BioFactors. 2014 Jul/Aug;40(4):448-457.

Tenascin-C is increased in atherothrombotic stroke patients and has an anti-inflammatory effect in the human carotid artery.

 

Clancy P, Lincz LF, Maguire J, McEvoy M, Koblar, SA, Golledge J.

 

Health practitioners And Researchers Together-Blood, Endothelium And Tissue (HART-BEAT), Australian Institute for Tropical Health and Medicine, School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland, Australia 4811

Hunter Haematology Research Group, Calvary Mater Newcastle Hospital, Edith Street, Waratah, NSW, Australia 2298

Faculty of Health and Medicine, School of Nursing and Midwifery, University of Newcastle, University Drive, Callaghan, NSW, Australia 2308

University of Newcastle, School of Medicine and Public Health, HMRI Building Lookout Rd, New Lambton, NSW, Australia 2305

Stroke Research Programme, School of Medicine, The Queen Elizabeth Hospital (TQEH) campus, Adelaide University, 28 Woodville Rd, Woodville Sth, South Australia, Australia 5011

The Vascular Biology Unit, Queensland Research Centre for Peripheral Vascular Disease, School of Medicine and Dentistry, James Cook University, Townsville, Queensland, Australia 4811

Department of Vascular and Endovascular Surgery, The Townsville Hospital, Townsville, Queensland, Australia 4811

 

Abstract

Tenascin-C (Tn-C) is an endogenous ligand of toll-like receptor-4 (TLR-4); a key signalling molecule associated with chronic inflammatory conditions. Both Tn-C and TLR-4 are increased in unstable human atheroma, but their effects on local inflammatory conditions have not been investigated. The aim of the present study was to investigate the association and functional implications of Tn-C/TLR-4 signalling in large artery atherosclerotic stroke.

Plasma Tn-C was measured by ELISA and found to be higher in recent stroke patients (n=336; median 12.77 µg/mL, inter-quartile range 10.23-15.74 µg/mL) than in controls (n=321; median 11.31 µg/mL, inter-quartile range 8.89-13.90 µg/mL), p<0.001. Plasma Tn-C was also independently positively associated with stroke (odds ratio for highest Tn-C quartile 2.27, 95% confidence interval 1.37-3.76). Assessment of Tn-C associated chronic cytokine secretion was performed in vitro using paired, human, macroscopically disease matched, carotid atheroma tissue biopsies obtained from five patients undergoing carotid endarterectomy. A 4 day incubation with specific Tn-C blocking antibodies (Abs) increased secretion of TLR-4 associated cytokines, interleukin (IL)-8, IL-1β, tumour necrosis factor (TNF)-α and C-C motif chemokine (CCL)3 and expression of TLR-4 in the tissue. These results suggest with Tn-C blockade another endogenous TLR-4 ligand upregulates TLR-4 expression and subsequent cytokine secretion. Titration of the Tn-C Abs also dose dependently increased secretion of IL-6, IL-8, IL-1β and CCL3 in mixed, healthy, primary vascular cell culture. In summary, circulating concentrations of Tn-C are higher in patients with a recent history of atherosclerotic stroke and may play an anti-inflammatory role by reducing pro-inflammatory cytokine release from atheroma.

KEYWORDS: anti-inflammatory; atheroma; stroke; tenascin-C; toll-like receptor-4

PMID: 24823872

 

Supplement:

A number of studies using synovial elements from rheumatoid arthritis demonstrated previously that Tn-C could up-regulate expression of a number of TLR-4 signalled cytokines, including IL-6, IL-8 and TNF-α (1-3). Mapping studies using recombinant fragments of each of the Tn-C domains narrowed the stimulating domain down to the C-terminal fibrinogen domain (FBG), surmising this domain must bind to TLR-4 (1). Our hypothesis drew from these studies. We hypothesized that blockade of the FBG domain of Tn-C using domain specific Abs would reduce cytokine secretion from cultured diseased human carotid atheroma tissue.

However, when we conducted this experiment we demonstrated the opposite effect. Unexpectedly, and to our surprise, cytokine expression increased with FBG blockade in the cultured atheroma tissue. Expression of the TLR-4 receptor in the tissue was also increased with FBG blockade.

TLR-4 is well known as a receptor for the bacterial endotoxin, lipopolysaccharide, the binding of which triggers the host innate immune response against the invading bacteria. However, TLR-4 also plays a role in the temporary inflammation needed for successful wound healing (4), the chronic inflammation of atherosclerosis (5) and potentially the depressed suicide brain (6). TLR-4 stimulation can lead to up-regulation of inflammatory cytokines, angiogenic growth factors, proteolytic enzymes such as matrix metalloproteinases (MMPs), and the thrombogenic protein, tissue factor, which are all proteins involved in wound healing and cardiovascular disease (CVD) (4). Both these processes are driven by TLR-4 ligands already present in the body i.e. endogenous ligands, and the overall signalling response through TLR-4 is a result of the interplay between the competing ligands.

Direct in vivo determination of vascular cellular signalling mechanisms that initiate disease in patients is not easily achieved. The authors have compromised and utilised cultured diseased human carotid atheroma tissue to investigate the role of Tn-C, angiotensin signalling peptides and anti-hypertensive drugs (7,8) in the up-regulation of a number of cytokines and MMPs that have been associated with both CVD and wound healing. These studies demonstrated that the ex vivo tissue signalling response to the anti-hypertensive drugs was in keeping with the systemic in vivo drug responses in people (7,8). However, the scarcity of human tissue that can be used for culture means this is not a viable in vitro methodology for long-term studies investigating a range of signalling pathways involved in disease initiation.

Dr Clancy has therefore developed a serum stimulated, mixed primary vascular cell culture system to assess vascular cell signalling responses in vitro. This methodology requires only the collection of a blood sample from patients making this a more sustainable methodology for future studies. This new vascular cell culture system also matched the in vivo systemic anti-hypertensive drug response (7) and supported the FBG blockade up-regulation of cytokines demonstrated in the cultured diseased atheroma tissue.

Originally, the vascular cell culture system was designed to support the atheroma studies. The methodology was developed from the observation that exposure of TF expressing smooth muscle cells (SMC) or fibroblasts (FIB) to blood initiates cell signalling responses, such as inflammatory cytokine release, within the vasculature (9) (Figure 1).

Due to the nature of the carotid endarterectomy procedure the types of vascular cells present in the original explant tissue included endothelial cells (ECs), SMCs, inflammatory cells that have infiltrated the atheroma from the blood and red blood cells (BCs) on the tissue surface. Thus, the initial model used these cell types stimulated by serum (Monolayer 2, Figure 1). Future studies will utilize other cell combinations, aiming to replicate differing vascular wound states in order to investigate vascular cell signalling and drug responses in vitro.

 

PC fig1

Figure 1. Tissue factor initiation of vascular wound healing. Blockade of factor VII stops up-regulation of inflammatory, tissue remodelling, angiogenic and thrombogenic proteins involved in vascular wound healing.

 

Importance of the study:

This innovative in vitro culture system developed by Dr Clancy can be used to assess the capacity of blood, from different patient cohorts, to activate signalling responses in the cells. This vascular cell culture system has also been demonstrated previously to emulate in vivo drug responses (7). This represents significant metholodological advancement in the assessment of human drug responses (particularly in combination) relating to inflammatory processes such as atherosclerosis, wound healing, pathogenic response and depression. In addition, current human culture studies are only able to assess CVD end points using explant samples. This is because tissue is only collected from patients at the time of reparative surgery (7,8,10,11) and so investigations into the early stages of CVD and disease initiation must rely on genetically modified mice. Using human samples rather than animals is a major advantage in the application of findings to treatments in humans.

 

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