Khalid Bajou

Involvement of PA/plasmin system in the processing of pro-MMP-9 and in the second step of pro-MMP-2 activation

© 1997 Federation of European Biochemical Societies.

The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent

Plasminogen activator inhibitor 1 (PAI‐1) is believed to control proteolytic activity and cell migration during angiogenesis. We previously demonstrated in vivo that this inhibitor is necessary for optimal tumor invasion and vascularization. We also showed that PAI‐1 angiogenic activity is associated with its control of plasminogen activation but not with the regulation of cell‐matrix interaction. To dissect the role of the various components of the plasminogen activation system during angiogenesis, we have adapted the aortic ring assay to use vessels from gene‐inactivated mice. The single deficiency of tPA, uPA, or uPAR, as well as combined deficiencies of uPA and tPA, did not dramatically affect microvessel formation. Deficiency of plasminogen delayed microves‐sel outgrowth. Lack of PAI‐1 completely abolished angio‐genesis, demonstrating its importance in the control of plasmin‐mediated proteolysis. Microvessel outgrowth from PAI‐1‐/‐ aortic rings could be restored by adding exogenous PAI‐1 (wild‐type serum or purified recombi‐nant PAI‐1). Addition of recombinant PAI‐1 led to a bell‐shaped angiogenic response clearly showing that PAI‐1 is proangiogenic at physiological concentrations and antiangiogenic at higher levels. Using specific PAI‐1 mutants, we could demonstrate that PAI‐1 promotes an‐giogenesis at physiological (nanomolar) concentrations through its antiproteolytic activity rather than by interacting with vitronectin.—Devy, L., Blacher, S., Grignet‐Debrus, C., Bajou, K., Masson, V., Gerard, R. D., Gils, A., Carmeliet, G., Carmeliet, P., Declerck, P. J., Noèl, A., Foidart, J. M. The pro‐ or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. FASEB J. 16, 147–154 (2002)

Contribution of host MMP-2 and MMP-9 to promote tumor vascularization and invasion of malignant keratinocytes

The matrix metalloproteinases (MMPs) play a key role in normal and pathological angiogenesis by mediating extracellular matrix degradation and/or controlling the biological activity of growth factors, chemokines, and/or cytokines. Specific functions of individual MMPs as anti‐ or proangiogenic mediators remain to be elucidated. In the present study, we assessed the impact of single or combined MMP deficiencies in in vivo and in vitro models of angiogenesis (malignant keratinocyte transplantation and the aortic ring assay, respectively). MMP‐9 was predominantly expressed by neutrophils in tumor transplants, whereas MMP‐2 and MMP‐3 were stromal. Neither the single deficiency of MMP‐2, MMP‐3, or MMP‐9, nor the combined absence of MMP‐9 and MMP‐3 did impair tumor invasion and vascularization in vivo. However, there was a striking cooperative effect in double MMP‐2:MMP‐9‐deficient mice as demonstrated by the absence of tumor vascularization and invasion. In contrast, the combined lack of MMP‐2 and MMP‐9 did not impair the in vitro capillary outgrowth from aortic rings. These results point to the importance of a cross talk between several host cells for the in vivo tumor promoting and angiogenic effects of MMP‐2 and MMP‐9. Our data demonstrate for the first time in an experimental model that MMP‐2 and MMP‐9 cooperate in promoting the in vivo invasive and angiogenic phenotype of malignant keratinocytes.

The antitumoral effect of endostatin and angiostatin is associated with a down-regulation of vascular endothelial growth factor expression in tumor cells

Endostatin and angiostatin are known as tumor‐derived angiogenesis inhibitors, but their mechanisms of action are not yet completely defined. We report here that endostatin and angiostatin, delivered by adenoviral vectors, reduced in vitro the neovessel formation in the mouse aortic ring assay by 85 and 40%, respectively. We also demonstrated in vivo that both endostatin and angiostatin inhibited local invasion and tumor vascularization of transplanted murine malignant keratinocytes, and reduced by 50 and 90% the development of highly vascularized murine mammary tumors. This inhibition of tumor growth was associated with a reduction of tumor vascularization. Expression analysis of vascular endothelial growth factor (VEGF) carried out in the mouse aortic ring model revealed a 3‐ to 10‐fold down‐regulation of VEGF mRNA expression in endostatin‐treated rings. A similar down‐regulation of VEGF expression at both mRNA and protein levels was also observed in the two in vivo cancer models after treatment with each angiogenesis inhibitor. This suggests that endostatin and angiostatin effects may be mediated, at least in part, by their ability to down‐regulate VEGF expression within the tumor. This work provides evidence that endostatin and angiostatin act on tumor cells themselves.

Host-derived plasminogen activator inhibitor-1 (PAI-1) concentration is critical for in vivo tumoral angiogenesis and growth

Plasminogen activator inhibitor type 1 (PAI-1) plays a key role in tumor progression and is believed to control proteolytic activity and cell migration during angiogenesis. We report here that host PAI-1, at physiological concentration, promotes in vivo tumor invasion and angiogenesis. In sharp contrast, inhibition of tumor vascularization was observed when PAI-1 was produced at supraphysiologic levels, either by host cells (transgenic mice overexpressing PAI-1) or by tumor cells (after transfection with murine PAI-1 cDNA). This study provides for the first time in vivo evidence for a dose-dependent effect of PAI-1 on tumor angiogenesis. Of great interest is the finding that PAI-1 produced by tumor cells, even at high concentration, did not overcome the absence of PAI-1 in the host, emphasizing the importance of the cellular source of PAI-1.

Mouse aortic ring assay: A new approach of the molecular genetics of angiogenesis

Angiogenesis, a key step in many physiological and pathological processes, involves proteolysis of the extracellular matrix. To study the role of two enzymatic families, serine-proteases and matrix metalloproteases in angiogenesis, we have adapted to the mouse, the aortic ring assay initially developed in the rat. The use of deficient mice allowed us to demonstrate that PAI-1 is essential for angiogenesis while the absence of an MMP, MMP-11, did not affect vessel sprouting. We report here that this model is attractive to elucidate the cellular and molecular mechanisms of angiogenesis, to identify, characterise or screen “pro- or anti-angiogenic agents that could be used for the treatment of angiogenesis-dependent diseases. Approaches include using recombinant proteins, synthetic molecules and adenovirus-mediated gene transfer.

Role of plasminogen activator-plasmin system in tumor angiogenesis

Abstract: New blood formation or angiogenesis has become a key target in therapeutic strategies aimed at inhibiting tumor growth and other diseases associated with neovascularization. Angiogenesis is associated with important extracellular remodeling involving different proteolytic systems among which the plasminogen system plays an essential role. It belongs to the large serine proteinase family and can act directly or indirectly by activating matrix metalloproteinases or by liberating growth factors and cytokines sequestered within the extracellular matrix. Migration of endothelial cells is associated with significant upregulation of proteolysis and, conversely, immunoneutralization or chemical inhibition of the system reduces angiogenesis in vitro. On the other hand, genetically altered mice developed normally without overt vascular anomalies indicating the possibility of compensation by other proteases in vivo. Nevertheless, they have in some experimental settings revealed unanticipated roles for previously characterized proteinases or their inhibitors. In this review, the complex mechanisms of action of the serine proteases in pathological angiogenesis are summarized alongside possible therapeutic applications.

Plasminogen activator inhibitor-1 protects endothelial cells from FasL-mediated apoptosis.

Plasminogen activator inhibitor-1 (PAI-1) paradoxically enhances tumor progression and angiogenesis; however, the mechanism supporting this role is not known. Here we provide evidence that PAI-1 is essential to protect endothelial cells (ECs) from FasL-mediated apoptosis. In the absence of host-derived PAI-1, human neuroblastoma cells implanted in PAI-1-deficient mice form smaller and poorly vascularized tumors containing an increased number of apoptotic ECs. We observed that knockdown of PAI-1 in ECs enhances cell-associated plasmin activity and increases spontaneous apoptosis in vitro. We further demonstrate that plasmin cleaves FasL at Arg144-Lys145, releasing a soluble proapoptotic FasL fragment from the surface of ECs. The data provide a mechanism explaining the proangiogenic activity of PAI-1.

Endothelial Cell Intracellular Ca2+ Concentration Is Increased Upon Breast Tumor Cell Contact and Mediates Tumor Cell Transendothelial Migration

Tumor cell extravasation is a determinant step in the process of hematogenous metastasis. The signal transduction pathways involved in the interactions between tumor cells and the vascular endothelium during transendothelial migration are still undefined. In the present study, we have investigated the influence of human breast adenocarcinoma cells (MCF7) on human umbilical vein endothelial cell (HUVEC) intracellular Ca2+ concentration ([Ca2+]i). We show that the contact between MCF7 cells and a confluent HUVEC monolayer induces an immediate and transient increase in HUVEC [Ca2+]i. This [Ca2+]i rise could not be elicited by tumor cell-conditioned medium, isolated tumor cell membranes, inert beads or normal breast epithelial cells, demonstrating the involvement of specific recognition mechanisms between MCF7 cells and HUVEC. Depletion of HUVEC intracellular Ca2+ stores by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin as well as the selective depletion of inositol 1,4,5-tri phosphate (IP3)-sensitive Ca2+ stores by prior activation of HUVEC using histamine resulted in a complete inhibition of tumor cell-induced [Ca2+]i elevation. Similar results were obtained when HUVEC monolayers were treated with the tyrosine kinase inhibitor herbimycin A, suggesting a role for tyrosine kinase-associated cell surface receptors in tumor cell-endothelial cell interactions. The depletion of HUVEC intracellular Ca2+ stores by thapsigargin was also shown to delay MCF7-induced endothelial cell disjunction, to prevent their spreading on the subendothelial extracellular matrix and transendothelial migration in vitro. These results suggest that transient changes in endothelial [Ca2+]i may govern multiple steps of tumor cell extravasation.

Human breast adenocarcinoma cell lines promote angiogenesis by providing cells with uPA-PAI-1 and by enhancing their expression

During angiogenesis, endothelial cells use uPA and PAI‐1 to migrate and degrade the basement membrane surrounding capillary blood vessels. Invasive tumor cells produce a large amount of uPA that could bind uPAR present at the endothelial cell surface to facilitate their invasion. To verify this hypothesis, endothelial cells were incubated with conditioned medium (CM) from two breast cancer cell lines (MCF7 and MDA MB 231 cells). Within a short incubation period (30 min) with both CM, an increase of uPA, PAI‐1 and uPA‐PAI‐1 complex was detected in endothelial cell layer as assessed by casein zymography, ELISA and uPA immunostaining. The extent of this enhancement was related to the levels of uPA secreted by tumor cells (high in MDA MB 231 cells and low in MCF7 cells). After 2 hr of incubation, the CM from both tumor cells upregulated uPA and PAI‐1 mRNA levels in endothelial cells in a time‐dependent manner. The uPA increase in the cell layer could not be attributable to an increase of uPAR level. Only the CM from highly invasive MDA MB 231 cells increased the angiogenic morphotype of endothelial cells assessed in a collagen gel. A single addition of amino‐terminal fragment of uPA (ATF) was able to abolish the angiogenic effect induced by MDA MB 231 cell CM. Our data demonstrate that the interactions occurring between breast tumor cells and endothelial cells can modulate tumor angiogenesis at least by two mechanisms: an increase of uPA and PAI‐1 cell surface‐binding and of their expression by endothelial cells.