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(Circulation Research. 2001;89:195.)
© 2001 American Heart Association, Inc.

Editorial

Drilling for Oxygen

Angiogenesis Involves Proteolysis of the Extracellular Matrix

Peter Libby, Uwe Schönbeck

From the Leducq Center for Cardiovascular Research, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Peter Libby, MD, Division of Cardiovascular Medicine, Department of Medicine, Brigham and Woman’s Hospital, 221 Longwood Ave, LMRC 307, Boston, MA 02115. E-mail plibby{at}rics.bwh.harvard.edu

Key Words: extracellular matrix • matrix metalloproteinases • endothelium • inflammation • angiogenesis

All acknowledge the importance of endothelial migration and proliferation in angiogenesis. However, the sprouting of extensions of established vascular channels in most tissues requires much more. The extracellular space does not consist of a vacuum, permitting free spread of endothelial sprouts during angiogenesis. Rather, a complex and often dense extracellular matrix invests the parenchymal cells and microvascular channels of most organs. The endothelial cells must penetrate this extracellular matrix to make a new vessel (Figure 1A). Immediately surrounding the endothelial cells, a basement membrane of type IV collagen, laminin, fibronectin, and many other matrix macromolecules presents the first obstacle to neovascular sprout formation (Figure 1B). That barrier breached, the nascent microvessel must then burrow through collagen fibrils, elastin, microfibrillar proteins, proteoglycans, and other constituents of the surrounding extracellular matrix (Figure 1C). How do the endothelial cells drill their way through these layered impediments to form a new vessel?

View larger version (52K): [in this window] [in a new window]   Figure 1. Formation of neovessels. A, To form new vessels, the endothelial cell layer (typically in a venule) must break down the subjacent basement membrane, composed of a complex mixture of many extracellular matrix components including type IV collagen, laminin, and fibronectin. B, Local action of proteolytic enzymes such as type IV collagenase (MMP-2) activated by MT1-MMP (MMP-14) aids dissolution of a patch of basement membrane. C, Other proteinases, including the MMPs that degrade fibrillar collagen (eg, MMP-1 and MMP-13) or proteoglycan core proteins (eg, MMP-9), assist the subsequent step in which endothelial cells force a path into the perivascular extracellular matrix composed of collagen fibrils, elastic fibers, and proteoglycans. Eventually, endothelial cells form a sprouting vessel by migration, furnishing blood supply to distal tissues.

Matrix Metalloproteinases: the Drill Set of the Endothelial Cell

To achieve this feat, endothelial cells use an endogenous proteolytic pathway. Many workers have addressed the role of proteases in angiogenesis through the years,¹ and the family of zinc-dependent enzymes known as matrix metalloproteinases (MMPs) has received substantial attention in this regard. Type IV collagenase, also called MMP-2 or gelatinase A, can help degrade the subendothelial basement membrane. Collagenases such as MMP-1, -8, and -13 can cleave the fibrils of collagen next encountered by the endothelial cell as it strives to form a new channel. Gelatinase B, denoted MMP-9, can break down the core proteins of proteoglycans and elastin. Together, the MMPs (now numbering more than 20) can catabolize all components of the extracellular matrix. Other elastolytic enzymes produced by endothelial cells include non–metalloelastases such as cathepsin S.

Matrix metalloproteinases, first synthesized as inactive precursors or zymogens, require processing, often by proteolysis, to attain full enzymatic function. The endothelial cell produces metal ion-independent proteinases such as plasminogen activators (both urokinase and tissue types). These serine proteinases convert plasminogen to plasmin, an enzyme not only involved in fibrinolysis but also capable of cleaving the MMP zymogens into their active forms. Recent data implicate a distinct subfamily of MMP, termed membrane-type MMP (MT-MMP), in the activation of collagenases and gelatinases. Indeed, MT1-MMP activation of MMP-2 occurs in endothelial cells undergoing angiogenesis,² and inhibition of MT1-MMP expressed by microvascular endothelial cells diminishes the formation of tubule-like structures in a three dimensional collagen gel.³ The modulation of MT-MMP via angiogenic factors further supports a role in angiogenesis for this enzyme subfamily. On the other hand, endothelium, like most other cells, can also synthesize tissue inhibitors of MMPs (TIMPs), the endogenous antagonists of this matrix-degrading family of enzymes. Thus, control of MMP activity exists on many levels: transcription, translations, posttranslational processing, and interaction with inhibitors including the TIMPs or other more recently recognized endogenous inhibitors, such as tissue factor pathway inhibitor-2.⁴

Inflammation, a Link Between MMPs and Angiogenesis

What mechanisms may evoke the action of MMPs at the site of angiogenesis? In diseased tissues, inflammation usually accompanies angiogenesis. Examples abound, but consider the synovia of a rheumatoid joint, atherosclerotic plaque, or healing infarcted tissue. We described overexpression of collagenase in microvessels of the human atherosclerotic plaque some years ago and pointed out the potential relevance of this enzyme in plaque angiogenesis.⁵ In inflamed tissues, leukocytes of various classes congregate and contribute to the angiogenic response. Classic studies implicated macrophages, the major effector leukocyte in innate immune responses and the hallmark of chronic inflammation, in the regulation of angiogenesis.^6,7 Macrophages can release MMPs, potentially making new vessel formation easier at sites of inflammation. Proinflammatory cytokines released by activated macrophages can also heighten MMP production by endothelial cells and enhance this vascular cell’s own matrix-degrading machinery.⁸ Mast cells can release chymase and tryptase, the serine proteinases capable of activating MMP zymogens.

Appreciation of the participation of T lymphocytes, the key cells of the adaptive immune response, in angiogenesis has emerged much more recently (Figure 2). Activated T cells can stimulate angiogenesis in human vascular endothelial cells,⁹ probably using several pathways. Activated T lymphocytes can elaborate angiogenic factors such as vascular endothelial growth factor (VEGF).^10,11 Interestingly, VEGF itself can elicit MMP production by endothelial cells.¹² Moreover, a central mediator of the T-helper cell type 1 response (Th1), CD154 (CD40 ligand), directly induces MMP production in macrophages, smooth muscle cells, and the endothelium.^9,13,14

View larger version (26K): [in this window] [in a new window]   Figure 2. Modulation of angiogenesis by T lymphocytes. T lymphocytes can regulate numerous aspects of neovessel formation, such as extracellular matrix degradation, migration, and proliferation. Th1- rather than Th2-derived cytokines dominate at sites of chronic inflammation such as atherosclerotic lesions and promote angiogenesis via several pathways, including the expression of angiogenic factors (eg, VEGF) or matrix-degrading enzymes (eg, MMP). Modulation of angiogenesis might occur directly or as an intricate interplay with other cell types, such as mononuclear phagocytes. Of note, Th2 cytokines as well as interferon- (IFN-) can inhibit the Th1-mediated formation of neovessels, demonstrating the complexity of interaction among T lymphocytes, endothelial cells, and other cell types in this process.

However, the effects of Th2 cytokines on regulation of angiogenesis have received even less attention. Stearns et al¹⁵ previously reported that the prototypical Th2 cytokine interleukin-10 (IL-10) can inhibit angiogenesis. In this issue of Circulation Research, Silvestre et al¹⁶ describe augmented angiogenesis in ischemic limb tissue in IL-10–deficient mice and implicate overproduction of MMPs in this phenomenon. They further suggest that augmented VEGF levels in the ischemic tissue of the IL-10–deficient animal may account for the increased angiogenesis.

Mechanisms of IL-10’s Inhibitory Effect on Angiogenesis?

The Silvestre study provides in vivo evidence for IL-10’s role as an inhibitor of MMP expression and angiogenesis, but many fundamental questions raised by these intriguing observations remain unanswered. What cell type is responsible for the enhanced MMP expression in the ischemic limb of the IL-10–deficient mouse? Do the tissue’s parenchymal cells (eg, striated muscle) produce the bulk of the gelatinases overproduced? In other words, does autolysis of extracellular matrix due to ischemic injury favor the formation of new vessel? Alternatively, do inflammatory cells recruited to the ischemic tissue release proteinases that prepare the terrain for endothelial cell outgrowth? Finally, MMPs derived directly from the sprouting endothelial cells could drill through the matrix, hastening angiogenesis. In all likelihood a combination of these mechanisms pertain. Only further studies such as in situ localization of proteinases can shed light on this important issue.

Silvestre et al¹⁶ evaluated levels of gelatinases, including MMP-2, the type IV collagenase implicated in lysis of the subendothelial basement membrane. However, they did not address the levels of interstitial collagenases, the MMPs likely needed to penetrate extracellular matrix in the tissue parenchyma (Figure 1C). Assessment of collagenases in this context carries particular interest, as VEGF regulates endothelial MMP-1 expression.¹² Furthermore, it would be of interest to assess the levels of inhibitors of MMPs, because the presence of active forms of MMPs would prove futile if not in stoichiometric excess over the ubiquitous TIMPs. Also, the mechanisms by which IL-10 limits MMP activity merit further inquiry. Is MMP gene transcription altered in the absence of IL-10? Can a less than 2-fold change in VEGF levels in the ischemic tissues account for the differences in MMP levels shown by Silvestre and colleagues? These unresolved issues notwithstanding, the study of Silvestre et al¹⁶ adds to the accumulating evidence that closely controlled metabolism of the extracellular matrix contributes decisively to normal cardiovascular structure and function and strengthens the link between inflammation and cardiovascular pathophysiology.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

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