Article pubs.acs.org/ac
Hyperglycemic Arterial Disturbed Flow Niche as an In Vitro Model of Atherosclerosis Phani K. Patibandla,†,‡ Aaron J. Rogers,†,‡ Guruprasad A. Giridharan,§ Manuel A. Pallero,∥ Joanne E. Murphy-Ullrich,∥ and Palaniappan Sethu*,†,‡ †
Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, United States ‡ Department of Biomedical Engineering, School of Engineering, University of Alabama at Birmingham, Birmingham, Alabama 35294, United States § Department of Bioengineering, Speed School of Engineering, University of Louisville, Louisville, Kentucky 40292, United States ∥ Departments of Pathology and Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35233, United States ABSTRACT: Type 2 diabetes significantly elevates the risk of cardiovascular disease. This can be largely attributed to the adverse effects of hyperglycemic conditions on normal endothelial cell (EC) function. ECs in both large and small vessels are influenced by hyperglycemic conditions, which increase susceptibility to EC dysfunction and atherosclerotic lesion formation. Fluid shear stress and flow patterns play an essential role in atherogenesis: lesions form only at locations where fluid flow behavior can be classified as “disturbed flow” (i.e., low shear stress recirculation and/ or retrograde flow). Since regions of disturbed flow are the focal points of atherosclerotic cardiovascular disease, we hypothesized that the combinatorial effects of high glucose and disturbed flow conditions elicit significantly different responses from ECs than high glucose alone. To validate our hypothesis, we used our endothelial cell culture model (ECCM) to establish vascular niches associated with “normal” and “disturbed” flow conditions typically seen in vivo along with physiological pressure and stretch. We subjected human aortic endothelial cells (HAECs) to hyperglycemic conditions under both “normal” and “disturbed” flow. Our results confirm significant and quantifiable differences in phenotypic and functional markers between cells cultured under conditions of “normal” and “disturbed flow” under hyperglycemic conditions suggesting that elevated glucose in conjunction with “disturbed” flow conditions results in significantly higher level of EC dysfunction. The ECCM can therefore be used as a physiologically relevant model to study early stage hyperglycemia induced atherosclerosis for basic research, drug discovery, and screening and toxicity studies.
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exposure of vascular endothelial cells (ECs) to glucose levels >1.5 g/L alters EC homeostasis and phenotype resulting in EC dysfunction in both large and small vessels.11 EC dysfunction is characterized by one or more of the following characteristics: decrease in nitric oxide (NO) bioavailability which results in reduced endothelial NO (eNO) mediated vasodilatation, impaired fibrinolytic ability, enhanced turnover and proliferation, increased production of growth factors, increased expression of adhesion molecules and inflammatory genes, elevated production of reactive oxygen species (ROS) and oxidative stress, and compromised adherens and tight junctions resulting in compromised barrier function.11 EC dysfunction is an initiating step in atherosclerosis, and hyperglycemia contributes to EC dysfunction. It has been well established that atherosclerotic lesions do not form randomly; rather, they preferentially occur at vascular niches proximal to
ype 2 diabetes is associated with a 2- to 4-fold increase in risk of cardiovascular disease.1 Glucose uptake by vascular cells is not insulin dependent, and therefore, an increase in systemic glucose levels can increase endothelial susceptibility to injury and dysfunction.2 The vascular endothelial layer plays a critical role in the modulation of vascular function. Endothelial cells (ECs), apart from serving as a physical barrier between blood and vascular smooth muscle cells (VSMCs), also perform several critical functions including modulation of vessel tone by regulating endothelial nitric oxide (eNO),3 endothelin-1,4 prostaglandin H2,5 and angiotensin II;6 regulation of vascular remodeling through control of growth factors such as fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), platelet derived growth factor (PDGF), and insulin-like growth factor-1 (IGF-1);7 maintenance of vessel integrity via formation of adherens and tight junctions,8 directing the response to inflammation via expression of adhesion molecules like LSelectin, an intercellular adhesion molecule (ICAM-1), and a vascular cell adhesion molecule (VCAM-1);9 and modulating the oxidative stress response via production of reactive oxygen species (ROS).10 High glucose (HG) conditions defined as © 2014 American Chemical Society
Received: September 2, 2014 Accepted: October 3, 2014 Published: October 3, 2014 10948
dx.doi.org/10.1021/ac503294p | Anal. Chem. 2014, 86, 10948−10954
Analytical Chemistry
Article
Figure 1. Experimental setup of the flow loop for the experiments is shown; arrows indicate the direction of fluid flow within the loop. Individual components include: (a) pulsatile pump, (b, c) one-way flow control valves, (d) flow sensor, (e) normal flow device, (f) disturbed flow device, (g) systemic compliance, (h) pressure sensor, (i) systemic resistance, (j) medium reservoir, and (k) one-way flow control valve.
majority of in vitro studies have evaluated ECs under static conditions19 or using flow conditions seen in straight sections of arteries20 which are less relevant to atherosclerotic cardiovascular disease development and progression. While an argument can be made that low shear stress, disturbed flow is similar to static conditions, the fact remains that ECs in vivo experience pressure and stretch that also play a significant role in maintaining EC phenotype and function. Therefore, recreating the hyperglycemic disturbed flow niche in conjunction with physiological pressure, stretch, and heart rate in vitro is essential to establish relevant model systems for research, drug discovery, and toxicity studies. In reviewing the literature, studies evaluating ECs under “normal flow” in vitro have commonly used parallel plate systems21 that deliver high or low shear stress, laminar flow without pulsatility or stretch. Disturbed flow conditions are generated using setups that have step heights22 or using cone-in-plate viscometers.23 While these geometries may be different from those seen in the body, the boundary layer separation and recirculation have similar profiles and shear stress values to locations seen in vivo. Furthermore, there are very few examples of systems that can accomplish concomitant stimulation with pressure, flow, stretch, and shear stress.24 Each of these systems mimics two or more aspects of
vessel branches and bends including several locations in the aortic arch, ascending and descending aorta, and coronary and carotid arteries.12 At these locations, the local flow behavior is characterized as “disturbed” and is associated with low shear stress recirculation, oscillation, or lateral flow (average: 10 dyn/cm2; maximum:
