Introduction Atherosclerosis is an inflammatory disease that develops preferentially at bends and branches of the vasculature exposed to disturbed flow and low shear stress (LSS). Shear stress modifies endothelial cell (EC) function by regulating proliferation, inflammation and other fundamental processes. Shear stress alters multiple transcriptional programs, including those regulated by the NF-κB family of transcription factors. Although some members of the NF-κB pathway are known to respond to shear, the influence of haemodynamics on the c-Rel NF-κB subunit and its role in atherogenesis are unknown, and are a focus of the current study.
Methods C57BL/6 wildtype and c-Rel knockout mice were used to quantify the expression of c-Rel, the inflammatory protein E-selectin, and EC proliferation at LSS and high shear stress (HSS) regions of the aorta. To establish the role of c-Rel in atherosclerosis, C57BL/6 wildtype and c-Rel knockout mice were treated with AAV-PCSK9 and fed a high fat diet for 6 weeks. Plaque burden was quantified using Oil Red O. In vitro studies were performed using human umbilical vein EC (HUVEC) or human coronary artery EC (HCAEC) exposed to LSS or HSS. c-Rel was silenced using siRNA and EC proliferation and inflammation were measured by PCNA staining and qRT-PCR, respectively. Gene expression was studied using a microarray (ClariomTM S).
Results En face staining of murine aortas revealed a striking enrichment of c-Rel at LSS regions (P<0.01). Similarly, c-Rel expression was significantly enhanced in HUVEC or HCAEC exposed to LSS compared to HSS (P<0.05). c-Rel genetic deletion in mice resulted in decreased proliferation and inflammation in EC at LSS sites, and it also decreased lesion area in AAV-PCSK9-treated mice (P<0.05). Moreover, depletion of c-Rel by siRNA in HUVEC resulted in decreased proliferation and inflammation under LSS (P<0.05). Microarray studies using HUVEC revealed that c-Rel activates genes implicated in inflammation and proliferation, including components of MAPK and non-canonical NF-κB pathways.
Our data demonstrate that c-Rel promotes EC pathophysiological changes at LSS regions and is a driver of atherosclerosis. Studies in cultured EC revealed that c-Rel activates numerous genes including components of MAPK and non-canonical NF-κB pathways, thereby providing a potential mechanism for its proinflammatory and proproliferative effects.
Conflict of interest None
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