Abstract
Rationale: Nonclassical mouse monocyte (CX3CR1 high , Ly-6C low ) patrolling along the vessels of the microcirculation is critical for endothelial homeostasis and inflammation. Because of technical challenges, it is currently not established how patrolling occurs in large arteries. Objective: This study was undertaken to elucidate the molecular, migratory, and functional phenotypes of patrolling monocytes in the high shear and pulsatile environment of large arteries in healthy, hyperlipidemic, and atherosclerotic conditions. Methods and Results: Applying a new method for stable, long-term 2-photon intravital microscopy of unrestrained large arteries in live CX3CR1-GFP (green fluorescent protein) mice, we show that nonclassical monocytes patrol inside healthy carotid arteries at a velocity of 36 μm/min, 3× faster than in microvessels. The tracks are less straight but lead preferentially downstream. The number of patrolling monocytes is increased 9-fold by feeding wild-type mice a Western diet or by applying topical TLR7/8 (Toll-like receptor) agonists. A similar increase is seen in CX3CR1 +/GFP /apoE −/− mice on chow diet, with a further 2- to 3-fold increase on Western diet (22-fold over healthy). In plaque conditions, monocytes are readily captured onto the endothelium from free flow. Stable patrolling is unaffected in CX3CR1-deficient mice and involves the contribution of LFA-1 (lymphocyte-associated antigen 1) and α 4 integrins. The endothelial damage in atherosclerotic carotid arteries was assessed by electron microscopy and correlates with the number of intraluminal patrollers. Abolishing patrolling monocytes in Nr4a1 −/− apoE −/− mice leads to pronounced endothelial apoptosis. Conclusions: Arterial patrolling is a prominent new feature of nonclassical monocytes with unique molecular and kinetic properties. It is highly upregulated in hyperlipidemia and atherosclerosis in a CX3CR1-independent fashion and plays a potential role in endothelial protection.
🔬 Techniques
🔭 Microscopes
✨ Fluorophores
🧪 Sample Preparation
🔬 Cell Lines
🏭 Microscope Brands
🔴 Lasers
🎨 Filters
💻 Software Details
🏛️ Research Organizations (ROR)
Affiliated research institutions:
📋 Methods
Animals
CX3CR1-GFP (green fluorescent protein) reporter mice were provided by S. Jung (Weizmann Institute, Israel) and backcrossed onto C57BL/6J until a genetic purity of >99% was achieved (SNP analysis by Dartmouse, Dartmouth College). ApoE −/− mice were obtained from Jackson Laboratory (stock number: 002052). CX3CR1-GFP and apoE −/− mice were crossed, resulting in CX3CR1 +/GFP apoE −/− mice. 21 Homogenous expression of CX3CR1-GFP leads to a functional protein knockout. 22 In experiments comparing CX3CR1 +/GFP and CX3CR1 GFP/GFP genotypes, littermates were used. Nr4a1 −/− apoE −/− mice were previously described. 20 All animal experiments were approved by the local animal ethics committee.
Intravital Live Cell Triggered Imaging System
Technical details of the ILTIS setup have been described before. 21 Preparation of the large arteries was done carefully with minimal handling to prevent surgery-related inflammation. The adventitia was not removed. Tissue was kept moist with phosphate-buffered saline at 35°C to 37°C. All imaging experiments were conducted on a Leica SP5 system using a water-dipping objective (Olympus XLUMPLFL 20X NA 0.95) and an objective heater. The system is composed of a DM6000 microscope, a Ti-Sapphire laser (Chameleon Ultra II, Coherent), tuned to 920 nm, a resonant scan head for fast scanning, and a Leica trigger box. Three NDD (nondescanned detector) detectors were used in most of the experiments with the following filter sets: 460/40 for second harmonics, 513/50 for GFP, and 640/40 for texas red. Videos were recorded in 512×256 pixel resolution (=455×227 μm) for 30 to 90 minutes. Under ketamine/xylazine anesthesia, the heart rate was typically at about 300 to 400 bpm. An Arduino-based circuit allowed image acquisition in triplets triggered by the signal of the pulse oxymeter attached to the thigh. Cell Tracking and Data Analysis After ILTIS postprocessing, which included frame selection and 2-dimensional registration using self-made plugins in ImageJ, 21 a final frame rate of 1 to 1.5 frames/s was achieved. In all movies analyzed, patrolling GFP + cells were detected using an absolute intensity threshold and tracked using Imaris autoregressive motion algorithm (Bitplane). Only tracks with a duration longer than 90 s were considered patrolling cells. To denoise residual motion artifacts of imaging, cell tracks were smoothed using Matlab as described previously. 21 For quantification, patrolling monocytes were tracked for 17 minutes in each movie unless indicated otherwise. Fast cells moving at over 100 μm/min were considered rolling. Further details are denoted in the Online Data Supplement .
Show full methods section
Animals
CX3CR1-GFP (green fluorescent protein) reporter mice were provided by S. Jung (Weizmann Institute, Israel) and backcrossed onto C57BL/6J until a genetic purity of >99% was achieved (SNP analysis by Dartmouse, Dartmouth College). ApoE −/− mice were obtained from Jackson Laboratory (stock number: 002052). CX3CR1-GFP and apoE −/− mice were crossed, resulting in CX3CR1 +/GFP apoE −/− mice. 21 Homogenous expression of CX3CR1-GFP leads to a functional protein knockout. 22 In experiments comparing CX3CR1 +/GFP and CX3CR1 GFP/GFP genotypes, littermates were used. Nr4a1 −/− apoE −/− mice were previously described. 20 All animal experiments were approved by the local animal ethics committee.
Intravital Live Cell Triggered Imaging System
Technical details of the ILTIS setup have been described before. 21 Preparation of the large arteries was done carefully with minimal handling to prevent surgery-related inflammation. The adventitia was not removed. Tissue was kept moist with phosphate-buffered saline at 35°C to 37°C. All imaging experiments were conducted on a Leica SP5 system using a water-dipping objective (Olympus XLUMPLFL 20X NA 0.95) and an objective heater. The system is composed of a DM6000 microscope, a Ti-Sapphire laser (Chameleon Ultra II, Coherent), tuned to 920 nm, a resonant scan head for fast scanning, and a Leica trigger box. Three NDD (nondescanned detector) detectors were used in most of the experiments with the following filter sets: 460/40 for second harmonics, 513/50 for GFP, and 640/40 for texas red. Videos were recorded in 512×256 pixel resolution (=455×227 μm) for 30 to 90 minutes. Under ketamine/xylazine anesthesia, the heart rate was typically at about 300 to 400 bpm. An Arduino-based circuit allowed image acquisition in triplets triggered by the signal of the pulse oxymeter attached to the thigh. Cell Tracking and Data Analysis After ILTIS postprocessing, which included frame selection and 2-dimensional registration using self-made plugins in ImageJ, 21 a final frame rate of 1 to 1.5 frames/s was achieved. In all movies analyzed, patrolling GFP + cells were detected using an absolute intensity threshold and tracked using Imaris autoregressive motion algorithm (Bitplane). Only tracks with a duration longer than 90 s were considered patrolling cells. To denoise residual motion artifacts of imaging, cell tracks were smoothed using Matlab as described previously. 21 For quantification, patrolling monocytes were tracked for 17 minutes in each movie unless indicated otherwise. Fast cells moving at over 100 μm/min were considered rolling. Further details are denoted in the Online Data Supplement .
📊 Figures
Figure 1.
Nonclassical monocytes patrol healthy arteries. A , Three-dimensional (3D) reconstruction showing a CX3CR1-GFP high (green fluorescent protein) monocyte (arrow) patrolling in the lumen of the external...
Figure 2.
Arterial patrolling induced by the TLR7/8 (Toll-like receptor) agonist R848. A , Patrollers in the carotid artery imaged before and after topical application of the TLR7/8 agonist R848. The red channe...
Figure 3.
Hyperlipidemia intensifies monocyte patrolling in arteries. A , The number of patrollers in the carotid artery was determined by intravital microscopy in CX3CR1 +/GFP apoE u2212/u2212 mice on chow die...
Figure 4.
Massive patrolling in arteries with atherosclerotic plaque. A , Three-dimensional (3D) reconstruction in top down ( top ) and axial ( bottom ) view and ( B ) representative frames from an intravital 2...
Figure 5.
Microkinetics and primary monocyte capture in atherosclerotic arteries. A , Velocity profiles of 4 individual cells patrolling in atherosclerotic vessels over time (time resolution 1 frame/s). Cells 1...
Figure 6.
Correlation of monocyte patrolling and endothelial damage during atherogenesis. A and B , Morphological signs of endothelial cell damage were assessed in control and apoE u2212/u2212 mice with chow or...
Figure 7.
Enhanced endothelial damage in atherosclerotic arteries of patroller-deficient Nr4a1 u2212/u2212 mice. A and B , Explanted aortas of apoE u2212/u2212 Nr4a1 knockout or control mice fed 6 wk Western di...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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