Processing of VEGF-C and -D by the Proprotein Convertases: Importance in Angiogenesis, Lymphangiogenesis, and Tumorigenesis

Transcription

1 Processing of VEGF-C and -D by the Proprotein Convertases: Importance in Angiogenesis, Lymphangiogenesis, and Tumorigenesis

2 ii Colloquium Digital Library of Life Sciences This e-book is an original work commissioned for the Colloquium Digital Library of Life Sciences, a curated collection of time-saving pedagogical resources for researchers and students who want to quickly get up to speed in a new area of life science/biomedical research. Each e-book available in Colloquium is an in-depth overview of a fast-moving or fundamental area of research, authored by a prominent contributor to the field. We call these resources Lectures because authors are asked to provide an authoritative, state-of-the-art overview of their area of expertise, in a manner that is accessible to a broad, diverse audience of scientists (similar to a plenary or keynote lecture at a symposium/meeting/colloquium). Readers are invited to keep current with advances in various disciplines, gain insight into fields other than their own, and refresh their understanding of core concepts in cell & molecular biology. For the full list of available Lectures, please visit: All Lectures available as PDFs on our website. Access is free for readers at institutions that license Colloquium. Please info@morganclaypool.com for more information.

3 iii Colloquium Series on Protein Activation and Cancer Majid Khatib, Ph.D., Research Director, INSERM, and University of Bordeaux, France This series is designed to summarize all aspects of protein maturation by convertases in cancer. Topics included deal with the importance of these processes in the acquisition of malignant phenotypes by tumor cells, induction of tumor growth, and metastasis. This series also provides the latest knowledge on the clinical significance of convertase expression and activity, and the maturation of their protein substrates in various cancers. The potential use of their inhibition as a therapeutic approach is also explored. For a full list of published and forthcoming titles:

4 Copyright 2014 by Morgan & Claypool All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means electronic, mechanical, photocopy, recording, or any other except for brief quotations in printed reviews, without the prior permission of the publisher. Processing of VEGF-C and -D by the Proprotein Convertases: Importance in Angiogenesis, Lymphangiogenesis, and Tumorigenesis Géraldine Siegfried and Abdel-Majid Khatib ISBN: paperback ISBN: ebook DOI: /C00097ED1V01Y201310PAC006 A Publication in the Morgan & Claypool Publishers series Colloquium Series on Protein Activation and Cancer Lecture #6 Series Editor: Majid Khatib, Research Director, INSERM and University of Bordeaux Series ISSN ISSN ISSN print electronic

5 Processing of VEGF-C and -D by the Proprotein Convertases: Importance in Angiogenesis, Lymphangiogenesis, and Tumorigenesis Géraldine Siegfried and Abdel-Majid Khatib University of Bordeaux, LAMC, UMR 1029, F-3400 and INSERM U1029 COLLOQUIUM SERIES ON PROTEIN ACTIVATION AND CANCER #6

6 vi Abstract The vascular endothelial growth factor (VEGF) family members that include VEGF-A, -B, -C, -D, and placental growth factor (PlGF), display distinct binding affinities for their receptors VEGFR-1, -2, and/or -3. In addition to their requirements in the initiation, development, and maintenance of blood and lymphatic vasculature, VEGFs and VEGFRs are upregulated during neoplasia and are involved in the remodeling of tumoral blood and lymphatic vasculature. By activating VEGFR-1 and VEGFR-2, both expressed on blood endothelial cells, VEGF-A promotes the formation of new tumoral blood vessels and thereby accelerates tumor growth. In contrast, upregulation of VEGF-C, a ligand for lymphatic endothelial VEGFR-3 as well as for VEGFR-2, induces the formation of tumor-associated lymphatic vessels and thus promotes the passive metastatic dissemination of tumor cells to regional lymph nodes. Of the VEGF family members, only VEGF-C and -D were found to be proteolytically processed by Furin-like enzymes. This processing controls the selective activation of VEGFR-2 and -3 signaling during tumor angiogenesis and lymphangiogenesis. Here, we provide an overview of angiogenesis processes and discuss the importance of VEGF-C and VEGF-D precursors processing by the proprotein convertases during the activation of VEGFR-2 and VEGFR-3 receptors and the mediation of their functions during angiogenesis, lymphangiogenesis, and tumorigenesis. Keywords VEGF-C, VEGF-D, proprotein convertases, angiogenesis, lymphangiogenesis

7 vii Contents Abbreviations... ix Acknowledgments... xi 1. Angiogenesis: A Global Overview New Vessels Formation Processes Intussusception and Sprouting Angiogenesis The Vascular System Structure of Vascular System The Lymphatic System Structure of Lymphatic System VEGF Family Members, Their Receptors, and Signaling Vascular Endothelial Growth Factors (VEGFs) Neuropilins Activation, Signaling, and Function of VEGFRs VEGFR1: A Negative Angiogenesis Regulator VEGFR2 Receptor Induction of Proliferation by VEGF-A Induction of Migration by VEGF-R2 Activation Regulation of Survival by VEGFR2 Activation Regulation of Vascular Permeability by VEGFR VEGFR3 Signaling and Function VEGFR Heterodimerization the Role of VEGF Family Members in Tumor Angiogenesis and Lymphangiogenesis VEGFs and VEGFRs in Tumor Angiogenesis VEGFs and VEGFRs in Tumor-Associated Lymphangiogenesis... 26

8 viii PROCESSING OF VEGF-C AND -D BY THE PROPROTEIN CONVERTASES 5. Maturation of VEGF-C and VEGF-D by the Proprotein Convertases VEGF-C Precursor Processing by the Proprotein Convertases ProVEGF-C Processing by the PCs and Normal Angiogenesis: Fin Zebrafish Model Processing of ProVEGF-C in Tumor Angiogenesis and Lymphangiogenesis Processing of VEGF-D by the PCs Concluding Remarks References Author Biographies... 53

9 ix Abbreviations Dll4 Delta like ligand 4 ECs endothelial cells MMPs metalloproteinases CEPs circulating bone marrow-derived endothelial precursor cells Prox1 homeobox transcription factor PTPs phosphotyrosine phosphatases DEP1 density enhanced phosphatase 1 VEPTP vascular endothelial PTP ERK extracellular signal-regulated kinase MAPK mitogen-activated protein kinase PI3K phosphoinositide 3 kinase PKB/AKT protein kinase B KDR kinase insert domain receptor Flk1 fetal liver kinase-1 SOS nucleotide-exchange factor son of sevenless TAMs tumor-associated macrophages CAFs carcinoma-associated fibroblasts PCs proprotein convertases VEGFs vascular endothelial growth factors prongf pro-nerve growth factor VHD VEGF homology domain NP neuropilin

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11 xi Acknowledgments This work was supported by grants from INCA, INSERM, LLCC, Region Aquitaine, and Bordeaux-1 University.

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13 1 c h a p t e r 1 Angiogenesis: A Global Overview 1.1 NEW VESSELS FORMATION PROCESSES Angiogenesis is controlled by a balance between stimulatory and inhibitory factors. The angiogenic switch occurs when this balance shifts in favor of positive stimuli [1, 2]. Indeed, induction of angiogenesis is mediated by various angiogenic factors including VEGFs, angiopoietin, FGF, and PDGF, that induce the proliferation and migration of endothelial cells (ECs) to newly formed vessels [3 5]. In response to chemotactic growth factors, the migration of ECs following degradation of extracellular matrix (ECM) by several matrix metalloproteinases (MMPs) plays an important role during angiogenesis. Produced by primitive blood vessels, this ECM was also found to be responsible for the recruitment of other cells (pericytes and smooth muscle cells) also involved in the formation and maturation of new vessels [3 6]. To date several negative regulators of angiogenesis were identified; however, little is known about their exact role during physiological angiogenesis. Of these, regulator thrombospondin, previously reported to be secreted by epithelial cells was found to inhibit tumor growth angiogenesis [7]. Lately, other anti-angiogenesis agents were also identified including endostatin, tumstatin, vasostatin, and lately, anti-vegf [8] Intussusception and Sprouting Angiogenesis The formation of new vessels not only requires endothelial cells migration, proliferation, but also their organization in three dimensions. The two major mechanisms necessary for vessel formation are intussusception and sprouting angiogenesis. Intussusception is the splitting of a preexisting vessel into two new smaller vessels by penetration of smooth muscle cells through the endothelial cell layer [9] (Figure 1). Whereas sprouting angiogenesis is characterized by the ability of endothelial cells derived from a preexisting vessel to acquire the capacity to invade the surrounding tissue by forming angiogenic sprouts (Figure 2). The latter is composed of a principal tip cells and trailing stalk cells (Figure 2). These cells were found to orientate and grow toward the source of stimulating factors [10]. During this angiogenesis

14 PROCESSING OF VEGF-C AND -D BY THE PROPROTEIN CONVERTASES FIGURE 1: Schematic representation of new vascular formation by intussusceptive growth processes. I Invagination of opposing capillary wall into the vessel lumen. II Contact between the two endothelial walls, III Endothelial bilayer and basal membranes (bm) are perforated centrally in order to form two new vessels with proliferating cells on each segment. The later are subsequently covered by fibroblasts (F) and pericytes (Pr). FIGURE 2: Schematic representation of endothelial sprouting. To generate new vessels, endothelial sprouts need to connect to other sprouts. The processes start with tip cells mobilization, and local matrix degradation (I), followed by the extension of the sprouts (II), initiation of junction formation between the new sprouts through Filopodias (III) and finally occurrence of endothelial cells proliferation and extension of the lumen (IV). Basal membrane (bm), endothelial cells (EC).

15 Angiogenesis: A Global Overview 3 processes, the deposition of smooth muscle cells and basement membrane following the lumen formation of the vessel stabilize the newly formed vessel [11]. Previously, these processes were reported to be regulated by various angiogenic factors, and the extent of sprouting was found to be negatively regulated by Delta-like ligand 4 (Dll4) and its Furin-like substrate receptor Notch [12, 13]. Other mechanisms were also suggested to be actively involved in the formation of new vessels. These include circulating bone marrow-derived endothelial precursor cells (CEPs or EPCs). However, although these cells were found to be active during angiogenesis immediately close to the endothelium, they were not found to be plainly incorporated into the endothelium, suggesting their probable minor and/or secondary role in angiogenesis [14 16]. 1.2 THE VASCULAR SYSTEM Essential for normal development and survival, the vascular system is also required for the delivery of nutrients and oxygen to all cells that constitute various organisms. During embryogenesis, mesodermal progenitor cells, known as hemangioblasts, generate both the precursors of EC (angioblasts) and primitive hematopoietic cells (Figure 3). Using endothelial tubes during vasculogenesis, the angioblasts aggregate in the embryo and yolk sac and form primary vascular plexus [3]. Whereas the primitive hematopoietic cells form blood islands in the visceral yolk sac [9, 10], angiogenesis also occurs in the adult as observed during wound and during pathological conditions, including diabetes, hypertension, and tumor progression (Figure 4) [1] Structure of Vascular System The vascular system displays various vessels with different structure that impact on their functions. The vessels that constitute the arteries and veins present three layers (Figure 5). These include the intima that contains endothelial cells, media formed with smooth muscle cells, and adventitia that contains fibroblasts, elastic laminae, and matrix. However, as compared to veins, arteries contain several sheets of smooth muscle cells that involve them in the control of vessel diameter and blood flow. Indeed, arteries are able to mediate high arterial pressure as compared to veins that are involved in the low-pressure section of the vascular system. The vessels that constitute the capillaries are involved in the mediation of the nutrients and oxygen exchanges with the surrounding tissue. These vessels are composed of one layer of endothelial cells (Figure 5). Based on their function and distribution in various organs, four types of capillaries are known:

16 4 PROCESSING OF VEGF-C AND -D BY THE PROPROTEIN CONVERTASES FIGURE 3: Embryonic endothelial cells generation. During embryonic development, mesodermderived precursor cells known as hemangioblasts are stimulated to give rise to hematopoietic and angioblasts (endothelial progenitor cells). A primary capillary network is formed by the process of vasculogenesis in which endothelial progenitors differentiate to form primitive blood vessels in the yolk sac and embryo (I). Later, new capillaries are formed by sprouting angiogenesis (II). These vessels are stabilized following the recruitment of other cells to form mature vasculature (e.g., pricytes and smooth muscle cells) (III).

17 Angiogenesis: A Global Overview FIGURE 4: Tumor angiogenesis. Schematic representation of new capillary sprouts generation from a pre-existing vessel towards the hypoxic region of a tumor. During these processes angiogenic factors secreted by tumor cells (I) that activate the vascular endothelium, induce proteases degradation of the basement membrane of endothelial cells. Endothelial cells migrate toward the angiogenic stimulus (II) and proliferate to form new vessels within the tumors (III).

18 PROCESSING OF VEGF-C AND -D BY THE PROPROTEIN CONVERTASES FIGURE 5: Blood vessel structures. There are three main types of blood vessels. I: arteries; that drive blood from the heart to tissues. II: veins; involved in the collection of blood from the small vessels and its return to heart. III: capillaries; small vessels that take blood through the organs and tissues. Arteries and veins present three layers (intima, media and adventitia). Capillaries are composed with one layer of endothelial cells. (1) endothelial cells found in the muscle, (2) endothelial cells of the retina and blood brain barrier, (3) endothelial cell layer of endocrine glands, (4) liver sinusoidal endothelial cells [3, 4]. 1.3 THE LYMPHATIC SYSTEM Initially, embryonic lymphatic endothelial cells were suggested to be derived from embryonic veins by sprouting [17]. Lately, recent studies confirmed this observation and revealed that lymphatic

19 Angiogenesis: A Global Overview 7 FIGURE 6: Lymphatic vessels origin. Lymphatic endothelial cells arise by sprouting from embryonic veins in the jugular and perimesonephric areas and migrate to form primary lymph sacs and primary lymphatic plexus. At embryonic day E10, the homeobox transcription factor Prox1 promotes endothelial cells differentiation into lymphatic endothelial cells (I). Subsequently, VEGFR-3 behaves as a chemotactic and growth factor for the newly formed lymphatic endothelial cells (II). Hematopoietic cells were also found to be involved in the separation of the vascular and lymphatic system (III).

20 PROCESSING OF VEGF-C AND -D BY THE PROPROTEIN CONVERTASES FIGURE 7: Structure of lymphatic vessels and function. Lymphatic vessels recruit smooth muscle cells and develop lymphatic valves that prevent any backflow of lymph. Increased interstitial fluid derived from vascular vessels induced tension on fibers of the ECM that permits its entry in the lymph capillary (I). The lymph progresses within the lymph vessel valves system (II). This fluid can be reabsorbed by the vascular system and return to blood circulation (III).

21 Angiogenesis: A Global Overview endothelial cell growth, migration, and survival are mediated by the vascular endothelial growth factors VEGF-C and VEGF-D and their receptor VEGFR-3 [17 22] (Figure 6). In addition, at the embryonic day E10, the homeobox transcription factor Prox1 responsible for the lymphatic endothelial cell fate, was found to be expressed in a subpopulation of vein endothelial cells and promotes their differentiation into lymphatic endothelial cells [23] (Figure 6). Subsequently, following VEGFR-3 expression, the latter is induced and activated by VEGF-C and behave as a chemotactic and growth factor for the newly formed lymphatic endothelial cells (Figure 6) [24]. Other findings have also implicated the hematopoietic cells in the separation of the vascular and lymphatic systems [25] (Figure 6) Structure of Lymphatic System Like the vascular system, the remodeling of the lymphatic vasculature includes sprouting of lymphatic capillaries from the primary lymphatic plexus, whereas deeper lymphatic vessels recruit smooth muscle cells and develop lymphatic valves. On the other hand, other studies proposed that lymphatic vessels are formed by fusion of mesenchymal lymphatic endothelial precursor cells or lymphangioblasts [26 28]. The structure of the lymphatic capillaries is distinct from the vascular capillaries. Indeed, the latter are composed of attenuated endothelial cells that form valves and lack continuous basal lamina and pericytes. In addition, in many areas, these endothelial cells are extensively overlapping and interact with several components of the ECM. As shown in Figure 7, increased interstitial fluid induced tension on fibers of the ECM space, which is transmitted via the anchoring filaments to endothelial cells and allows them to distend from each other, that permits the entry of interstitial fluid, macromolecules, and cells [27, 28] (Figure 7). The interstitial fluid found in the extracellular space is derived from the plasma found in the vascular capillaries. This fluid is reabsorbed by the vascular system and return to blood circulation [29] (Figure 7). Different from the cardiovascular system, the lymphatic system has no central pump in mammals and using microvalves, is involved in only 10% of the interstitial fluid reabsorption that constitutes the lymph. The lymph is then transported to the blood circulatory system through progressively larger lymphatic vessels (Figure 7). The lymphatic system also plays an important role in the immune system. The presence of pathogens lymph nodes was found to elicit an immune response due to the presence of the antigen-presenting cells, including B and T lymphocytes [30].