Acetylated-low density lipoprotein (Ac-LDL) is taken up by macrophages and endothelial cells via the "scavenger cell pathway" of LDL metabolism. In this report, aortic and microvascular endothelial cells internalized and degraded 7-15 times more [1251]-Ac-LDL than did smooth muscle cells or pericytes. Bound [12Sl]-Ac-LDL was displaced by unlabeled Ac-LDL, but not unmodified LDL. The ability to identify endothelial cells based on their increased metabolism of Ac-LDL was examined using Ac-LDL labeled with the fluorescent probe 1,1 '-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). When cells were incubated with 10 pg/ml DiI-Ac-LDL for 4 h at 37°C and subsequently examined by fluorescence microscopy, capillary and aortic endothelial cells were brilliantly fluorescent whereas the fluorescent intensity of retinal pericytes and smooth muscle cells was only slightly above background levels. DiI-Ac-LDL at the concentration used for labeling cells had no effect on endothelial cell growth rate. When primary cultures of bovine adrenal capillary cells were labeled with 10 pg/ml of DiI-Ac-LDL for 4 h at 37°C, then trypsinized and subjected to fluorescence-activated cell sorting, pure cultures of capillary endothelial cells could be obtained. Utilizing this method, large numbers of early passage microvascular endothelial cells can be obtained in significantly less time than with conventional methods.A major problem in the study of microvascular endothelial cells is the identification of the desired cell population and the subsequent isolation of pure cultures. Our currently used method of establishing pure cultures of capillary endothelial cells involves many weeks of "weeding out" nonendothelial cells (l). The weeding technique involves the assumption that the morphology of capillary endothelial cells is similar to other endothelial cells and is thus directed at isolating colonies with those characteristics. Several markers for endothelial cells are routinely used for confirmation that established cell lines are of endothelial origin. These include the presence of factor VIII related antigen (2, 3) and angiotensin converting enzyme (4, 5). Microvascular endothelial cells differ from large vessel endothelial cells in their requirement for additional growth factors and modified surfaces for optimal growth (l), and their response to tumor factors (l, 6).The receptor-mediated uptake of low density lipoprotein (LDL ~) by cells has been studied in detail (for a review see reference 7). An alternative pathway for the metabolism of chemically modified lipoproteins has also been described (8) and has been termed the "scavenger cell pathway" of LDL metabolism, due to its occurrence in rodent and canine macrophages (9-11) and human monocytes (12). Various chemical methods for modification of LDL have been used to modify the charge of amino groups on LDL including acetylation (8), acetoacetylation (9), and malondialdehyde treatment (12). These modified lipoproteins are taken up by ~Abbreviations us...

Angiogenesis, the recruitment of new blood vessels, is an essential component of the metastatic pathway. These vessels provide the principal route by which tumor cells exit the primary tumor site and enter the circulation. For many tumors, the vascular density can provide a prognostic indicator of metastatic potential, with the highly vascular primary tumors having a higher incidence of metastasis than poorly vascular tumors. Tumor angiogenesis is regulated by the production of angiogenic stimulators including members of the fibroblast growth factor and vascular endothelial growth factor families. In addition, tumors may activate angiogenic inhibitors such as angiostatin and endostatin that can modulate angiogenesis both at the primary site and at downstream sites of metastasis. The potential use of these and other natural and synthetic angiogenic inhibitors as anticancer drugs is currently under intense investigation. Such agents may have reduced toxicity and be less likely to generate drug resistance than conventional cytotoxic drugs. Clinical trials are now underway to develop optimum treatment strategies for antiangiogenic agents.

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Chemotaxis is an important component of wound healing, development, immunity and metastasis, yet the signalling pathways that mediate chemotaxis are poorly understood. Platelet-derived growth factor (PDGF) acts both as a mitogen and a chemoattractant. Upon stimulation, the tyrosine kinase PDGF receptor-beta (PDGFR-beta) autophosphorylates and forms a complex that includes SII2(Src homology 2)-domain-containing proteins such as the phosphatidylinositol-specific phospholipase C-gamma, Ras-GTPase-activating protein (GAP), and phosphatidylinositol-3-OH kinase. Specific tyrosine-to-phenylalanine substitutions in the PDGFR-beta can prevent binding of one SH2-domain-containing protein without affecting binding of other receptor-associated proteins. Here we use phospholipase C-gamma and PDGFR-beta mutants to map specific tyrosines involved in both positive and negative regulation of chemotaxis towards the PDGF-BB homodimer. Our results indicate that a delicate balance of migration-promoting (phospholipase C-gamma and phosphatidylinositol-3-OH kinase) and migration-suppressing (GAP) activities are recruited by the PDGFR-beta to drive chemotaxis towards PDGF-BB.

Capillary endothelial cells from rats, calves, and humans, have been carried in long-term culture. Bovine capillary endothelial cells have been cloned and maintained by serial passage for longer than 8 months. This prolonged culture was accomplished by using tumor-conditioned medium, gelatin-coated plates, and a method of enriching cells However, long-term culture and cloning of capillary endothelial cells have not previously been possible. Del Vecchio et al. (5) reported the isolation of capillary endothelial cells from the adrenal cortex of rats. These cells, however, did not grow and could not be maintained in vitro for more than a few weeks. By modifying Del Vecchio's method and by using tumor-conditioned medium, gelatin-coated plates, and a method of enriching for capillary endothelial cells in primary culture, we have been able to culture capillary endothelial cells obtained from bovine adrenal glands, human adrenal glands, foreskin, and spleen, and rat heart, lung, kidney, and adrenal tissue. We now report long-term culture of human and bovine capillary endothelial cells and the cloning of bovine capillary endothelial cells. bisected and the adrenal cortex was extricated from the capsule. The adrenal cortex was cut into 1-mm pieces. These were centrifuged in phosphate-buffered saline and then incubated in 0.5% collagenase (Worthington) at room temperature for 1 hr in a polyethylene centrifuge tube. This suspension was pipetted with a large-bore pipette against the wall of the centrifuge tube to break the tissue into smaller pieces. The suspension was then passed through a nylon-covered funnel (Nitex Mono Screen Cloth, HC3-110, Tetko, Elmsford, NY). Only capillary segments smaller than 110 ,um passed through the filter. These were centrifuged at 650 rpm for 7 min at 40C and the collagenase was discarded. The pellet was resuspended in 10 ml of Dulbecco's modified Eagle's medium supplemented with 10% calf serum and washed three times. The final pellet of cells was resuspended in 10 ml of Dulbecco's modified Eagle's medium and plated into four gelatinized dishes (60 mm). These plates were incubated at 37°C. Capillary segments and endothelial cell aggregates were the first to adhere to the substratum. Adrenal cortical cells and fibroblasts continued to float, and most were removed by aspirating the supernatant between 1 and 3 hr after plating. After the first aspiration of supernatant, tumor-conditioned medium was added and replaced every 2 days. Capillary fragments usually contained from one to four endothelial cells. As these capillary segments spread onto the coated plastic substratum, each segment became a microcolony with a characteristic appearance that persisted during the next 2-5 days (see Fig. 1 a and b). METHODSCloning. To enrich the primary cultures for bovine capillary endothelial cells and to remove contaminating nonendothelial

The role of angiogenesis in tumor growth has been studied continuously for over 45 years. It is now appreciated that angiogenesis is also essential for the dissemination and establishment of tumor metastases. In this review, we focus on the role of angiogenesis as a necessity for the escape of tumor cells into the bloodstream and for the establishment of metastatic colonies in secondary sites. We also discuss the role of tumor lymphangiogenesis as a means of dissemination of lymphatic metastases. Appropriate combination therapies may be used in the future to both prevent and treat metastatic disease through the rational use of anti-angiogenic and anti-lymphangiogenic therapies in ways that are informed by the current and future work in the field.

Angiostatin, a fragment of plasminogen, has been identified and characterized as an endogenous inhibitor of neovascularization. We show that angiostatin treatment of endothelial cells in the absence of growth factors results in an increased apoptotic index whereas the proliferation index is unchanged. Angiostatin also inhibits migration and tube formation of endothelial cells. Angiostatin treatment has no effect on growth factor-induced signal transduction but leads to an RGD-independent induction of the kinase activity of focal adhesion kinase, suggesting that the biological effects of angiostatin relate to subversion of adhesion plaque formation in endothelial cells.

PTEN is a well-characterized tumour-suppressor gene that is lost or mutated in about half of metastatic castration-resistant prostate cancers and in many other human cancers. The restoration of functional PTEN as a treatment for prostate cancer has however proven difficult. Here, we show that PTEN mRNA can be reintroduced into PTEN-null prostate cancer cells in vitro and in vivo via its encapsulation in polymer-lipid hybrid nanoparticles coated with a poly(ethylene glycol) shell. The nanoparticles are stable in serum, elicit low toxicity, enable high PTEN mRNA transfection in prostate cancer cells, and lead to significant inhibition of tumour growth when delivered systemically in multiple mouse models of prostate cancer. We also show that the restoration of PTEN function in PTEN-null prostate cancer cells inhibits the PI3K-AKT pathway and enhances apoptosis. Our findings provide proof-of-principle evidence of the restoration of mRNA-based tumour suppression in vivo .