2Department of Chemical and Petroleum Engineering, University of Calgary, Calgary, Canada
3Department of Physiology and Pharmacology, University of Calgary, Calgary, Canada
4Department of Radiology, University of Calgary, Calgary, Canada
5Department of Clinical Neurosciences, University of Calgary, Calgary, Canada
6Centre for Bioengineering Research and Education, University of Calgary, Calgary, Canada
7Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
8Seaman Family MR Research Centre, Foothills Medical Centre, Calgary, Canada
To determine the initial feasibility of using magnetic resonance (MR) imaging to detect early atherosclerosis, we investigated inflammatory cells labeled with a positive contrast agent in an endothelial cell–based testing system. The human monocytic cell line THP-1 was labeled by overnight incubation with a gadolinium colloid (Gado CELLTrack) prior to determination of the in vitro release profile from T1-weighted MR images. Next, MR signals arising from both a synthetic model of THP-1/human umbilical vein endothelial cell (HUVEC) accumulation and the dynamic adhesion of THP-1 cells to activated HUVECs under flow were obtained. THP-1 cells were found to be successfully—but not optimally—labeled with gadolinium colloid, and MR images demonstrated increased signal from labeled cells in both the synthetic and dynamic THP-1/HUVEC models. The observed THP-1 contrast release profile was rapid, suggesting the need for an agent that is optimized for retention in the target cells for use in further studies. Detection of labeled THP-1 cells was accomplished with no signal enhancement from unlabeled cells. These achievements demonstrate the feasibility of targeting early atherosclerosis with MR imaging, and suggest that using an in vitro system like the one described provides a rapid, efficient, and cost-effective way to support the development and evaluation of novel MR contrast agents.
Diagnosis and intervention in the first stages of atherosclerosis could potentially offset economic and clinical costs of cardiovascular disease; however, there are no effective methods currently available for the detection of fatty streaks or early plaques. MR is an imaging modality suitable for use as a screening tool for early atherosclerosis (1). In principle, it should be possible to achieve useful images by employing MR contrast agents that promote signal enhancement in early lesions compared with the background obtained from surrounding healthy tissue (2-4). A physiologically relevant model system for contrast agent testing would facilitate the development of screening methods in a manner that can target eventual translation to clinical protocols.
Inflammation plays a key role in the progression of atherosclerotic diseases (5); thus, targeting components of the vascular inflammatory response for imaging is an interesting approach for development of noninvasive methods to detect early atherosclerosis. This report presents the results of an initial study in which a commercially available positive MR contrast agent was used to label human monocytes in a series of cell-based assays to determine the feasibility of detecting early atherosclerosis using labeled inflammatory cells. Results demonstrate proof-of-concept for this approach, and also illustrate the utility of employing an in vitro culture system to support the development and evaluation of novel MR contrast agents.Materials and methods Cell culture
The human acute monocytic leukemia cell line THP-1 (ATCC; Manassas VA, USA) and pooled human umbilical vein endothelial cells (HUVECs; Lonza, Walkersville MD, USA) were cultured according to the suppliers’ directions. Both cell types are readily available, easy to culture, and useful for studying early atherosclerotic processes in vitro. Notably, THP-1 cells possess the ability to differentiate into macrophages (6), the inflammatory cells found in atherosclerotic plaques. HUVECs are commonly used to study endothelial physiology in vitro (7,8), including interactions with monocytes under flow conditions (9,10).
HUVECs were plated at ~3500 cells/cm2 on glass microscope slides precoated with 0.1% gelatin (BD Difco, Mississauga, ON, Canada) and grown to confluence prior to undertaking the monocyte contact experiments described.Cell Gd labeling
THP-1 cells, at an initial density of 7 × 105 cells/mL, were incubated for 17 h at 37°C and 5% CO2 with the gadolinium (Gd) colloid Gado CELLTrack (BioPAL, Worcester, MA, USA) at a Gd concentration of 0.08 mg/mL in Roswell Park Memorial Institute (RPMI) medium (ATCC). Cell density and viability counts were made via trypan blue staining on a hemacytometer (Fisher Scientific, Ottawa, ON, Canada), and used to inform subsequent experiments.In vitro Gd release
To determine the retention of the contrast agent over time within THP-1 cells, an in vitro release study was conducted. The release profile was established using six time points (10, 20, 40, 80, 160, and 320 min), from triplicate analyses. Following contrast agent incubation, 1 × 106 cells were pelleted by centrifugation at 150× g in a swinging bucket rotor (Centra CL3R, Fisher Scientific) for 3 min, the supernatant removed, and the cells resuspended in 1.5 mL fresh prewarmed RPMI. For each time point, the cells were repelleted and 80% of the media (1.2 mL) removed by pipet for MR analysis. This volume was the maximum that could be collected while being sure not to disturb/remove any cells. Prewarmed fresh media (1.2 mL) was added to the samples and used to resuspend the pellet. This volume allowed the assumption of perfect sink mass transfer conditions (11) to evaluate contrast agent release. At the terminal time point, the cells were collected via centrifugation and resuspended in 300 µL 1% agarose (Bio-Rad, Mississauga, ON, Canada) in RPMI for MR assessment of contrast agent remaining associated with the cells. Supernatant time point samples were analyzed in a 96-well plate (300 µL/well), and release was assessed by determination of media MR relaxivity (R1).THP-1 cell adhesion under flow to activated HUVECs in vitro
Similar to methods described in Reference 10, confluent HUVECs cultured on glass slides were treated with tumor necrosis factor-α (TNF-α; Sigma-Aldrich, Oakville, ON, Canada) at 7 ng/mL for 4 h to activate monocyte adhesion receptor expression (an inflammatory response relevant to athero sclerosis). These activated HUVECs were then transferred to a parallel-plate flow chamber (Figure 1) with a flow channel formed by a 4.6 cm × 1.2 cm cutout section from a 0.025 cm–thick silicone rubber gasket (Specialty Manufacturing, Saginaw, MI, USA) placed between the HUVEC slide and a ported acrylic top plate. The chamber was assembled with small clamps and connected to Norprene tubing (Cole Parmer, Montreal, QC, Canada), and flow was generated by a syringe pump (KD Scientific, Holliston, MA, USA). A heat lamp was used to maintain the experimental setup at ~37°C.