Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands
XIAOXIA Z. WEST, NIKOLAY L. MALININ, ALONA A. MERKULOVA, MIRA TISCHENKO, BETHANY A. KERR, ERNEST C. BORDEN, EUGENE A. PODREZ, ROBERT G. SALOMON & TATIANA V. BYZOVA
Reciprocity of inflammation, oxidative stress and neovascularization is emerging as an important mechanism underlying numerous processes from tissue healing and remodelling to cancer progression 1 2. Whereas the mechanism of hypoxia-driven angiogenesis is well understood 3 4, the link between inflammation-induced oxidation and de novo blood vessel growth remains obscure. Here we show that the end products of lipid oxidation, ω-(2-carboxyethyl)pyrrole (CEP) and other related pyrroles 5, are generated during inflammation and wound healing and accumulate at high levels in ageing tissues in mice and in highly vascularized tumours in both murine and human melanoma. The molecular patterns of carboxyalkylpyrroles are recognized by Toll-like receptor 2 (TLR2), but not TLR4 or scavenger receptors on endothelial cells, leading to an angiogenic response that is independent of vascular endothelial growth factor. CEP promoted angiogenesis in hindlimb ischaemia and wound healing models through MyD88-dependent TLR2 signalling. Neutralization of endogenous carboxyalkylpyrroles impaired wound healing and tissue revascularization and diminished tumour angiogenesis. Both TLR2 and MyD88 are required for CEP-induced stimulation of Rac1 and endothelial migration. Taken together, these findings establish a new function of TLR2 as a sensor of oxidation-associated molecular patterns, providing a key link connecting inflammation, oxidative stress, innate immunity and angiogenesis.
Angiogenesis can either promote host defence and tissue repair or exacerbate organ dysfunction resulting in disease. In many pathologies, angiogenesis and inflammation 6 are intimately related. Inflammatory cells release proangiogenic growth factors, including vascular endothelial growth factor (VEGF) 7, which facilitate neovascularization. Newly formed blood vessels enhance inflammatory cell recruitment, thereby promoting chronic inflammation. Leukocytes, in particular myeloid cells, are guided by 8 and contribute to 9 oxidative stress and the generation of oxidative products, including hydroxy-ω-oxoalkenoic acids and their esters ( Supplementary Fig. 1). When present in oxidized phospholipids, these molecules are recognized by the scavenger receptor CD36 and contribute to atherosclerotic progression and platelet hyper-reactivity 10 11. Hydrolysis followed by reaction of the resulting unesterified hydroxy-ω-oxoalkenoic acids with proteins, or reaction of the esterified hydroxy-ω-oxoalkenoic acids with proteins followed by hydrolysis, gives rise to a family of carboxyalkylpyrrole protein adducts (CAPs), among them CEP and similarly modified compounds ( Supplementary Fig. 1). These adducts, present in oxidized low-density lipoprotein, accumulate in atherosclerotic plaques and are found in the retina 12, where they promote choroidal neovascularization and age-related macular degeneration 5 13.
These adducts, CEP in particular, are transiently present during wound healing, reaching a maximum 3 days after injury before returning to original levels when the wound has healed (Fig. 1a, b and Supplementary Fig. 2a–c). This increase coincides with the recruitment of bone-marrow-derived cells ( Supplementary Fig. 2b), which generate additional oxidants 9. A substantial proportion of CEP (about 60% at 3 days, about 50% at 7 days) is present in F4/80+ macrophages (Fig. 1c) but not in Gr-1+ neutrophils ( Supplementary Fig. 2c). High levels of CEP coincide with intense wound vascularization, suggesting a role for CEP in wound angiogenesis (Fig. 1a, b). In contrast to wounds, in pathological states CEP levels were continuously elevated. In melanoma, showing excessive vascularization and inflammation (assessed by CD31 and CD68 staining, respectively), CEP levels were elevated sixfold (Fig. 1d). Similarly, in murine melanoma, CEP levels were elevated ninefold ( Supplementary Fig. 3). In contrast to wound and tumour tissues, CEP in uninjured muscle was confined to arteriolar smooth muscle cells (Fig. 1e). CEP accumulation increased in ageing tissues (Fig. 1f). These data suggest a role of CEP in inflammation-associated vascularization.
When tested on endothelial cells (ECs) from human umbilical vein, mouse lung or aorta, CEP had a proangiogenic effect comparable to that of VEGF, as evaluated in various assays (Fig. 2 and Supplementary Figs 4 and 5). Similarly to VEGF, the effect of CEP was mediated by integrins ( Supplementary Fig. 5b). The proangiogenic effect was dependent on the presence of pyrrole adducts, and the protein moiety did not influence the effect of CEP, because adducts coupled to mouse serum albumin, human serum albumin or a dipeptide were equally effective (Fig. 2a and Supplementary Fig. 4).
However, in contrast to VEGF, stimulation of ECs with CEP did not result in phosphorylation of VEGF receptor 2 (VEGFR2) ( Supplementary Fig. 6). Moreover, CEP-induced effects both in vitro and in vivo were not decreased by the VEGFR kinase inhibitor AAL-993 (ref. 14) at a concentration sufficient to block VEGF-A effects (Fig. 2b, c); vehicle alone had no effect (data not shown). In a wound healing assay, CEP accelerated vascularization and wound closure; this effect was unimpeded by AAL-993, which delayed wound closure in control animals (Fig. 2d). Thus, CEP activates the proangiogenic responses independently of VEGF/VEGFR2 signalling. Adducts from the same family of CAPs ( Supplementary Fig. 1), represented by carboxypropylpyrrole (CPP), were also proangiogenic (Fig. 2a and Supplementary Figs 4a, b and 5a), indicating that the ECs respond to the molecular pattern rather than to a particular chemical moiety.
To identify receptors mediating CEP-induced angiogenesis, we tested the role of CD36 and SR-BI scavenger receptors, as CD36 recognizes precursors of CAPs 11. Both CEP and CPP adducts were as effective on CD36−/− and SR-BI−/− ECs as they were on wild-type cells ( Supplementary Fig. 7a–d), indicating that scavenger receptors are not involved in the recognition of these adducts.
Because ECs respond to the molecular pattern of CAP, which is a characteristic feature of oxidative stress, we proposed the involvement of TLRs in CEP-induced angiogenesis. TLRs recognize several damage-associated molecular patterns, including pathogen-associated molecular patterns (reviewed in refs 15, 16) and ligands of host origin 17 18 19 20 21 22, contributing to immune defence and to sterile inflammation, respectively. We focused on TLR2 and TLR4 because they are expressed on the endothelium, are implicated in angiogenesis 23 and are known to recognize a broad range of protein and lipid ligands 24. Anti-TLR2, but not antibodies against TLR4, inhibited CEP-induced, but not VEGF-induced, tube formation (Fig. 2e) and EC migration ( Supplementary Fig. 8a). To further address the role of TLR2 in CEP-driven angiogenesis, ECs from TLR2−/− mice were compared with those from TLR2+/+ controls. TLR2−/− ECs did not respond to treatment with CEP or CPP in several in vitro assays; in contrast, VEGF-triggered responses were not affected by the lack of TLR2 (Fig. 2f and Supplementary Figs 8b and 9a). To confirm the role of TLR2 in proangiogenic responses in ECs, we tested the TLR2 synthetic ligand Pam3CSK4 (ref. 25). This ligand induced robust sprouting of ECs from aortic rings of TLR2+/+ mice but not in TLR2−/− mice (Fig. 2g). Pam3CSK4 was as effective as CEP or VEGF in tubulogenesis and cell adhesion assays, and the effect of Pam3CSK4 was eliminated by TLR2 antibodies ( Supplementary Figs 9b and 10a). To investigate the relative roles of CEP and VEGF, we examined the effect of a VEGFR inhibitor, AAL-993, or TLR2 deletion in wound healing. Inhibiting either pathway had a similar effect and in combination they led to additive inhibition of wound closure ( Supplementary Fig. 10b).
Having established the role of TLR2 in the proangiogenic effects of CEP on ECs, we speculated that CEP administration might promote vascularization in ischaemic or injured tissues. In a hindlimb ischaemia model, CEP-dipeptide injections resulted in 11-fold increase of the pyrrole adduct in muscle tissue ( Supplementary Fig. 11a), which promoted the revascularization of the ischaemic limb (Fig. 3a) and restructuring of collateral blood vessels bypassing the ligated femoral artery ( Supplementary Fig. 11b). Exogenous CEP affected vascularization in a TLR2-dependent manner (Fig. 3a). Consequently, CEP injection increased blood flow in TLR2+/+ mice but not in TLR2−/− mice (Fig. 3b). In a wound model, exogenous CEP accelerated wound closure and vascularization in TLR2+/+ mice but not in TLR2−/− mice (Fig. 3c). Moreover, tumours implanted in TLR2−/− mice showed markedly decreased vascularization and increased areas of necrosis ( Supplementary Fig. 12).
To distinguish the effect of CEP on ECs from that on leukocytes, TLR2+/+ and TLR2−/− mice were transplanted with TLR2+/+ bone marrow. In wound assays, injection of CEP into TLR2+/+ > TLR2+/+ chimaeras accelerated wound closure, whereas TLR2+/+ > TLR2−/− animals healed more slowly and CEP had no effect on wound closure (Fig. 3d). The difference was associated with a 2.5-fold increase in vasculature by treatment of TLR2+/+ > TLR2+/+ but not TLR2+/+ > TLR2−/− animals with CEP (Fig. 3e). Even in the absence of exogenous CEP, vascular area was diminished in wounds of TLR2+/+ > TLR2−/− chimaeras in comparison with TLR2+/+ > TLR2+/+ animals (Fig. 3f). Moreover, melanoma vascularization in TLR2+/+ > TLR2−/− animals was reduced relative to that in TLR2+/+ > TLR2+/+ or TLR2−/− > TLR2+/+ chimaeras (Fig. 3g). Thus, TLR2 on non-haematopoietic cells mediated vascularization induced by exogenous CEP and it also contributed to wound and tumour angiogenesis in the absence of an exogenous adduct.
Because CEP is accumulated at high levels during wound healing (Fig. 1 and Supplementary Fig. 2) and in tumours (Fig. 1d and Supplementary Fig. 3), we addressed the contribution of endogenously generated adducts to the process of vascularization in these models. Intravenous administration of neutralizing antibodies against CEP 13, but not control antibodies, resulted in a more than twofold decrease in wound recovery (Fig. 4A). Vascularization of wounds was also inhibited by anti-CEP but not control antibodies, demonstrating the significance of endogenously generated CEP in wound angiogenesis (Fig. 4B and Supplementary Fig. 13). Administration of anti-CEP, but not control antibodies, diminished the progression and vascularization of melanoma, and this effect was additive to that of the VEGFR inhibitor (Fig. 4C). Thus, the CEP/TLR2 axis drives VEGF-independent angiogenesis in a variety of pathological conditions.
The key mechanism underlying the broad ligand specificity of TLR2 (ref. 24) is its heterodimerization with other members of the TLR family, particularly TLR1 and TLR6 (ref. 26). In a tube formation assay, anti-TLR1 and anti-TLR2, but not anti-TLR6 blocking antibodies, diminished the CEP-induced angiogenic response (Fig. 4D), indicating the involvement of TLR2/TLR1 heterodimers in CEP recognition. It seems that CEP interacts directly with TLR2, because recombinant TLR2 bound the CEP protein adduct but not the carrier protein alone ( Supplementary Fig. 14a). Similarly to other TLR2 ligands, CEP stimulated NF-κB in a cell-based assay ( Supplementary Fig. 14b).
Next, we evaluated the role of MyD88, a mediator of TLR2 signalling, in the proangiogenic activity of CEP. CEP-induced EC sprouting is dependent on MyD88 because MyD88−/− cells did not respond to stimulation by CEP (Fig. 4E). VEGF-induced angiogenesis was not affected by the lack of MyD88 (Fig. 4E). Considering that CEP-induced angiogenesis is integrin-dependent, we focused on mediators of integrin and TLR signalling. Rac1 mediates integrin-dependent migration in vascular development 27 and is reported to be activated downstream of TLRs 28. Accordingly, we assessed the loading of Rac1 with GTP in response to treatment with CEP. GTP-bound Rac1 is increased by treatment of TLR2+/+, but not TLR2−/− or MyD88−/− cells, with CEP (Fig. 4F). Thus, lipid oxidation products, represented by CEP, promote the angiogenic responses of ECs by activating TLR2 signalling in a MyD88-dependent manner, resulting in Rac1 activation, which in turn facilitates integrin function.
Taken together, our results establish a novel mechanism of angiogenesis that is independent of hypoxia-triggered VEGF expression. The products of lipid oxidation are generated as a consequence of oxidative stress and are recognized by TLR2, possibly in a complex with TLR1 on ECs, and promote angiogenesis in vivo, thereby contributing to accelerated wound healing and tissue recovery. If high levels of CEP and its analogues accumulate in tissues, it may lead to excessive vascularization, for example, in tumours. Contribution of the CEP/TLR2 axis to angiogenesis varies in different physiological settings, possibly depending on the extent of oxidative stress. CEP-driven angiogenesis may be an attractive therapeutic target, especially in cancers resistant to anti-VEGF therapy. Inflammation and oxidation-driven angiogenesis may occur in other pathologies, for example atherosclerosis, in which arterial thickening can depend on its microvasculature. In these settings there is an extensive generation of oxidative products that might promote atherogenesis through TLR2. Indeed, it was shown that TLR2−/− mice are protected from atherosclerosis, and this effect could be mediated by cells other than those derived from bone marrow 29. Thus, along with pathogen-associated and danger-associated molecular patterns, TLR2 recognizes an oxidation-associated molecular pattern. This function of TLR2 as a sensor of oxidative stress reveals the shortcut link between innate immunity, oxidation and angiogenesis.
Immunostaining was completed to assess the presence of endogenous CEP in various tissues. Tube formation assays, Matrigel plug assays, wound healing assays, aortic ring assays, tumour implantation models, and a hindlimb ischaemia model evaluated the angiogenic effects of CEP and TLR2. TLR2+/+ and TLR2−/− mice were used for the above assays and in bone marrow transplantation studies. MyD88−/− mice and a commercially available Rac1 activation assay were used to examine the CEP/TLR2 signalling pathway.
Full methods accompany this paper on the web.
N.L.M., E.A.P. and T.V.B. designed experiments. In vivo and ex vivo experiments were performed by X.Z.W. with a help from A.A.M. and M.T. In vitro experiments were performed by N.L.M. with help from B.A.K. Melanoma samples and their analysis was done by E.C.B. Synthesis of CAPs was performed by R.G.S. The data were analysed and plotted by X.Z.W. The manuscript was written by N.L.M. and T.V.B.
We thank L. Hong for the help with synthesis of CEP and CPP adducts, Y. Cui for expressing and purifying anti-CEP monoclonal antibodies, and J. Crabb for providing anti-CEP monoclonal antibody hybridoma cells. This work was supported by NIH grants HL073311, HL071625, CA126847 to T.V.B., HL077213 to E.A.P. and GM021249 to R.G.S. and an American Heart Association grant (10SDG4300062) to N.L.M.
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