Divergent JAM-C Expression Accelerates Monocyte-Derived Cell Exit from Atherosclerotic Plaques

<div><p>Atherosclerosis, caused in part by monocytes in plaques, continues to be a disease that afflicts the modern world. Whilst significant steps have been made in treating this chronic inflammatory disease, questions remain on how to prevent monocyte and macrophage accumulation in atherosclerotic plaques. Junctional Adhesion Molecule C (JAM-C) expressed by vascular endothelium directs monocyte transendothelial migration in a unidirectional manner leading to increased inflammation. Here we show that interfering with JAM-C allows reverse-transendothelial migration of monocyte-derived cells, opening the way back out of the inflamed environment. To study the role of JAM-C in plaque regression we used a mouse model of atherosclerosis, and tested the impact of vascular JAM-C expression levels on monocyte reverse transendothelial migration using human cells. Studies in-vitro under inflammatory conditions revealed that overexpression or gene silencing of JAM-C in human endothelium exposed to flow resulted in higher rates of monocyte reverse-transendothelial migration, similar to antibody blockade. We then transplanted atherosclerotic, plaque-containing aortic arches from hyperlipidemic ApoE^-/- mice into wild-type normolipidemic recipient mice. JAM-C blockade in the recipients induced greater emigration of monocyte-derived cells and further diminished the size of atherosclerotic plaques. Our findings have shown that JAM-C forms a one-way vascular barrier for leukocyte transendothelial migration only when present at homeostatic copy numbers. We have also shown that blocking JAM-C can reduce the number of atherogenic monocytes/macrophages in plaques by emigration, providing a novel therapeutic strategy for chronic inflammatory pathologies.</p></div

Trafficking profiles of monocytes on activated HUVECs in a flow assay system.

<p>Scheme of monocyte adhesion, TEM and rTEM. (A) Monocytes captured from flow adhere to HUVEC luminal surfaces before transmigrating into the abluminal compartment (Step-1). The period of time occupied by the monocyte can be measured (Step-2) before the monocyte undergoes rTEM (Step-3). Blockade of JAM-C or increased vascular JAM-C expression induced higher levels of rTEM. (B) Blocking JAM-C 225.3 (white circles) had no effect on primary-TEM compared to conditions using an isotype control (grey circles). Blocking JAM-C 225.3 (white inverted triangles) led to increased rTEM of abluminal monocytes compared to isotype controls (grey inverted triangles). Data are presented as the mean of three fields ±SEM. Data shown is representative of three independent experiments.(N = 3). Decreased vascular JAM-C expression leads to a reduced occupation time of transmigrated monocytes. (C) Transmigrated monocytes on HUVECs with down-regulated JAM-C by siRNA transfection (JAM-C-neg) spent shorter intervals in the abluminal compartment (white triangles) compared to a sham transfection control (JAM-C-WT) (grey triangle). Data shown is representative of two independent experiments (N = 2). JAM-C blockade reduced the occupation interval of transmigrated monocyte in the abluminal compartment. (D) The time spent by individual monocyte in the abluminal compartment was assessed using 225.3 (white inverted triangles). This was shorter than co-cultures treated with an isotype control (grey inverted triangles). Data shown is representative of two independent experiments (N = 2). Increasing expression of JAM-C leads to a reduced occupation time of transmigrated monocytes. (E) HUVECS expressing JAM-C-1.8x (white triangles) had no effect on primary-TEM compared to EGFP-control transfected HUVECs (grey triangles). HUVECS expressing JAM-C-1.8x (white squares) led to increased rTEM of abluminal monocytes compared to the controls (grey squares). Data are presented as the mean of three fields ±SEM. Data shown is representative of three independent experiments (N = 3). Increasing expression of JAM-C also reduced the occupation interval of transmigrated monocyte in the abluminal compartment. (F) The time spent by individual monocytes in the abluminal compartment showed a reduction with increased expression of JAM-C-1.8x (white squares) and JAM-C-6.6x (white diamonds) compared to controls (grey squares). JAM-C-1.8x and JAM-C-6.6x had 1.8- and 6.6-times more expression than control JAM-C-WT respectively. Median values are marked. Mann–Whitney test was used for all dot plots statistical analyses. Data shown is representative of two independent experiments (N = 2).</p

JAM-C expression in lesion-free and atherosclerotic carotid arteries.

<p><b>(A)</b> Carotid artery sections of 10-wk old, lesion-free (left panels) and 6-mths old atherosclerotic ApoE^-/- mice (middle panels) stained for JAM-C and PECAM-1 expression. JAM-C (green) and PECAM-1 (red) immunostaining of carotid arteries shows JAM-C expression on smooth muscle cells (arrowheads) and endothelial cells of carotid arteries of 6-mths old, atherosclerotic ApoE^-/- mice (middle panels, arrows). No JAM-C staining was observed when an isotype control antibody was used (right panels). DAPI staining was used for nuclear counterstain (blue). Bars correspond to 100-um (upper panels); 200-um (lower panels). (B) Quantification of endothelial JAM-C expression of healthy (10 wks) and atherosclerotic carotid arteries (6 mths). Pixel intensity of endothelial associated JAM-C was measured using Fiji (ImageJ) software. Data presented as mean values ±SEM. (C) JAM-C expression in wild-type carotid arteries. Carotid artery sections of 10-wks old wild-type C57BL6/J mice stained for JAM-C and PECAM-1 expression. JAM-C (green) and PECAM-1 (red) immunostaining of carotid arteries shows JAM-C expression only on smooth muscle cells (arrowheads) and not endothelial cells (intima, arrows). Bars correspond to 100-um. (D) JAM-C expression in lesion-free and atherosclerotic aortic arches. Aortic arch sections of 16-wks old, atherosclerotic ApoE^-/- mice fed on Western-type diet (WD, left panel) and 20-wks old, lesion-free WT mice fed on normal chow diet (Chow, right panel) stained for JAM-C (green) and PECAM-1 (red) expression. JAM-C expression on endothelial cells of neointima in aortic arches of atherosclerotic ApoE^-/- mice (left panel, arrows). DAPI staining was used for nuclear counterstain (blue). Bars correspond to 100-μm. Images are representative of multiple stains done in at least two separate mice.</p

Monocytes migrate at higher velocities on HUVECs expressing increasing levels of JAM-C.

<p>The velocity of individual migrating monocytes was measured in the (A, B) abluminal and (C, D) abluminal compartment of HUVEC co-cultures under flow. (A, C) No significant effect was seen with monocytes on HUVECs with JAM-C-neg (white diamonds) compared to the sham transfected control JAM-C-WT HUVECs (grey diamonds) in both the (A) luminal and (C) abluminal compartment. Monocyte velocity was measured on JAM-C-WT (grey circles) JAM-C-1.8x (grey inverted triangles) and JAM-C-6.6x (grey triangles) HUVECs in the (B) luminal and (D) abluminal compartment. Blockade of JAM-C function using the 225.3 and isotype control were marked as ‘+’ and ‘-’ respectively. ‘N’ numbers indicate the number of monocytes analyzed per group. JAM-C-1.8x and JAM-C-6.6x had 1.8- and 6.6-times more expression than wild type levels (JAM-C-WT) respectively. Median values are marked. Mann–Whitney test was used for all dot plots statistical analyses (NS = not significant). Data shown is representative of two independent experiments (N = 2).</p

Monocyte bead recruitment assay.

<p>To assess monocyte recruitment, all recipients (anti-JAM-C, WT, or IgG control (not shown) were injected with fluorescent latex beads 48-hrs before sacrifice, which are taken up by circulating monocytes. Beads were identified by FITC+ fluorescence in the transplanted aortic arch sections, with representative sections shown for (A) anti-JAM-C antibody or (B) PBS buffer-control recipients. Beads are indicated with white arrowheads. (C) Beads were counted in aortic arch lesions and quantified by averaging #beads per arch section over at least 4 slides in the transplanted arch, similar to morphometrics. (D) Labeling efficiency of monocytes with the FITC beads was measured by blood draw 24-hrs after beads injection. Blood monocytes were analyzed for labeling efficiency by the percentage of FITC⁺ monocytes. Data are presented as the mean of three sections ±SEM (N = 6, NS = not significant).</p

Increased atherosclerotic lesion regression following blockade of JAM-C is concurrent with increased leukocyte emigration from plaques.

<p>48-hrs prior to transplantation or immediate sacrifice, all donor ApoE^-/- mice fed 12-wks of WD were injected with EdU (2mg/kg, i.p) to incorporate into DNA of monocyte precursors proliferating in the bone marrow. EdU-labeled circulating monocytes then enter the circulation and are recruited into atherosclerotic plaques. (A) Emigration from the plaques was quantified as the decrease in the number of EdU⁺ cells per section in the aortic arch plaques in baseline mice, compared to after transfer to the normolipidemic recipients. (B) Donor mice sacrificed at the transplant time point show robust EdU⁺ labeling in the aortic arch lesions. Recipient mice sacrificed 4-days after transplantation show significant reduction of EdU⁺ cells, indicating emigration during regression. (C) Recipient mice in which JAM-C was blocked via injections (JAM-C antibody) demonstrate even higher leukocyte egress than recipients treated with the isotype control (not shown) or (D) PBS buffer control. Lesions are outlined from endothelium with dashed white line. Data are presented as the mean value ±SEM (N = 6). ** = P<0.01 compared to baseline measurements. § = P<0.05 compared to WT and IgG recipients.</p

JAM-C expression level regulates monocyte retention and rTEM.

<p>As observed in both animal models and human disease, levels of vascular JAM-C can be decreased or increased during inflammation. JAM-C expression above and below homeostatic levels increased rTEM of monocytes.</p

Ab blockade of JAM-C reduces plaques in a transplantation model of atherosclerosis.

<p>(A) Scheme illustrating how atherosclerotic arches are generated in ApoE^-/- donor mice preceding transplantation into treated or untreated normolipidemic WT mice. (B) Timeline of treatment and monocyte tracing in the aortic arches to establish recruitment and emigration profiles. Aortic arches were harvested from donor ApoE^-/- mice (baseline) and transplanted or not into WT recipient mice treated with PBS buffer control, anti-JAM-C (JAM-C antibody) or an isotype control antibody (isotype control). Tissue sections were stained for CD68⁺ and visualized with Vector Red substrate. (C) Representative images from each group are shown. Morphometrics were analyzed using the ImageProPlus7 program with at least 2 measured areas per slide. To account for changes in the axial length of the arch, 4–7 indexes of sections were taken per arch, and 1 slide from each index was analyzed so that at least 4 slides were analyzed for each aortic arch vessel, and the mean value was used as the summary parameter. (D) Total plaque area, (E) CD68⁺ area, and (F) CD68⁺ as a percentage of the total plaque area were used as parameters of the lesion morphometrics. Data are presented as the mean ±SEM (N = 6). P values marked *—*** were calculated compared to baseline measurements. Both § and §§ = p<0.01 were compared to WT and IgG recipients.</p

Flow cytometry data from blood monocytes.

<p>WBCs were counted by a hemocytometer after red blood cell lysis and stained with leukocyte markers from donor mice (Baseline) and recipient mice groups (JAM-C antibody, PBS buffer-control or an isotype control). (A) Total monocytes were gated for CD45⁺/CD115^hi and expressed as % of total WBCs. (B) Total neutrophils were defined as CD45⁺/CD115^lo/Ly6C^hi and expressed as % of WBCs. Data are presented as the mean ±SEM (N = 6). (C) Monocytes were analyzed for Ly6C expression to differentiate Ly6C^hi and Ly6C^lo populations. NS = not significant. P values marked ** were calculated compared to baseline measurements. Both § and §§ = p<0.01 compared to PBS buffer-control and isotype control recipients.</p