receptor autoradiographic evidence of specific brain natriuretic peptide binding sites in the porcine subfornical organ

Specific binding sites of brain natriuretic peptide (BNP), a newly discovered peptide in the subfornical organ (SFO) of porcine brain were investigated, following incubation of related tissue sections with 125I-BNP, then using autoradiography and an image analysis coupled with computer-assisted microdensitometry. Specific 125I-BNP binding sites were found to be localized in the SFO, an area densely labeled by 125I-alpha-rat atrial natriuretic peptide and 125I-(Sar1,Ile8)-angiotensin II. Specific 125I-BNP binding to the SFO was displaced by unlabeled BNP, with a high affinity, and was calculated to be Ka = 0.385 x 10(-9) M and Bmax = 40.1 fmol/mg using a LIGAND computer program. Acquisition of these present findings enhances our knowledge of the physiology of BNP, atrial natriuretic peptides and angiotensin II system in the SFO.     

No association of GSK3beta gene (GSK3B) with Japanese schizophrenia.

Several lines of evidence indicate that glycogen synthase kinase-3beta (GSK3beta) is one of the candidates for schizophrenia-susceptibility factor. However, it has not been reported the association analysis between GSK3beta gene (GSK3B) and Japanese schizophrenia based on linkage disequilibrium (LD). We provide an association analysis using relatively large samples (381 schizophrenia, and 352 controls) after determination of "tag single nucleotide polymorphisms (SNPs)." In this LD mapping, we selected and genotyped for eight polymorphisms (seven SNPs and one diallelic (CAA)(n) repeat), which covered the entire region of GSK3B, and determined two "tag SNPs." In the following association analysis using these two "tag SNPs," we could not find association with Japanese schizophrenia. Furthermore, we also include subgroup analysis considering age-at-onset and subtypes, neither could we find associations. Because our samples provided quite high power, these results indicate that GSK3B may not play a major role in Japanese schizophrenia.     

Oxygen radical production by neutrophils from patients with bacterial infection and rheumatoid arthritis

The production of three kinds of oxygen radicals (superoxide, hydrogen peroxide, and hydroxyl radicals) by neutrophils from patients with bacterial infection or rheumatoid arthritis was measured. The stimulators used in this study were opsonized zymosan (1 mg/ml), phorbol myristate acetate (20 ng/ml), A23187 (1M, and platelet activating factor (1M). Oxygen radical production by neutrophils from patients with rheumatoid arthritis was not significantly different from that of the control group. Hydrogen peroxide production by the neutrophils from patients with bacterial infection was significantly enhanced by only opsonized zymosan, but the production of the other kinds of oxygen radicals was not. Cytochalasin B reduced the production of hydrogen peroxide induced by opsonized zymosan more markedly than that of any other kind of oxygen radical. The measurement of hydrogen peroxide is suggested to be the most accurate indicator of the enhancement of intracellular production of oxygen radicals by neutrophils during infection.     

Morphological study of disordered myelination and the degeneration of nerve fibers in the spinal cord of mice lacking complex gangliosides

Gangliosides, a family of glycosphingolipids that contain sialic acid, are abundant on the neuronal cell membranes, but their precise functions in the central nervous system remain largely undefined. In a previous study of GalNAc-T(-/-) mice engineered to lack beta1,4-N-acetylgalactos-aminyltransferase (GM2/GD2 synthase) to abolish any, complex gangliosides, we observed the reduction of nerve conduction velocity but did not find any obvious morphological change in the brain. In the present study, we observed morphological changes in the nerve fiber tracts of the spinal cord in these mice. In GalNAc-T(-/-) mice, the number of degenerated axons was markedly increased in the dorsal funiculus, tract of Lissauer, and dorsolateral funiculus of the cervical segment of the spinal cord as well as the dorsal funiculus and tract of Lissauer of the lumbar segment of the spinal cord. There were also increased numbers of unmyelinated fibers in GalNAc-T(-/-) mice. Loosened myelin sheaths and myelin sheaths separated from axons by wide spaces were also observed in GalNAc-T(-/-) mice. These results provide a morphological basis for the previously observed reduction in the nerve conduction velocity and suggest that complex gangliosides are essential for the maintenance of myelin and the integrity of nerve fibers of the spinal cord.     

Role of apoptosis controlled by cytochrome c released from mitochondria for luteal function in human granulosa cells

PROBLEM: The aim of this study was to investigate the relationship between apoptosis by the mitochondrial pathway and luteal function in human granulosa cells. METHOD OF STUDY: Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization and embryo transfer. After the addition of RU486, cells were stained with a mitochondria-specific fluorescent dye, MitoTracker Red CM x Ros. Using flow cytometry and National Institute of Health image, the mitochondrial fluorescent area was measured. After staining with Hoechst 33258 dye, the number of apoptotic bodies per 1000 cells were counted at random on photomicrographs. Homogenates were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against cytochrome c or caspase-3. RESULTS: The incidence of apoptotic bodies increased and the mitochondrial membrane potential decreased time dependently. The opposite effect was observed dose dependently with RU486 treatment. Western blot analysis showed increased cytochrome c expression, after treatment with 1-2 microg/mL of RU486 which then decreased with 5-10 microg/mL of RU486. Caspase-3 expression increased dose dependently with RU486. CONCLUSIONS: These results suggest that the activation of caspase-3 caused by cytochrome c released from mitochondria plays an important role in apoptosis-related luteal function in human granulosa cells.