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JAM‐C deficiency primes endothelial cells for a pro‐inflammatory state
Author(s) -
Reglero Natalia,
Beal Robert,
Cabrera Claudia,
Pickworth Catherine,
Golding Matthew,
Whiteford James,
Nightingale Tom,
Imhof Beat,
Barnes Michael R,
Nourshargh Sussan
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.165.10
Subject(s) - microbiology and biotechnology , tight junction , chemokine , gene knockdown , transfection , immune system , endothelium , inflammation , endothelial stem cell , interleukin 8 , chemistry , immunology , biology , in vitro , gene , biochemistry , genetics
Endothelial cells (ECs) line the inner surface of all vascular walls and as such form a vital barrier between blood and interstitial tissues. Key to this function is the ability of ECs to form cell‐cell junctions that support the formation of a confluent EC monolayer, providing an effective gate‐keeper for the regulated vascular crossing of blood borne proteins and circulating immune cells. Junctional adhesion molecule‐C (JAM‐C) is a component of EC tight junctions and plays a key role in controlling numerous EC vascular and immune functions. Of note, previous work from our laboratory has shown that certain inflammatory conditions (e.g. ischemia‐reperfusion injury) can elicit reduced expression of EC JAM‐C, a phenomenon that can lead to disrupted neutrophil transendothelial cell migration (TEM) dynamics such as neutrophil reverse TEM (rTEM) ( Woodfin et al., Nature Immunol., 2011; Colom et al., Immunity, 2015 ). To gain a better understanding of the role of JAM‐C in EC phenotype and functions, we conducted an unbiased gene expression analysis of ECs subjected to control or JAM‐C siRNA. Briefly, HUVECs were transfected with control or JAM‐C specific siRNA by nucleofection, with the latter achieving a consistently high (~90%) knockdown (KD) efficiency. Of note, JAM‐C ( JAM3 ) KD did not affect the expression of other key EC junctional markers such as VE‐Cadherin or JAM‐A. The study revealed that JAM‐C KD HUVECs exhibit a pro‐inflammatory gene expression profile under basal conditions. Several genes implicated in leukocyte migration, such as the chemokine IL8 (p=6 × 10 − 5 fold change (FC) = 5.1) the adhesion molecules VCAM‐1 (p=9.4 × 10 − 6 FC= 16.7) and ICAM‐1 (p=0.0014; FC= 3.7) , were significantly upregulated in JAM‐C KD cells. Furthermore, pathway analysis of the gene array data by Ingenuity (IPA) software indicated upregulation of numerous gene clusters associated with cell functions such as cellular homeostasis, leukocyte trafficking and several inflammatory signalling pathways. Regulation of key genes (e.g. IL8, VCAM‐1 ) was further validated by qPCR and at protein level. On‐going works are addressing the functional implications of these findings. Collectively, the present results have identified the crucial role of JAM‐C in maintaining ECs in a quiescent state, indicating that loss of EC JAM‐C could prime ECs for an enhanced and/or potentially aberrant inflammatory response. These findings provide novel insights into the consequences of reduced expression of JAM‐C under pathological conditions, such as that noted in response to ischemia‐reperfusion injury. Support or Funding Information This work was supported by generous funds from the The Wellcome Trust and Marie Curie Actions.