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95 Investigating fibroblast involvement in vascular inflammation using co-culture models
  1. Kelly Wright1,
  2. John Haycock2,
  3. Sheila Francis3
  1. 1The University of Sheffield,.Kroto Research Institute, University of Sheffield, Sheffield, UK
  2. 2The University of Sheffield
  3. 3Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield


Introduction Coronary heart disease is caused by the accumulation of fatty deposits within the vascular lining. This process often goes undetected until a patient becomes symptomatic (e.g. myocardial infarction); consequently, current treatments are primarily aimed at symptomatic relief. However, it is necessary to understand the inflammatory processes that underpin atherogenesis, enabling the development of novel therapeutic strategies. ICAM-1 is up-regulated during inflammation to aid leukocyte extravasation and is expressed by numerous cell types. Moreover, the involvement of the adventitial fibroblast is becoming increasingly popular, thus ICAM-1 was used as a marker to study the augmentation of the inflammatory response in HUVEC/HUASMC/NHDF co-cultures.

Methods 25 U/mL human recombinant TNF-α was used to stimulate HUVEC, HUASMC and NHDF in 2D monoculture and co-culture arrangements, fixing samples every 3 hours for up to 12 hours before immunolabelling for ICAM-1. T-tests were used to determine any significant differences in ICAM-1 expression, and the relationship between cell ratio and ICAM-1 expression was examined.

Results ICAM-1 expression was significantly up-regulated upon the introduction of TNF-α under all conditions in HUVEC (figure 1). However, baseline expression was increased when co-cultured with both NHDF (2.0 vs 1.3, p<0.001) and HUASMC (6.5 vs 1.3, p<0.001). This meant that ICAM-1 expression at 12 hours was also significantly higher in co-culture with NHDF (8.3 vs 5.2, p<0.001) and HUASMC (11.0 vs 5.2, p<0.001). Moreover, there was a moderate relationship between HUVEC ICAM-1 expression and the cell ratio when in co-culture with NHDF, where decreasing NHDF resulted in decreased ICAM-1 in HUVEC (R2=0.45).

TNF-α caused an increase in ICAM-1 expression in NHDF under monoculture conditions (Fig. 2); this up-regulation was significantly reduced in co-culture conditions with HUVEC (1.7 vs 5.1, p<0.001). A similar trend was observed when in co-culture with HUASMC (2.7 vs 5.1, p<0.001), except the baseline expression of ICAM-1 was also increased (2.8 vs 1.1, p<0.001).

Constitutive production of ICAM-1 was observed in HUASMC, whereby the introduction of TNF-α or additional cell types resulted in no significant differences after 12 hours (not shown).

Discussion/conclusion It appears that a complex communication network exists between the endothelial cell, smooth muscle cell and fibroblast to control the vascular inflammatory response that underpins atherogenesis. Whilst the smooth muscle cell enhanced ICAM-1 expression on endothelial cells prior to the addition of TNF-α, the largest change in ICAM-1 appeared when the endothelial cells were co-cultured with fibroblasts. Though these experiments were carried out in 2D, evidence suggests that the fibroblast may contribute significantly to the expression of ICAM-1 in vasculature. Developing a 3D model to study this further will yield the significance of these cellular interactions to the atherogenic inflammatory response.

  • Inflammation
  • co-culture
  • ICAM-1

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