Michael S. Pepper

Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis

Metastasis is a frequent and lethal complication of cancer. Vascular endothelial growth factor‐C (VEGF‐C) is a recently described lymphangiogenic factor. Increased expression of VEGF‐C in primary tumours correlates with dissemination of tumour cells to regional lymph nodes. However, a direct role for VEGF‐C in tumour lymphangiogenesis and subsequent metastasis has yet to be demonstrated. Here we report the establishment of transgenic mice in which VEGF‐C expression, driven by the rat insulin promoter (Rip), is targeted to β‐cells of the endocrine pancreas. In contrast to wild‐type mice, which lack peri‐insular lymphatics, RipVEGF‐C transgenics develop an extensive network of lymphatics around the islets of Langerhans. These mice were crossed with Rip1Tag2 mice, which develop pancreatic β‐cell tumours that are neither lymphangiogenic nor metastatic. Double‐transgenic mice formed tumours surrounded by well developed lymphatics, which frequently contained tumour cell masses of β‐cell origin. These mice frequently developed pancreatic lymph node metastases. Our findings demonstrate that VEGF‐C‐induced lymphangiogenesis mediates tumour cell dissemination and the formation of lymph node metastases.

Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene.

Expression of the polyoma virus middle T (mT) oncogene in vivo is associated with a profound subversion of normal vascular development, which results in the formation of endothelial tumors (hemangiomas). In an attempt to understand the molecular mechanisms responsible for this phenomenon, we have investigated, in an in vitro system, the morphogenetic properties of endothelial cells expressing this oncogene. mT-expressing endothelioma (End) cells grown within fibrin gels formed large hemangioma-like cystic structures. All End cell lines examined expressed high levels of fibrinolytic activity resulting from increased production of urokinase-type plasminogen activator and decreased production of plasminogen activator inhibitors. Neutralization of excess proteolytic activity by exogenously added serine protease inhibitors corrected the aberrant in vitro behavior of End cells and allowed the formation of capillary-like tubules. These results suggest that tightly controlled proteolytic activity is essential for vascular morphogenesis and that physiological protease inhibitors play an important regulatory role in angiogenesis.

Molecular characterization of lymphatic endothelial cells

The lymphatic microvasculature is uniquely adapted for the continuous removal of interstitial fluid and proteins and is an important entry point for leukocytes and tumor cells. Specialized functions of lymphatics suggest differences in the molecular composition of the lymphatic and blood vascular endothelium. However, the extent to which the two cell types differ is still unclear, and few molecules that are truly specific to lymphatic endothelial cells have been identified to date. We have isolated primary lymphatic and blood microvascular endothelial cells from human skin by immunoselection with the lymphatic marker LYVE-1 and demonstrate that the two cell lineages express distinct sets of vascular markers and respond differently to growth factors and extracellular matrix. Comparative microarray analysis of gene-expression profiles revealed a number of unique molecular properties that distinguish lymphatic and blood vascular endothelium. The molecular profile of lymphatic endothelium seems to reflect characteristic functional and structural features of the lymphatic capillaries. Classification of the differentially expressed genes into functional groups revealed particularly high levels of genes implicated in protein sorting and trafficking, indicating a more active role of lymphatic endothelium in uptake and transport of molecules than previously anticipated. The identification of a large number of genes selectively expressed by lymphatic endothelium should facilitate the discovery of hitherto unknown lymphatic vessel markers and provide a basis for the analysis of the molecular mechanisms accounting for the characteristic functions of lymphatic capillaries.

Regulation of Angiopoietin-2 mRNA Levels in Bovine Microvascular Endothelial Cells by Cytokines and Hypoxia

Angiopoietin-2 (Ang2) is a ligand for the endothelial cell tyrosine kinase receptor Tie2 and counteracts blood vessel maturation/stability mediated by angiopoietin-1 (Ang1), the other known ligand of Tie2. Using degenerate oligonucleotides and reverse transcriptase-polymerase chain reaction, we have screened bovine microvascular endothelial (BME), aortic, lymphatic, pulmonary artery, and transformed fetal aortic endothelial cells, as well as rat smooth muscle cells for Ang1 and Ang2 expression. Except for high Ang2 mRNA levels found in BME cells, none of the endothelial cell types studied expressed appreciable levels of Ang1 or Ang2 mRNAs, whereas smooth muscle cells expressed both Ang1 and Ang2. BME cell Ang2 mRNA levels were increased by vascular endothelial growth factor (1.9- to 2.9-fold), basic fibroblast growth factor (1.6- to 2-fold), both cytokines in combination (2.9- to 4-fold), and hypoxia (3.1- to 5.6-fold) and were decreased by Ang1 (31% to 70%) or transforming growth factor-ss1 (64% to 81%). Ang2 also decreased (60% to 82%) BME cell Ang2 mRNA. mRNA levels for the Tie1 or Tie2 receptors were only slightly modulated under the conditions described above. These findings suggest that the angiogenic effect of a number of regulators may be achieved in part through the regulation of an autocrine loop of Ang2 activity in microvascular endothelial cells.

Manipulating Angiogenesis From Basic Science to the Bedside

Considerable progress has been made recently in understanding the molecular mechanisms of angiogenesis, which like most other biological processes is the result of subtle and often complex interactions between molecules that have regulatory (eg, cytokines and their receptors) and effector (eg, extracellular matrix, integrins, and proteases) functions. The title of this review was chosen to reflect a recent trend in which knowledge acquired through a molecular/cell biological approach is being rapidly transferred to the clinical setting. As a result, by manipulating angiogenesis either positively or negatively, considerable therapeutic benefit can now be envisaged in physiological and pathological settings in which neovascularization is a prominent component.

Angiogenesis: a paradigm for balanced extracellular proteolysis during cell migration and morphogenesis.

Extracellular proteolysis is required for matrix degradation and the regulation of cytokine activity during angiogenesis, and this is dependent on a cohort of proteases and protease inhibitors produced by endothelial and nonendothelial cells. The plasminogen activator (PA)/plasmin system has been extensively investigated in these processes, and descriptive studies have demonstrated that urokinase-type PA (uPA), uPA receptor (uPAR) and PA inhibitor-1 (PAI-1) are expressed by endothelial cells during angiogenesis in vivo. In vitro studies have led to the notion that normal capillary morphogenesis is dependent on a protease-antiprotease equilibrium. These findings are discussed in the context of recent observations on uPA-, uPAR-, PAI-1 and plaminogen-deficient mice, in which developmental and physiological angiogenesis appear to occur normally. This has led to a reevaluation of the role of the PA/plasmin system during angiogenesis. In particular, these observations raise the possibility that the role of this system may be limited to situations in which endothelial cells encounter and must degrade fibrin in order to form new capillary sprouts.

Proteolytic balance and capillary morphogenesis

Angiogenesis is the process by which new capillary blood vessels are formed from preexisting vessels. A number of components of this morphogenetic process, including endothelial cell invasion and capillary lumen formation, are believed to be dependent on tightly controlled proteolytic degradation of the extracellular matrix. The critical importance of an appropriate balance between proteases and protease inhibitors in these processes is suggested by two sets of observations. Firstly, that extracellular matrix invasion and capillary lumen formation are inhibited in the presence of an excess of protease inhibitors. Secondly, that when unchecked by protease inhibitors, excessive proteolysis is incompatible with normal capillary morphogenesis. These results clearly suggest that a precisely regulated proteolytic balance is necessary for normal capillary morphogenesis.

VEGF is a chemoattractant for FGF-2-stimulated neural progenitors

Mmigration of undifferentiated neural progenitors is critical for the development and repair of the nervous system. However, the mechanisms and factors that regulate migration are not well understood. Here, we show that vascular endothelial growth factor (VEGF)-A, a major angiogenic factor, guides the directed migration of neural progenitors that do not display antigenic markers for neuron- or glia-restricted precursor cells. We demonstrate that progenitor cells express both VEGF receptor (VEGFR) 1 and VEGFR2, but signaling through VEGFR2 specifically mediates the chemotactic effect of VEGF. The expression of VEGFRs and the chemotaxis of progenitors in response to VEGF require the presence of fibroblast growth factor 2. These results demonstrate that VEGF is an attractive guidance cue for the migration of undifferentiated neural progenitors and offer a mechanistic link between neurogenesis and angiogenesis in the nervous system.

Vascular endothelial growth factor (VEGF)‐C synergizes with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity

Vascular endothelial growth factor‐C (VEGF‐C) is a recently characterized member of the VEGF family of angiogenic polypeptides. We demonstrate here that VEGF‐C is angiogenic in vitro when added to bovine aortic or lymphatic endothelial (BAE and BLE) cells but has little or no effect on bovine microvascular endothelial (BME) cells. As reported previously for VEGF, VEGF‐C and basic fibroblast growth factor (bFGF) induced a synergistic in vitro angiogenic response in all three cells lines. Unexpectedly, VEGF and VEGF‐C also synergized in the in vitro angiogenic response when assessed on BAE cells. Characterization of VEGF receptor (VEGFR) expression revealed that BME, BAE, and BLE cell lines express VEGFR‐1 and ‐2, whereas of the three cell lines assessed, only BAE cells express VEGFR‐3. We also demonstrate that VEGF‐C increases plasminogen activator (PA) activity in the three bovine endothelial cell lines and that this is accompanied by a concomitant increase in PA inhibitor‐1. Addition of α2‐antiplasmin to BME cells co‐treated with bFGF and VEGF‐C partially inhibited collagen gel invasion. These results demonstrate, first, that by acting in concert with bFGF or VEGF, VEGF‐C has a potent synergistic effect on the induction of angiogenesis in vitro and, second, that like VEGF and bFGF, VEGF‐C is capable of altering endothelial cell extracellular proteolytic activity. These observations also highlight the notion of context, i.e., that the activity of an angiogenesis‐regulating cytokine depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell. J. Cell. Physiol. 177:439–452, 1998.

αvβ3 and αvβ5 integrin antagonists inhibit angiogenesis in vitro

Although angiogenesis is believed to require cell–extracellular matrix interactions which are mediated in part via integrins αvβ3 and αvβ5, a formal demonstration that αvβ3 and αvβ5 are involved in endothelial-cell invasion and capillary-like tube formation is still required. This has arisen from the cellular complexities which occur in vivo and the difficulty in finding appropriate in vitro model systems. Here we have used a three-dimensional assay which employs bovine aortic and microvascular endothelial cells, to show that αvβ3 and αvβ5 regulate angiogenesis in vitro. We cloned and characterized 350–450 bp regions of the bovine homologues of αv, β3 and β5, covering much of the β-propeller and A-domain regions, and show that they are >95% identical to their human orthologues. We used cyclic peptides EMD 121974, 85189 and 66203, which selectively inhibit αvβ3 and αvβ5, but not gpIIbIIIa or α5β1, to probe in vitro angiogenesis induced by angiogenic cytokines in three-dimensional fibrin or collagen gels. We found that these peptides are potent inhibitors of endothelial cell invasion and differentiation induced by vascular endothelial growth factor-A or fibroblast growth factor-2 but do not affect the unstimulated cells in 3D culture. Inhibition was greatest when cells were grown on fibrin, but also occurred on collagen I which is not a recognized ligand for αvβ3. These findings demonstrate the requirement for endothelial cell αvβ3 and αvβ5 integrins during angiogenesis in vitro, and are in accord with the proposed therapeutic application of αvβ3 and αvβ5 antagonists.