Site-Specific Athero-Susceptible Endothelial Phenotype Shows Prominent Adaptive ER-Stress and Unfolded Protein Responses in Vivo
Peter F. Davies and Mete Civelek*, Departments of Pathology and Laboratory Medicine and Bioengineering, University of Pennsylvania, Philadelphia, PA19104, USA, *Present address: Department of Medicine, University of California, Los Angeles, CA, USA
Please address correspondence to:
Dr. Peter F. Davies
Director, Institute for Medicine and Engineering
University of Pennsylvania
1010 Vagelos Laboratories
3340 Smith Walk
Philadelphia, PA19104, USA
Atherosclerosis is not a diffuse disease; it has been noted for centuries that lesion development is associated with arterial curvatures, asymmetries, and branches where the non-uniform arterial geometry generates patterns of blood flow that are considerably more complex than elsewhere. Since it is well established that endothelial cells are highly sensitive to flow/shear stress, a biomechanical contribution to localized susceptibility is likely. Athero-susceptible endothelium in vivo expresses a different repertoire of cell phenotypes than that in nearby protected locations . Identification of important differences in gene and protein expression and the mechanisms responsible requires both global profiling and classic cell and molecular approaches. Recently, the chronic activation of a common signature of cellular stress in endothelium has emerged as a potential underlying contributor to athero-susceptibility.
Endoplasmic Reticulum (ER)-Stress and the Unfolded Protein Response (UPR)
ER stress is an adaptive protective mechanism that arises because of excessive protein biosynthesis or interference with normal protein folding mechanisms in the ER lumen in response to multiple kinds of cellular stress. Excessive newly synthesized and/or misfolded polypeptides in the ER lumen exceed its protein folding capacity. It results in the activation of the unfolded protein response (UPR), a ubiquitous adaptive cell response that assists cell survival in an adverse environment by activating a set of intracellular signaling pathways. The UPR signals a coordinated transcriptional up-regulation of ER chaperones and folding enzymes to promote the correct assembly of unfolded polypeptides and prevent incompletely folded proteins from aggregating.
In the unstressed state, the ER chaperone binding protein, BiP (also known as heat shock protein A5, HSPA5 and glucose related protein 78, GRP78) binds to each of three ER stress transducers. These are ER transmembrane proteins each having an ER-luminal domain for the sensing of unfolded proteins and a cytosolic domain for signaling. While bound, BiP maintains the inactive state of the transducers; however, when an imbalance occurs in the luminal flux of newly synthesized unfolded or misfolded peptides as a result of cell stress, the UPR is activated. To bind unfolded/misfolded polypeptides in the ER lumen, BiP dissociates from the chaperones causing their phosphorylation. Activation of ATF6a (activating transcription factor 6a), IRE1a (inositol requiring kinase 1a), and PERK (protein kinase-like ER kinase) and the downstream consequences of their activation constitute the UPR. The products of the activated UPR transducers converge as transcriptional regulators in the nucleus to upregulate ER chaperones and UPR transducer synthesis and to ubiquitinate unfolded proteins for degradation through the proteosome; both processes relieve ER stress accumulation of unfolded proteins and restore homeostasis. Failure to restore ER protein equilibrium to a normal range leads to apoptosis through transcriptional induction of the transcription factor CHOP (C/ERB homologous protein), inflammation through activation of NFkB, and generation of reactive oxygen species (ROS) through excessive protein oxidation in the ER.
The links between site-specific endothelial ER-stress in vivo, hemodynamics, and athero-susceptibility have converged in 3 recent studies.
Unbiased global genomics 
In a multi-site study in 45 normal adult swine, endothelium in susceptible regions of the aortic arch (AA), proximal brachiocephalic artery, aorto-renal branch region, and abdominal aorta were analyzed relative to protected sites of the common carotid artery, descending thoracic aorta (DT), and the distal renal artery. All athero-susceptible regions are associated with complex disturbed blood flow. From this multi-site study the most abundant common feature of the endothelium of all athero-susceptible regions was the upregulation of genes associated with ER processing of proteins, ER stress, and the UPR. Differential gene expression analysis identified 133 genes, 73% of which are involved in ER protein processing and folding and which form a highly connected and coordinated network of genes upregulated in the susceptible regions. Three independent and unbiased pathway mining approaches – Gene Ontology using the program DAVID, Gene Set Enrichment Analysis (GSEA), and Ingenuity Pathway Analysis – identified ER stress and the unfolded protein response to be over-represented functional categories in athero-susceptible endothelium including genes that function in protein folding, synthesis, and post-translational protein modification.
To validate the global genomics analyses, endothelial cell proteins were isolated from AA and DT and also from the athero-susceptible aorto-renal branch and the protected distal renal artery. At each athero-susceptible disturbed flow site, BiP transcript and/or protein expression was significantly upregulate. Western blot demonstrated significantly elevated phospho ATF6a, phospho IRE1a and its target, spliced XBP-1. However, the third transducer pathway PERK was not activated. Overall, this study, approached without preconceived expectations of differential expression of genes and proteins associated with ER stress/UPR, strongly suggests that stresses associated with flow disturbance in vivo elicit partial activation of the UPR, an ER response common to other forms of stress, and that chronic stress is a signature for athero-susceptible endothelial phenotype in vivo.
Flow characteristics in vitro induce BiP activation 
Using an in vitro model to simulate human arterial shear stress waveforms, athero-susceptible or atheroprotective flow was applied to human endothelial cells. BiP (GRP78) was found to be significantly upregulated in a sustained manner under athero-susceptible, but not atheroprotective flow up to 24 hours. This response was dependent on both sustained activation of p38, as well as integrin α2β1. Increased BiP expression correlated with the activation of the ER stress sensing element promoter by athero-susceptible flow as a marker of the UPR. Shear stress regulation of BiP was through increased protein stability when compared to other flow regulated proteins, such as connexin-43 and vascular cell adhesion molecule (VCAM)-1. Increased endothelial expression of BiP was also observed in athero-susceptible versus atheroprotective regions of C57BL6 mice. The study supports a role for the hemodynamic environment in preferentially inducing BiP and the UPR in athero-susceptible regions before lesion development.
Spliced XBP-1 chaperone pathway of UPR 
Spliced XBP-1 (sXBP-1) encodes the XBP-1 transcription factor that translocates to the nucleus to activate selective pro-apoptotic target genes as one of the 3 transduction arms of the UPR response. Following the observation of endothelial expression of the XBP-1 pathway of UPR in branching regions of apoE-/- mice arteries and in atherosclerotic lesions that developed there, this study reported that athero-susceptible flow waveforms induced XBP-1 splicing in cultured endothelial cells. Over-expression of (activated) sXBP-1 induced apoptosis in cultured human endothelial cells. To extend the findings to an in vivo assay for atherogenesis, adenoviral-mediated over-expression of sXBP-1 was induced in an apoE-/- murine aortic isograft model. In these animals, enhanced intimal hyperplasia and atherosclerosis developed in normally protected regions of the aorta suggesting that when the XBP-1 UPR pathway is greatly over-stimulated, the adaptive protective function of UPR reverts to a pathological imbalance. While over-expression was not entirely limited to the endothelium in the isograft model, the data are supportive for a prominent role for endothelial sXBP-1.
These three different but complementary approaches to endothelial ER stress provide compelling evidence for the existence of site-specific chronic adaptive UPR in endothelial cells in vivo, and that the hemodynamic environment associated with sites of athero-susceptibility plays a significant role.
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