Tumor angiogenesis promoted by ex vivo differentiated endothelial progenitor cells is effectively inhibited by an angiogenesis inhibitor, TK1-2.

Neovascularization plays a critical role in the growth and metastatic spread of tumors and involves recruitment of circulating endothelial progenitor cells (EPC) from bone marrow as well as sprouting of preexisting endothelial cells. In this study, we examined if EPCs could promote tumor angiogenesis and would be an effective cellular target for an angiogenesis inhibitor, the recombinant kringle domain of tissue-type plasminogen activator (TK1-2). When TK1-2 was applied in the ex vivo culture of EPCs isolated from human cord blood, TK1-2 inhibited adhesive differentiation of mononuclear EPCs into endothelial-like cells. In addition, it inhibited the migration of ex vivo cultivated EPCs and also inhibited their adhesion to fibronectin matrix or endothelial cell monolayer. When A549 cancer cells were coimplanted along with ex vivo cultivated EPCs s.c. in nude mice, the tumor growth was increased. However, the tumor growth and the vascular density of tumor tissues increased by coimplanted EPCs were decreased upon TK1-2 treatment. Accordingly, TK1-2 treatment reduced the remaining number of EPCs in tumor tissues and their incorporation into the host vascular channels. In addition, overall expression levels of vascular endothelial growth factor (VEGF) and von Willebrand factor in tumor tissues were decreased upon TK1-2 treatment. Interestingly, strong VEGF expression by implanted EPCs was decreased by TK1-2. Finally, we confirmed in vitro that TK1-2 inhibited VEGF secretion of EPCs. TK1-2 also inhibited endothelial cell proliferation and migration induced by the conditioned medium of EPCs. Therefore, we concluded that EPCs, as well as mature endothelial cells, could be an important target of TK1-2.

Outgrowing endothelial progenitor-derived cells display high sensitivity to angiogenesis modulators and delayed senescence.

Outgrowing endothelial progenitor-derived cells (EPDCs) originate from a novel hierarchy of endothelial progenitor cells. In this study, EPDCs isolated from human cord blood were examined for phenotype and functional features upon aging. Young or aged EPDCs were similar to human umbilical vein endothelial cells (HUVECs), in exhibiting typical endothelial phenotypes. However, EPDCs were more sensitive to angiogenesis inducers or inhibitors in proliferation and migration. In addition, EPDCs underwent senescence markedly slowly with sustained endothelial NO synthase expression and activation, and their ability to undergo capillary morphogenesis was retained throughout longterm culture. Thus, these results suggest that a homogenous population of EPDCs derived from clonogenic expansion may provide an effective vasculogenesis tool.

RGD-peptide presents anti-adhesive effect, but not direct pro-apoptotic effect on endothelial progenitor cells.

Circulating endothelial progenitor cells (EPCs) contribute to neovascularization in tumor or ischemic tissues by multi-step events, including adhesion, migration, chemoattraction, and differentiation to endothelial cells. Anti-angiogenic RGD-peptides have been shown to directly induce apoptosis in human umbilical vein endothelial cells (HUVECs) and T cells. Here, we examined the effects of RGD-peptides on EPCs in terms of adhesive differentiation and apoptosis. When mononuclear cells (MNCs) isolated from human cord blood were cultured on fibronectin-coated plates for 7 days, RGD-peptide treatment decreased dose-dependently the number of adherent cells double positive for DiI-ac-LDL uptake and UEA-1 binding. The cells treated with RGD peptide were also stained less strongly by vWF or KDR antibody by immunofluorescence staining. Immobilization of the RGD-peptide promoted cell adhesion, but resulted in a deficiency in the development of ability of ac-LDL uptake and UEA-1 binding, showing an antagonistic effect. Accordingly, ex vivo-cultivated EPCs expressed integrin alpha5, alphav, beta1, beta3, and beta5, and antibodies to integrins alpha5, alphav, and beta1 decreased the number of adherent cells. However, viability of total MNCs containing early EPCs was not affected by RGD-peptide. In addition, neither an increase in apoptotic cell death nor a direct activation of caspase-3 by RGD-peptide was detected in ex vivo-cultivated EPCs, unlike in HUVECs. Interestingly, RGD-peptide rather enhanced Bcl-2 expression in ex vivo-cultivated EPCs and the EPCs themselves with a high Bcl-2/Bax ratio are comparatively resistant to apoptosis. Therefore, these results suggest that RGD-peptides may inhibit EPC differentiation by anti-adhesive effect, but not by a direct pro-apoptotic effect.

Expression of the non-glycosylated kringle domain of tissue type plasminogen activator in Pichia and its anti-endothelial cell activity.

The two-kringle domain of tissue-type plasminogen activator (TK1-2) has been identified as a potent angiogenesis inhibitor by suppressing endothelial cell proliferation, in vivo angiogenesis, and in vivo tumor growth. Escherichia coli-derived, non-glycosylated TK1-2 more potently inhibits in vivo tumor growth, whereas Pichia expression system is more efficient for producing TK1-2 as a soluble form, albeit accompanying N-glycosylation. Therefore, in order to avoid immune reactivity and improve in vivo efficacy, we expressed the non-glycosylated form of TK1-2 in Pichia pastoris and evaluated its activity in vitro. When TK1-2 was mutated at either Asn(117) or Asn(184) by replacing with Gln, the mutated proteins produced the glycosylated form in Pichia, of which sugar moiety could be deleted by endoglycosidase H treatment. When both sites were replaced by Gln, the resulting mutant produced a non-glycosylated protein, NQ-TK1-2. Secreted NQ-TK1-2 was purified from the culture broth by sequential ion exchange chromatography using SP-sepharose, Q-spin, and UNO-S1 column. The purified NQ-TK1-2 migrated as a single protein band of approximately 20 kDa in SDS-PAGE and its mass spectrum showed one major peak of 19,950.71 Da, which is smaller than those of two glycosylated forms of wild type TK1-2. Functionally, the purified NQ-TK1-2 inhibited endothelial cell proliferation and migration stimulated by bFGF and VEGF, respectively. Therefore, the results suggest that non-glycosylated TK1-2 useful for the treatment of cancer can be efficiently produced in Pichia, with retaining its activity.

Inhibition of endothelial cell proliferation by the recombinant kringle domain of tissue-type plasminogen activator.

Tissue-type plasminogen activator (tPA) is a multidomain serine protease that converts the zymogen plasminogen to plasmin. tPA contains two kringle domains which display considerable sequence identity with those of angiostatin, an angiogenesis inhibitor. TK1-2, a recombinant kringle domain composed of t-PA kringles 1 and 2 (Ala(90)-Thr(263)), was produced by both bacterial and yeast expression systems. In vitro, TK1-2 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, and epidermal growth factor. It did not inhibit proliferation of non-endothelial cells. TK1-2 also inhibited in vivo angiogenesis in the chick embryo chorioallantoic membrane model. These results suggest that the recombinant kringle domain of t-PA is a selective inhibitor of endothelial cell growth and identifies this molecule as a novel anti-angiogenic agent.