Ryosaburo Takaki

Platelet factor 4 blocks the binding of basic fibroblast growth factor to the receptor and inhibits the spontaneous migration of vascular endothelial cells

Platelet factor 4 (PF-4) blocked the binding of basic fibroblast growth factor (bFGF) to the plasma membrane receptor. Five micrograms/ml of PF-4 completely blocked the specific binding of bFGF to the receptor of NIH 3T3 cells. Endogenously produced bFGF regulates the spontaneous migration of bovine aortic endothelial (BAE) cells as an autocrine factor (Sato and Rifkin, 1988). PF-4 inhibited the spontaneous migration of BAE cells in a reversible and dose dependent manner. The inhibition reached maximum at 5 micrograms/ml of PF-4, where the binding of bFGF to the receptor was completely blocked.

The stimulatory effect of PDGF on vascular smooth muscle cell migration is mediated by the induction of endogenous basic FGF.

The migration of arterial smooth muscle cells from the media to the intima is a crucial event for the development of the atherosclerotic lesion, and platelet derived growth factor (PDGF) is thought to play an important role in this process. Here we report that the spontaneous migration of bovine smooth muscle (BSM) cells is dependent on endogenously produced basic fibroblast growth factor (bFGF). PDGF stimulates the migration of BSM cells and its effect is abolished by affinity purified anti-bFGF antibody. PDGF induces bFGF mRNA in BSM cells. These results indicate that the effect of PDGF on the migration of BSM cells may be mediated by the induction of endogenous bFGF.

Autocrinological role of basic fibroblast growth factor on tube formation of vascular endothelial cells in vitro

When bovine capillary endothelial (BCE) cells plated on type I collagen gel were covered with a second layer of collage gel, BCE cells reorganized into a network of capillary-like structures. In the presence of affinity purified anti-basic fibroblast growth factor (bFGF) antibody, this reorganization was inhibited. By using a computerized image analyzer, the formation of network structures and the effect of anti-bFGF antibody was quantitated. The inhibitory effect of anti-FGF antibody was dose-dependent and maximal inhibition was observed at 2.0 micrograms/ml of antibody. Exogenously added bFGF potentiated network formation of BCE cells and coadministration of bFGF abrogated the inhibitory effect of anti-bFGF antibody. Platelet factor 4, which blocks the binding of bFGF to its receptor, inhibited network formation. These results indicate that bFGF produced by endothelial cells regulates angiogenesis as an autocrine factor.

The endocrine pancreas of spontaneously diabetic db db mice: microangiopathy as revealed by transmission electron microscopy

Abnormalities in ultrastructures of islet capillaries were detected in db/db mice, with the visual inspection and morphometry of electron micrographs. The observed changes are: (1) capillary scarcity; (2) increase in the mean and diversity of capillary size; (3) pericapillary edema and fibrosis; (4) hypertrophy of the pericyte and abundance therein of actin-like microfilaments; and (5) luminal irregularity. Changes (2), (3) and (4) are conceived to indicate hyperperfusion, capillary hypertension and secondary vascular response. In particular, such pericyte changes were found to be shared by other organs whose capillaries are susceptible to diabetic complications.

Long-term culture of pancreatic islet cells with special reference to the β-cell function

SummaryIslet cells of adult rat pancreas, dissociated with EDTA-Dispase, were cultivated in Microtest wells for over 2 months. In our cultures, islet cells were free-floating and cohered with each other to reorganize histotypic aggregates resembling the nondissociated islets. Morphologically, excellent preservation of islet cells in the aggregates was confirmed during the culture using both light and electron microscopy. The function of islet β-cells, as demonstrated by the synthesis and release of insulin, also was retained throughout the culture period. Islet β-cells cultured for long periods exhibited better response to the shortterm stimulation of theophylline than to a high concentration of glucose, as observed in the islets of fetal or newborn rat pancreas.

The Role of Thymic Immunity and Insulitis in the Development of Streptozocin-induced Diabetes in Mice

This article is concerned with the role of thymic immunity in the development of diabetes experimentally induced by multiple injections of subdiabetogenic doses of streptozocin (STZ). Euthymic + / +, + /nu, and athymic nu/nu mice of CD-1 and BALB/cAJcl origin were studied. Daily intraperitoneal (i.p.) injections of 30 mg /kg body wt of STZ for 5 consecutive days in CD-1 + / + and + /nu mice resulted in hyperglycemia and mononuclear cell infiltrations of islets (insulitis). The CD-1 nu/nu mice developed neither insulitis nor hyperglycemia after the same treatment. In the nu/nu mice, when thymic immunity was restored by thymus grafting, both insulitis and hyperglycemia developed, thus demonstrating that thymic immunity was a prerequisite for the development of insulitis and hyperglycemia. There was a positive correlation among the degrees of thymic immunity, insulitis, and hyperglycemia in CD-1 + /nu,nu/nu with thymus grafts, and nu/nu mice, indicating that thymic immunity may amplify the diabetogenic effect of STZ by eliciting insulitis. In contrast, in BALB/cAJcl mice, a nonsusceptible strain to insulitis, no significant differences in plasma glucose levels were observed between the + /nu and nu/nu or between the nu/nu and thymus-grafted nu/nu mice. Furthermore, no significant difference was found in plasma testosterone levels between the + /nu and nu/nu mice of both CD-1 and BALB/cAJcI origin. In conclusion, our data indicate that thymic immunity enhances the diabetogenic effect of STZ by eliciting insulitis in susceptible mice.

Isolation and primary culture of adult human hepatocytes

SummaryBiopsy tissue of adult human liver was gently dissociated with collagenase followed by Dispase. By repeated low g centrifugation, a large number of almost pure, viable hepatocytes was obtained. This is the first report of a successful procedure for obtaining adult human hepatocytes for study in tissue culture. The isolated cells have the typical morphology of liver parenchyma, and these characteristics persist throughout the period of culturing. Evidence of their function is indicated by albumin synthesis. This procedure is now being used to study human hepatocyte functions in vitro and the effects of a variety of agents including carcinogens and viruses.

Histologic and immunofluorescent studies on the site of origin of glucagon in mammalian pancreas.

The present study was designed to investigate the site of origin of glucagon in the adult rabbit pancreas. Tissue fragments, fixed with Carnoys solution and embedded in paraffin, were sectioned 4 µ in thickness. In a section stained with Gomoris chromium hematoxylin-phloxine, several islets were marked and photographed for comparison with immunofluorescent staining. The same section was then stained by an indirect immunofluorescent method using antiglucagon serum to identify glucagon-containing cells after transfer through xylene, decreasing concentrations of alcohol and buffered saline in succession. The islets previously marked were again observed and photographed under the fluorescent microscope. The specificity of the fluorescent staining was confirmed by the block test, absorption test and others. Good agreement between histologic and immunofluorescent staining of identical sections indicated the α cell origin of glucagon in the rabbit pancreas.

Cellular Interaction Between Mouse Pancreatic α-Cell and β-Cell Lines: Possible Contact-Dependent Inhibition of Insulin Secretion

The endocrine cells in the pancreatic islet have cellular communication between the heterotypic cells as well as the homotypic cells. The present study was conducted to elucidate the cellular interaction between pancreatic α cells and β cells utilizing differentiated mouse cell lines (i.e., αTC clone 6 and βTC cells). Co-culture of these two cell lines on a gyratory shaker generated numerous cellular aggregates of homogenous size within 48 h. Immunohistochemical staining for insulin and glucagon demonstrated that βTC cells were located in the central core of each aggregate, while αTC cells formed a mantle layer surrounding the βTC cells. This segregation was observed regardless of the ratios of the two cell types employed. Although glucagon at concentrations of 10−8 M or higher stimulated Insulin secretion from βTC cells in both monolayer and aggregates, an increase in the ratio of αTC/βTC cells in aggregate cultures was accompanied by a decrease in secreted insulin and a rise in intracellular insulin content of the βTC component. The inhibitory effect of αTC cells on βTC insulin secretion was not limited to aggregate culture, since insulin secretion from βTC cells was also suppressed, and intracellular insulin content increased, by co-culture of αTC with βTC cells in monolayer. On the other hand, the secreted and intracellular insulin of βTC cells was not affected by αTC cells in a Transwell™ co-culture system in which direct cell-to-cell contacts were prevented by a semipermeable membrane that permitted chemical communication via medium metabolites. These data suggest that the insulin secretion from βTC cells may be inhibited possibly as a result of the contact with αTC cells.

Enhancement of excision-repair efficiency by conditioned medium from density-inhibited cultures in V79 Chinese hamster cells: Evidence for excision repair as an error-free repair process

Conditioned medium from density-inhibited V79 Chinese hamster cell cultures, given as a post-treatment to UV-irradiated homologous cells, was demonstrated to reduce the lethal action of ultraviolet light by temporarily blocking DNA replication. Since the increased survival was not affected by various non-toxic concentrations of caffeine, such protective effect would be attributable to the prolonged intervention of excision repair before DNA replication during the post-treatment period. The influence of conditioned medium on the UV-induced mutation at the ouabain-resistance locus was also examined and a significant decrease in mutation frequency was noted. The observed reduction in killing and mutation as a result of post-incubation in conditioned medium, which delays DNA replication, would be interpreted as evidence that conditioned medium provides a longer period of time for an error-free excision-repair process, leaving lesion in DNA available for error-prone post-replication repair.