Shear-Sensing by C-Reactive Protein: Linking Aortic Stenosis and Inflammation

Figures

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  Figure 1.. Extensional flow apparatus and computational fluid dynamics (CFD) calculations of the flow field generated. A, Schematic drawing of the utilized extensional flow apparatus. B, Image of extensional flow device. C, Flow streamlines in the middle cross-section of the infusing syringe, capillary tube, and withdrawing syringe colored according to flow velocity. Results were obtained using CFD simulation with a plunger velocity of 8 mm s−1 corresponding to a flow rate of 8 mL min−1. D, Variations in shear rate along the length of the capillary tube at different distances to the central axis of the capillary tube obtained using CFD simulation (0 mm, central axis; 0.17 mm, closest to the capillary wall). Results were obtained by setting the flow rate to 8 mL min−1. PE indicates polyethylene.

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  Figure 2.. Shear dissociates pCRP (pentameric C-reactive protein) into mCRP (monomeric C-reactive protein), resulting in aggregate formation. A, Human pCRP subjected to shear dissociates into its monomeric subunits, as demonstrated by native Coomassie sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Human pCRP (2.5 mg/mL) was subjected to extensional shear stress for 200 passes and subsequently separated on a native SDS-PAGE.17 Unsheared pCRP (pCRP) did not show any staining at the size of mCRP (25 kDa, indicated in the protein ladder by the black arrowhead). However, pCRP sheared for 200 passes (sheared CRP [C-reactive protein]) showed a second band at the same size as the mCRP control (mCRP). The sheared CRP solution was separated into a pellet with decreased solubility (Pellet) and supernatant (Super). The pellet showed only minor staining at the size of the pCRP control (red arrowhead) and a strong band at ≈23 kDa. The soluble portion of the sheared sample (Super) showed up exclusively at the size of the control pCRP. The black arrow located above the 250-kDa marker indicates the border between the stacking and the separating gel. B, Further identification of mCRP…

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  Figure 3.. Biophysical measurements verified pCRP (pentameric C-reactive protein) dissociation and monomer formation under shear. A, Tryptophan (TRP) fluorescence was used to distinguish between different CRP (C-reactive protein) isoforms. The inset shows a globular monomeric subunit of pCRP with TRP residues highlighted in orange (PDB ID: 7TBA).13 With increasing numbers of passes (p) through the extensional flow field, TRP emission spectra decreased from pCRP toward mCRP (monomeric C-reactive protein). B, TRP fluorescence intensity at peak wavelength (λ=370 nm) was measured and compared for the different treatments by ANOVA and Tukey post hoc test (n=4), biological replicates. C and D, 8-anilino-1-naphthalenesulfonic acid (ANS) binding was used to further verify the conformational change in CRP. Shear-dissociated CRP demonstrated an increase in ANS fluorescence and, finally, (1000 passes) reached similar emission spectra and peak values to those of the control mCRP. ANS fluorescence intensity at peak wavelength (λ=500 nm) was measured and compared for the different treatments by ANOVA and Dunnett T3 test (n=5–6), biological replicates. E through G, Dynamic light scattering (DLS) size…

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  Figure 4.. Human CRP (C-reactive protein) exposed to high shear was deposited as mCRP (monomeric C-reactive protein) in a mouse model of arterial stenosis. Graphical representation (A) and intraoperative images (B) of the carotid artery stenosis model, as described previously.41 B, The stenosis model with the right coronary artery (RCA) exposed. A consistent 80% stenosis was induced by applying a 6-0 braided polyester fiber suture circumferentially (black arrowhead) around a 150-µm needle. The direction of blood flow is indicated by the black arrow throughout B through D. C, Computational fluid dynamics (CFD) calculated wall shear rates in the artery stenosis model. The color scheme and scale indicate the respective wall shear rate. D and F, Representative immunohistochemistry of longitudinal sections of the carotid artery. The stenosis-inducing thread (black arrowhead) and the flow direction (black arrow) are indicated. Slides were incubated with either antihuman mCRP antibody (9C9; D) or antihuman pCRP (pentameric C-reactive protein) antibody (1D6; F) and visualized with secondary antibody conjugated to HRP, followed by incubation with 3,3’-diaminobenzidine peroxidase substrate. The scale…

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  Figure 5.. Circulating mCRP (monomeric C-reactive protein) is significantly reduced after transcatheter aortic valve implantation (TAVI). A, Schematic drawing of stenotic aortic valve with an illustration of the associated high shear stress. B through H, Clinical and experimental parameters of patients with TAVI before (red) and 1 month after TAVI (blue) are shown. B, Aortic mean gradients were significantly reduced after TAVI (n=37). P values were calculated using 2-tailed, matched-pairs signed rank tests (Wilcoxon). Dots represent biological replicates. C, Shear rates were calculated as described in detail in the methods section for patients before and after TAVI, demonstrating a significant reduction in mean shear rate (n=37, biological replicates). Paired 2-tailed t-tests were applied. D, High-sensitivity (hs) CRP (C-reactive protein) from patients before and 1 month after TAVI. pCRP (pentameric C-reactive protein) in patient blood was measured for 22 patients (n=22, biological replicates) by hsCRP assay, demonstrating no significant difference before and after TAVI, using paired nonparametric Wilcoxon matched-pairs signed rank text. E and F, Flow cytometry of microvesicles (MVs)…

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  Figure 6.. Shear-dissociated CRP (C-reactive protein) exhibits proinflammatory effects on endothelial cells and platelets. A, schematic drawing of the microfluidic model that recapitulates the shear conditions of aortic valve stenosis with direct flow into µ-slide channels covered with monolayers of human aortic valve endothelial cells (HAoVECs). B, Representative immunofluorescence images of human aortic valve endothelial cell monolayers incubated under static conditions (16 h) with 50-µg/mL pCRP (pentameric C-reactive protein; static pCRP) or mCRP (monomeric C-reactive protein; static mCRP). In addition, 50-µg/mL pCRP was subjected to flow for 16 h through an 80% stenosis microfluidic chip (5000 s−1) and subsequent perfusion over a monolayer of human aortic valve endothelial cells (sheared CRP). Representative expressions of the adhesion molecules, ICAM-1 (intercellular adhesion molecule 1) (green) and P-selectin (red), are shown (static control [no CRP], static pCRP, static mCRP, and sheared pCRP; n=16), and each is merged with DAPI staining. C and D, Quantification of B. mCRP increased the expression of ICAM-1 (C) and P-selectin (D) compared with static pCRP and static control (no CRP…