Tetsuichiro Inai

Inhibition of Vascular Endothelial Growth Factor (VEGF) Signaling in Cancer Causes Loss of Endothelial Fenestrations, Regression of Tumor Vessels, and Appearance of Basement Membrane Ghosts

Angiogenesis inhibitors are receiving increased attention as cancer therapeutics, but little is known of the cellular effects of these inhibitors on tumor vessels. We sought to determine whether two agents, AG013736 and VEGF-Trap, that inhibit vascular endothelial growth factor (VEGF) signaling, merely stop angiogenesis or cause regression of existing tumor vessels. Here, we report that treatment with these inhibitors caused robust and early changes in endothelial cells, pericytes, and basement membrane of vessels in spontaneous islet-cell tumors of RIP-Tag2 transgenic mice and in subcutaneously implanted Lewis lung carcinomas. Strikingly, within 24 hours, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and patency and blood flow ceased in some vessels. By 7 days, vascular density decreased more than 70%, and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells. Vessels in Lewis lung tumors, which lacked endothelial fenestrations, showed less regression. In both tumors, pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels acquired a more normal phenotype. Vascular basement membrane persisted after endothelial cells degenerated, providing a ghost-like record of pretreatment vessel number and location and a potential scaffold for vessel regrowth. The potent anti-vascular action observed is evidence that VEGF signaling inhibitors do more than stop angiogenesis. Early loss of endothelial fenestrations in RIP-Tag2 tumors is a clue that vessel phenotype may be predictive of exceptional sensitivity to these inhibitors.

Rapid vascular regrowth in tumors after reversal of VEGF inhibition

Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less alpha-SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.

Claudin-1 contributes to the epithelial barrier function in MDCK cells

Tight junctions (TJs) create a paracellular permeability barrier and also act as a fence preventing intermixing of proteins and lipids between the apical and basolateral plasma membranes. Recently, claudin-1 has been identified as an integral membrane protein localizing at TJs, and introduced claudin-1 can form TJ-like networks in fibroblasts. To investigate the function of claudin-1, MDCK cells were transfected with a mammalian expression vector containing myc-tagged mouse claudin-1, and four stable clones were obtained. The myc-tagged claudin-1 precisely colocalized with both occludin and ZO-1 at cell-cell contact sites, indicating that exogenous claudin-1 was properly targeted to the TJs. Immunoblot analysis revealed that overexpression of claudin-1 increased expression of ZO-1 but not of occludin or ZO-2. The barrier functions of these cells were evaluated by transepithelial electrical resistance (TER) and paracellular flux. Claudin-1-expressing cells exhibited about four times higher TER than wild-type MDCK cells. Consistent with the increase of TER, the cells overexpressing claudin-1 showed reduced paracellular flux, estimated at 4 and 40 kD FITC-dextrans. These results suggest that claudin-1 is involved in the barrier function at TJs.

Immunohistochemical detection of an enamel protein-related epitope in rat bone at an early stage of osteogenesis.

Monoclonal antibody MI315 was produced against hamster tooth germ homogenate by in vitro immunization. It was found that MI315 reacted with enamel matrix, ameloblasts, and bone matrix at an early stage of osteogenesis. Decalcified tissues of rat femurs and mandibles were examined with MI315 using indirect immunofluorescence. In endochondral ossification of femurs, immunoreactivity was found in bone extracellular matrix (ECM) deposited on the surface of the cartilage core of primary spongiosa, but not in the cartilage core itself. In intramembranous ossification of 0-day-old rat mandibles, intense immunofluorescence was detected in bone ECM and a few young osteocytes, but not in osteoblasts. Immunoreactivity in bone ECM of 2-day-old rats decreased and almost disappeared from bone ECM of 4-day-old rats. Although in nondecalcified sections of 0-day-old rats, negligible immunofluorescence was detected in bone ECM which showed positive staining in decalcified tissues, the immunostaining appeared after decalcification using ethylenediaminetetraacetic acid (EDTA). These results indicate that a substance(s), which had a common epitope with an enamel-derived protein(s), existed in immature bone ECM of both endochondral and intramembranous ossification, and that it might be masked by bone mineral. Monoclonal antibody MI315 is a useful tool to investigate the time- and position-specific changes in osteogenesis and amelogenesis.

Primary and secondary culture of rat ameloblasts in serum-free medium

SummaryEnamel is the hardest tissue in vertebrates. Ameloblasts are derived from epithelial cells and are responsible for enamel formation. They secrecte enamel matrix components in which amelogenins are the major proteins, the biochemical properties of which are well known. However, little is known about the characteristics of ameloblasts themselves or about the functions of amelogenins. In this study, we developed a novel primary and secondary culture system for ameloblasts using a monoclonal antibody which recognized amelogenin (En3). The cell layer on dentine removed from rat mandibular incisors was isolated and cultured in low calcium, serum-free medium. Primary culture was performed on collagen-coated culture plates and typically, two types of cells appeared. One major type changed morphology after the addition of a high concentration of calcium to the medium. Expression of amelogenin was shown as cytoplasmic particles in these cells using En3. In the secondary culture, expression of amelogenins was also observed. In this system, the cells grew and maintained the expression of amelogenin for about 3 weeks.

Influence of fluoride on secretory pathway of the secretory ameloblast in rat incisor tooth germs exposed to sodium fluoride

Abstract Fluoride, which is an environmental toxicant, is a potent inducer of mottled enamel in humans and rats. To define the influence of fluoride on the secretory pathway in enamel fluorosis, mottled enamel was induced in the incisor tooth germs of rats by subcutaneous injections of sodium fluoride for 4 days, and then morphological and cytochemical changes of the secretory ameloblast were examined in the tooth germs with HRP-labeled lectin (Con A, GS-I, SBA and PNA) and En3 antibody labeling amelogenins. The accumulation of small vesicles on the route of the secretory pathway between the rER and the Golgi apparatus, disorder of Golgi stacks, and formation of abnormal large granules in distal cytoplasm were seen in the secretory ameloblast. Lectin staining patterns of the secretory ameloblast indicated the disturbance of the vesicular transport between the rER and the Golgi apparatus, and disorganization of the Golgi stack. Immunolabeling of the cell showed disruption of the sorting and fusion process on the secretory pathway. These results suggest that the fluoride disturbs the intracellular transport in the synthesis-secretory pathway of the ameloblast, and that this effect of fluoride on the synthesis-secretory pathway participates in the formation of enamel fluorosis.

Recombinant LD78 protein, a member of the small cytokine family, enhances osteoclast differentiation in rat bone marrow culture system

Cytokine LD78 is a member of the small inducible protein family involved in cell growth, wound healing and inflammation. However, its exact function is not known. In this study, we demonstrated that recombinant LD78 alpha and its variant LD78 beta proteins stimulate osteoclast-like cell formation in rat bone marrow cultures in the presence of 1 alpha, 25-dihydroxyvitamin D3. This enhancing activity was independent of prostaglandin synthesis. This is the first report which describes the effect of proteins involved in the small inducible protein family on the differentiation of osteoclasts.

The protoplasmic or exoplasmic face association of tight junction particles cannot predict paracellular permeability or heterotypic claudin compatibility.

Claudins constitute tight junction (TJ) strands and regulate paracellular permeability, which varies in the epithelial cells of various organs. Heterotypic claudin compatibility and/or the association of TJ particles to either the protoplasmic (P) or exoplasmic (E) face may be related to paracellular permeability. This study examined the relationship between the TJ morphology, heterotypic claudin compatibility and paracellular permeability using claudin-10b- or claudin-15-expressing HEK293 cells and MDCK I cells. Claudin-10b or -15 expressed in TJ-free HEK293 cells formed E-face- or P-face-associated TJ particles, respectively. The coculture of claudin-1-expressing HEK293 cells and either claudin-10b- or claudin-15-expressing HEK293 cells showed that claudin-10b and -15 were not compatible with claudin-1. The expression of claudin-10b or -15 in high-resistance MDCK I cells did not alter the expression of endogenous claudins except for claudin-3 and dramatically reduced transepithelial electrical resistance by increasing the permeability of Na(+) but it did not change that of Cl(-). The expression of claudin-10b or -15 in MDCK I cells either decreased or increased the flux of 4 kDa dextran, respectively. The coculture of MDCK I cells and either claudin-10b- or claudin-15-expressing MDCK I cells showed claudin-10b to be partly compatible, while claudin-15 was incompatible with the endogenous claudins in MDCK I cells. These results indicate that the TJ morphology cannot predict the properties of either paracellular permeability or heterotypic claudin compatibility.

Claudin-7 Expressed on Lateral Membrane of Rat Epididymal Epithelium does not Form Aberrant Tight Junction Strands

Claudins are integral membrane proteins at tight junctions (TJs) and form TJ strands. In the present study, we found that claudin‐7 was localized along the entire lateral membranes of epididymal epithelium, including the apical junctional region throughout the epididymis, but claudin‐8 was restricted to the apical junctional region. This finding raises the possibility that aberrant TJ strands may be formed on lateral membranes. Thus, we focused on examining whether TJ strands exist on lateral membranes of epididymal epithelium. Freeze‐fracture electron microscopy showed that aberrant TJ strands were observed in only a few principal cells in all segments of the epididymis except for the initial segment, indicating that the occurrence of aberrant strands is very rare. Aberrant TJ strands were smooth and not subdivided into individual particles in the protoplasmic face, and complementary grooves in the extracellular face were almost free of particles. Aberrant TJ strands in the distal caput and corpus epididymis were accompanied by many vesicle‐like structures but those in the proximal caput and cauda epididymis were not. These results suggest that most of claudin‐7 in lateral membranes may exist in a nonpolymerized form and may play some different roles other than the formation of TJ strands, for example, in the formation of a pool of claudin proteins or in the reinforcement of cell adhesion. Anat Rec, 1431‐1438, 2007.

Intron Retention Generates a Novel Isoform of CEACAM6 That May Act as an Adhesion Molecule in the Ectoplasmic Specialization Structures Between Spermatids and Sertoli Cells in Rat Testis

Abstract By differential display technique followed by RT-PCR and DNA sequence analyses, we isolated carcinoembryonic antigen-related cell adhesion molecule 6 (Ceacam6) and its novel spliced variant Ceacam6-Long (Ceacam6-L) from rat testis. Ceacam6-L mRNA was generated by retention of 67 nucleotide-length third intron in Ceacam6 gene. Ceacam6-L is a member of an immunoglobulin superfamily and encodes a protein of 50 kDa with a signal sequence at the N-terminus, one immunoglobulin (Ig)-like domain, three IgCAM domains, a transmembrane region, and a short intracellular region. Expression analyses by RT-PCR and Northern blot showed that Ceacam6-L was exclusively expressed in rat testis and first detectable at 5 wk during postnatal development of testis. We performed immunoblot analyses and immunohistochemistry using the anti-CEACAM6-L antibody. Confocal laser scanning microscopy revealed that CEACAM6-L was not present at blood-testis barrier junctions between Sertoli cells but localized at the interface between Sertoli cells and germ cells, possibly to work as an adhesion molecule in the apical compartment of the seminiferous epithelium. At stages VII–VIII, at which all of the elongated spermatids migrated to the luminal surface of the seminiferous tubules, CEACAM6-L was found to locate at the concave side of elongated spermatid heads, following the curvature of their sickle-shaped nuclei, suggesting that CEACAM6-L might be involved in the anchoring of spermatids to Sertoli cells and spermiation. We concluded that CEACAM6-L might be a novel adhesion molecule constructing the apical ectoplasmic specialization in testis.