Basic Science for Clinicians Molecular Biology and Immunology for Clinicians 20 Angiogenesis and Vascular Growth Factors and Signals Leonard H. Sigal If we accept the perfectly reasonable premise that the mass of inflammatory tissue in rheumatoid arthritis (and psoriatic arthritis or any other inflammatory joint disease) requires oxygen and nutrition to survive and grow, we are confronted with a novel concept for therapy: If we can block the nutritional supply of the pannus, we can suppress or prevent its growth and the subsequent destruction of the joint. Thus, an understanding of how new blood vessels nourish the inflammatory mass could be pivotal in successfully treating our patients. Angiogenesis is the process whereby new blood vessels enter the site of inflammation or growing malignancy to supply the invading tissue. Many growth factors and local tissue conditions help to determine blood vessel growth, there being proand antiangiogenetic influences. Thus, this is fertile ground for therapeutic molecular manipulations. (J Clin Rheumatol 2002;8:281–283) Key words: Angiogenesis, Rheumatoid arthritis, Metalloproteinases hat is the cause of rheumatoid arthritis? We can speculate, which is fun, and as long as what you suggest is plausible, how can I argue? But I do not know—no one does. We will return to this issue, at least some of the potential therapeutic aspects, in a future installment on “tolerance.” For the time being, let’s return to the often-repeated observation that the pannus of rheumatoid arthritis can be thought of as a locally invasive nonmetastatic malignancy— presumably driven by local cytokines and growth factors. Think about this analogy; what allows tumors, especially metastases, to W Sigal • Angiogenesis Growth Factors and Signals grow and expand is blood vessels. Without the influx of new blood vessels, the tumor mass would rapidly expand beyond its blood flow’s ability to supply nutrition and oxygen and the tumor would die. It has been estimated that diffusion of nutrients can adequately supply a tumor’s growth to no more than 1 to 2 mm3. Like an army that has been cut off from its supply lines, a tumor must have (induce) new blood vessels or it will die. Thus, in understanding how pannus grows and invades, we should have a better understanding of how pannus is supplied—and this supply is a poten- tial Achilles’ heel, a possible way of suppressing pannus formation or expansion, thereby decreasing cartilage and bone destruction. So, we turn our attention to angiogenesis, the growth of blood vessels, and the growth factors and other signals that control this development. Invasion of new blood vessels starts with modification of the extracellular matrix (ECM) of the tissue being entered; this modification is accomplished by a number of enzymes, including matrix metalloproteinases (MMPs). These include MMP1 (also known as collagenase 1, which cleaves collagen type II), MMP2 (also known as the 72 kD gelatinase or matrilysin, which cleaves collagen type X), MMP9 (also known as type IV collagenase, type V collagenase, gelatinase B, and invasin, which cleaves collagen type IV) and cathepsins, among others. The Division of Rheumatology and Connective Tissue Research, Department of Medicine, Department of Pediatrics, and Department of Molecular Genetics & Microbiology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey. Address correspondence to: Leonard H. Sigal, MD, 1 Robert Wood Johnson Place—MEB 484, New Brunswick, NJ 08903-0019 U.S.A. E-mail: sigallh@umdnj.edu. Copyright © 2002 by Lippincott Williams & Wilkins, Inc. DOI: 10.1097/01.RHU.0000030734.22246.25 281 ECM modification is followed by migration of a tube of endothelial cells with subsequent endothelial cell proliferation and maturation, capillary differentiation, and ultimate anastomosis of the capillary tube to more proximal supplying vessels. But what drives all of this? There are a variety of proangiogenic factors produced by a number of different cells that elicit this influx of vessels. Recall the principle of yin and yang discussed in a previous entry in this series. For every biologic stimulus, there is an opposing force that tries to impede it. So it is with angiogenesis; there are inhibitors of angiogenesis, as well. And what is the place of angiogenesis in a normal adult? New blood vessels are needed in normal adults in the female genital tract once a month and in wound healing, but no place else—not in the eye (vascular disease in diabetes) or in tumors or in inflammatory tissue in the joint. Something has gone awry locally in each of the latter examples. The main focus of this discussion will be a family of proteins called vascular endothelial growth factor (VEGF), which was previously also known as vascular permeability factor (VPF). VEGF is a homodimer of 34 to 42 kD proteins linked by a disulfide bond. Its two names describe the main effects of VEGF: It is an endothelial cell mitogen and it helps control vascular permeability and thus the ability of fluid to leave the intravascular space and to enter surrounding tissues. VEGF is a family of five proteins, produced by a series of alternative mRNA splicings of a single gene transcript, of between 121 and 165 amino acid residues. All but the smallest VEGF maintain the ability to bind to heparan sulfate (keep this in mind as we proceed); this is a crucial property. VEGF is the first member of a family of proteins including placental growth factor (P1GF) and VEGF-A, -B, -C, -D, and -E. Another very potent angiogenic factor 282 is basic fibroblast growth factor (bFGF2), which also binds avidly to heparan sulfate-containing glycosaminoglycans. High levels of VEGF, b-FGF2, and P1GF have been identified in rheumatoid synovial fluids. The level of VEGF-A in synovial fluid correlates with the concentration of polymorphonuclear (PMNs) cells in the fluid which, along with studies showing elevated levels of VEGF121 mRNA in the PMNs in RA synovial fluid, suggests that PMNs may be a significant source of VEGF in the rheumatoid joint. Synovial tissue monocytes and fibroblasts or synoviocytes as well as vascular smooth muscle cells all are also potential sources of VEGF in the inflamed joint. Local cytokines can induce VEGF production by these cells. For example, production is induced by IL-1␤ from aortic smooth muscle cells and synoviocytes, transforming growth factor (TGF) ␤ in epithelial cells and fibroblasts, FGF2 and plateletderived growth factor (PDGF) in vascular smooth muscle cells, and TNF-␣ from monocytes purified from peripheral blood mononuclear cells (PBMC); TNF-␣ does not, however, stimulate VEGF production by synovial fibroblasts. Epidermal growth factor (EGF) is another angiogenic factor, made in vitro by glioblastoma cells. Additional triggers include prostaglandins and engagement of the CD40 surface marker on synoviocytes; in vitro synoviocyte synthesis of VEGF is stimulated by exposure to peripheral blood monocytes and PMNs (the former more so than the latter) from RA patients. Endotoxin also stimulates VEGF production. Of note, there are people whose cells make low levels of VEGF upon such stimulation and others who are “high producers.” Polymorphisms within the DNA promoter for VEGF have been found, but no correlation with clinical status has thus far been established. IL-4 and IL-10 both downregulate VEGF, thus acting as antian- giogenic factors. TNF-␣ also acts as an antiangiogenic factor. Despite the fact that TNF-␣ induces the production of VEGF, it downregulates the VEGF R-I and R-II receptors on endothelial cells, thus helping to control angiogenesis. In vivo, one of the most potent stimuli of VEGF production is hypoxia. Inflamed joints are quite hypoxic, perhaps because of decreased blood flow due to pressure from local swelling. Hypoxia synergizes with IL-1␤ and TGF-␤ in stimulating VEGF production. Addition of cobalt to cell culture mimics the effects of hypoxia; this system has been used to demonstrate that RA PBMC make more VEGF than do PBMC from normal controls. Hypoxic stimulation of VEGF production is at least in part mediated by a heterodimeric transcription factor known as hypoxia inducible factor- 1 (HIF-1). HIF-1 binds to a DNA sequence within the hypoxia response element (HRE) that helps control VEGF transcription. Of note, this DNA sequence was first identified as an enhancer sequence within the response element for the erythropoietin gene, erythropoietin being another protein whose production is elicited by hypoxia. The HIF-1 heterodimer is made up of an HIF-1␣ chain and an HIF-1␤ chain. There are at least two other ␣ chains, HIF-2␣ and HIF-3␣, suggesting alternative control pathways for angiogenesis. The transcription factors AP1 and SP1 also bind cooperatively with HIF-1 in the hypoxia response. But wait, it gets more complicated. There are other natural products that are involved in the control of angiogenesis. Proangiogenic factors include tissue factor (TF), platelet activating factor (PAF), granulocyte-colony stimulating factor (G-CSF), hepatocyte growth factor (HGF)/scatter factor (SF), and a protein known as angiopoietin-1. (Now for one of my digressions: the suffix -poietin is de- Journal of Clinical Rheumatology • Volume 8, Number 5 • October 2002 rived from the Greek word poiesis for “a creation” and is also the root for the words poems, poetry, and poet.) On the other side of the equation are inhibitors of angiogenesis, including angiostatin (a peptide derived via proteolytic cleavage, from plasminogen), endostatin (cleavage product of type XVIII collagen), vasostatin (derived from calcireticulin), a 16 kDa fragment derived from prolactin, thrombospondin, 2 methoxyestradiol, interferons ␣ and ␤, IL-12, and tissue inhibitors of metalloproteinase (TIMPs). Last, but by no means least, in the inhibitor category is angiopoietin-2, which binds to the same receptor as does angiopoietin-1. Angiopoietin-1 is an agonist, and angiopoietin-2 an antagonist at a receptor known as Tie-2 (tyrosine kinase with immunoglobulin-like loops and EGF homology domains— do not blame me, I do not make up these acronyms). The balance of effects of angiopoietin-1 and angiopoietin-2 on a single receptor is reminiscent to me of the IL-1 balance with IL-1ra, which has been brought from the bench to the clinic within the last months. So, there are all sorts of proteins and peptides to work with, receptors to block, cytokines and growth factors whose expression you can enhance or suppress to modify angiogenesis. But here is one more thing to consider. VEGF and FGF-2, among other proangiogenic factors, are known to bind to heparan sulfatecontaining glycosaminoglycans (HS-GAG). In fact, the ECM is a capacious storage site for these heparan-binding growth factors, with specific oligosaccharide sequences on the HS-GAG binding Sigal • Angiogenesis Growth Factors and Signals the growth factors. This binding protects the growth factors from proteolysis and inhibits the diffusion of the factors away from the intended site of action. The same MMPs noted above degrade the ECM, liberating intact growth factors at the same time that the modified ECM allows endothelial cell tubules to enter. Of note, the HSGAGs seem to enhance growth factor binding to their cell surface receptors. Endothelial cells bear GAGs, for example, syndecan and ryudecan, that bind growth factors and efficiently present them to specific receptors on the same cell. Synoviocytes express perlecan, a specialized HS-GAG; about 25% of the proteoglycans expressed by synoviocytes include HS oligosaccharides. Without HS-GAGs, FGF-2 does not bind to its receptor and has no biologic activity. In in vitro studies, ECM binding competes favorably with cellular receptor binding. Seventy percent of exogenous FGF-2 bound to the ECM compared with 7% binding to endothelial cells in culture. Heparanase releases growth factors from the ECM, and heparanase activity has been identified in RA synovial fluids, derived from PBMC, PMN, or endothelial cells. Degradation of the ECM liberates other bound growth factors, including P1GF, TNF-␣, IL-1, a series of Th1-derived cytokines (IL-2, IL-12, ␥ interferon), the Th-2 cytokine IL-4, and the chemokines IL-8, monocyte chemotactic factor-1 (MCP-1), and midkine. Thus, the levels of pro- and antiangiogenetic factors are dependent on many factors: synthesis by resident and newly arriving cells, stimulation or suppression of this synthesis by influencing cytokines and other growth factors, stimulation by hypoxia, destruction by proteolytic enzymes, liberation of stashed factors upon degradation of the ECM by MMPs and heparanase, inhibition of the MMPs by the presence of TIMPs. Each one of these influences presents a potential target for modification of angiogenesis. If you block angiogenesis, then you retard or hinder pannus formation, thus preventing bone and cartilage damage and erosion. Clinical studies of a variety of methods to control angiogenesis have been started or are planned, including: antibodies to VEGF; inhibitors of MMPs; inhibitors of angiogenic factor receptor tyrosine kinase activity; inhibitors of bFGF and TNF-␣; monoclonal antibodies that block integrins on the surface of endothelial cells; anti-angiogenic cytokines, like IL-12 and interferon-␣; fragmin or a synthetic heparinoid pentose polysulfate (PPS) that bind bFGF; and inhibitors of endothelial cell proliferation. Although one can control inflammation with current medications, including TNF-␣ and IL-1 blockades, it may well be that specific angiogenesis blockade is necessary to really control RA; that is, until we identify the root cause of RA, the antigen(s) that are the initial focus of the inflammation in our rheumatoid patients’ joints. SUGGESTED READING 1. Brenchley PEC. Antagonising angiogenesis in rheumatoid arthritis. Ann Rheum Dis 2001; 60(Suppl 3):iii71– 4. 2. Brenchley PEC. Angiogenesis in inflammatory joint disease: a target for therapeutic intervention. Clin Exp Immunol 2000;121:426 –9. 3. Koch AE. Angiogenesis: implications for rheumatoid arthritis. Arthritis Rheum 1998;41:951– 62. 4. Talks KL, Harris AL. Current status of antiangiogenic factors. Brit J Haematol 2000;109:477– 89. 283