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Paclitaxel is an alkaloid that inhibits endothelial cell proliferation, motility, and tube formation at nanomolar concentrations. Cationic liposome preparations have been shown to target blood vessels. We wished to explore the possibility that paclitaxel encapsulated in cationic liposomes carries paclitaxel to blood vessels and thereby provides an antiangiogenic effect. We used a humanized SCID mouse melanoma model, which allowed us to analyze tumor growth and tumor angiogenesis in an orthotopic tumor model. Here, human melanoma cells grow on human dermis and are in part nourished by human vessels. We show that paclitaxel encapsulated in liposomes prevents melanoma growth and invasiveness and improves survival of mice. Moreover, liposome-encapsulated paclitaxel reduces vessel density at the interface between the tumor and the human dermis and reduces endothelial cell mitosis to background levels. In contrast, equimolar concentrations of paclitaxel solubilized in Cremophor EL(R) had only insignificant effects on tumor growth and did not reduce the mitotic index of endothelium in vivo, although the antiproliferative effect of solubilized paclitaxel in Cremophor EL(R)in vitro was identical to that seen with liposome-coupled paclitaxel. In conclusion, we present a model of how to exploit cytotoxic effects of compounds to prevent tumor growth by using cationic liposomes for targeting an antiproliferative drug to blood vessels.

ABSTRACT.Purpose: In this study we established a protocol for transfection of human corneal endothelial and human retinal pigment epithelial cells. This protocol was used for immortalization of human corneal endothelial cells. Methods: Transfection was performed by means of electroporation. For immortalization a plasmid encoding large and small SV40 T-antigen was used.Results: The established electroporation protocol was suitable for both cell types. This protocol was used for transfection of human corneal endothelial cells with a plasmid containing the early region of SV40. The transfected cultures exibited an increased life-span before they entered crisis. One culture recovered from crisis and was cultivated for 300 population doublings. The cells exhibited an in vivo-like morphology usually lost during cell culture. Conclusions: We describe for the first time a culture of SV40 transfected human corneal endothelial cells which recovered from crisis and can therefore be regarded as immortalized.

Paclitaxel encapsulated in cationic liposomes (EndoTAG‐1) significantly impairs tumor growth by a significant reduction of functional tumor microcirculation and induction of endothelial cell apoptosis. The aim of the study was to analyze whether platelet activation within the tumor microcirculation contributes to the antivascular effects of vascular targeting chemotherapy using EndoTAG‐1. In vitro, FACS analysis revealed a significant activation of platelets upon treatment with EndoTAG‐1. In vivo, using A‐Mel‐3 tumors in Syrian Golden hamsters equipped with dorsal skinfold chamber preparations, the contribution of platelets to the antivascular effects of EndoTAG‐1 was evaluated by fluorescence and laser‐scanning microscopy. Immediately after a single treatment with EndoTAG‐1 or cationic liposomes devoid of paclitaxel, an increase of platelet adherence in tumor microvessels was observed. This was accompanied by an acute impairment of the microcirculation within the treated tumors leading to reduced tumor perfusion. After repetitive therapy, an increase of platelet adherence and subsequent tumor microvessel occlusions occurred only after treatment with EndoTAG‐1. Comparing to “tumor free” normal tissue controls these microthromboses were tumor selective. Significantly disbalancing the coagulation system within tumors by targeted induction of microthromboses within the tumor microcirculation appears to be an important mechanism of EndoTAG‐1 therapy. © 2007 Wiley‐Liss, Inc.