Aging-induced isoDGR-modified fibronectin activates monocytic and endothelial cells to promote atherosclerosis

Highlights

• IsoDGR-modified plasma proteins are associated with coronary artery diseases (CAD).

• IsoDGR:integrin binding on leukocytes and endothelium induces outside-in signalling, and monocyte and endothelium activation.

• IsoDGR-modified fibronectin may initiate vascular inflammation in atherosclerotic cardiovascular disease.

Abstract

Background and aims

Aging is the primary risk factor for cardiovascular disease (CVD), but the mechanisms underlying age-linked atherosclerosis remain unclear. We previously observed that long-lived vascular matrix proteins can acquire ‘gain-of-function’ isoDGR motifs that might play a role in atherosclerotic pathology.

Methods

IsoDGR-specific mAb were generated and used for ELISA-based measurement of motif levels in plasma samples from patients with coronary artery diseases (CAD) and non-CAD controls. Functional consequences of isoDGR accumulation in age-damaged fibronectin were determined by bioassay for capacity to activate monocytes, macrophages, and endothelial cells (signalling activity, pro-inflammatory cytokine expression, and recruitment/adhesion potential). Mice deficient in the isoDGR repair enzyme PCMT1 were used to assess motif distribution and macrophage localisation in vivo.

Results

IsoDGR-modified fibronectin and fibrinogen levels in patient plasma were significantly enhanced in CAD and further associated with smoking status. Functional assays demonstrated that isoDGR-modified fibronectin activated both monocytes and macrophages via integrin receptor ‘outside in’ signalling, triggering an ERK:AP-1 cascade and expression of pro-inflammatory cytokines MCP-1 and TNFα to drive additional recruitment of circulating leukocytes. IsoDGR-modified fibronectin also induced endothelial cell expression of integrin β1 to further enhance cellular adhesion and matrix deposition. Analysis of murine aortic tissues confirmed accumulation of isoDGR-modified proteins co-localised with CD68⁺ macrophages in vivo.

Conclusions

Age-damaged fibronectin features isoDGR motifs that increase binding to integrins on the surface of monocytes, macrophages, and endothelial cells. Subsequent activation of ‘outside-in’ signalling elicits a range of potent cytokines and chemokines that drive additional leukocyte recruitment to the developing atherosclerotic matrix.

Introduction

Despite recent advances in therapeutic and preventive strategies, cardiovascular disease (CVD) remains a devastating disorder associated with extremely high morbidity and mortality across the world [1]. Previous research has now clearly demonstrated that aging is the primary risk factor for this complex, multifactorial disorder. Early events in CVD pathogenesis include endothelial dysfunction, vascular inflammation, and atherosclerotic plaque formation, ultimately leading to luminal narrowing and thrombotic occlusion of blood vessels [2,3]. While these key processes have now been investigated in detail, it remains unclear how tissue aging initiates the events required for later progression to overt pathology.

We recently reported that extensive deamidation of the amino acid sequence NGR (Asn-Gly-Arg) in extracellular matrix (ECM) proteins results in ‘gain-of-function’ conformational switching to isoDGR (isoAsp-Gly-Arg) motifs [[4], [5], [6], [7]] that can mimic integrin-binding RGD ligands [[8], [9], [10]]. Atypical isoDGR modifications have previously been detected in fibronectin, laminin, tenascin C, and several other ECM proteins derived from human carotid plaque tissues [[4], [5], [6], [7]], suggesting that these molecules may be capable of enhancing leukocyte binding to the atherosclerotic matrix. Long-lived ECM components are well recognized to accumulate degenerative protein modifications (DPMs) including spontaneous deamidation [[11], [12], [13], [14], [15], [16], [17]], under the influence of microenvironmental stresses, flanking amino acid sequences, and genetic factors [13,17]. DPMs are typically associated with loss of protein function and have long been regarded as critical events in human aging and degenerative disorders, but recent data have suggested that DPMs may also induce ‘gain of function’ structural changes that could play equally important roles in human pathology [[4], [5], [6], [7], [8], [9], [10]]. While age is the largest risk factor for many human chronic diseases including CVD, the mechanisms by which advancing age promotes atherosclerosis and CVD have remained obscure and the therapeutic opportunities these may provide are currently unknown. Having previously observed that long-lived ECM proteins undergo age-dependent spontaneous deamidation to generate isoDGR motifs, we postulate that age-related structural damage to key vascular proteins may represent the ‘missing link’ in common pathologies affecting the elderly. In particular, we hypothesize that atypical isoDGR:integrin interactions may serve as a key early trigger for endothelial dysfunction, macrophage activation, monocyte recruitment, and vascular inflammation leading to atherosclerotic plaque development.

Fibronectin is among the first matrix proteins deposited at sites of atherosclerosis [18,19]. Human fibronectin features an RGD integrin binding motif, as well as 4 NGR motifs that can be modified by spontaneous asparagine deamidation to isoDGR. Indeed, we have previously detected 4 characteristic isoDGR motifs in fibronectin derived from human carotid atherosclerotic plaques (at residues 263–265, 367–369, 501–503 and 1432–1434) [7]. In the current study, we generated hybridoma cells that produce isoDGR-specific mAb and aimed to determine the effects of age-damaged fibronectin on monocyte and macrophage function in the early stages of CVD pathogenesis. We observed that interaction of isoDGR motifs with integrins on the surface of monocytes and macrophages triggered an ERK:AP-1 signalling cascade that induced expression of a range of potent chemotactic mediators that promoted further leukocyte infiltration of the assembling atherosclerotic matrix. Biochemical and functional assays also demonstrated that isoDGR-modified FN promoted endothelial activation via expression of integrin β1, which enhanced adhesion of blood leukocytes and facilitated protein deposition resembling intimal thickening in atherosclerosis. Crucially, administration of isoDGR-specific mAb was sufficiently to potently inhibit all of the pathological features assessed in vitro, suggesting that antibody blockade of this structure may represent a viable therapeutic option. Consistent with in vitro results, we observed in vivo accumulation of isoDGR-modified proteins co-localised with CD68⁺ macrophages in aortic tissues of mouse model. In addition, we detected significant enrichment of isoDGR motifs in plasma fibronectin from CVD patients analysed ex vivo, indicating isoDGR may be a predictive biomarker of CVD.