April 2010 | Vol 7 | N.º 4 | CNIC-19 [PDF (572K)]

Inflammation-driven angiogenesis: cellular and molecular players

Vanessa Moreno, Pilar Gonzalo and Alicia G. Arroyo
Department of Vascular Biology and Inflammation, Centro Nacional de Investigaciones Cardiovasculares (CNIC). Melchor Fernández Almagro 3. 28029 Madrid. Spain.

Correspondence:
Alicia G. Arroyo, Department of Vascular Biology and Inflammation, Centro Nacional de Investigaciones Cardiovasculares (CNIC), C/ Melchor Fernández Almagro 3, 28029 Madrid, Spain.
Email sgonzalez@cnic.es

ABSTRACT
Angiogenesis, the formation of new capillaries from the pre-existing vasculature, is critical during embryonic development. In adults, angiogenesis is often coupled to inflammation, but many aspects of this interplay remain unexplored. Inflammatory factors can activate endothelial cells, leading to the formation of a nascent sprout. The growth of a new vessel at this site requires invasion of the surrounding tissue by specialised endothelial tip cells expressing specific proteases, in particular, membrane type 1-matrix metalloproteinase (MT1-MMP). Proper tissue repair after acute inflammation requires regression of the newly formed vasculature because the persistence of this vasculature, which is often aberrant and highly permeable, would favour perpetuation of the inflammatory process. Interventions to either block capillary sprouting or induce vascular normalisation, therefore, are possible strategies to promote tissue repair and reduce chronic inflammation. Understanding the cellular and molecular players involved in these processes is relevant to both the pathogenesis and the treatment of chronic inflammatory disorders such as atherosclerosis.

Introduction
The primitive vascular plexus is formed in the embryo from the differentiation of mesangioblasts via the morphogenetic program of vasculogenesis¹. This primitive plexus needs further remodelling to form a mature and functional vascular tree, and processes like pruning and intussusception are relevant to this early vascular remodelling1. After these primary vasculogenic events occur, further expansion of the vascular tree is mostly accomplished by angiogenesis, in particular, by capillary sprouting. Endothelial progenitor cells (EPCs) also play a role in vascular expansion in certain contexts, but capillary sprouting remains the primary mechanism for neovascularisation in the adult organism^2,3
Several molecular pathways are involved in the different processes required for the formation of a mature, stable and functional vasculature during development4. Vascular endothelial growth factor (VEGF) is the key inducer of angiogenesis in a variety of contexts and acts by activating endothelial cells and initiating the events required for sprouting. The functions of VEGF are mostly transduced through VEGF receptor (VEGFR)-2, one of two VEGF tyrosine kinase receptors, but gradients of soluble VEGFR-1 (Flt-1) modulate VEGFR-2 activation and provide local cues that also influence capillary sprouting5. VEGF gradients in tissues and differential expression of VEGFRs are important factors for defining the differential behaviour of endothelial cell types (e.g., tip, stalk and phalanx) that can be found in the growing vessel.

Inflammation–Angiogenesis
It was initially observed that the induction of capillary sprouting in the adult is often accompanied by the presence of an inflammatory infiltrate³. Accumulated evidence has since shown that angiogenesis in the adult is characterised by an interplay between endothelial and inflammatory cells, particularly monocytes/macrophages. Interestingly, although VEGF is secreted by endothelial cells, leukocytes—in particular, cells of the myeloid lineage—are an important source of this angiogenic factor and other proangiogenic cytokines (TNFα), chemokines (CCL2/MCP-1 and IL-8), growth factors and proteases that might induce or modulate the vascular response^6,7. Supporting such a role, monocytes/macrophages have been shown to contribute to the formation of tubular endothelial structures in Matrigel models, to promote VEGFR-3-mediated angiogenesis and lymphangiogenesis, to produce chemokines and proangiogenic factors that induce lymphangiogenesis, and to contribute to ischemia-induced lung angiogenesis in vivo^8-11. The molecular mechanisms by which mononuclear cells and circulating EPCs are recruited to the incipient sprout and contribute to vascularisation are not fully defined¹², although proteases are thought to play important roles².
Related to the interplay between inflammatory cells and endothelial cells during angiogenesis, several chronic inflammatory disorders are associated with an expanded vasculature. Psoriasis is a chronic inflammatory disease of the skin and features a highly vascularised dermis, while rheumatoid arthritis affects the joints and, at advanced stages, is characterised by the formation of a highly vascularised and destructive tissue called pannus. Furthermore, atherosclerosis is a chronic inflammatory disease involving the formation of arterial plaques in which newly formed vessels originating from the vasa vasorum in the adventitia penetrate the neointima and can contribute to plaque instability^13,14. Thus, it is becoming clear that angiogenesis in these inflammatory contexts is not just a collateral effect of these diseases but is an important contributor to their pathogenesis and evolution.

Endothelial tip cells and inflammation: the MT1-MMP-mediated invasive program
Quiescent endothelial cells of the vascular lumen are long-lived and have a very low turnover rate, with only 0.01% dividing in a healthy adult¹⁵. Several stimuli can activate these quiescent endothelial cells and induce key events for the initiation of capillary sprouting: weakening of cell-cell interactions, detachment from the extracellular matrix (ECM), and invasion into the subendothelial tissue. This activation is fine-tuned and needs to be localised for productive capillary sprouting, but it results in changes in the cellular and molecular features of the activated endothelial cells, which may become specialised endothelial tip cells¹⁵. Tip cells are highly polarised and proliferate poorly, and they are especially suited to navigating into the surrounding tissue. This contrasts with the other two endothelial cell types found at the capillary sprout: stalk cells, which are proliferative, non-migratory and mainly involved in lumen formation; and phalanx cells, which have a stable, quiescent phenotype important for vascular homeostasis¹⁵. These three types of endothelial cells can respond to distinct soluble factors and respond differently to the same factor; for example, VEGF induces the migration of tip cells, proliferation of stalk cells and survival of phalanx cells¹⁶. VEGF gradients and Dll4/Notch signalling are crucial for specification of the tip cell phenotype^15,17,18; in fact, tip cells express VEGFR-2, VEGFR-3, and Delta-like 4 (Dll4), along with angiomotin and other molecules^18-20. These specialised navigational cells also express the enzymatic machinery required for invasion through tissue barriers, including the subendothelial basement membrane, interstitial matrix, and provisional fibrin matrix often deposited during acute inflammation. Several matrix metalloproteinases (MMPs) participate in different steps of the angiogenic response, including MT1-MMP, MT2-MMP, MT3-MMP, MMP-2, MMP-3, MMP-7, MMP-9 and MMP-13 and a distintegrin and metalloproteinase (ADAM) family proteins ADAM-10, ADAM-15 ADAM-17 and ADAMTS121,22. The contribution of these proteases to tissue invasion by endothelial tip cells, however, remains undefined.

Several lines of evidence indicate that MT1-MMP is a major player in the invasive behaviour of endothelial tip cells. In addition to being the main endothelial fibrinolysin and collagenase responsible for endothelial cell sprouting in 3D matrices and for capillary sprouting in vivo^23,24, MT1-MMP also contributes to tunnel formation for EC guidance and capillary stabilisation in 3D matrices²⁵. In addition, a computational model points to MT1-MMP as the main mediator of ECM proteolysis by endothelial tip cells²⁶. More importantly, as pericyte-endothelial cell interactions inhibit MT1-MMP expression, MT1-MMP is restricted to the endothelial tip cells of nascent vessels that lack mural cells²⁷. All these data suggest that MT1-MMP is an important regulator of matrix remodelling at the tip of the developing sprout; however, the precise role and regulation of MT1-MMP in endothelial tip cells in vivo remain unexplored.

Within the context of inflammation-induced angiogenesis, recent reports have shown that the endothelial tip cell phenotype is induced not only by the Notch pathway but also by certain inflammatory factors via an alternative pathway¹⁵. TNFα, NF-κB, bradykinin and sphingosine-1-phosphate can promote the full tip cell phenotype or at least the formation of the characteristic filopodia and lamellipodia present on these migratory and invasive cells^28-30. Interestingly, some of these mediators can also modulate MT1-MMP expression in endothelial cells^31,32. Our group has shown that the inflammatory mediators nitric oxide (a downstream effector of bradykinin), prostaglandin E2 (PGE2) and the chemokine MCP-1/CCL2 increase the cell surface clustering and activity of MT1-MMP in human endothelial cells. Moreover, these factors also require MT1-MMP in order to induce efficient capillary tube formation^32-34. Our work has shown that MT1-MMP is a general, but not universal, requirement for capillary formation, suggesting that MT1-MMP activity is likely to be especially relevant in specific inflammatory scenarios³⁵.
Thus, MT1-MMP is regulated by certain inflammatory factors and seems to be a key player in the invasive function of endothelial tip cells. Furthermore, we have recently shown that in myeloid cells, MT1-MMP regulates Rac1 signalling, which is known to be critical for angiogenesis^36,37. These findings leave several questions open for investigation. For instance, it will be important to define the signalling pathways triggered by inflammatory factors (likely involving Rac1/actin polymerisation^38,39) that lead to the differentiation of endothelial tip cells expressing active MT1-MMP and to identify and characterise MT1-MMP cellular substrates (degradome), post-translational modifications, molecular partners, and induced signals in tip cells. It will also be of interest to search for novel molecular players involved in this context and to develop in vitro and in vivo models designed to dissect the interplay between inflammation and endothelial tip cells under different pathophysiological conditions. This information will help to increase our understanding of the initiation and progression of chronic inflammatory disorders.

Vascular integrity and normalisation
In the later stages of the angiogenic response, the newly formed capillaries are stabilised by various mechanisms involving specific mediators and signalling pathways that promote the restoration of quiescence. This includes the inactivation of migratory and invasive phenotypes, re-establishment of endothelial cell-cell junctions, deposition of a new basement membrane, and recruitment of mural cells. This stabilisation is necessary for normal functioning of the newly formed vessels and is an essential requirement for the supply of oxygen and nutrients during development and organ growth. At the early stages of an inflammatory response, the newly formed vessels at the inflammatory focus are beneficial because they favour the influx of inflammatory cells and factors responsible for resolution of the inflammatory injury. Normal tissue repair, however, requires regression of this new vasculature after the acute phase. The proteases MMP-1 and MMP-10 participate in this regression, and monocytes/macrophages can also play a role^40,41.
If vascular regression is not accomplished, the persistent vasculature is often aberrant, highly permeable and poorly functional. This results in plasma leakage, increased tissue pressure and chronic hypoxia, thus inducing a positive feedback loop for capillary sprouting as observed in tumours and inflammatory pathologies¹⁶. These observations suggest that strategies to promote the normalisation of this aberrant vasculature would help to slow the progression of these diseases. This has been demonstrated in the context of tumours and age-related macular degeneration^14,16. In a mouse model, the deletion of VEGF in myeloid cells induced vascular normalisation and increased the sensitivity of the tumours to chemotherapy, thus demonstrating the contribution of inflammatory components to this process⁴². A similar situation occurs in chronic inflammatory contexts such as atherosclerotic plaques, in which the aberrant vasculature favours plasma leakage and leukocyte recruitment that, together with haemorrhage, can contribute to the transition from a stable to a vulnerable plaque¹⁴.
Thus, it is of the utmost importance to understand the mechanisms that contribute to vascular stabilisation and integrity and that might be stimulated to induce the normalisation of aberrant vessels. Among the factors involved in vascular integrity, stabilisation and normalisation, we consider the following to be of particular interest^16,43,44:
1. Junctional complexes. Endothelial cells have two specialised junctional regions similar to the adherens and tight junctions (AJs and TJs) found in epithelial cells. TJs control paracellular traffic of substances through the endothelial monolayer and are formed by the interaction of claudins and other proteins such as occludins and JAMs. The function and regulation of these TJ components are dependent on their interactions with cytoskeletal proteins such as ZO-1, actin-binding partners and signalling molecules. AJs are primarily established by VE-cadherin interactions and their association with intracellular proteins such as p120, β-catenin, plakoglobin, actin-binding proteins, kinases, and phosphatases. AJs are responsible for the establishment and maintenance of endothelial integrity and quiescence (for review see 43).
2. PKA/Epac/Rap1. Increased levels of cAMP reduce endothelial permeability. Epac is a cAMP-activated guanine nucleotide exchange factor for the small GTPase Rap1, which in turn promotes the organisation of cell-cell contacts through its impact on various signalling pathways and processes, including VE-cadherin adhesion⁴³.
3. Tie-2. This angiopoietin receptor plays a dual role in cell-matrix and cell-cell adhesion interactions by forming a variety of distinct molecular complexes at these sites^{45 46}.
4. Mural cell recruitment. Stabilisation of vessels in vivo requires coverage of the naked capillary by mural cells (pericytes or smooth muscle cells), and these cells are often lacking in the aberrant vasculature of tumours and chronic inflammatory sites. Studies in both mouse models and human disease have shown that signals triggered by platelet-derived growth factor (PDGF)-B/PDGF receptor and transforming growth factor (TGF)β/TGFβ receptors (endoglin, ALK-1 and ALK-5) are especially important for the recruitment of these cells⁴³.
5. Proline-hydroxylase-2 (PHD2). Studies recently performed by Carmeliet’s group have shown that oxygen sensors are important for the maintenance of endothelial quiescence and that mice heterozygous for PHD2 show a normalised vasculature in induced tumours⁴⁷.
With regard to the putative role of proteases in this process, some findings indicate that regulation of MT1-MMP is required for vascular integrity. For example, MT1-MMP is inhibited by tissue inhibitor of metalloproteinases (TIMP)-3 released by pericytes in the final steps of capillary sprouting⁴⁸, and we have observed that endothelial cell monolayers on certain ECM substrates relocate MT1-MMP to cell-cell junctions. Notably, the ternary complex formed by MT1-MMP, α3β1 integrin, and Tspan CD151 at these sites maintains MT1-MMP in an inactive state^49,50. These data suggest that MT1-MMP inactivation is required for basement membrane deposition and vessel stabilisation. The role of MT1-MMP in vivo, however, may be more complex as the absence of MT1-MMP in mural cells results in microvasculature defects at certain locations in null mice⁵¹. We are currently setting up approaches and tools to use in conjunction with in vitro and in vivo models (e.g., atherosclerosis, rheumatoid arthritis) to explore the regulation of MT1-MMP in aberrant vasculature within chronic inflammatory foci and to investigate whether modulation of MT1-MMP expression or activity influences vascular normalisation. We also plan to search for new players (MT-MMPs and related molecules) involved in this process.

From basic research to clinical opportunities
Major efforts have been made in recent years to develop new therapeutic tools to control diverse pathologies, and angiogenesis has attracted attention as a promising target for the treatment of cancer and other diseases characterised by hypervascularisation. Anti-VEGF antibodies have been approved for clinical trials for several types of cancer and age-related macular degeneration¹⁴; however, systemic administration of anti-VEGF is not free of side effects, and there is interest in local delivery and the generation of more selective drugs. Placental growth factor (PlGF) is a promising target in this regard as it is mainly linked to pathological angiogenesis and its blockade results in effective tumour reduction and the stabilisation of plaques in atherosclerotic models^52,53.
New avenues have been opened by the concept of using vascular normalisation as a strategy to improve the response of tumours to chemotherapy or to slow the evolution of disorders such as atherosclerosis. New drugs such as PHD2 inhibitors or agents that promote any of the pathways involved in vascular integrity may have beneficial effects. In general, combined therapies are preferable; for example, vascular normalising agents could be used together with chemotherapy drugs for the treatment of cancer or with anti-inflammatory agents such as statins for the treatment of atherosclerosis¹⁴.
The ongoing lines of research in our lab are directed at identifying and characterising new molecular players whose modulation would either specifically block capillary sprouting by endothelial tip cells, avoiding effects on quiescent vasculature, or normalise the aberrant vasculature generated in chronic inflammatory disorders. In addition, given that MT1-MMP is expressed in tip cells, anti-MT1-MMP antibodies coupled to paramagnetic nanoparticles might provide a means of imaging early angiogenic events (similar to the approach described for αvβ3 integrin⁵⁴), enabling identification of those patients most likely to benefit from anti-angiogenic therapy. The expression of MT1-MMP in endothelial tip cells, mural cells and macrophages could potentially be exploited to deliver drugs or other agents selectively to nascent vessels or the inflammatory infiltrate and could also be manipulated in a cell type-specific manner to treat distinct disorders⁵⁵. Our efforts to identify the MT1-MMP degradome (the total collection of substrates) in endothelial cells could also shed light on the actions of transmembrane proteins whose release into the plasma might be used as a surrogate marker of endothelial MT1-MMP activity and, therefore, of active angiogenesis (e.g., within atherosclerotic plaques).