Osteoglycin’s embracement of VEGF receptor-2 limits angiogenesis and collateralization

This editorial refers to ‘Loss of osteoglycin promotes angiogenesis in limb ischaemia mouse models via modulation of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 signalling pathway’ by Gao et al., pp. 70–80. The circulatory system regulates adequate distribution of blood and delivery of oxygen and nutrients to the tissues. When arterial blood flow is interrupted a rapid neovascularization response occurs by expansion of collateral blood vessels and angiogenic sprouting in the hypoxic tissue. VEGF-A plays a central role in hypoxia-induced angiogenesis.1 According to insight obtained from developing tissues, VEGF-A binds to VEGF receptor-2 dimers on the protruding filopodia of sprouting endothelial tips. The receptor is subsequently internalized, after which intracellular signalling evokes a proper sprouting response.2 However, clinical studies in which VEGF was used to induce angiogenesis in chronically ischemic heart or limb have been unsuccessful.3,4 Apparently these tissues are refractory to VEGF stimulation. It is generally assumed that the extracellular matrix harbours information that regulates and guides endothelial sprouting. Matrix association of VEGF-A via its heparin-binding domain can direct the invading sprout, while angiogenesis inhibitors such as thrombospondin and matrikines (proteolytically generated matrix fragments that modulate angiogenesis) do the opposite. In this issue of Cardiovascular Research, Wu et al.5 report on a potentially important role of osteoglycin in limiting the cellular response to VEGF. Osteoglycin inhibits the VEGF-VEGFR2 interaction in healthy tissue, while its absence in the ischemic mouse hindleg enhances both sprouting angiogenesis and collateralization. Osteoglycin (gene symbol: OGN), also called mimecan (UniProt entry name: MIME), is a member of the small leucine-rich proteoglycans (SLRPs) and is expressed in most cells of the body.6 Many SLRPs including osteoglycin can bind to matrix proteins, can expose a binding site to collagen fibrils, and can act as a paracrine factor that modulates cell signalling, e.g. by interacting with specific tyrosine kinase receptors. Like other SLRPs, osteoglycin is secreted and acts extracellularly. While there is one OGN gene, which is translated into one precursor protein of 298 amino acids, the protein undergoes posttranslational modification by glycosylation, in particular keratan sulfation, and proteolytic cleavage resulting in various products ranging from 12 to 50 kD.7,8 The spectrum of actions is further extended by the fact that differentially spliced OGN mRNAs are transcribed, which not only are translated into the precursor protein, but also may interact directly with other RNAs or proteins.7,8 Osteoglycin has been connected to many types of disease through changes in its gene expression and antigen and/or circulating protein levels. Such pathologies include various types of cardiovascular and neurological disease, eye and bone disease and cancer (recently summarized by Deckx et al.8) Lung and adipose tissue also express osteoglycin, which can be released into the circulation. Indeed, circulating osteoglycin has been reported to act on the regulation of food intake by inducing hypothalamic cytokines.9 In various healthy and cancer cells, elevated osteoglycin expression was paralleled by a reduced proliferation, while osteoglycin deletion enhanced proliferation. Interest in the role of osteoglycin in cardiovascular disease was initiated by an integrated genomic analysis that identified osteoglycin as a major candidate regulator of left ventricular mass in the rat. This conclusion was further supported by a small genomic study in man that confirmed such role for osteoglycin and by in vitro experiments showing decreased OGN transcription in phenylephrine-induced cardiomyocyte hypertrophy.10 In mouse myocardial infarction tissue, osteoglycin deletion increased rupture-related mortality, while increased expression of osteoglycin was associated with proper collagen maturation and deposition.11 In this way, osteoglycin protects against cardiac rupture and limits adverse remodelling after myocardial infarction. Additional studies in man showed an association between osteoglycin expression and collagen deposition in human myocardial infarction tissue. Furthermore, circulating osteoglycin has been suggested as a biomarker for heart failure.11 Additional studies implied differential expression of osteoglycin in atherosclerotic vessels,12 but another study debated a functional role of this factor in atherosclerosis.13 Osteoglycin mRNA was negatively related to the extent of aortic hypertrophy in rats, while osteoglycin deletion caused an increase in angiotensin II stimulated proliferation of cultured smooth muscle. Interestingly, osteoglycin was also identified as a differentially expressed gene that was downregulated in collateral arteries after rabbit femoral artery ligation.14 A role in collateralization was further suggested by a recent study in 559 consecutive patients with stable angina and angiographic total occlusion of at least one major coronary artery. This study showed that an increased serum concentration of osteoglycin is associated with poor angiographic coronary collateralization.15 Schematic representation of the proposed effect of osteoglycin on angiogenesis and collateralization. In vascular smooth muscle cells (SMC, purple) and endothelial cells (EC, red), the small leucine-rich proteoglycan osteoglycin (OGN) is synthesized and accumulates in the vascular extracellular matrix. By binding to the VEGF binding part of the VEGF receptor-2, it prevents VEGFR2 signalling in endothelial cells (upper part). After arterial occlusion OGN expression drops, while VEGF-A transcription is markedly enhanced by hypoxia. This allows binding of VEGF to its receptor and internalization of the complex, the signalling of which facilitates sprouting angiogenesis and collateral formation. NRP1, neuropilin-1; VEGF, VEGF-A. Hence, the picture arises of osteoglycin being a matrikine which controls the activity of VEGF-VEGFR2 signalling and which is rapidly downregulated by hypoxia, while VEGF-A is upregulated simultaneously in this condition. As usual for such interesting data, they also generate additional questions. First, while the OGN mRNA drops rapidly and almost completely under hypoxia, the decrease in osteoglycin protein is less pronounced and proceeds steadily over 2 weeks. Since a major effect on neovascularization is already visible within 4 days when osteoglycin antigen dropped by 50%, one may question whether different forms of osteoglycin are involved. These might differ in the degree and type of glycosylation as well as in their proteolytic processing. Western blot analysis did not show a change in molecular mass of osteoglycin, but one cannot exclude that this 34kD form represents a less active form than a freshly synthesized one with strong VEGFR2 blocking properties. On the other hand, if osteoglycin competitively inhibits the VEGF-VEGFR2 interaction, the simultaneous induction of VEGF by hypoxia can make a drop of 50% in osteoglycin level sufficient to stimulate endothelial sprouting. As different concentrations of VEGF-A are required to stimulate proliferation and migration/invasion of cultured endothelial cells, osteoglycin may play a role in balancing these responses. A VEGFR2 inhibitor bound to the matrix would be an ideal starting point for paving a track for new sprouts. Proteolytic degradation of the matrix associated osteoglycin, e.g. by migrating macrophages, would remove the inhibitor and prepare a track in which VEGF-VEGFR2 signalling would be privileged. However, it is clear that firm follow-up experiments are needed to fully understand the nature and mode of action of the active osteoglycin. Furthermore, one wonders whether OGN transcription remains decreased in chronically hypoxic tissues or that an adaptation occurs by which this VEGFR2 inhibitor becomes re-expressed and will interfere with revascularization. This also begs the question regarding the factors that regulate OGN transcription. It is evident that hypoxia does decrease OGN mRNA, but it is not yet known whether this depends on hypoxia inducible factors or other mechanisms. Furthermore Kampmann et al.14 reported that FGF-2 reduces OGN transcription by smooth muscle cells. As FGF-1 and FGF-2 are often used to culture endothelial cells, the use of these FGFs may have masked endothelial osteoglycin production in other studies. It is also interesting to note that p53 binding enhanced transcription of bovine and human OGN, which links osteoglycin expression to inhibition of the cell cycle and angiogenesis.16 This suggests a mutual interaction between cell division and OGN on the one hand and OGN and VEGF-induced proliferation on the other. Finally, the effect of osteoglycin on arteriogenesis is intriguing. Initial studies on collateralization pointed to the importance of shear stress and inflammatory cells, in particular monocytes, as drivers of collateral formation. Subsequent studies also reported an important role of VEGF and neuropilin-1. VEGF binding to VEGFR2 stimulates complex formation between VEGFR2 and its co-receptor neuropilin-1. Lanahan et al.17 showed that neuropilin-1 in complex with VEGFR2 is required to maintain VEGFR2 phosphorylation in its cytoplasmic domain (Tyr1175) and for the cellular uptake of the VEGF/VEGFR2/neuropilin1 complex, which is required for proper ERK activation. As the interaction of osteoglycin with VEGFR2 prevents VEGF-A binding, complex formation with neuropilin-1 will also be disturbed. The expression of osteoglycin in various tissues and the many different functions that have been attributed to osteoglycin may complicate application of its general downregulation. Therefore, local modulation of its expression or action needs first to be considered. Furthermore, more knowledge is needed regarding osteoglycin’s functions and which molecular forms are involved in its various functions. In this respect, the existence of different forms of osteoglycin may also provide an advantage as it may allow tailored fruitful application of this interesting molecule. Conflict of interest: none declared.