EDITORIAL COMMENT
Intrusion Through the Fragile Back DoorImmature Plaque Microvessels as Entry Portals for Leukocytes and Erythrocytes in Atherosclerosis*
Sebastian F. Mause, MD , and
Christian Weber, MD,*
Institute for Molecular Cardiovascular Research (IMCAR), Medical Faculty, RWTH Aachen University, Aachen, Germany
Department of Cardiology, Pulmonology and Vascular Medicine, Medical Faculty, RWTH Aachen University, Aachen, Germany
* Reprint requests and correspondence: Dr. Christian Weber, Institut für Molekulare Herz-Kreislaufforschung, Universitätsklinikum Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany (Email: cweber{at}ukaachen.de).
Key Words: coronary atherosclerosis • angiogenesis • microvascular leakage • junctions • ultrastructure
Large and healthy human arteries possess in varying degrees a nourishing microvasculature, consisting of first- and second-order vasa vasorum, which is confined to the adventitia and outer media. However, as already noted in 1876 by Koester (1), atherosclerotic modified blood vessels are associated with an "ectopic" vascularization of the intima and inner media. Barger et al. (2) proposed that proliferation of the adventitial microvasculature into the intima enables atherosclerotic plaques to develop beyond a critical size by supplying oxygen and nutrients. Morphometric analysis identified that the majority of intimal microvessels originate indeed from adventitial vasa vasorum, whereas microvessels derived from the vessel lumen are rarely detectable, although present in particular in the shoulder region of atherosclerotic lesions (3,4).
The molecular mechanisms responsible for ectopic microvessel formation are related predominantly to tissue hypoxia (5). Once vessel wall thickness exceeds a certain depth as a consequence of, for example, intimal thickening, oxygen levels in the intima will be critically diminished. Alternatively, hypoxia might be present as a result of preceding dysfunction of the adventitial microvasculature. Accordingly, increased neovascularization could be observed in hypercholesteremic animal models before the establishment of manifest vascular lesions and before impaired vasorelaxation in response to bradykinin (5,6). Hypoxia-dependent neovascularization is significantly mediated by the hypoxia-inducible factor (HIF)-1 , a transcription factor that has been substantially detected in inflammatory and hypoxic areas of human atherosclerotic lesions and that promotes the expression of multiple genes controlling angiogenesis such as vascular endothelial growth factor (VEGF), angiopoietin (Ang)-2, nitric oxide synthase, and matrix metalloproteinase-2 (7,8). Independent of hypoxia, inflammatory reactions and oxidative stress may support ectopic neovascularization and extensive vascular remodeling (9). It is believed that rather than being the initiators of the process, inflammatory cells would amplify and perpetuate the ongoing angiogenic process originally triggered by hypoxia. Possible mechanisms involve augmented production of acidic fibroblast growth factor and secretion of the chemokine interleukin-8 by activated plaque macrophages, delivery of basic fibroblast growth factor by mast cells found in proximity to microvessels, or increased generation of reactive oxygen species (9–11).
The study by Sluimer et al. (12) in this issue of the Journal shows that microvessel density in the adventitia and intima increased with plaque progression in coronary arteries. Intriguingly, an abnormality of intraplaque microvascular endothelial cells (ECs) with incomplete endothelial junctions and membrane detachment could be identified. As in addition, altered ECs were spatially linked to monocyte and mast cell accumulation, this study substantiates the concept of leaky and dysfunctional plaque microvessels. According to such a concept, these nascent vessels may constitute key entry points for cellular and soluble lesion components, implicating a permissive role of plaque microvessels for the progression of atherosclerosis. These portals may gain importance particularly in advanced lesions, because the dense fibrous caps are practically not penetrable by blood cells. Indeed, previous histopathological studies have shown a close association between areas of increased intimal vascularization and accentuated accumulation of monocytes/macrophages, mast cells, and T cells, indicative of ongoing inflammatory reactions and leaky microvessels. This association was shown to be prominent in patients with symptomatic atherosclerosis and in the shoulder regions of plaques, which are known to be prone to rupture (4,13,14). On a molecular level, extravasation of inflammatory cells from plaque microvessels may be supported by modified expression of endothelial adhesion and junctional molecules. It has been shown that vascular endothelial cadherin, which is of pivotal importance for the control of endothelial cell contacts and negatively interferes with VEGF-stimulated cell proliferation, is down-regulated within intimal microvessels (15). This was associated with a discontinuity of the endothelial lining and increased recruitment of inflammatory cells. Moreover, expression of intercellular adhesion molecule-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin was found to be intensified on ECs of the intimal microvasculature (9). Further studies are needed to complement and validate phenotypic observations on a molecular level and to more precisely render the determinants of microvessel leakiness, in particular junctional molecules regulating endothelial permeability and leukocyte transmigration (16).
Similar to leukocytes, a temporospatial association of plaque microvessels with extravasation of erythrocytes has been documented, consistent with intimal neoangiogenesis as a source for focal intraplaque hemorrhages. These microhemorrhages and subsequent phagocytosis of cholesterol-rich erythrocytes are related to an increase in free cholesterol and lipid peroxidation within the plaque, hemoglobin release and iron deposition contributing to oxidative stress, as well as activation of macrophages linked to the release of tissue lytic factors (17). Together with a local enrichment of VEGF and ongoing inflammation, this can create a highly reactive microenvironment, which is counteractive to efficient maturation of nascent plaque microvessels. It is tempting to speculate that such a cycle of events with hypoxia, accelerated angiogenesis, local inflammation, and matrix remodeling finally promotes plaque expansion and plaque vulnerability.
Mural cells, in particular pericytes, may play a crucial role in microvascular maturation, stabilization, and function via extensive reciprocal communication with endothelium cells. For instance, Ang-1, expressed substantially by pericytes, has been shown to mediate vessel maturation via interaction with Tie-2 (18). Furthermore, the transforming growth factor-β signaling machinery may assist in vascular smooth muscle cell differentiation and platelet-derived growth factor released from ECs can support proliferation and migration of mural cells during vessel wall stabilization (19). Scrutinizing the presence of mural cells around microvessels, Sluimer et al. (12) surprisingly observed no differences in the scarce mural cell coverage between normal and atherosclerotic coronary arteries. Together with the documented lack of regional disparity within the same artery, these results may question the common understanding of mural cells crucially supporting vascular integrity. However, such a conclusion should be drawn with caution, because data on mural cell coverage of microvessels is not fully consistent (13), and concerted studies evaluating the molecular and functional properties of mural cells communicating with the microvascular endothelium are lacking in the context of atherosclerosis (Fig. 1).
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Figure 1 Immaturity of Plaque Microvessels
Plaque microvessels show a compromised structural integrity and a modified expression profile of adhesion and junctional molecules, thus permitting extravasation of inflammatory cells and soluble factors as well as the occurrence of microhemorrhages. The resulting reactive microenvironment may support further plaque growth and plaque vulnerability. For further details, please see text. Ang = angiopoietin; FGF = fibroblast growth factor; Hb = hemoglobin; HGF = hepatocyte growth factor; HIF = hypoxia-inducible factor; PDGF = platelet-derived growth factor; VE = vascular endothelial; VEGF = vascular endothelial growth factor. Figure illustration by Rob Flewell.
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Functional animal studies have corroborated the proposed link between intimal neovascularization and atherosclerosis; however, no consensus has yet emerged on the causality of such an association. It has been shown that endothelium-specific inhibitors of angiogenesis (endostatin, TNP-470) significantly reduced plaque area in hypercholesterolemic apolipoprotein (apo) E–/– mice (20). Conversely, administration of recombinant VEGF, known to modulate vascular permeability, promoted atherosclerosis in apoE/apoB-100–deficient mice (21). Interestingly, most of the subsequent small animal studies suggesting a proatherogenic and destabilizing effect of VEGF failed to show an increased angiogenesis in the vessel wall (9,11), indicative of angiogenic-independent effects of the dichotomous VEGF. Accordingly, VEGF has been shown to trigger up-regulation of endothelial VCAM-1 and redistribution of platelet endothelial cell adhesion molecule-1, thus augmenting inflammatory leukocyte adhesion and transmigration (11). Of note, studies on VEGF and its receptors in animal models yielded contradictory findings with regard to atheroprogression, which may be partly reconciled by linking biological effects of VEGF to different concentrations within the microenvironment (9,22). Whereas low VEGF concentrations may exert atheroprotective effects, higher local VEGF levels can result in formation of aberrant and leaky vessels.
Because of the emerging evidence linking ectopic neovascularization and immature microvessels with atherosclerosis, reinforced inflammation, and plaque destabilization and because of the promising results of antiangiogenic treatment strategies in cancer disorders and macular degeneration (23,24), antiangiogenic therapy in atherosclerotic diseases becomes increasingly appealing. Keeping in mind the adverse cardiovascular consequences of systemically applying high doses of the anti-VEGF antibody bevacizumab in cancer patients, the establishment of a new therapeutic paradigm in cardiovascular diseases targeting plaque microvasculature with the aim of decelerating or even inverting plaque growth and adverse inflammatory reactions demands: 1) further evaluation of molecular determinants of immaturity and of the critical balance of proangiogenic and antiangiogenic factors in the plaque microenvironment; 2) technical advances in noninvasive and invasive imaging strategies for diagnosis, risk stratification, and treatment surveillance; and 3) amelioration of application techniques achieving site-specific delivery for agents that are angio-modulating rather than antiangiogenic. The findings of Sluimer et al. (12) corroborate the concept that the mere inhibition of intimal neoangiogenesis should not be the concrete therapeutic goal, but rather the induction of accelerated microvessel maturation and vessel robustness. A normalized, less leaky vasculature, characterized by a quiescent endothelium, a restrictive basement membrane, and sufficient coverage by pericytes, may establish a more favorable microenvironment by diminishing extravasation of inflammatory cells, deposition of lipoproteins, and the occurrence of adverse microhemorrhages and thus intercepting the vicious cycle leading to vascular remodeling and plaque destabilization. Possible strategies for curbing the immaturity of microvessels comprise a temporospatial control of VEGF levels, promotion of Ang-1–Tie-2 pathway loops, facilitation of pericyte recruitment and organization, or therapy with progenitor cells giving rise to smooth muscle-like cells (18,25,26).
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Footnotes
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Dr. Weber is a cofounder and shareholder of Carolus Therapeutics Inc., and has received funding from the German Research Foundation DFG.
* Editorials published in the Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology. 
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References
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Neovascular expression of VE-cadherin in human atherosclerotic arteries and its relation to intimal inflammation Cardiovasc Res 1999;43:1003-1017.[Abstract/Free Full Text] 16. Weber C, Fraemohs L, Dejana E. The role of junctional adhesion molecules in vascular inflammation Nat Rev Immunol 2007;7:467-477.[CrossRef][Web of Science][Medline] 17. Kockx MM, Cromheeke KM, Knaapen MW, et al. Phagocytosis and macrophage activation associated with hemorrhagic microvessels in human atherosclerosis Arterioscler Thromb Vasc Biol 2003;23:440-446.[Abstract/Free Full Text] 18. Thurston G, Suri C, Smith K, et al. Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1 Science 1999;286:2511-2514.[Abstract/Free Full Text] 19. Armulik A, Abramsson A, Betsholtz C. Endothelial/pericyte interactions Circ Res 2005;97:512-523.[Abstract/Free Full Text] 20. Moulton KS, Vakili K, Zurakowski D, et al. 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