Xenograft of microencapsulated Sertoli cells for the cell therapy of type 2 diabetes mellitus in spontaneously diabetic nonhuman primates: preliminary data.

Insulin resistance in type 2 diabetes mellitus (T2DM) may be due to a chronic inflammation of the visceral adipose tissue (VAT) leading to local and systemic increases in proinflammatory cytokines. Microencapsulated porcine Sertoli cells (MC-pSC), by provision of immunomodulatory and trophic factors, have been successfully used to reduce such inflammation in rodent animal models of type 1 diabetes with no complications or deleterious side effects. Herein, we have begun to investigate this novel and safe therapeutic approach in the spontaneously obese nonhuman primate with spontaneous, insulin-dependent T2DM. After MC-pSC intraperitoneal injection we have evaluated, throughout a 6-month follow-up period, daily ad libitum fed glucose levels, daily exogenous insulin supplementation, biweekly body weight measurements, periodic fasting blood glucose concentrations, glycated hemoglobin (HbA1c) levels, glucose tolerance tests (GTT), and fluorescence-activated cell sorting cytometry (FACS) assessment of peripheral blood mononuclear cells. Very preliminarily, we have observed a slight reduction in fasting (FPG) and mean nonfasting (NF) plasma glucose levels. We found minimal changes, only in 1 animal, in daily exogenous insulin requirements and HbA1c levels. Flow cytometric analysis was associated with decrease in CD8(+) cells only in 1 recipient with a reduction in mean regulatory T Cells (Treg), whereas interestingly, decrease of B lymphocytes was observed in both animals. These results may suggest that this novel MC-SC-based transplantation protocol might possibly impact the metabolic status of T2DM in higher mammals that are close to humans.

Eicosanoid regulation of angiogenesis in human prostate carcinoma and its therapeutic implications.

Cancer of the prostate is the most commonly diagnosed cancer in America. There are several lines of evidence implicating the involvement of arachidonate 12-lipoxygenase, an enzyme metabolizing arachidonic acid to form 12(S)-hydroxyeicosatetraenoic acid (HETE), in prostate cancer progression. First, as prostate cancer reaches a more advanced stage, the level of 12-lipoxygenase expression is increased. Second, overexpression of 12-lipoxygenase in human prostate cancer cells stimulates angiogenesis and tumor growth. Third, an inhibitor of 12-lipoxygenase has been found effective against metastatic prostate tumor growth, and the inhibition of 12-lipoxygenase is related with the reduction of tumor angiogenesis. Collectively, these studies suggest that 12-lipoxygenase regulates tumor angiogenesis in prostate cancer and that inhibition of 12-lipoxygenase is a novel therapeutic approach for the treatment of prostate cancers.

Thromboxane A(2) regulation of endothelial cell migration, angiogenesis, and tumor metastasis.

Prostaglandin endoperoxide H synthases and their arachidonate products have been implicated in modulating angiogenesis during tumor growth and chronic inflammation. Here we report the involvement of thromboxane A(2), a downstream metabolite of prostaglandin H synthase, in angiogenesis. A TXA(2) mimetic, U46619, stimulated endothelial cell migration. Angiogenic basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) increased TXA(2) synthesis in endothelial cells three- to fivefold. Inhibition of TXA(2) synthesis with furegrelate or CI reduced HUVEC migration stimulated by VEGF or bFGF. A TXA(2) receptor antagonist, SQ29,548, inhibited VEGF- or bFGF-stimulated endothelial cell migration. In vivo, CI inhibited bFGF-induced angiogenesis. Finally, development of lung metastasis in C57Bl/6J mice intravenously injected with Lewis lung carcinoma or B16a cells was significantly inhibited by thromboxane synthase inhibitors, CI or furegrelate sodium. Our data demonstrate the involvement of TXA(2) in angiogenesis and development of tumor metastasis.

Expression and function of the high affinity alphaIIbbeta3 integrin in murine melanoma cells.

In resting platelets integrin alphaIIbbeta3 is constitutively expressed in an inactive state and it does not recognize soluble proteins. Platelet activation results in a conformational change of the low-affinity alphaIIbbeta3 to a high-affinity state which then recognizes plasma fibrinogen. The ectopic expression of alphaIIbbeta3 integrin in rodent and human cells derived from solid tumors is well documented, although little is known about its affinity state in these tumor cells. In this study we analysed expression and function of high-affinity alphaIIbbeta3 in murine metastatic melanoma B16a cells by using a mAb that specifically recognizes high-affinity alphaIIbbeta3 (PAC-1). These tumor cells while in suspension bound PAC-1 and fibrinogen. Immunofluorescent studies of B16a cells indicated that high-affinity alphaIIbbeta3 is associated with the Golgi complex and the cell surface. Stimulation of B16a cells with a PKC-activator, 12(S)-HETE, induced translocation of the high-affinity integrin from an intracellular pool to the plasma membrane, which resulted in increased tumor cell adhesion to fibronectin. In addition to participating in 12(S)-HETE-stimulated adhesion of B16a cells, the high-affinity alphaIIbbeta3 integrin is also involved in tumor cell invasion through a reconstituted basement membrane. In conclusion, results from this study suggest that in non-megakaryocytic lineage B16a cells alphaIIbbeta3 is constitutively expressed in a high-affinity state, and that this conformation participates in tumor cell adhesion and invasion.

The high affinity alphaIIb beta3 integrin is involved in invasion of human melanoma cells.

Integrins play an important role in mediating tumor cell-extracellular matrix (ECM) and tumor cell-endothelial cell interactions. The integrin alphaIIb beta3 (GPIIb-IIIa) is expressed on the surface of platelets in an inactive state and requires a conformational change to recognize extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and others. In this study, we questioned whether human melanoma cells express the alphaIIb beta3 integrin. Reverse transcription-PCR/Southern blotting, Northern blotting, and dot blotting demonstrated the presence of the platelet-type alphaIIb beta3 integrin in human melanoma WM 983B, WM 983A, and WM 35 cells. AP-2, a monoclonal antibody (mAb) to alphaIIb beta3, positively stained two human melanoma specimens, indicating expression of this integrin in vivo. Phorbol 12-myristate 13-acetate and 12(S)-hydroxyeicosatetraenoic acid, two activators of protein kinase C, stimulated adhesion of melanoma cells to immobilized fibronectin and PAC-1, a mAb to alphaIIb beta3. PAC-1 specifically recognizes the conformationally active form of platelet alphaIIb beta3. Phorbol 12-myristate 13-acetate-stimulated adhesion of WM 983B cells to PAC-1 was completely blocked by an RGD peptide, thus providing evidence that tumor cell adhesion to PAC-1 is mediated via the alphaIIb beta3 integrin but not the Fc receptor. Confocal immunofluorescent studies demonstrated that fibronectin-adherent melanoma cells possess an intracellularly localized pool of high-affinity alphaIIb beta3. Invasion of WM 983B cells through fibronectin was stimulated by 12(S)-hydroxyeicosatetraenoic acid, and this stimulated invasion was blocked by the mAb PAC-1. The data suggest that melanoma cells express the high-affinity alphaIIb beta3 integrin, which is involved in tumor invasion.

Human prostate carcinoma cells express functional alphaIIb(beta)3 integrin.

The integrin alphaIIb(beta)3 was initially believed to be expressed only in cells from the megakaryocytic lineage, such as platelets or HEL cells. In this study, we report for the first time that human prostate carcinoma PC-3 and DU-145 cells express alphaIIb(beta)3. Reverse transcription-PCR from HEL (positive control), PC-3, and DU-145 cells amplified a predicted alphaIIb fragment that hybridized to the full-length alphaIIb cDNA probe. DNA sequencing of the PCR fragments revealed 100% sequence homology to the corresponding extracellular domain of platelet alphaIIb but minimal sequence homology to integrins (alpha)v or a5. An RNase protection assay was used to confirm the results from reverse transcription-PCR. An antisense riboprobe to alphaIIb mRNA hybridized to total RNA from HEL, PC-3, and DU-145 cells, suggesting that alphaIIb mRNA is transcribed in these tumor cells. In situ hybridization on surgical specimens from human prostate tumor tissue stained positive with an antisense riboprobe to alphaIIb mRNA. The expression of alphaIIb(beta)3 protein in PC-3 and DU-145 cells was demonstrated by Western and dot blotting and flow cytometry with monoclonal antibodies (mAbs) to alphaIIb (MAB 1990), beta3, and alphaIIb(beta)3 (AP-2). A protein kinase C activator, phorbol 12-myristate 13-acetate, increased the adhesion of PC-3 cells to PAC-1, a mAb specific to the high-affinity state of alphaIIb(beta)3, by more than 80-fold. The invasion of DU-145 cells through a reconstituted basement membrane was blocked 40-50% by mAbs AP-2 or PAC-1. These data collectively suggest that: (a) prostate tumor cells express alphaIIb(beta)3; (b) surface expression of alphaIIb(beta)3 integrin is regulated by protein kinase C; and (c) mAbs to this receptor inhibit invasion of prostate cancer cells through a reconstituted basement membrane.

Autocrine motility factor signals integrin-mediated metastatic melanoma cell adhesion and invasion.

The binding of autocrine motility factor (AMF) to its cell surface receptor, gp78, stimulates tumor cell motility. In this report, we provide evidence that stimulation of gp78 by either AMF or a monoclonal antibody to gp78 (3F3A) increases adhesion and spreading of metastatic murine melanoma (B16a) cells on fibronectin. This gp78-regulated increase is mediated by up-regulation of surface alphaIIbbeta3++ and alpha5beta1 integrin receptors. In addition, AMF treatment of B16a cells increased translocation of alphaIIbbeta3 and alpha5beta1 from the cytoplasm to the cell surface. However, alphaIIbbeta3 and alpha5beta1 demonstrate separate and unique staining patterns at the surface of B16a cells in response to stimulation of gp78. Furthermore, stimulation of B16a cells with AMF increased their invasion through Matrigel. This stimulated invasion was inhibited by antibodies to alphaIIbbeta3 but not by antibodies to alpha5beta1. The increased integrin surface expression and function in response to AMF was blocked by N-benzyl-N-hydroxy-5-phenylpentanamide, an inhibitor of 12-lipoxygenase, and calphostin C, an inhibitor of protein kinase C. The results demonstrate that AMF stimulates integrin-mediated B16a cell adhesion, spreading, and invasion, and these events are regulated by a signaling pathway involving 12-lipoxygenases and protein kinase C.