Plasma Levels of Endothelial Microparticles Bearing Monomeric C-reactive Protein are Increased in Peripheral Artery Disease

Abstract

C-reactive protein (CRP) as an indicator of cardiovascular disease (CVD) has shown limited sensitivity. We demonstrate that two isoforms of CRP (pentameric, pCRP and monomeric, mCRP) present in soluble form or on microparticles (MPs) have different biological effects and are not all measured by clinical CRP assays. The high-sensitivity CRP assay (hsCRP) did not measure pCRP or mCRP on MPs, whereas flow cytometry did. MPs derived from endothelial cells, particularly those bearing mCRP, were elevated in peripheral artery disease (PAD) patients compared to controls. The numbers of mCRP(+) endothelial MPs did not correlate with hsCRP measurements of soluble pCRP, indicating their independent modulation. In controls, statins lowered mCRP(+) endothelial MPs. In a model of vascular inflammation, mCRP induced endothelial shedding of MPs and was proinflammatory, while pCRP was anti-inflammatory. mCRP on endothelial MPs may be both an unmeasured indicator of, and an amplifier of, vascular disease, and its detection might improve risk sensitivity.

Keywords: CRP; Inflammation; Microparticle; Monomeric CRP; Pentameric CRP; Peripheral artery disease; hsCRP.

Fig. 1

Detection of CRP isoforms and MPs by the hsCRP assay. a CRP detection in unmanipulated plasma (first column) or after passage over phosphocholine-coupled beads (second column, CRP depletion). The third column was 2 μg/ml pCRP added to CRP-depleted plasma and the fourth column was 2 μg/ml urea-solubilized mCRP added to the CRP-depleted plasma. b Levels of CRP in plasma with MPs removed (second column), or in MPs purified from the plasma (third column). (n=3)

Fig. 2

Endothelial-derived MPs (and those bearing mCRP) measured by flow cytometry. (a) The number of lactadherin⁺ CD144⁺ particles was increased in PAD patients. The number of endothelial MPs bound with (b) mCRP, but not (c) pCRP, was increased for PAD patients. (n=11 for controls, n=18 for PAD patients, *p ≤ 0.05)

Fig. 3

Correlation of hsCRP measurements with the number of endothelial MPs. There was no correlation (R=0.196 linear) between the elevated pCRP plasma levels measured by hsCRP in PAD patients compared to the number of endothelial derived MPs bearing mCRP (n=18)

Fig. 4

Separation of normal controls by statin treatment. (a) The number of endothelial mCRP⁺ MPs in controls on or off statins. (b) The correlation (R=0.85) between the number of endothelial mCRP⁺ MPs and LDL-c in the plasma. No correlations were found for (c) endothelial pCRP ⁺ MPs and LDL-c (R=0.02) or (d) endothelial mCRP ⁺ MPs and triglycerides (R=0.17). (n=5 for controls, n=6 for controls + statin treatment, *p ≤ 0.05)

Fig. 5

Characterization of cells and MPs after transendothelial migration (TEM). a The number of M1 or M2 macrophages counted four days post-migration with the indicated treatment above the endothelial monolayer (pCRP and mCRP). (n=5; *p ≤ 0.05 and ***p ≤ 0.001). Migrated macrophages treated with pCRP were CD206 positive, whereas those treated with mCRP were CD86 positive. b The average signal (mean pixel density) was measured for cytokines using protein arrays. Protein levels of IFN-γ were higher after mCRP than after pCRP treatment, whereas levels of IL-13 were higher after pCRP treatment (n=4; p=0.02). c and d The number of MPs from endothelial cells (EC), with the presence or absence of mononuclear cells (MNLs). c Solubilized mCRP bound to MPs generated in the TEM assay. d mCRP, but not MCP-1 alone, induced the generation of MPs in the TEM assay similar to TNF stimulation. (n=3)