Angiogenesis is the process of forming new blood vessels from existing blood vessels, formed in vasculogenesis. It is a highly complex process involving extensive interplay between cells, soluble factors, and the extracellular matrix (ECM). Angiogenesis is critical during normal physiological development, but it also occurs in adults during inflammation, wound healing, ischemia, and in pathological conditions such as rheumatoid arthritis, hemangioma, and tumor growth. Proteolysis has been indicated as one of the first and most sustained activities involved in the formation of new blood vessels. Numerous proteases including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase domain (ADAM), a disintegrin and metalloproteinase domain with throbospondin motifs (ADAMTS), and cysteine and serine proteases are involved in angiogenesis. This article focuses on the important and diverse roles that these proteases play in the regulation of angiogenesis.

MMPs Matrix metalloproteinases (MMPs) are a large multigene family of zinc-dependent endopeptidases. The collective MMP family is capable of degrading all known ECM macromolecules. MMP activity is regulated at the level of transcription, post-translationally by proteolytic cleavage, and by endogenous inhibitors known as tissue inhibitors of metalloproteinases (TIMPs). The role of matrix metalloproteinases and TIMPs in several pathological conditions including angiogenesis, tumor growth, and metastasis has been investigated and very well described.

Matrix metalloproteinases contain five conserved domains/sequence motifs: Signal peptide sequence, which directs the enzyme into the rough endoplasmic reticulum during synthesis Propeptide domain, which is cleaved to activate the enzyme Catalytic domain, which contains the conserved Zn2+ binding region and mediates enzyme activity Hemopexin domain, which provides the substrate specificity Small hinge region, which allows the hemopexin domain to bring the substrate to the active core of the catalytic domain

There is also a subfamily of the matrix metalloproteinases, the membrane-type MMPs (MT-MMPs) which contain an additional transmembrane domain and a short cytoplasmic domain. After activation of MMPs by removal of the propeptide domain, their activity is regulated by TIMPs. TIMPs specifically and reversibly inhibit the activity of MMPs. So far there have been identified four members of the family, TIMP1–4. All TIMPs contain twelve conserved cystein residues, which form six disulfide bonds. The C-terminal domains of TIMPs are highly variable and confer their specificity towards preferred MMP targets.

ADAM/ADAMTS

ADAMs comprise a family of integral membrane as well as secreted glycoproteins which are related to snake venom metalloproteinases and MMPs. Like MMPs, ADAMs are composed of multiple conserved domains. They contain propeptide, metalloproteinase, disintegrin-like, cystein-rich, and epidermal growth factor like domains, although variations in domain composition have been observed in non-animal organisms. Membrane anchored ADAMs contain a transmembrane and cytoplasmic domain. The domains contained within the ADAMs family have been characterized, uncovering their functional and structural roles. ADAMs contain a consensus sequence which has three histidine residues that bind to the catalytically essential zinc ion. The propeptide is removed through cleavage by a furin type protease yielding the active enzyme. The propeptide of most MMPs is cleavable by proteases such as trypsin, plasmin, chymotrypsin and other MMPs. ADAMs participate in a wide variety of cell surface remodeling processes, including ectodomain shedding, regulation of growth factor availability and mediating cell-matrix interactions. ADAM17 and ADAM15 have recently been identified in endothelial cells (EC).

ADAMTS are a subfamily of ADAM related metalloproteinases that contain at least one thrombospondin type I sequence repeat motif (TSR). They are secreted proteins; and the TSR facilitates their localization to the ECM placing it in close proximity to their substrates. Functionally, ADAMTS can be divided into three groups: procollagen aminopeptidase, aggrecanase, and ADAMTS13 which cleaves von Willebrand factor. Unlike with MMPs, TIMPs are more selective in their ability to inhibit ADAMs and ADAMTSs. TIMP3 is able to inhibit ADAM17 and 12 as well as ADAMTS4 and 5. ADAM8 and ADAM9 are not susceptible to inhibition by TIMPs.

Other proteolytic enzymes Many additional classes of enzymes have been identified that facilitate angiogenesis. They include serine, aspartic, and cysteine-type proteases. A highly characterized example of the serine protease family is the plasminogen activator-plasmin system, which has been shown to be involved in vascular remodelling. Tissue plasminogen activator (tPA), and urokinase plasminogen activator (urokinase, uPA) are serine proteases which cleave and activate plasminogen. The activated form of plasminogen, plasmin, is a wide-ranging protease capable of acting on various ECM components including fibrin, collagens, laminin, fibronectin, and proteoglycans. Additionally, plasmin also is able to activate various other MMPs.

In humans, the group of cathepsin cysteine proteases or cysteine cathepsins comprises 11 family members, cathepsins B, C, F, H, L1, L2, K, O, S, W, and X/Z. Cysteine cathepsins are synthesized as inactive zymogens and activated by proteolytic removal of their propeptide. These enzymes are primarily localized in lysosomes and function in terminal protein degradation and processing. Cathepsins also can be secreted by cells, associate with the cell surface, and degrade the ECM. A study of all 11 members of the cathepsin family highlights their importance in tumorigenesis and tumor associated angiogenesis. Examination of cathepsin activity by using chemical probes and in vivo imaging techniques demonstrated an increase in cathepsin activity in the angiogenic blood vessels and invasive fronts of carcinoma in the RIP-Tag2 transgenic mouse model of pancreatic islet tumor genesis.

Aminopeptidases function as exopeptidases which remove amino acids from the amino-terminus of proteins. Aminopeptidase N (CD13/APN) is highly expressed on the endothelium of growing vessels. Inhibitors of CD13/APN dramatically impair tumor growth.

Ectodomain shedding

It has become clear in the past years that ectodomain shedding is an initial step for the activation of specific receptors such as Notch, ErbB-4 and the angiopoietin receptor Tie-1. Notch-1 signaling is essential for endothelial differentiation, and tumor angiogenesis, while the angiopoietin receptor Tie-1 facilitates embryonic blood vessel formation. Upon binding of their ligands, Notch-1 and Tie-1 undergo proteolytic cleavage of the ectodomains by ADAM17 and ADAM10. This cleavage frees the cytoplasmic fragment for cellular signaling. In the case of Notch-1, it transfers to the nucleus.

Many cytokines and growth factors are synthesized as membrane-bound proforms which undergo proteolytic shedding for activation. The ephrins EPH receptor A2 and A3 are shed by ADAM10, creating cleaved soluble Eph receptors, which inhibit tumor angiogenesis in mice. Additional examples are the proteolytic shedding of soluble E-selectin, shedding of urokinase receptor (uPAR) by MMP-12 creating soluble uPAR which has chemotactic properties for leukocytes and progenitor cells, and the shedding of interleukin-6 receptors by ADAM10 and ADAM17 which facilitates interleukin-6 signaling in endothelial cells. Semaphorin 4D is cleaved from its membrane-bound form by MT1-MMP (MMP-14) in tumor cells; it then interacts with plexin B1 on endothelial cells, promoting pro-angiogenic chemotaxis. Shedding of a membrane-anchored cytokine or growth factor by ADAM proteinases may be relevant for various signal transduction events. Alternatively, shedding may be required for the ligand to diffuse to distant receptors. Shedding may be required for the down regulation of signals by removing signaling ligands, or cleavage and release of receptors. Release of the receptor may also generate soluble receptors which act as decoys by sequestering ligands. These findings indicate that ectodomain shedding is a ubiquitous process facilitating a wide variety of cellular events involved in angiogenesis. Because potent biological modifiers are generated, it is likely controlled by highly regulated mechanism. Along with ADAMs and MT-MMPs, membrane-bound serine proteases also may play a role in ectodomain shedding.

Proteolytic degradation of the extracellular matrix (ECM)

The formation of capillaries from pre-existing blood vessels requires the remodeling of both the peicapillary membrane of the parent venule, as well as the local and distal ECM. At the onset of angiogenesis endothelial cells (EC) must remodel three different barriers in order to migrate and invade the target tissue. First is the basement membrane between the endothelium and vascular smooth muscle cells or pericytes, followed by the fibrin gel formed from fibrinogen that is leaked from the vasculature, and finally the extracellular matrix in the target tissue. The vascular basement membrane is composed of type IV collagen, type XV collagen, type XVIII collagen, laminins, entactin, heparan sulfate proteoglycans, perlecan, and osteonectin. All of these components of the basement membrane are substrates for MMP-2, 3, 7, and 9, among others. Inhibitors of MMP activity have spotlighted the importance of these proteins in controlling angiogenesis. After working their way through the basement membrane, EC must invade through a dense fibrin gel which is polymerized from fibrinogen derived from the vascular bed. Plasmin, an effective fibrinolysin produced by tPA or uPA, was thought to be essential in this process, but plasminogen deficient mice do not display major defects of neovascularization in fibrin rich tissues. These findings highlight the diverse amount of proteolytic enzymes ECs use to remodel the ECM. For example, MMP-3, 7, 8, 12 and 13 can cleave fibrinogen.

MMP activity is one of the earliest and most sustained processes that take place during angiogenesis. By studying the transition from an avascular to a vascular tumor Fang et al. were able to identify the key role of MMP-2 in angiogenesis. MMP-2 expression and activity was increased in angiogenic tumors as compared with avascular tumors, and the addition of antisense oligonucleotides targeting MMP-2 inhibits the initiation of angiogenesis maintaining the avascular phenotype. This data along with other reports suggest that MMP activity is necessary to initiate the earliest stages of angiogenesis and tumor development. The creation of MMP deficient mice has provided important insight into the role of MMPs in the regulation of angiogenesis. For example, MMP-2 knockout mice develop normally but display significant inhibition of corneal angiogenesis.

Proteolytic fragments as regulators of angiogenesis