The chemokine receptor CX 3 CR1 coordinates monocyte recruitment and endothelial regeneration after arterial injury

Abstract Regeneration of arterial endothelium after injury is critical for the maintenance of normal blood flow, cell trafficking, and vascular function. Using mouse models of carotid injury, we show that the transition from a static to a dynamic phase of endothelial regeneration is marked by a strong increase in endothelial proliferation, which is accompanied by induction of the chemokine CX 3 CL1 in endothelial cells near the wound edge, leading to progressive recruitment of Ly6Clo monocytes expressing high levels of the cognate CX 3 CR1 chemokine receptor. In Cx3cr1‐deficient mice recruitment of Ly6Clo monocytes, endothelial proliferation and regeneration of the endothelial monolayer after carotid injury are impaired, which is rescued by acute transfer of normal Ly6Clo monocytes. Furthermore, human non‐classical monocytes induce proliferation of endothelial cells in co‐culture experiments in a VEGFA‐dependent manner, and monocyte transfer following carotid injury promotes endothelial wound closure in a hybrid mouse model in vivo. Thus, CX 3 CR1 coordinates recruitment of specific monocyte subsets to sites of endothelial regeneration, which promote endothelial proliferation and arterial regeneration.

Common carotid arteries were extracted using microsurgical scissors and a Zeiss Stemi DV4 Sport surgical microscope by separating the internal and external carotid artery just above the bifurcation. Special care was taken not to handle or stress the injury site. To ensure proper staining with antibodies the arteries were cut open and fixed longitudinally under a Zeiss Stemi 2000-C stereo microscope, using microsurgical scissors and forceps. For Flow cytometry analysis mice were euthanized and injured, contralateral carotid arteries were excised without fixation.
Tissue was digested using Collagenase Type 2 (Worthington Biochemical) and single cell suspension was prepared.

Immunohistochemistry, Confocal laser scanning microscopy and image processing
The carotid arteries were placed on microscopic slides. Nonspecific binding of antibodies was prevented using blocking buffer (0.3% Triton X-100, 5% normal serum in PBS) at room temperature for 4 hours. After blocking slides were stained with the primary antibody (4°C, O/N) followed by washing and overnight staining steps with secondary antibody conjugates (4°C) described in appendix table S1. Finally, tissue slides were washed, counterstained with DAPI and mounted with tissue mounting medium (Fluorescent mounting medium, DAKO). Samples were imaged with 3 fluorescent microscope (Observer Z1, Carl Zeiss AG) or confocal microscope (Leica DM IRB2, Leica microsystems).
Confocal microscopy was performed using a Leica DM IRB2 microscope equipped with TCS SP2 AOBS scan head and 40x and 63x Oil immersion objectives. To characterise monocyte subsets based on GFP expression confocal images were acquired using different amplification levels of green detector (amplifier gain) which allows to distinguish between GFP hi and GFP lo cells as described previously (Auffray et al, 2007). Amplifier gain for green detector was set to 600V (Low amplifier gain) to identify GFP hi cells. After acquisition of the image amplifier gain was set to 800V (High amplifier gain) and the same area was imaged again to detect cells expressing low levels of GFP. Leica confocal software (LCS Lite) was used to create Z-stacks of confocal images, which were later merged together for 3D reconstruction.

Human endothelial cell culture and monocyte isolation
Human CD14 ++ CD16 neg and CD14 + CD16 ++ monocytes were isolated from blood rings of healthy donors (Blood bank, Medizinische Hochschule Hannover) using CD14 microbeads and CD16 monocyte isolation kit (Mitenyi Biotec) respectively according to manufacturer's instructions. The purity of the isolated cells was more than 90% routinely tested by flow cytometry. Human Aortic Endothelial cells (HAEC) were purchased from Lonza and cultured as per manufacturer's instructions.

in vitro co-culture studies
For proliferation assay, semi-confluent HAECs were serum starved for 12-16 hours, stimulated with recombinant human TNF-α (25µg/ml, Peprotech) and recombinant human IFN-γ (20µg/ml, Peprotech) to induce CX 3 CL1 expression and co-cultured with CD14 ++ CD16 neg or CD14 + CD16 ++ human monocytes. 24 hours after incubation 4 10µM BrdU was applied to the cultures and incorporation of it in EC was measured by flow cytometry.
For proliferation assays cells were incubated with 10µM BrdU for 12 hours. Cultured cells were collected, stained using BrdU flow kit (BD Pharmingen) according to manufacturer's instructions and analyzed by flow cytometry.
For the Transwell assay, Monocytes and EC's were cultured separated by a transwell (Polycarbonate membrane,12mm diameter inserts, 0.4µm pore size from Corning Inc) for 48 hours. For VEGF neutralization assays, EC's were pre-treated with antihuman VEGF neutralizing antibody (100ng/mL, Affinity purified goat IgG, Catalog no: AF-293-NA, R&D systems) for the duration of the co-culture (48 hrs). For conditioned medium experiments, supernatants from 48hr co-cultures were harvested, spun at 12000 xg to remove cell debris and diluted 1:1 with EBM2+0.5% FCS. The diluted supernatants were used for the conditioned medium experiments.

Flow cytometry
After blocking of non-specific binding with anti-mouse CD16/32 (TruStain fcX, Biolegend) cells were stained with appropriate primary and secondary antibodies or