Sustained expression of MCP-1 by low wall shear stress loading concomitant with turbulent flow on endothelial cells of intracranial aneurysm

Abstract

Introduction: Enlargement of a pre-existing intracranial aneurysm is a well-established risk factor of rupture. Excessive low wall shear stress concomitant with turbulent flow in the dome of an aneurysm may contribute to progression and rupture. However, how stress conditions regulate enlargement of a pre-existing aneurysm remains to be elucidated.

Results: Wall shear stress was calculated with 3D-computational fluid dynamics simulation using three cases of unruptured intracranial aneurysm. The resulting value, 0.017 Pa at the dome, was much lower than that in the parent artery. We loaded wall shear stress corresponding to the value and also turbulent flow to the primary culture of endothelial cells. We then obtained gene expression profiles by RNA sequence analysis. RNA sequence analysis detected hundreds of differentially expressed genes among groups. Gene ontology and pathway analysis identified signaling related with cell division/proliferation as overrepresented in the low wall shear stress-loaded group, which was further augmented by the addition of turbulent flow. Moreover, expression of some chemoattractants for inflammatory cells, including MCP-1, was upregulated under low wall shear stress with concomitant turbulent flow. We further examined the temporal sequence of expressions of factors identified in an in vitro study using a rat model. No proliferative cells were detected, but MCP-1 expression was induced and sustained in the endothelial cell layer.

Conclusions: Low wall shear stress concomitant with turbulent flow contributes to sustained expression of MCP-1 in endothelial cells and presumably plays a role in facilitating macrophage infiltration and exacerbating inflammation, which leads to enlargement or rupture.

Fig. 1

CFD analysis of intracranial aneurysm from three human patients. The value of wall shear stress (WSS) is indicated in the color phase

Fig. 2

Induction of genes in cultured endothelial cells (ECs) loaded on low WSS and turbulent flow. RNA was purified from cultured ECs loaded on 3.0 Pa, 0.05 Pa, or 0.05 Pa with turbulent flow (TF), and gene expressions were analyzed by RT-PCR analysis. All bars indicate mean ± SEM (n = 3). ^* p < 0.05 compared with 3.0 Pa-loaded group

Fig. 3

Detection of proliferative cells in intracranial aneurysm lesions of rat. a, Proliferative cells in the small intestine of rats after intraperitoneal injection of EdU (80mg/kg) were detected by click reaction (green color). Merged images with nuclear staining by DAPI are shown. Magnified images are shown in the lower panels. Scale bar = 50μm. b and c, Proliferative cells in intracranial arterial walls of rats before (day 0, b) or after (day 28, c) aneurysm induction were labeled after intraperitoneal injection of EdU. Immunostaining for α-smooth muscle actin (SMA) is shown to visualize the arterial walls. Scale bar = 50μm

Fig. 4

Induction of MCP-1 in the endothelial cell layer during intracranial aneurysm formation in rats. a, Representative image to show the anatomical structure of the bifurcation site of the intracranial artery. A merged image of immunohistochemistry for smooth muscle marker, α-smooth muscle actin (SMA, red), an endothelial cell marker, CD31 (green), and nuclear staining by DAPI (blue) is shown. White arrows indicate the direction of blood flow. Scale bar = 50μm. b and c, Expression of MCP-1 (b) and CX3CL1 (c) in the endothelial cell layer of arterial walls during IA formation. Immunohistochemistry for MCP-1 (green in b), CX3CL1 (green in c), SMA (red), and for the merged images with DAPI (blue) are shown. Scale bar = 50μm