Deletion of tenascin-C gene exacerbates atherosclerosis and induces intraplaque hemorrhage in Apo-E-deficient mice

Abstract

Aims

Tenascin-C (TNC), a matricellular protein, is up-regulated in atherosclerotic plaques. We investigated whether the deletion of TNC gene affects the development of atherosclerosis in a murine model.

Methods

TNC−/−/apo E−/− mice were generated and used for atherosclerosis studies. We compared these results to those observed in control groups of apo E−/− mice.

Results

The en face analysis of aortic area showed that the mean aortic lesion area of the double knockout (KO) mice was significantly higher than that of control mice at different times after feeding of atherogenic diet; the accumulation of lesional macrophages and lipids was significantly higher. Analysis of cell adhesion molecules revealed that vascular cell adhesion molecule-1 (VCAM-1), but not intercellular adhesion molecule-1, was up-regulated 1 week after feeding of atherogenic diet in the double KO mouse as compared to apo E−/− mouse. Cell culture studies revealed that the expression of VCAM-1 in endothelial cells isolated from the double KO mouse is more sensitive to the tumor necrosis factor α stimulation than the cells isolated from apo E−/− mice. Cell adhesion studies showed that the adherence of RAW monocytic cells to the endothelial cells was significantly enhanced in the cultured endothelial cells from the TNC gene-deleted cells. Following the prolonged feeding of an atherogenic diet (28–30 weeks), the aortic and carotid atherosclerotic lesions frequently demonstrated large grossly visible areas of intraplaque hemorrhage in the double KO mice compared to control.

Conclusions

These data unveil a protective role for TNC in atherosclerosis and suggest that TNC signaling may have the potential to reduce atherosclerosis, in part by modulating VCAM-1 expression.

Introduction

Atherosclerosis is a chronic inflammatory disease affecting the large- and medium-sized arteries. This disease process involves diverse cell types whose activities are regulated by soluble as well as insoluble molecules found in the plaque. One such class of insoluble molecules is matricellular proteins; these are extracellular matrix proteins that regulate cell–matrix interactions and cell functions, and they do not seem to have a direct structural role. Their expression is high during embryogenesis, but almost absent during normal postnatal life; however, they are reexpressed in response to injury in adult tissue.

Tenascin-C (TNC) is a member of the matricellular protein family. This protein is expressed in a rigidly controlled temporospatial pattern in the developing fetus, yet is undetectable or found only in low levels in the corresponding regions of the intact adult organs (for general discussion, see Midwood and Orend [1]). Multiple functions have been assigned to TNC based on in vitro studies, which may or may not truly reflect the activity of this protein in vivo.

We have previously shown that TNC is expressed in human vein grafts [2], and balloon catheterization induces arterial expression of new TNC isoforms [3], [4]. Further, we showed that TNC is expressed in macrophage-rich regions of human atherosclerotic plaque [5]. Others have reported that TNC is an essential factor for neointimal hyperplasia after aortotomy [6]. Using cultured cells, we showed that factors which are important in neointima formation, such as angiotensin II [7] and PDGF-BB [8], regulate the expression of TNC in vascular smooth muscle cells (SMCs). To understand the mechanism of TNC activity in vitro, we cloned various domains of this molecule and showed that TNC interacts with SMCs through its fibrinogen-like domain and that this domain is important for the migration of cultured SMCs [9]. In addition, we reported that the epidermal growth factor domain of TNC induces apoptosis of SMCs [10]. Despite these findings, the precise role of TNC in atherosclerosis in vivo remains unclear.

Based on the expression pattern of TNC in vivo and in vitro and the activity of TNC in vitro, we hypothesized that TNC expression may affect atherosclerosis. To explore this idea, we generated double knockout (KO) mice lacking TNC and apo E (TNC−/−/apo E−/−) and assessed the quantity and phenotype of atherosclerotic lesions in these mice by comparing them to apo E−/− mice. We found that deletion of TNC gene accelerated lesion development in apo E−/− mice in the aortic sinuses, the brachiocephalic artery, and the whole aorta. Remarkably, we found frequent gross and large areas of intraplaque hemorrhage in TNC−/−/apo E−/− mice which were absent in apo E−/− mice. Furthermore, we noted that absence of TNC gene led to rapid up-regulation of vascular cell adhesion molecule-1 (VCAM-1) in vivo, which correlated with increased content of lesional macrophages, as compared to apo E−/− mice. In vitro studies supported a role for TNC in regulating interaction of monocytes with endothelial cells. Therefore, our data suggest that TNC expression may have a protective role in atherosclerosis in part by controlling plaque inflammation.