Exploring the Role of Smooth Muscle GRP78 in Angiotensin II-Induced Vascular Amyloid Deposition and Remodeling

Abstract

Cardiovascular disease (CVD) is the leading cause of death in the United States, and hypertension has been recognized as a major contributor to its manifestation and progression. Vascular smooth muscle cells control the tone and elasticity of vessels and their dysfunction in hypertension contributes to arterial remodeling and subsequent end organ damage. Evidence has indicated that the Endoplasmic Reticulum (ER) Unfolded Protein Response (UPR) may be compromised in hypertension, while the contribution of protein aggregate formation (a main driver of UPR activation) is undefined. Glucose regulated protein-78 (GRP78), a residential ER chaperone, acts to aid in the proper folding of nascent peptides during translation, while also acting as the primary signal transducer for UPR. We hypothesize overexpression of GRP78 can protect against Angiotensin II induced protein aggregation in vascular smooth muscle cells (VSMCs) to reduce pathological ER UPR signaling and hypertensive vascular remodeling. To test this hypothesis, we investigated protein aggregate induction by Ang II stimulation as well as ER UPR activation, and if overexpression of the ER-resident chaperone glucose regulated protein 78 (GRP78) could protect against these as well as VSMC remodeling markers: hypertrophy, collagen production and inflammation. Utilizing pre-amyloid oligomer (PAO) immunofluorescence staining to identify Ang II induction of amyloid in VSMCs, we found amyloid accumulation was maximal at 48h post stimulation, which could be prevented with adenoviral overexpression of GRP78. Ang II significantly induced ER stress markers p-IRE1α, p-PERK and cleaved ATF6 in VSMCs. Overexpression of GRP78 was able to attenuate these ER stress responders. Interestingly, shotgun proteomic analysis of triton X-100 insoluble aggregate fractions revealed proteostasis machinery enriched in Ang II treated VSMC aggregates (HSP70, VCP, CryAB), which were attenuated with GR78 overexpression. To investigate pathological VSMC remodeling markers, we found that Ang II induced VSMC collagen production, immune cell adhesion, VCAM-1 expression, and hypertrophy (via protein synthesis) which was attenuated by GRP78 overexpression. Utilizing a VSMC-promoter derived GRP78 overexpression mouse model (GRP78TG SM22α Cre-/- or GRP78TG SM22α Cre+/-), we investigated the effect of ER stress inhibition on Ang II induced vascular remodeling. Importantly, hypertrophy and fibrosis in the aorta and the cardiac vasculature were assessed by Masson’s Trichrome and Sirius red staining and found to be increased in Cre-/- mice, while Cre+/- were significantly protected. These effects were accompanied with a significant reduction in Ang II-induced aortic amyloid burden (PAO) and ER UPR signaling. Blood pressure was monitored via tail cuff which revealed GRP78 Cre+/- mice were not protected against Ang II induced hypertension. Overall, these findings indicate that VSMC protein aggregation activates the ER stress response and contribute to hypertensive vascular remodeling. Furthermore, therapeutically targeting this mechanism via overexpression of GRP78 may elude new pharmacological interventions for arterial stiffness, while addressing fundamental questions about the mechanisms involved.