Roberto Montesano

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.

Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a recently characterized endothelial-specific mitogen which is angiogenic in vivo. Here we demonstrate that VEGF is angiogenic in vitro: when added to microvascular endothelial cells grown on the surface of three-dimensional collagen gels, VEGF induces the cells to invade the underlying matrix and to form capillary-like tubules, with an optimal effect at approximately 2.2nM (100ng/ml). When compared to basic fibroblast growth factor (bFGF) at equimolar (0.5nM) concentrations, VEGF was about half as potent. The most striking effect was seen in combination with bFGF: when added simultaneously, VEGF and bFGF induced an in vitro angiogenic response which was far greater than additive, and which occurred with greater rapidity than the response to either cytokine alone. These results demonstrate that like bFGF, VEGF induces an angiogenic response via a direct effect on endothelial cells, and that by acting in concert, these two cytokines have a potent synergistic effect on the induction of angiogenesis in vitro. We suggest that the synergism between VEGF and bFGF plays an important role in the control of angiogenesis in vivo.

Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells

Extracellular proteolysis is believed to be an essential component of the angiogenic process. The effects of VEGF, a recently described angiogenic factor, were assessed on PA activity and PA and PAI-1 mRNA levels in microvascular endothelial cells. u-PA and t-PA activity were increased by VEGF in a dose-dependent manner, with maximal induction at 30 ng/ml. u-PA and t-PA mRNAs were increased 7.5- and 8-fold respectively after 15 hours, and PAI-1 mRNA 4.5-fold after 4 hours exposure to VEGF. At equimolar concentrations (0.5 nM), VEGF was a more potent inducer of t-PA mRNA than bFGF, while bFGF was a more potent inducer of u-PA and PAI-1 mRNAs. In addition, VEGF induced u-PA and PAI-1 mRNAs with kinetics similar to those previously demonstrated for bFGF. These results demonstrate the regulation of PA and PAI-1 production by VEGF in microvascular endothelial cells and are in accord with the hypothesis that extracellular proteolysis, appropriately balanced by protease inhibitors, is required for normal capillary morphogenesis.

Tumor-promoting phorbol esters induce angiogenesis in vitro

A crucial event during angiogenesis is the invasion of the perivascular extracellular matrix by sprouting endothelial cells. To investigate the possible role of proteases in endothelial cell invasiveness in vitro, bovine microvascular endothelial cells (BMEC) grown on collagen gels were treated with phorbol myristate acetate (PMA), a tumor promoter that markedly increases their production of collagenase and plasminogen activator. Whereas control BMEC were confined to the surface of the gels, PMA-treated BMEC invaded the underlying collagen matrix, where they formed an extensive network of capillary-like tubular structures. This phenomenon, which mimics some of the events occurring during angiogenesis in vivo, required protein synthesis and intercellular contact, was accompanied by collagen degradation, and was prevented by the metalloprotease inhibitor 1,10-phenanthroline.

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.

Rapid transformation of white adipocytes into fat-oxidizing machines

Adenovirus-induced hyperleptinemia rapidly depletes body fat in normal rats without increasing free fatty acids and ketogenesis, implying that fat-storing adipocytes are oxidizing the fat. To analyze the ultrastructural changes of adipocytes accompanying this functional transformation, we examined the fat tissue by electron microscopy. After 14 days of hyperleptinemia, adipocytes had become shrunken, fatless, and encased in a thick basement-membrane-like matrix. They were crowded with mitochondria that were much smaller than those of brown adipocytes. Their gene expression profile revealed striking up-regulation of peroxisome proliferator-activated receptor γ coactivator 1α (an up-regulator of mitochondrial biogenesis not normally expressed in white fat), increased uncoupling proteins-1 and -2, and down-regulation of lipogenic enzymes. Phosphorylation of both acetyl CoA carboxylase and AMP-activated protein kinase was increased, thus explaining the increase in fatty acid oxidation. The ability to transform adipocytes into unique fat-burning cells may suggest novel therapeutic strategies for obesity.

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.

Expression of an activated Notch4(int-3) oncoprotein disrupts morphogenesis and induces an invasive phenotype in mammary epithelial cells in vitro

The protein encoded by the Notch4 gene is a member of the Notch/lin‐12 family of transmembrane receptor proteins, which have been shown to control cell fate determination and cell differentiation in a wide variety of organisms. Expression of Notch4(int‐3), a truncated form of Notch4 having most of its extracellular domain deleted, as a transgene in mice induces the formation of poorly differentiated mammary carcinomas. To establish whether Notch4(int‐3) has the capacity of subverting normal epithelial architecture, we assessed the effect of Notch4(int‐3) expression on the in vitro morphogenetic properties of TAC‐2 mammary epithelial cells. When grown in three‐dimensional collagen gels in the presence of hydrocortisone, both wild‐type and LacZ‐transfected TAC‐2 cells formed alveolar‐like structures composed of polarized epithelial cells surrounding a central lumen. In contrast, TAC‐2 cells programmed to express Notch4(int‐3) formed compact cell aggregates devoid of tissue‐specific organization. In addition, when grown on the surface of a collagen gel, Notch4(int‐3)‐expressing TAC‐2 cells invaded the underlying matrix, whereas TAC‐2 LacZ cells remained strictly confined to the gel surface. Expression of Notch4(int‐3) in TAC‐2 cells also disrupted contact‐inhibition of cell proliferation, resulting in cell multilayering. Our results suggest that the ability of Notch4(int‐3) to subvert normal epithelial morphogenesis and to promote invasion of the extracellular matrix contributes significantly to its tumorigenic potential. Int. J. Cancer 86:652–659, 2000.

Generation and Characterization of Telomerase-Transfected Human Lymphatic Endothelial Cells with an Extended Life Span

The study of lymphatic endothelial cells and lymphangiogenesis has, in the past, been hampered by the lack of lymphatic endothelial-specific markers. The recent discovery of several such markers has permitted the isolation of lymphatic endothelial cells (LECs) from human skin. However, cell numbers are limited and purity is variable with the different isolation procedures. To overcome these problems, we have transfected human dermal microvascular endothelial cells (HDMVECs) with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have produced a cell line, hTERT-HDLEC, with an extended lifespan. hTERT-HDLEC exhibit a typical cobblestone morphology when grown in culture, are contact-inhibited, and express endothelial cell-specific markers. hTERT-HDLEC also express the recognized lymphatic markers, Prox-1, LYVE-1 and podoplanin, as well as integrin alpha9, but do not express CD34. They also form tube-like structures in three-dimensional collagen gels when stimulated with vascular endothelial growth factors -A and -C. Based on these currently recognized criteria, these cells are LEC. Surprisingly, we also found that the widely studied HMEC-1 cell line expresses recognized lymphatic markers; however, these cells are also CD34-positive. In summary, the ectopic expression of hTERT increases the life span of LECs and does not affect their capacity to form tube-like structures in a collagen matrix. The production and characterization of hTERT-HDLEC will facilitate the study of the properties of lymphatic endothelium in vitro.