models that attempt to recreate this environment ex vivo. The goal of this review is to summarize our current knowledge in this area and identify some of the key unresolved challenges and questions requiring further study.
Atherosclerosis is an inflammatory disease that preferentially forms at hemodynamically compromised regions of altered shear stress patterns. Endothelial cells (EC) and smooth muscle cells (SMC) undergo phenotypic modulation during atherosclerosis. An in vitro coculture model was developed to determine the role of hemodynamic regulation of EC and SMC phenotypes in coculture. Human ECs and SMCs were plated on a synthetic elastic lamina and human-derived atheroprone, and atheroprotective shear stresses were imposed on ECs. Atheroprone flow decreased genes associated with differentiated ECs (endothelial nitric oxide synthase, Tie2, and Kruppel-like factor 2) and SMCs (smooth muscle alpha-actin and myocardin) and induced a proinflammatory phenotype in ECs and SMCs (VCAM-1, IL-8, and monocyte chemoattractant protein-1). Atheroprone flow-induced changes in SMC differentiation markers were regulated at the chromatin level, as indicated by decreased serum response factor (SRF) binding to the smooth muscle alpha-actin-CC(a/T)(6)GG (CArG) promoter region and decreased histone H(4) acetylation. Conversely, SRF and histone H(4) acetylation were enriched at the c-fos promoter in SMCs. In the presence of atheroprotective shear stresses, ECs aligned with the direction of flow and SMCs aligned more perpendicular to flow, similar to in vivo vessel organization. These results provide a novel mechanism whereby modulation of the EC phenotype by hemodynamic shear stresses, atheroprone or atheroprotective, play a critical role in mechanical-transcriptional coupling and regulation of the SMC phenotype.
Dash A, Simmers MB, Deering TG, Berry DJ, Feaver RE, Hastings NE, Pruett TL, LeCluyse EL, Blackman BR, Wamhoff BR. Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro. Am J Physiol Cell Physiol 304: C1053-C1063, 2013. First published March 13. 2013 doi:10.1152/ajpcell.00331.2012.-In vitro primary hepatocyte systems typically elicit drug induction and toxicity responses at concentrations much higher than corresponding in vivo or clinical plasma Cmax levels, contributing to poor in vitro-in vivo correlations. This may be partly due to the absence of physiological parameters that maintain metabolic phenotype in vivo. We hypothesized that restoring hemodynamics and media transport would improve hepatocyte architecture and metabolic function in vitro compared with nonflow cultures. Rat hepatocytes were cultured for 2 wk either in nonflow collagen gel sandwiches with 48-h media changes or under controlled hemodynamics mimicking sinusoidal circulation within a perfused Transwell device. Phenotypic, functional, and metabolic parameters were assessed at multiple times. Hepatocytes in the devices exhibited polarized morphology, retention of differentiation markers [E-cadherin and hepatocyte nuclear factor-4␣ (HNF-4␣)], the canalicular transporter [multidrug-resistant protein-2 (Mrp-2)], and significantly higher levels of liver function compared with nonflow cultures over 2 wk (albumin ϳ4-fold and urea ϳ5-fold). Gene expression of cytochrome P450 (CYP) enzymes was significantly higher (fold increase over nonflow: CYP1A1: 53.5 Ϯ 10.3; CYP1A2: 64.0 Ϯ 15.1; CYP2B1: 15.2 Ϯ 2.9; CYP2B2: 2.7 Ϯ 0.8; CYP3A2: 4.0 Ϯ 1.4) and translated to significantly higher basal enzyme activity (device vs. nonflow: CYP1A: 6.26 Ϯ 2.41 vs. 0.42 Ϯ 0.015; CYP1B: 3.47 Ϯ 1.66 vs. 0.4 Ϯ 0.09; CYP3A: 11.65 Ϯ 4.70 vs. 2.43 Ϯ 0.56) while retaining inducibility by 3-methylcholanthrene and dexamethasone (fold increase over DMSO: CYP1A ϭ 27.33 and CYP3A ϭ 4.94). These responses were observed at concentrations closer to plasma levels documented in vivo in rats. The retention of in vivo-like hepatocyte phenotype and metabolic function coupled with drug response at more physiological concentrations emphasizes the importance of restoring in vivo physiological transport parameters in vitro.hemodynamics; hepatocyte; metabolism; organotype; phenotype HEPATOTOXICITY AND BIOAVAILABILITY issues comprise over 60% of drug failures during clinical trials (45) and are a major cause of postmarketing withdrawal (23), pointing to the need to develop more efficient and predictive preclinical test systems. Simple cellular and subcellular assays used to screen compound libraries offer the advantage of higher throughput but are often unable to capture complex biological effects that may require a physiological context for drug interactions with cells. Primary in vitro hepatocyte models widely used to study liver disease, drug metabolism, and toxicity are extensively reviewed in the literature (16,42). The ability to test the metabolic f...
Objective-The initiation of atherosclerosis is in part dependent on the hemodynamic shear stress environment promoting a proinflammatory phenotype of the endothelium. Previous studies demonstrated increased expression of ER stress protein and unfolded protein response (UPR) regulator, GRP78, within all vascular cells in atherosclerotic lesions and its regulation in the endothelium by several atherosclerotic stressors; however, regulation of GRP78 by shear stress directly has not been established. Method and Results-Using an in vitro model to simulate human arterial shear stress waveforms, atheroprone or atheroprotective flow was applied to human endothelial cells. GRP78 was found to be significantly upregulated (3-fold) in a sustained manner under atheroprone, but not atheroprotective flow up to 24 hours. This response was dependent on both sustained activation of p38, as well integrin ␣21. Increased GRP78 correlated with the activation of the ER stress sensing element (ERSE1) promoter by atheroprone flow as a marker of the UPR. Shear stress regulated GRP78 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 GRP78 was also observed in atheroprone versus atheroprotective regions of C57BL6 mice. Conclusions-This study supports a role of the hemodynamic environment in preferentially inducing GRP78 and the UPR in atheroprone regions, before lesion development, and suggests a potential atheroprotective (ie, prosurvival), compensatory effect in response to ER stress within atherosclerotic lesions. Key Words: endothelial Ⅲ GRP78 Ⅲ shear stress Ⅲ atherosclerosis Ⅲ unfolded protein response A therosclerosis is a focal inflammatory disease that develops preferentially in areas of disturbed flow, where variations in shear stress have been shown to alter the phenotypes of endothelial cells toward either an atheroprone or atheroprotective state in vitro and in vivo. 1,2 Therefore, hemodynamic-induced shear stress provides a major mechanical signal, which causes the overlying endothelium to become at risk for the promotion of atherosclerosis.Of many proteins of interest, the chaperone protein, glucose regulated protein 78 (GRP78), a common marker for endoplasmic reticulum (ER) stress, is preferentially expressed in advanced atherosclerotic lesions 3 and on the fibrous cap surface in ApoE-KO mice. 4 Further, cell-surface associated GRP78 has been speculated to serve a protective role in atheroprone environments by inhibiting tissue factor through direct binding to the endothelium overlying the plaque. 5 Hyperhomocysteinemia is associated with increased risk of cardiovascular disease possibly by limiting the antioxidant activity and causing ER stress, leading to the activation of GRP78. 4 ER stress is further linked to oxidative stress through peroxynitrite-induced GRP78 expression. 3 ER stress is present at every stage of atherosclerosis, even preceding free cholesterol accumulation. 6 This su...
GK. PDGF-DD, a novel mediator of smooth muscle cell phenotypic modulation, is upregulated in endothelial cells exposed to atherosclerosis-prone flow patterns. Am J Physiol Heart Circ Physiol 296: H442-H452, 2009. First published November 21, 2008 doi:10.1152/ajpheart.00165.2008.-Platelet-derived growth factor (PDGF)-BB is a well-known smooth muscle (SM) cell (SMC) phenotypic modulator that signals by binding to PDGF ␣␣-, ␣-, and -membrane receptors. PDGF-DD is a recently identified PDGF family member, and its role in SMC phenotypic modulation is unknown. Here we demonstrate that PDGF-DD inhibited expression of multiple SMC genes, including SM ␣-actin and SM myosin heavy chain, and upregulated expression of the potent SMC differentiation repressor gene Kruppel-like factor-4 at the mRNA and protein levels. On the basis of the results of promoter-reporter assays, changes in SMC gene expression were mediated, at least in part, at the level of transcription. Attenuation of the SMC phenotypic modulatory activity of PDGF-DD by pharmacological inhibitors of ERK phosphorylation and by a small interfering RNA to Kruppel-like factor-4 highlight the role of these two pathways in this process. PDGF-DD failed to repress SM ␣-actin and SM myosin heavy chain in mouse SMCs lacking a functional PDGF -receptor. Importantly, PDGF-DD expression was increased in neointimal lesions in the aortic arch region of apolipoprotein C-deficient (ApoE Ϫ/Ϫ ) mice. Furthermore, human endothelial cells exposed to an atherosclerosis-prone flow pattern, as in vascular regions susceptible to the development of atherosclerosis, exhibited a significant increase in PDGF-DD expression. These findings demonstrate a novel activity for PDGF-DD in SMC biology and highlight the potential contribution of this molecule to SMC phenotypic modulation in the setting of disturbed blood flow. shear stress; disturbed blood flow; smooth muscle myosin heavy chain; smooth muscle ␣-actin ATHEROSCLEROSIS IS A COMPLEX disease characterized by the accumulation of lipid and cholesterol deposits within the walls of blood vessels, as well as intimal proliferation and extracellular matrix deposition by phenotypically modulated smooth muscle (SM) cells (SMCs) (1). SMCs within human atherosclerotic lesions and experimental atherosclerosis exhibit a distinct morphological change compared with medial SMCs within the normal vessel wall (32). Along with this morphological change, phenotypically modulated SMCs exhibit decreased expression of a variety of contractile genes, including SM ␣-actin, SM myosin heavy chain (MHC), SM22␣, smoothelin, and h1-calponin (32). Advanced atherosclerotic lesions are characterized by a large lipid and necrotic core covered by a fibrous cap, and the thickness and mechanical properties of these lesions are key determinants of the probability of plaque rupture (3, 12, 13), thrombosis, and subsequent acute myocardial infarction or stroke, the leading causes of death in developed countries (3,12,13). Although the precise factors and mechanisms that con...
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