Serotonin potentiates high-glucose-induced endothelial injury: the role of serotonin and 5-HT(2A) receptors in promoting thrombosis in diabetes.

To clarify the involvement of 5-hydroxytryptamine (5-HT) in promotion of thrombogenesis in diabetes, we examined the inhibitory effect of sarpogrelate, a 5-HT(2A) receptor antagonist, on thrombus formation in diabetic rats. In streptozotocin-induced diabetic rats, polyethylene tube-induced thrombus formation was enhanced compared with that in normal rats. The thrombogenesis was inhibited by sarpogrelate; cilostazol, a PDE3 inhibitor; and aspirin, a COX inhibitor, by 75.8%, 42.3%, and 34.3%, respectively. The inhibition by sarpogrelate was more pronounced in diabetic rats than normal ones. High glucose and 5-HT increased the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and combination of both high glucose and 5-HT further potentiated the effect. Sarpogrelate but not aspirin inhibited the increase in VCAM-1 expression induced by high glucose and 5-HT. These findings suggest that 5-HT mediates the enhanced thrombogenesis in diabetes and suggests that a 5-HT(2A) receptor antagonist may have novel therapeutic potential for the treatment of diabetic complications.

A new immunofluorostaining method using red fluorescence of PerCP on formalin-fixed paraffin-embedded tissues.

Immunofluorostaining, a versatile tissue staining method, is used in biomedical research because of its clear contrast and precise quantification of positive signals. However, its application in clinical diagnosis has been limited. A major obstacle is high fluorescent background of formalin-fixed, paraffin-embedded tissue sections (paraffin sections). On paraffin sections, strong and broad fluorescence of the section overlapping that of conventional fluorescent dyes such as fluorescein isothiocyanate (FITC) prevents detection of target immunofluorescence. To circumvent the background, we selected an albuminous dye, peridinin chlorophyll a protein (PerCP), for immunostaining of human tumor sections with tumor-reactive monoclonal antibodies. Red fluorescence of PerCP clearly distinguished the tumor region within the yellow-green autofluorescence of the section. Furthermore, it was possible to observe tissue morphology simultaneously without any counterstaining; autofluorescence served as counterstaining in this method. Digital quantification of PerCP-stained image intensity correlated (r2>0.99) well with extracted PerCP amount, indicating the usefulness of image quantification. We conclude that this new and simple immunofluorostaining method can be applied to pathological diagnosis of a wide range of conditions, including cancer.

Establishment and evaluation of cancer-specific human monoclonal antibody GAH for targeting chemotherapy using immunoliposomes.

To establish human monoclonal antibodies suitable for targeting chemotherapy, we prepared a panel of human-mouse hybridomas, using mouse myelomas and lymphocytes of regional lymph nodes excised from cancer patients, and selected antibodies on the basis of their specificity of binding to the surface of viable cancer cells derived from fresh cancer tissues. A selected antibody, named GAH, was found to react with viable cancer cells from 21/22 stomach and 13/20 colon cancer tissues. As for further analysis, complementary DNAs encoding GAH were cloned and recombinant GAH (rGAH) was obtained from established CHO cells transfected with GAH expression vectors. rGAH selectively stained cancer cells in human tissue sections from 13/14 stomach, 4/11 colon, 5/11 mammary, and 0/7 lung cancers, while no positive staining was observed in those of non-tumor and various normal specimens. Notably, using confocal fluorescence microscopy, rGAH was not only bound to the surface of cancer cells, but was also internalized by the cells. The potential of rGAH for intracellular drug delivery was subsequently evaluated using rGAH-conjugated, doxorubicin (DXR)-encapsulated immunoliposomes. The immunoliposomes were also internalized into the cancer cells and finally DXR was delivered to the cell nucleus. Furthermore, the immunoliposomes could inhibit the growth of DXR-insensitive stomach cancer cells (B37) in an in vivo model. These results suggest that a GAH-utilized liposome-targeting technique will provide a potent and useful cancer chemotherapy with broad applications for cancer patients.

Antitumor effect of MCC-465, pegylated liposomal doxorubicin tagged with newly developed monoclonal antibody GAH, in colorectal cancer xenografts.

MCC-465 is an immunoliposome-encapsulated doxorubicin. The liposome is tagged with polyethylene glycol and the F(ab')2 of a monoclonal antibody named GAH, a human antibody obtained by the hybridoma technique. The epitope recognized by GAH is not well characterized, but human gastric, colorectal, and mammary cancer cells were GAH-positive, while the normal counterparts were GAH-negative. Pegylated liposome doxorubicin (PLD) and MCC-465 did not show significant antitumor activity against GAH-negative Caco-2 xenografts. On the other hand, MCC-465 exhibited significantly superior antitumor effects against GAH-positive WiDr-Tc and SW837 xenografts, compared with PLD. Immunohistochemistry with GAH revealed that 94% (100 of 106) of surgical specimens of colorectal cancer were GAH-positive. These results warrant a phase I clinical trial of MCC-465 for patients with metastatic colorectal cancer.