VPH-06068; No of Pages 6 Vascular Pharmacology xxx (2014) xxx–xxx
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Flow-induced regulation of brain endothelial cells in vitro XiaoOu Mao a, Lin Xie a, Rose B. Greenberg a, Jack B. Greenberg a, Botao Peng a, Isabelle Mieling a, Kunlin Jin a,b, David A. Greenberg a,⁎ a b
Buck Institute for Research on Aging, Novato, CA, United States Department of Pharmacology and Neuroscience, University of North Texas, Fort Worth, TX, United States
a r t i c l e
i n f o
Article history: Received 9 January 2014 Received in revised form 31 January 2014 Accepted 18 February 2014 Available online xxxx Chemical compounds studied in this article: Mevastatin (PubChem CID: 64715) Atorvastatin (PubChem CID: 60823) Simvastatin (PubChem CID: 54454) Losartan (PubChem CID: 3961) Valsartan (PubChem CID: 60846)
a b s t r a c t Endothelial cell (EC) function and susceptibility to vascular disease are regulated by flow; this relationship has been modeled in systemic, but not cerebrovascular, EC culture. We studied the effects of unidirectional flow of medium, produced by orbital rotation of cultures, on morphology and protein expression in bEnd.3 mouse brain ECs. Flow altered the expression of key transcription factors and gasotransmitter-synthesizing enzymes, and increased NO production. Statins and angiotensin receptor blockers reproduced the effect of flow on endothelial nitric oxide synthase expression. Thus, flow modified brain EC properties and function in vitro, with similarities and possible differences compared to previous studies on systemic ECs. Thus, the effect of flow on brain ECs can be modeled in vitro and may assist the investigation of mechanisms of cerebrovascular disease. © 2014 Elsevier Inc. All rights reserved.
Keywords: Endothelial cell Flow Nitric oxide synthase Statin Angiotensin receptor blocker
1. Introduction Endothelial cell (EC) dysfunction, characterized by impaired vasoregulatory, antithrombotic, anti-inflammatory, and antiproliferative activity, is an early feature of vasodegenerative disease due to atherosclerosis, hypertension and diabetes [1]. A clinical correlate is defective endothelium-dependent, nitric oxide (NO)-mediated vasodilation [2], which is also observed in animal models of vascular disease [3], and appears to result from reduced generation of NO by endothelial NO synthase (eNOS) [4]. eNOS-mediated NO production is likewise impaired at sites of predilection for atherosclerosis [5], which include arterial branch points and curvatures associated with diminished or
Abbreviations: EC, endothelial cell; eNOS, endothelial NO synthase; DiI-Ac-LDL, 1,1′dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate-labeled acetylated lowdensity lipoprotein; HO1, heme oxygenase 1; iNOS, inducible NO synthase; nNOS, neuronal NO synthase; NFκB, nuclear factor κB; CBS, cystathionine-β-synthase; CSE, cystathionine–γ-lyase; HO2, heme oxygenase 2; KLF2, Krüppel-like factor 2; DAPI, 4′,6diamidino-2-phenylindole; vWF, von Willebrand factor; DAF-DA, 4,5-diaminofluorescein diacetate; HUVEC, human umbilical vein endothelial cell. ⁎ Corresponding author at: 8001 Redwood Boulevard, Novato, CA 94945, United States. Tel.: +1 415 209 2087; fax: +1 415 209 2030. E-mail address: [email protected] (D.A. Greenberg).
disturbed blood flow [6], and can be restored by drugs used to treat vascular disease [7]. Thus, flow-sensitive endothelial eNOS activity provides a benchmark for investigating the pathophysiology and pharmacotherapy of vascular disease. Effects of flow on EC function have been modeled in vitro using cultures maintained under various flow conditions. Flow of culture medium over EC monolayers can be induced using a cone-and-plate or parallel-plate apparatus or by growing cells on a mechanical platform shaker [8]. Cultures can thereby be exposed to unidirectional, laminar flow and associated shear stress [9], under which conditions they exhibit features of normal EC function, including alignment in the direction of flow [10], transcription factor induction [11], gene expression [12], and eNOS activation [13], that are absent in stationary EC cultures. Most studies of the interaction between flow and EC function in vitro have used venous or aortic ECs, but other sites, such as the cerebral circulation, are also important targets of disease. In addition, the cerebral circulation may have unique features that influence EC function and disease pathophysiology or treatment [14–16]. To examine these issues and characterize brain EC responses to flow and drugs in vitro, we investigated the effects of stationary versus orbitally rotating culture conditions and selected cardiovascular drugs on the expression of eNOS and other key proteins in the bEnd.3 mouse brain EC line [17].
http://dx.doi.org/10.1016/j.vph.2014.02.003 1537-1891/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Mao X, et al, Flow-induced regulation of brain endothelial cells in vitro, Vascul Pharmacol (2014), http://dx.doi.org/ 10.1016/j.vph.2014.02.003
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2. Material and methods 2.1. Cell culture bEnd.3 mouse brain ECs (derived by transformation with polyoma middle T antigen; #CRL-2299) were purchased from ATCC (Manassas, VA). Cells were cultured in 100-mm uncoated plastic dishes in humidified 95% air/5% CO2 at 37 °C and DMEM containing 10% FBS and 1 × penicillin/streptomycin, with medium changed 2–3 times per week. Near-confluent cultures were split 1:4–6 and given 4 h for attachment before further manipulation. 2.2. DiI-labeled acetylated low-density lipoprotein uptake DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate)-labeled acetylated low-density lipoprotein (DiI-Ac-LDL; Kalen Biomedical, Montgomery Village, MD) was added at 20–100 μg/ml and cultures were incubated for 2 h at 37 °C [18]. Medium was removed, cultures washed with phosphate-buffered saline, and DiI detected by fluorescence microscopy with excitation at 514 nm and emission at 565 nm using a Gemini XPS Fluorescence Microplate Reader (Molecular Devices, Sunnyvale, CA). 2.3. Flow experiments Cultures on 100-mm plates were placed on an orbital shaker (Barnstead Lab-Line 1314R; VWR International, Radnor, PA) in an incubator containing humidified 95% air/5% CO2 at 37 °C. For stationary cultures, the orbital shaker was not turned on. For orbitally shaken cultures used to model unidirectional flow, the shaker was set at 80 rpm. In some experiments a reciprocal shaker (ADV 3750 Recipro Shaker; VWR, Radnor, PA; setting 100) was used to model bidirectional flow. Cultures were maintained under these conditions for 7 days, with 1–2 medium changes, before being assayed.
were HRP-conjugated donkey anti-rabbit and sheep anti-mouse (GE Healthcare, Pittsburgh, PA) and donkey anti-goat (Santa Cruz) IgG (1:10,000–15,000). 2.6. Immunocytochemistry Cells were cultured in 4- or 24-well plates containing 500 μl/well of medium and fixed by adding 500 μl/well of 4% paraformaldehyde (PFA) for 15 min at room temperature. Supernatant was aspirated and 500 μl/well of PFA was added for 15 min at room temperature. Cultures were washed 3 times with 1 × PBS, and blocking buffer (2% horse serum, 1% BSA and 0.1% Triton 100 in 1 × PBS) was added for 1 h at room temperature. Cultures were incubated with primary antibody at 4 °C overnight, washed 3 times with 1 × PBS at room temperature, incubated with secondary antibody for 1 h at room temperature, and washed 3 times with 1 × PBS. 4′,6-Diamidino-2-phenylindole (DAPI) was added to stain nuclei. Primary antibodies were rabbit polyclonal anti-von Willebrand factor (vWF; 1:500) (Abcam) and rabbit polyclonal anti-F-actin (1:500) (Millipore). Secondary antibody was donkey anti-rabbit IgG (1:400) (Millipore). 2.7. NOS activity assay NOS activity was measured using a commercial kit (Sigma #FCANOS1). Cells grown on 100-mm dishes were detached with 0.25% trypsin, centrifuged at 1000 rpm for 5 min and resuspended in 5 ml of medium. Resuspended cells (105/200 μl/well) were added to black, clear-bottomed, 96-well culture plates and maintained in humidified 95% air/5% CO2 at 37 °C for 4 h. Medium was removed and replaced with 190 μl of reaction buffer (Sigma #R2525), 10 μl of arginine substrate solution, and 0.1 μl of 4,5-diaminofluorescein diacetate (DAF-2 DA) solution. Cells were incubated for 2 h at room temperature in the dark, and fluorescence was measured with excitation at 470 nm and emission at 510 nm, using a Gemini XPS Fluorescence Microplate Reader (Molecular Devices). Controls included omitting cells, substrate, or DAF-2 DA.
2.4. Drug treatment 2.8. Statistics Mevastatin, atorvastatin, simvastatin, losartan, and valsartan were from Sigma (St. Louis, MO). Stationary cultures were treated as described above, except drugs were added at the onset of the 7-day culture period and with each medium change.
Each experiment was repeated at least 3 times with different platings of cells. Statistical significance was determined by t-tests with a threshold of p b 0.05.
2.5. Western blotting
3. Results
Protein (30 μg) was run on 4–12% SDS-polyacrylamide gels at 200 V for 35–40 min and transferred to polyvinylidene fluoride membranes at 75 V for 90–120 min at room temperature. Membranes were incubated with 5% non-fat milk in 1 × PBS-Tween 20 (PBST) for 45 min at room temperature, then with primary antibody in 5% non-fat milk at 4 °C overnight. Membranes were washed 3 times for 10 min each with 1 × PBST, and secondary antibody in 5% non-fat milk was added for 1 h at room temperature. Membranes were washed again, treated with Western Lightning Ultra (PerkinElmer, Waltham, MA) for 2 min, and blotted dry. Protein was measured by densitometry with Quantity One (Biorad, Hercules, CA), with mouse monoclonal anti-β-actin (1:10,000; Sigma) used to detect differences in loading. Primary antibodies were rabbit polyclonal anti-heme oxygenase 1 (HO1; 1:2000) and anti-inducible NOS (iNOS; 1:200) (Abcam, Cambridge, MA); rabbit polyclonal anti-neuronal NOS (nNOS; 1:1000) and anti-nuclear factor κB (NFκB; 1:1000) (Cell Signaling, Danvers, MA); rabbit polyclonal anti-eNOS (1:2000) and anti-Ser1177phospho-eNOS (Ser1177P-eNOS; 1:1000) (Millipore, Billerica, MA); mouse monoclonal anti-cystathionine-β-synthase (CBS; 1:1000), anticystathionine-γ-lyase (CSE; 1:500), and anti-heme oxygenase 2 (HO2; 1:200) (Santa Cruz Biotechnology, Santa Cruz, CA); and rabbit polyclonal anti-Krüppel-like factor 2 (KLF2; 1:400) (Sigma). Secondary antibodies
Stationary bEnd.3 cultures showed phenotypic attributes of ECs, including expression of von Willebrand factor (vWF) and uptake of DiI-Ac-LDL (Fig. 1A). Under these conditions, cells grew without directional alignment (Fig. 1B). In contrast, cells grown for 7 days on an orbital shaker platform, which produced unidirectional (circular) flow of culture medium, were aligned in the direction of rotation at the periphery (but not the center) of culture dishes (Fig. 1B), consistent with similarly rotated bovine aortic ECs [8] and the relationship between shear stress and orbital radius [8,9]. Cells grown on a reciprocal shaker, which produced direction-changing flow, remained unaligned (Fig. 2A), as observed for stationary cultures. Cell alignment in orbitally rotated cultures was also evident in cultures immunostained for F-actin (Fig. 1C), which reorganizes in response to shear stress to promote EC elongation and alignment [19]. The beneficial effect of flow-induced shear stress on EC function is mediated partly by altered activity of transcription factors [20], including induction of KLF2 [11] and suppression of NFκB [21]. Accordingly, we compared expression of these proteins in stationary and orbitally rotated bEnd.3 cultures. Rotation increased KLF2 expression to 341 ± 113% (p b 0.05, n = 6) and reduced the ratio of nuclear to cytoplasmic NFκB to 17 ± 7% (p b 0.05, n = 4) of values in stationary cultures (Fig. 3).
Please cite this article as: Mao X, et al, Flow-induced regulation of brain endothelial cells in vitro, Vascul Pharmacol (2014), http://dx.doi.org/ 10.1016/j.vph.2014.02.003
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Fig. 1. Endothelial properties and effect of unidirectional flow on bEnd.3 cells. (A) Cells express von Willebrand factor (vWF, green) and take up DiI-labeled acetylated LDL (DiI-Ac-LDL, red). (B) Cells cultured under stationary conditions (left) showed no predominant alignment, whereas cells at the periphery of orbitally rotated cultures (right) aligned in the direction of flow. (C) Alignment of cells in rotated cultures was also demonstrable by staining for F-actin (green). DAPI (blue) was used to stain nuclei in (A) and (C).
eNOS-derived NO has been implicated in the vasodilatory, antithrombotic, anti-inflammatory, and antiproliferative effects of ECs [22] and defective expression or post-translational activation of eNOS is thought to contribute to EC dysfunction in vascular diseases [23]. Orbital rotation increased eNOS expression to 253 ± 60% (p b 0.05, n = 3) and phosphoactivated (Ser1177P)-eNOS to 200 ± 32% (p b 0.05, n = 3) of values in stationary cultures (Fig. 4A), which is comparable to the shear-induced increase in eNOS mRNA in human umbilical vein EC (HUVEC) [11] and abdominal aorta EC [24] cultures. In contrast, reciprocal shaking had no effect on eNOS levels (Fig. 2B). Orbital rotation also reduced expression of iNOS, to 25 ± 8% of that in stationary cultures (p b 0.005, n = 5), while nNOS was undetectable. The rotationinduced increase in Ser1177P-eNOS levels was fully reversible (Fig. 4B). Finally, orbital rotation of cultures approximately doubled NO production (Fig. 4C). Two other gasotransmitters—CO and H2S—also have important roles in cerebrovascular function [25] and are atheroprotective in animal models [26,27]. Therefore, we measured expression of the CO- and H2S-synthesizing enzymes in stationary and orbitally rotated bEnd.3
cultures. Expression of all four enzymes increased in rotated cultures: HO1 to 184 ± 14% (p b 0.05, n = 3), HO2 to 292 ± 47% (p b 0.005, n = 3), CBS to 128 ± 3% (p b 0.05, n = 3), and CSE to 176 ± 12% (p b 0.01, n = 5) of values in stationary cultures (Fig. 5). To evaluate if bEnd.3 cells are regulated by cardiovascular drugs as well as flow, we examined whether such drugs increased eNOS expression in stationary cultures. The benefit of statins in atherosclerosis, originally attributed to reduction of circulating low-density lipoprotein cholesterol levels, also involves direct effects on ECs, including upregulation of eNOS [28]. As shown in Fig. 6A, several statins stimulated eNOS expression: mevastatin (0.1 μM) to 216 ± 28% (p b 0.05, n = 3), atorvastatin (1 μM) to 369 ± 122% (p b 0.05, n = 3), and simvastatin (0.1 μM) to 386 ± 68% (p b 0.01, n = 3) of control. Some antihypertensive drugs also appear to protect against cardiovascular disease not only by lowering blood pressure, but also by direct effects on ECs [29]. These include angiotensin AT1 receptor blockers [30], which likewise enhanced eNOS expression in our cultures: losartan (1 μM) to 253 ± 22% (p b 0.01, n = 4) and valsartan (1 μM) to 382 ± 121% (p b 0.05, n = 4) of control (Fig. 6B).
Please cite this article as: Mao X, et al, Flow-induced regulation of brain endothelial cells in vitro, Vascul Pharmacol (2014), http://dx.doi.org/ 10.1016/j.vph.2014.02.003
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Fig. 2. Effects of direction-changing flow on bEnd.3 cells in culture. (A) Cells cultured with direction-changing flow failed to align. (B) Compared to cells cultured under stationary conditions (−, left), cells cultured with direction-changing flow (↔, right) showed no difference in eNOS expression. Blot shown is representative of 5 experiments.
4. Discussion We report that unidirectional medium flow confers upon cultured bEnd.3 mouse brain ECs properties similar to those conferred by blood flow in vivo, including alignment, altered expression or distribution of transcription factors, and changes in expression, phosphoactivation, or activity of gasotransmitter-synthesizing enzymes. eNOS activity and associated NO production promote normal endothelial function through vasoregulatory, antithrombotic, anti-inflammatory, and antiproliferative effects [22]. Certain cardiovascular drugs also promote normal endothelial function and two such drug classes—statins and angiotensin receptor blockers—increased eNOS expression in our cultures as well. bEnd.3 cells were derived by retroviral infection of primary mouse brain ECs with polyoma virus middle T oncogene [17]. Our cells exhibited EC properties including vWF expression and DiI-Ac-LDL uptake. In addition, orbital shaking of bEnd.3 cultures produced alignment of cell
Fig. 3. Western analysis of KLF2 expression and of NFκB expression in cytosol (NFκB-c) and nuclei (NFκB-n) in bEnd.3 cells cultured under stationary (−, left) and orbitally rotated (+, right) conditions. Blots shown are representative of 4–6 experiments; densitometric quantification is given in Results.
Fig. 4. Effects of unidirectional flow on NOS expression and NO production in bEnd.3 cultures. (A) Western analysis of eNOS, Ser1177P-eNOS (P-eNOS), iNOS and nNOS expression in bEnd.3 cells cultured under stationary (−, left) and orbitally rotated (+, right) conditions. Blots are representative of 3–6 experiments; densitometric quantification is given in Results. (B) Reversal of the effect of unidirectional flow on P-eNOS expression after return of cultures to stationary conditions for 3 days. Blots shown are representative of 3 experiments; densitometric quantification is given in Results. (C) NO production, detected by DAF-2, in bEnd.3 cells cultured under stationary (circles) and orbitally rotated (squares) conditions. Data are mean ± SEM from 3 cultures per point. *p b 0.05 compared to stationary cultures.
Fig. 5. Effects of unidirectional flow on expression of gasotransmitter-synthesizing enzymes in bEnd.3 cultures. Western analysis of (A) CO-synthesizing enzymes HO1 and HO2 and (B) H2S-synthesizing enzymes CBS and CSE in bEnd.3 cells cultured under stationary (−, left) and orbitally rotated (+, right) conditions. Blots are representative of 3–5 experiments; densitometric quantification is given in Results.
Please cite this article as: Mao X, et al, Flow-induced regulation of brain endothelial cells in vitro, Vascul Pharmacol (2014), http://dx.doi.org/ 10.1016/j.vph.2014.02.003
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flow has been reported in HUVEC [44] and human aortic EC [45] cultures, and we found this in bEnd.3 cells as well. We also observed increased HO2 expression in rotated cultures, which appears to be a new finding. H2S, another EC gasotransmitter [46], is synthesized by CBS and CSE. CBS expression, which has not been associated with shear flow to our knowledge, was increased in our rotated cultures. CSE expression was also increased, which contrasts with a report that laminar shear had no effect, whereas oscillatory shear decreased CSE levels in bovine aortic ECs [47]. Of interest, genetic deletion of HO1 [48], CBS [49], or CSE [50] promotes atherosclerosis in mice. Pharmacologic reversal of endothelial dysfunction and reduced eNOS activity has therapeutic benefit, as shown for statins [28] and inhibitors of the renin–angiotensin axis [29]. We found that statins increased eNOS expression in bEnd.3 cultures, as reported for bovine aortic ECs [51], and that angiotensin receptor blockers had a similar effect, as noted previously in rat aortic rings [52]. 5. Conclusions Orbital rotation of bEnd.3 brain EC cultures, which produces unilateral flow over the cell surface, confers morphologic and biochemical features of normal EC function. This includes induction of enzymes involved in synthesis of three gasotransmitters—NO, CO and H2S—with key roles in EC physiology and vascular disease. At least some of these effects can be replicated by cardiovascular drugs, which likely contributes to their therapeutic effects [28,53]. Our results highlight similarities and possible differences—e.g., iNOS suppression and CSE induction by flow—between cerebrovascular ECs and ECs from other vascular beds, and may therefore be relevant to regional differences in the epidemiology, mechanisms, and treatment response [15,54] of vascular disease. Acknowledgment This study was supported by the Buck Institute for Research on Aging. Fig. 6. Effects of statins and angiotensin receptor blockers (ARBs) on eNOS expression in bEnd.3 cells cultured under stationary conditions. Blots are representative of 3–4 experiments; drug concentrations and densitometric quantification of blots are given in Results.
growth and actin filaments, consistent with shear-induced cytoskeletal reorganization [19]. If orbital rotation of EC cultures is a useful model of blood flow over ECs in vivo, it should recapitulate features of the in vivo state. Previous studies have modeled the effect of orbital rotation on wall shear stress [9] and demonstrated the influence of shear on cell alignment, protein expression, intracellular signaling, or granulocyte adhesion of HUVECs [8,31–34] and rat epididymal ECs [35]. Flow-induced shear is thought to induce vasoprotection partly by activation of KLF2 and reduced activation of NFκB, with consequent effects on their transcriptional targets [20]. Changes in expression or disposition of these factors have been shown in HUVECs [11,36], and also occurred in our cultures. eNOS-derived NO is pivotal in conferring a physiological, vasoprotective EC phenotype. Thus, eNOS-knockout mice exhibit hypertension, a thrombogenic state, vascular inflammation, and vascular smooth muscle proliferation [37]. Orbital rotation of bEnd.3 cultures increased levels of eNOS and Ser1177-phospho-eNOS, which are upregulated by flow-related shear [38,39]; Ser1177-phosphorylation of eNOS [40] was reversible. In contrast, the proinflammatory isoform iNOS, which is induced in ECs of atherosclerotic arteries [41], was downregulated by rotation. This differs from the shear-induced increase in iNOS reported in rat lung and human dermal ECs [42]. The net effect of altered NOS expression in our bEnd.3 cells was increased NO production, consistent with the effect of flow in HUVECs [13]. CO, produced by HO1 and HO2, contributes to normal endothelial function and is vasoprotective [43]. Induction of HO1 expression by
References [1] Endemann DH, Schiffrin EL. Endothelial dysfunction. J Am Soc Nephrol 2004;15:1983–92. [2] Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 1992;340:1111–5. [3] Yang R, Powell-Braxton L, Ogaoawara AK, Dybdal N, Bunting S, Ohneda O, et al. Hypertension and endothelial dysfunction in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 1999;19:2762–8. [4] Huang PL. Unraveling the links between diabetes, obesity, and cardiovascular disease. Circ Res 2005;96:1129–31. [5] Oemar BS, Tschudi MR, Godoy N, Brovkovich V, Malinski T, Luscher TF. Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis. Circulation 1998;97:2494–8. [6] Ku DN, Giddens DP, Zarins CK, Glagov S. Pulsatile flow and atherosclerosis in the human carotid bifurcation. Positive correlation between plaque location and low oscillating shear stress. Arteriosclerosis 1985;5:293–302. [7] Endres M, Laufs U, Huang Z, Nakamura T, Huang P, Moskowitz MA, et al. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci U S A 1998;95:8880–5. [8] Dardik A, Chen L, Frattini J, Asada H, Aziz F, Kudo FA, et al. Differential effects of orbital and laminar shear stress on endothelial cells. J Vasc Surg 2005;41:869–80. [9] Thomas JM, Chakraborty A, Sharp MK, Berson RE. Spatial and temporal resolution of shear in an orbiting petri dish. Biotechnol Prog 2011;27:460–5. [10] Tzima E, Irani-Tehrani M, Kiosses WB, Dejana E, Schultz DA, Engelhardt B, et al. A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 2005;437:426–31. [11] Dekker RJ, van Soest S, Fontijn RD, Salamanca S, de Groot PG, VanBavel E, et al. Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Kruppel-like factor (KLF2). Blood 2002;100:1689–98. [12] Brooks AR, Lelkes PI, Rubanyi GM. Gene expression profiling of human aortic endothelial cells exposed to disturbed flow and steady laminar flow. Physiol Genomics 2002;9:27–41. [13] Noris M, Morigi M, Donadelli R, Aiello S, Foppolo M, Todeschini M, et al. Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions. Circ Res 1995;76:536–43. [14] Guo S, Zhou Y, Xing C, Lok J, Som AT, Ning M, et al. The vasculome of the mouse brain. PLoS One 2012;7:e52665.
Please cite this article as: Mao X, et al, Flow-induced regulation of brain endothelial cells in vitro, Vascul Pharmacol (2014), http://dx.doi.org/ 10.1016/j.vph.2014.02.003
6
X. Mao et al. / Vascular Pharmacology xxx (2014) xxx–xxx
[15] Jashari F, Ibrahimi P, Nicoll R, Bajraktari G, Wester P, Henein MY. Coronary and carotid atherosclerosis: similarities and differences. Atherosclerosis 2013;227:193–200. [16] Steelman SM, Humphrey JD. Differential remodeling responses of cerebral and skeletal muscle arterioles in a novel organ culture system. Med Biol Eng Comput 2011;49:1015–23. [17] Montesano R, Pepper MS, Mohle-Steinlein U, Risau W, Wagner EF, Orci L. Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene. Cell 1990;62:435–45. [18] Voyta JC, Via DP, Butterfield CE, Zetter BR. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 1984;99:2034–40. [19] McCue S, Noria S, Langille BL. Shear-induced reorganization of endothelial cell cytoskeleton and adhesion complexes. Trends Cardiovasc Med 2004;14:143–51. [20] Boon RA, Horrevoets AJ. Key transcriptional regulators of the vasoprotective effects of shear stress. Hamostaseologie 2009;29:39–43. [21] Nagel T, Resnick N, Dewey Jr CF, Gimbrone Jr MA. Vascular endothelial cells respond to spatial gradients in fluid shear stress by enhanced activation of transcription factors. Arterioscler Thromb Vasc Biol 1999;19:1825–34. [22] Tousoulis D, Kampoli AM, Tentolouris C, Papageorgiou N, Stefanadis C. The role of nitric oxide on endothelial function. Curr Vasc Pharmacol 2012;10:4–18. [23] Chatterjee A, Black SM, Catravas JD. Endothelial nitric oxide (NO) and its pathophysiologic regulation. Vascul Pharmacol 2008;49:134–40. [24] Rossi J, Jonak P, Rouleau L, Danielczak L, Tardif JC, Leask RL. Differential response of endothelial cells to simvastatin when conditioned with steady, non-reversing pulsatile or oscillating shear stress. Ann Biomed Eng 2011;39:402–13. [25] Leffler CW, Parfenova H, Jaggar JH, Wang R. Carbon monoxide and hydrogen sulfide: gaseous messengers in cerebrovascular circulation. J Appl Physiol 2006;100: 1065–76. [26] Otterbein LE, Zuckerbraun BS, Haga M, Liu F, Song R, Usheva A, et al. Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nat Med 2003;9:183–90. [27] Wang Y, Zhao X, Jin H, Wei H, Li W, Bu D, et al. Role of hydrogen sulfide in the development of atherosclerotic lesions in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 2009;29:173–9. [28] Balakumar P, Kathuria S, Taneja G, Kalra S, Mahadevan N. Is targeting eNOS a key mechanistic insight of cardiovascular defensive potentials of statins? J Mol Cell Cardiol 2012;52:83–92. [29] Patarroyo Aponte MM, Francis GS. Effect of Angiotensin-converting enzyme inhibitors and Angiotensin receptor antagonists in atherosclerosis prevention. Curr Cardiol Rep 2012;14:433–42. [30] Michel MC, Foster C, Brunner HR, Liu L. A systematic comparison of the properties of clinically used angiotensin II type 1 receptor antagonists. Pharmacol Rev 2013;65:809–48. [31] Kraiss LW, Weyrich AS, Alto NM, Dixon DA, Ennis TM, Modur V, et al. Fluid flow activates a regulator of translation, p70/p85 S6 kinase, in human endothelial cells. Am J Physiol Heart Circ Physiol 2000;278:H1537–44. [32] Kraiss LW, Alto NM, Dixon DA, McIntyre TM, Weyrich AS, Zimmerman GA. Fluid flow regulates E-selectin protein levels in human endothelial cells by inhibiting translation. J Vasc Surg 2003;37:161–8. [33] Ley K, Lundgren E, Berger E, Arfors KE. Shear-dependent inhibition of granulocyte adhesion to cultured endothelium by dextran sulfate. Blood 1989;73:1324–30. [34] Pearce MJ, McIntyre TM, Prescott SM, Zimmerman GA, Whatley RE. Shear stress activates cytosolic phospholipase A2 (cPLA2) and MAP kinase in human endothelial cells. Biochem Biophys Res Commun 1996;218:500–4.
[35] Yun S, Dardik A, Haga M, Yamashita A, Yamaguchi S, Koh Y, et al. Transcription factor Sp1 phosphorylation induced by shear stress inhibits membrane type 1-matrix metalloproteinase expression in endothelium. J Biol Chem 2002;277:34808–14. [36] Parmar KM, Larman HB, Dai G, Zhang Y, Wang ET, Moorthy SN, et al. Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2. J Clin Invest 2006;116:49–58. [37] Atochin DN, Huang PL. Endothelial nitric oxide synthase transgenic models of endothelial dysfunction. Pflugers Arch 2010;460:965–74. [38] Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 1999;399:601–5. [39] Uematsu M, Ohara Y, Navas JP, Nishida K, Murphy TJ, Alexander RW, et al. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress. Am J Physiol 1995;269:C1371–8. [40] Moncada S, Higgs EA. Nitric oxide and the vascular endothelium. Handb Exp Pharmacol 2006:213254. [41] Wilcox JN, Subramanian RR, Sundell CL, Tracey WR, Pollock JS, Harrison DG, et al. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol 1997;17:2479–88. [42] Ozawa N, Shichiri M, Iwashina M, Fukai N, Yoshimoto T, Hirata Y. Laminar shear stress up-regulates inducible nitric oxide synthase in the endothelium. Hypertens Res 2004;27:93–9. [43] Leffler CW, Parfenova H, Jaggar JH. Carbon monoxide as an endogenous vascular modulator. Am J Physiol Heart Circ Physiol 2011;301:H1-11. [44] De Keulenaer GW, Chappell DC, Ishizaka N, Nerem RM, Alexander RW, Griendling KK. Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: role of a superoxide-producing NADH oxidase. Circ Res 1998;82:1094–101. [45] Chen XL, Varner SE, Rao AS, Grey JY, Thomas S, Cook CK, et al. Laminar flow induction of antioxidant response element-mediated genes in endothelial cells. A novel anti-inflammatory mechanism. J Biol Chem 2003;278:703–11. [46] Vandiver M, Snyder SH. Hydrogen sulfide: a gasotransmitter of clinical relevance. J Mol Med (Berl) 2012;90:255–63. [47] Go YM, Lee HR, Park H. H2S inhibits oscillatory shear stress-induced monocyte binding to endothelial cells via nitric oxide production. Mol Cells 2012;34:449–55. [48] Yet SF, Layne MD, Liu X, Chen YH, Ith B, Sibinga NE, et al. Absence of heme oxygenase-1 exacerbates atherosclerotic lesion formation and vascular remodeling. FASEB J 2003;17:1759–61. [49] Zhang D, Jiang X, Fang P, Yan Y, Song J, Gupta S, et al. Hyperhomocysteinemia promotes inflammatory monocyte generation and accelerates atherosclerosis in transgenic cystathionine beta-synthase-deficient mice. Circulation 2009;120:1893–902. [50] Mani S, Li H, Untereiner A, Wu L, Yang G, Austin RC, et al. Decreased endogenous production of hydrogen sulfide accelerates atherosclerosis. Circulation 2013;127:2523–34. [51] Laufs U, Gertz K, Dirnagl U, Bohm M, Nickenig G, Endres M. Rosuvastatin, a new HMG-CoA reductase inhibitor, upregulates endothelial nitric oxide synthase and protects from ischemic stroke in mice. Brain Res 2002;942:23–30. [52] Wenzel P, Daiber A, Oelze M, Brandt M, Closs E, Xu J, et al. Mechanisms underlying recoupling of eNOS by HMG-CoA reductase inhibition in a rat model of streptozotocin-induced diabetes mellitus. Atherosclerosis 2008;198:65–76. [53] Schlimmer N, Kratz M, Bohm M, Baumhakel M. Telmisartan, ramipril and their combination improve endothelial function in different tissues in a murine model of cholesterol-induced atherosclerosis. Br J Pharmacol 2011;163:804–14. [54] Frohlich ED. Local hemodynamic changes in hypertension: insights for therapeutic preservation of target organs. Hypertension 2001;38:1388–94.
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Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph
Flow-induced regulation of brain endothelial cells in vitro XiaoOu Mao a, Lin Xie a, Rose B. Greenberg a, Jack B. Greenberg a, Botao Peng a, Isabelle Mieling a, Kunlin Jin a,b, David A. Greenberg a,⁎ a b
Buck Institute for Research on Aging, Novato, CA, United States Department of Pharmacology and Neuroscience, University of North Texas, Fort Worth, TX, United States
a r t i c l e
i n f o
Article history: Received 9 January 2014 Received in revised form 31 January 2014 Accepted 18 February 2014 Available online xxxx Chemical compounds studied in this article: Mevastatin (PubChem CID: 64715) Atorvastatin (PubChem CID: 60823) Simvastatin (PubChem CID: 54454) Losartan (PubChem CID: 3961) Valsartan (PubChem CID: 60846)
a b s t r a c t Endothelial cell (EC) function and susceptibility to vascular disease are regulated by flow; this relationship has been modeled in systemic, but not cerebrovascular, EC culture. We studied the effects of unidirectional flow of medium, produced by orbital rotation of cultures, on morphology and protein expression in bEnd.3 mouse brain ECs. Flow altered the expression of key transcription factors and gasotransmitter-synthesizing enzymes, and increased NO production. Statins and angiotensin receptor blockers reproduced the effect of flow on endothelial nitric oxide synthase expression. Thus, flow modified brain EC properties and function in vitro, with similarities and possible differences compared to previous studies on systemic ECs. Thus, the effect of flow on brain ECs can be modeled in vitro and may assist the investigation of mechanisms of cerebrovascular disease. © 2014 Elsevier Inc. All rights reserved.
Keywords: Endothelial cell Flow Nitric oxide synthase Statin Angiotensin receptor blocker
1. Introduction Endothelial cell (EC) dysfunction, characterized by impaired vasoregulatory, antithrombotic, anti-inflammatory, and antiproliferative activity, is an early feature of vasodegenerative disease due to atherosclerosis, hypertension and diabetes [1]. A clinical correlate is defective endothelium-dependent, nitric oxide (NO)-mediated vasodilation [2], which is also observed in animal models of vascular disease [3], and appears to result from reduced generation of NO by endothelial NO synthase (eNOS) [4]. eNOS-mediated NO production is likewise impaired at sites of predilection for atherosclerosis [5], which include arterial branch points and curvatures associated with diminished or
Abbreviations: EC, endothelial cell; eNOS, endothelial NO synthase; DiI-Ac-LDL, 1,1′dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate-labeled acetylated lowdensity lipoprotein; HO1, heme oxygenase 1; iNOS, inducible NO synthase; nNOS, neuronal NO synthase; NFκB, nuclear factor κB; CBS, cystathionine-β-synthase; CSE, cystathionine–γ-lyase; HO2, heme oxygenase 2; KLF2, Krüppel-like factor 2; DAPI, 4′,6diamidino-2-phenylindole; vWF, von Willebrand factor; DAF-DA, 4,5-diaminofluorescein diacetate; HUVEC, human umbilical vein endothelial cell. ⁎ Corresponding author at: 8001 Redwood Boulevard, Novato, CA 94945, United States. Tel.: +1 415 209 2087; fax: +1 415 209 2030. E-mail address: [email protected] (D.A. Greenberg).
disturbed blood flow [6], and can be restored by drugs used to treat vascular disease [7]. Thus, flow-sensitive endothelial eNOS activity provides a benchmark for investigating the pathophysiology and pharmacotherapy of vascular disease. Effects of flow on EC function have been modeled in vitro using cultures maintained under various flow conditions. Flow of culture medium over EC monolayers can be induced using a cone-and-plate or parallel-plate apparatus or by growing cells on a mechanical platform shaker [8]. Cultures can thereby be exposed to unidirectional, laminar flow and associated shear stress [9], under which conditions they exhibit features of normal EC function, including alignment in the direction of flow [10], transcription factor induction [11], gene expression [12], and eNOS activation [13], that are absent in stationary EC cultures. Most studies of the interaction between flow and EC function in vitro have used venous or aortic ECs, but other sites, such as the cerebral circulation, are also important targets of disease. In addition, the cerebral circulation may have unique features that influence EC function and disease pathophysiology or treatment [14–16]. To examine these issues and characterize brain EC responses to flow and drugs in vitro, we investigated the effects of stationary versus orbitally rotating culture conditions and selected cardiovascular drugs on the expression of eNOS and other key proteins in the bEnd.3 mouse brain EC line [17].
http://dx.doi.org/10.1016/j.vph.2014.02.003 1537-1891/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Mao X, et al, Flow-induced regulation of brain endothelial cells in vitro, Vascul Pharmacol (2014), http://dx.doi.org/ 10.1016/j.vph.2014.02.003
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2. Material and methods 2.1. Cell culture bEnd.3 mouse brain ECs (derived by transformation with polyoma middle T antigen; #CRL-2299) were purchased from ATCC (Manassas, VA). Cells were cultured in 100-mm uncoated plastic dishes in humidified 95% air/5% CO2 at 37 °C and DMEM containing 10% FBS and 1 × penicillin/streptomycin, with medium changed 2–3 times per week. Near-confluent cultures were split 1:4–6 and given 4 h for attachment before further manipulation. 2.2. DiI-labeled acetylated low-density lipoprotein uptake DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate)-labeled acetylated low-density lipoprotein (DiI-Ac-LDL; Kalen Biomedical, Montgomery Village, MD) was added at 20–100 μg/ml and cultures were incubated for 2 h at 37 °C [18]. Medium was removed, cultures washed with phosphate-buffered saline, and DiI detected by fluorescence microscopy with excitation at 514 nm and emission at 565 nm using a Gemini XPS Fluorescence Microplate Reader (Molecular Devices, Sunnyvale, CA). 2.3. Flow experiments Cultures on 100-mm plates were placed on an orbital shaker (Barnstead Lab-Line 1314R; VWR International, Radnor, PA) in an incubator containing humidified 95% air/5% CO2 at 37 °C. For stationary cultures, the orbital shaker was not turned on. For orbitally shaken cultures used to model unidirectional flow, the shaker was set at 80 rpm. In some experiments a reciprocal shaker (ADV 3750 Recipro Shaker; VWR, Radnor, PA; setting 100) was used to model bidirectional flow. Cultures were maintained under these conditions for 7 days, with 1–2 medium changes, before being assayed.
were HRP-conjugated donkey anti-rabbit and sheep anti-mouse (GE Healthcare, Pittsburgh, PA) and donkey anti-goat (Santa Cruz) IgG (1:10,000–15,000). 2.6. Immunocytochemistry Cells were cultured in 4- or 24-well plates containing 500 μl/well of medium and fixed by adding 500 μl/well of 4% paraformaldehyde (PFA) for 15 min at room temperature. Supernatant was aspirated and 500 μl/well of PFA was added for 15 min at room temperature. Cultures were washed 3 times with 1 × PBS, and blocking buffer (2% horse serum, 1% BSA and 0.1% Triton 100 in 1 × PBS) was added for 1 h at room temperature. Cultures were incubated with primary antibody at 4 °C overnight, washed 3 times with 1 × PBS at room temperature, incubated with secondary antibody for 1 h at room temperature, and washed 3 times with 1 × PBS. 4′,6-Diamidino-2-phenylindole (DAPI) was added to stain nuclei. Primary antibodies were rabbit polyclonal anti-von Willebrand factor (vWF; 1:500) (Abcam) and rabbit polyclonal anti-F-actin (1:500) (Millipore). Secondary antibody was donkey anti-rabbit IgG (1:400) (Millipore). 2.7. NOS activity assay NOS activity was measured using a commercial kit (Sigma #FCANOS1). Cells grown on 100-mm dishes were detached with 0.25% trypsin, centrifuged at 1000 rpm for 5 min and resuspended in 5 ml of medium. Resuspended cells (105/200 μl/well) were added to black, clear-bottomed, 96-well culture plates and maintained in humidified 95% air/5% CO2 at 37 °C for 4 h. Medium was removed and replaced with 190 μl of reaction buffer (Sigma #R2525), 10 μl of arginine substrate solution, and 0.1 μl of 4,5-diaminofluorescein diacetate (DAF-2 DA) solution. Cells were incubated for 2 h at room temperature in the dark, and fluorescence was measured with excitation at 470 nm and emission at 510 nm, using a Gemini XPS Fluorescence Microplate Reader (Molecular Devices). Controls included omitting cells, substrate, or DAF-2 DA.
2.4. Drug treatment 2.8. Statistics Mevastatin, atorvastatin, simvastatin, losartan, and valsartan were from Sigma (St. Louis, MO). Stationary cultures were treated as described above, except drugs were added at the onset of the 7-day culture period and with each medium change.
Each experiment was repeated at least 3 times with different platings of cells. Statistical significance was determined by t-tests with a threshold of p b 0.05.
2.5. Western blotting
3. Results
Protein (30 μg) was run on 4–12% SDS-polyacrylamide gels at 200 V for 35–40 min and transferred to polyvinylidene fluoride membranes at 75 V for 90–120 min at room temperature. Membranes were incubated with 5% non-fat milk in 1 × PBS-Tween 20 (PBST) for 45 min at room temperature, then with primary antibody in 5% non-fat milk at 4 °C overnight. Membranes were washed 3 times for 10 min each with 1 × PBST, and secondary antibody in 5% non-fat milk was added for 1 h at room temperature. Membranes were washed again, treated with Western Lightning Ultra (PerkinElmer, Waltham, MA) for 2 min, and blotted dry. Protein was measured by densitometry with Quantity One (Biorad, Hercules, CA), with mouse monoclonal anti-β-actin (1:10,000; Sigma) used to detect differences in loading. Primary antibodies were rabbit polyclonal anti-heme oxygenase 1 (HO1; 1:2000) and anti-inducible NOS (iNOS; 1:200) (Abcam, Cambridge, MA); rabbit polyclonal anti-neuronal NOS (nNOS; 1:1000) and anti-nuclear factor κB (NFκB; 1:1000) (Cell Signaling, Danvers, MA); rabbit polyclonal anti-eNOS (1:2000) and anti-Ser1177phospho-eNOS (Ser1177P-eNOS; 1:1000) (Millipore, Billerica, MA); mouse monoclonal anti-cystathionine-β-synthase (CBS; 1:1000), anticystathionine-γ-lyase (CSE; 1:500), and anti-heme oxygenase 2 (HO2; 1:200) (Santa Cruz Biotechnology, Santa Cruz, CA); and rabbit polyclonal anti-Krüppel-like factor 2 (KLF2; 1:400) (Sigma). Secondary antibodies
Stationary bEnd.3 cultures showed phenotypic attributes of ECs, including expression of von Willebrand factor (vWF) and uptake of DiI-Ac-LDL (Fig. 1A). Under these conditions, cells grew without directional alignment (Fig. 1B). In contrast, cells grown for 7 days on an orbital shaker platform, which produced unidirectional (circular) flow of culture medium, were aligned in the direction of rotation at the periphery (but not the center) of culture dishes (Fig. 1B), consistent with similarly rotated bovine aortic ECs [8] and the relationship between shear stress and orbital radius [8,9]. Cells grown on a reciprocal shaker, which produced direction-changing flow, remained unaligned (Fig. 2A), as observed for stationary cultures. Cell alignment in orbitally rotated cultures was also evident in cultures immunostained for F-actin (Fig. 1C), which reorganizes in response to shear stress to promote EC elongation and alignment [19]. The beneficial effect of flow-induced shear stress on EC function is mediated partly by altered activity of transcription factors [20], including induction of KLF2 [11] and suppression of NFκB [21]. Accordingly, we compared expression of these proteins in stationary and orbitally rotated bEnd.3 cultures. Rotation increased KLF2 expression to 341 ± 113% (p b 0.05, n = 6) and reduced the ratio of nuclear to cytoplasmic NFκB to 17 ± 7% (p b 0.05, n = 4) of values in stationary cultures (Fig. 3).
Please cite this article as: Mao X, et al, Flow-induced regulation of brain endothelial cells in vitro, Vascul Pharmacol (2014), http://dx.doi.org/ 10.1016/j.vph.2014.02.003
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Fig. 1. Endothelial properties and effect of unidirectional flow on bEnd.3 cells. (A) Cells express von Willebrand factor (vWF, green) and take up DiI-labeled acetylated LDL (DiI-Ac-LDL, red). (B) Cells cultured under stationary conditions (left) showed no predominant alignment, whereas cells at the periphery of orbitally rotated cultures (right) aligned in the direction of flow. (C) Alignment of cells in rotated cultures was also demonstrable by staining for F-actin (green). DAPI (blue) was used to stain nuclei in (A) and (C).
eNOS-derived NO has been implicated in the vasodilatory, antithrombotic, anti-inflammatory, and antiproliferative effects of ECs [22] and defective expression or post-translational activation of eNOS is thought to contribute to EC dysfunction in vascular diseases [23]. Orbital rotation increased eNOS expression to 253 ± 60% (p b 0.05, n = 3) and phosphoactivated (Ser1177P)-eNOS to 200 ± 32% (p b 0.05, n = 3) of values in stationary cultures (Fig. 4A), which is comparable to the shear-induced increase in eNOS mRNA in human umbilical vein EC (HUVEC) [11] and abdominal aorta EC [24] cultures. In contrast, reciprocal shaking had no effect on eNOS levels (Fig. 2B). Orbital rotation also reduced expression of iNOS, to 25 ± 8% of that in stationary cultures (p b 0.005, n = 5), while nNOS was undetectable. The rotationinduced increase in Ser1177P-eNOS levels was fully reversible (Fig. 4B). Finally, orbital rotation of cultures approximately doubled NO production (Fig. 4C). Two other gasotransmitters—CO and H2S—also have important roles in cerebrovascular function [25] and are atheroprotective in animal models [26,27]. Therefore, we measured expression of the CO- and H2S-synthesizing enzymes in stationary and orbitally rotated bEnd.3
cultures. Expression of all four enzymes increased in rotated cultures: HO1 to 184 ± 14% (p b 0.05, n = 3), HO2 to 292 ± 47% (p b 0.005, n = 3), CBS to 128 ± 3% (p b 0.05, n = 3), and CSE to 176 ± 12% (p b 0.01, n = 5) of values in stationary cultures (Fig. 5). To evaluate if bEnd.3 cells are regulated by cardiovascular drugs as well as flow, we examined whether such drugs increased eNOS expression in stationary cultures. The benefit of statins in atherosclerosis, originally attributed to reduction of circulating low-density lipoprotein cholesterol levels, also involves direct effects on ECs, including upregulation of eNOS [28]. As shown in Fig. 6A, several statins stimulated eNOS expression: mevastatin (0.1 μM) to 216 ± 28% (p b 0.05, n = 3), atorvastatin (1 μM) to 369 ± 122% (p b 0.05, n = 3), and simvastatin (0.1 μM) to 386 ± 68% (p b 0.01, n = 3) of control. Some antihypertensive drugs also appear to protect against cardiovascular disease not only by lowering blood pressure, but also by direct effects on ECs [29]. These include angiotensin AT1 receptor blockers [30], which likewise enhanced eNOS expression in our cultures: losartan (1 μM) to 253 ± 22% (p b 0.01, n = 4) and valsartan (1 μM) to 382 ± 121% (p b 0.05, n = 4) of control (Fig. 6B).
Please cite this article as: Mao X, et al, Flow-induced regulation of brain endothelial cells in vitro, Vascul Pharmacol (2014), http://dx.doi.org/ 10.1016/j.vph.2014.02.003
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Fig. 2. Effects of direction-changing flow on bEnd.3 cells in culture. (A) Cells cultured with direction-changing flow failed to align. (B) Compared to cells cultured under stationary conditions (−, left), cells cultured with direction-changing flow (↔, right) showed no difference in eNOS expression. Blot shown is representative of 5 experiments.
4. Discussion We report that unidirectional medium flow confers upon cultured bEnd.3 mouse brain ECs properties similar to those conferred by blood flow in vivo, including alignment, altered expression or distribution of transcription factors, and changes in expression, phosphoactivation, or activity of gasotransmitter-synthesizing enzymes. eNOS activity and associated NO production promote normal endothelial function through vasoregulatory, antithrombotic, anti-inflammatory, and antiproliferative effects [22]. Certain cardiovascular drugs also promote normal endothelial function and two such drug classes—statins and angiotensin receptor blockers—increased eNOS expression in our cultures as well. bEnd.3 cells were derived by retroviral infection of primary mouse brain ECs with polyoma virus middle T oncogene [17]. Our cells exhibited EC properties including vWF expression and DiI-Ac-LDL uptake. In addition, orbital shaking of bEnd.3 cultures produced alignment of cell
Fig. 3. Western analysis of KLF2 expression and of NFκB expression in cytosol (NFκB-c) and nuclei (NFκB-n) in bEnd.3 cells cultured under stationary (−, left) and orbitally rotated (+, right) conditions. Blots shown are representative of 4–6 experiments; densitometric quantification is given in Results.
Fig. 4. Effects of unidirectional flow on NOS expression and NO production in bEnd.3 cultures. (A) Western analysis of eNOS, Ser1177P-eNOS (P-eNOS), iNOS and nNOS expression in bEnd.3 cells cultured under stationary (−, left) and orbitally rotated (+, right) conditions. Blots are representative of 3–6 experiments; densitometric quantification is given in Results. (B) Reversal of the effect of unidirectional flow on P-eNOS expression after return of cultures to stationary conditions for 3 days. Blots shown are representative of 3 experiments; densitometric quantification is given in Results. (C) NO production, detected by DAF-2, in bEnd.3 cells cultured under stationary (circles) and orbitally rotated (squares) conditions. Data are mean ± SEM from 3 cultures per point. *p b 0.05 compared to stationary cultures.
Fig. 5. Effects of unidirectional flow on expression of gasotransmitter-synthesizing enzymes in bEnd.3 cultures. Western analysis of (A) CO-synthesizing enzymes HO1 and HO2 and (B) H2S-synthesizing enzymes CBS and CSE in bEnd.3 cells cultured under stationary (−, left) and orbitally rotated (+, right) conditions. Blots are representative of 3–5 experiments; densitometric quantification is given in Results.
Please cite this article as: Mao X, et al, Flow-induced regulation of brain endothelial cells in vitro, Vascul Pharmacol (2014), http://dx.doi.org/ 10.1016/j.vph.2014.02.003
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flow has been reported in HUVEC [44] and human aortic EC [45] cultures, and we found this in bEnd.3 cells as well. We also observed increased HO2 expression in rotated cultures, which appears to be a new finding. H2S, another EC gasotransmitter [46], is synthesized by CBS and CSE. CBS expression, which has not been associated with shear flow to our knowledge, was increased in our rotated cultures. CSE expression was also increased, which contrasts with a report that laminar shear had no effect, whereas oscillatory shear decreased CSE levels in bovine aortic ECs [47]. Of interest, genetic deletion of HO1 [48], CBS [49], or CSE [50] promotes atherosclerosis in mice. Pharmacologic reversal of endothelial dysfunction and reduced eNOS activity has therapeutic benefit, as shown for statins [28] and inhibitors of the renin–angiotensin axis [29]. We found that statins increased eNOS expression in bEnd.3 cultures, as reported for bovine aortic ECs [51], and that angiotensin receptor blockers had a similar effect, as noted previously in rat aortic rings [52]. 5. Conclusions Orbital rotation of bEnd.3 brain EC cultures, which produces unilateral flow over the cell surface, confers morphologic and biochemical features of normal EC function. This includes induction of enzymes involved in synthesis of three gasotransmitters—NO, CO and H2S—with key roles in EC physiology and vascular disease. At least some of these effects can be replicated by cardiovascular drugs, which likely contributes to their therapeutic effects [28,53]. Our results highlight similarities and possible differences—e.g., iNOS suppression and CSE induction by flow—between cerebrovascular ECs and ECs from other vascular beds, and may therefore be relevant to regional differences in the epidemiology, mechanisms, and treatment response [15,54] of vascular disease. Acknowledgment This study was supported by the Buck Institute for Research on Aging. Fig. 6. Effects of statins and angiotensin receptor blockers (ARBs) on eNOS expression in bEnd.3 cells cultured under stationary conditions. Blots are representative of 3–4 experiments; drug concentrations and densitometric quantification of blots are given in Results.
growth and actin filaments, consistent with shear-induced cytoskeletal reorganization [19]. If orbital rotation of EC cultures is a useful model of blood flow over ECs in vivo, it should recapitulate features of the in vivo state. Previous studies have modeled the effect of orbital rotation on wall shear stress [9] and demonstrated the influence of shear on cell alignment, protein expression, intracellular signaling, or granulocyte adhesion of HUVECs [8,31–34] and rat epididymal ECs [35]. Flow-induced shear is thought to induce vasoprotection partly by activation of KLF2 and reduced activation of NFκB, with consequent effects on their transcriptional targets [20]. Changes in expression or disposition of these factors have been shown in HUVECs [11,36], and also occurred in our cultures. eNOS-derived NO is pivotal in conferring a physiological, vasoprotective EC phenotype. Thus, eNOS-knockout mice exhibit hypertension, a thrombogenic state, vascular inflammation, and vascular smooth muscle proliferation [37]. Orbital rotation of bEnd.3 cultures increased levels of eNOS and Ser1177-phospho-eNOS, which are upregulated by flow-related shear [38,39]; Ser1177-phosphorylation of eNOS [40] was reversible. In contrast, the proinflammatory isoform iNOS, which is induced in ECs of atherosclerotic arteries [41], was downregulated by rotation. This differs from the shear-induced increase in iNOS reported in rat lung and human dermal ECs [42]. The net effect of altered NOS expression in our bEnd.3 cells was increased NO production, consistent with the effect of flow in HUVECs [13]. CO, produced by HO1 and HO2, contributes to normal endothelial function and is vasoprotective [43]. Induction of HO1 expression by
References [1] Endemann DH, Schiffrin EL. Endothelial dysfunction. J Am Soc Nephrol 2004;15:1983–92. [2] Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 1992;340:1111–5. [3] Yang R, Powell-Braxton L, Ogaoawara AK, Dybdal N, Bunting S, Ohneda O, et al. Hypertension and endothelial dysfunction in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 1999;19:2762–8. [4] Huang PL. Unraveling the links between diabetes, obesity, and cardiovascular disease. Circ Res 2005;96:1129–31. [5] Oemar BS, Tschudi MR, Godoy N, Brovkovich V, Malinski T, Luscher TF. Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis. Circulation 1998;97:2494–8. [6] Ku DN, Giddens DP, Zarins CK, Glagov S. Pulsatile flow and atherosclerosis in the human carotid bifurcation. Positive correlation between plaque location and low oscillating shear stress. Arteriosclerosis 1985;5:293–302. [7] Endres M, Laufs U, Huang Z, Nakamura T, Huang P, Moskowitz MA, et al. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci U S A 1998;95:8880–5. [8] Dardik A, Chen L, Frattini J, Asada H, Aziz F, Kudo FA, et al. Differential effects of orbital and laminar shear stress on endothelial cells. J Vasc Surg 2005;41:869–80. [9] Thomas JM, Chakraborty A, Sharp MK, Berson RE. Spatial and temporal resolution of shear in an orbiting petri dish. Biotechnol Prog 2011;27:460–5. [10] Tzima E, Irani-Tehrani M, Kiosses WB, Dejana E, Schultz DA, Engelhardt B, et al. A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 2005;437:426–31. [11] Dekker RJ, van Soest S, Fontijn RD, Salamanca S, de Groot PG, VanBavel E, et al. Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Kruppel-like factor (KLF2). Blood 2002;100:1689–98. [12] Brooks AR, Lelkes PI, Rubanyi GM. Gene expression profiling of human aortic endothelial cells exposed to disturbed flow and steady laminar flow. Physiol Genomics 2002;9:27–41. [13] Noris M, Morigi M, Donadelli R, Aiello S, Foppolo M, Todeschini M, et al. Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions. Circ Res 1995;76:536–43. [14] Guo S, Zhou Y, Xing C, Lok J, Som AT, Ning M, et al. The vasculome of the mouse brain. PLoS One 2012;7:e52665.
Please cite this article as: Mao X, et al, Flow-induced regulation of brain endothelial cells in vitro, Vascul Pharmacol (2014), http://dx.doi.org/ 10.1016/j.vph.2014.02.003
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[15] Jashari F, Ibrahimi P, Nicoll R, Bajraktari G, Wester P, Henein MY. Coronary and carotid atherosclerosis: similarities and differences. Atherosclerosis 2013;227:193–200. [16] Steelman SM, Humphrey JD. Differential remodeling responses of cerebral and skeletal muscle arterioles in a novel organ culture system. Med Biol Eng Comput 2011;49:1015–23. [17] Montesano R, Pepper MS, Mohle-Steinlein U, Risau W, Wagner EF, Orci L. Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene. Cell 1990;62:435–45. [18] Voyta JC, Via DP, Butterfield CE, Zetter BR. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 1984;99:2034–40. [19] McCue S, Noria S, Langille BL. Shear-induced reorganization of endothelial cell cytoskeleton and adhesion complexes. Trends Cardiovasc Med 2004;14:143–51. [20] Boon RA, Horrevoets AJ. Key transcriptional regulators of the vasoprotective effects of shear stress. Hamostaseologie 2009;29:39–43. [21] Nagel T, Resnick N, Dewey Jr CF, Gimbrone Jr MA. Vascular endothelial cells respond to spatial gradients in fluid shear stress by enhanced activation of transcription factors. Arterioscler Thromb Vasc Biol 1999;19:1825–34. [22] Tousoulis D, Kampoli AM, Tentolouris C, Papageorgiou N, Stefanadis C. The role of nitric oxide on endothelial function. Curr Vasc Pharmacol 2012;10:4–18. [23] Chatterjee A, Black SM, Catravas JD. Endothelial nitric oxide (NO) and its pathophysiologic regulation. Vascul Pharmacol 2008;49:134–40. [24] Rossi J, Jonak P, Rouleau L, Danielczak L, Tardif JC, Leask RL. Differential response of endothelial cells to simvastatin when conditioned with steady, non-reversing pulsatile or oscillating shear stress. Ann Biomed Eng 2011;39:402–13. [25] Leffler CW, Parfenova H, Jaggar JH, Wang R. Carbon monoxide and hydrogen sulfide: gaseous messengers in cerebrovascular circulation. J Appl Physiol 2006;100: 1065–76. [26] Otterbein LE, Zuckerbraun BS, Haga M, Liu F, Song R, Usheva A, et al. Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nat Med 2003;9:183–90. [27] Wang Y, Zhao X, Jin H, Wei H, Li W, Bu D, et al. Role of hydrogen sulfide in the development of atherosclerotic lesions in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 2009;29:173–9. [28] Balakumar P, Kathuria S, Taneja G, Kalra S, Mahadevan N. Is targeting eNOS a key mechanistic insight of cardiovascular defensive potentials of statins? J Mol Cell Cardiol 2012;52:83–92. [29] Patarroyo Aponte MM, Francis GS. Effect of Angiotensin-converting enzyme inhibitors and Angiotensin receptor antagonists in atherosclerosis prevention. Curr Cardiol Rep 2012;14:433–42. [30] Michel MC, Foster C, Brunner HR, Liu L. A systematic comparison of the properties of clinically used angiotensin II type 1 receptor antagonists. Pharmacol Rev 2013;65:809–48. [31] Kraiss LW, Weyrich AS, Alto NM, Dixon DA, Ennis TM, Modur V, et al. Fluid flow activates a regulator of translation, p70/p85 S6 kinase, in human endothelial cells. Am J Physiol Heart Circ Physiol 2000;278:H1537–44. [32] Kraiss LW, Alto NM, Dixon DA, McIntyre TM, Weyrich AS, Zimmerman GA. Fluid flow regulates E-selectin protein levels in human endothelial cells by inhibiting translation. J Vasc Surg 2003;37:161–8. [33] Ley K, Lundgren E, Berger E, Arfors KE. Shear-dependent inhibition of granulocyte adhesion to cultured endothelium by dextran sulfate. Blood 1989;73:1324–30. [34] Pearce MJ, McIntyre TM, Prescott SM, Zimmerman GA, Whatley RE. Shear stress activates cytosolic phospholipase A2 (cPLA2) and MAP kinase in human endothelial cells. Biochem Biophys Res Commun 1996;218:500–4.
[35] Yun S, Dardik A, Haga M, Yamashita A, Yamaguchi S, Koh Y, et al. Transcription factor Sp1 phosphorylation induced by shear stress inhibits membrane type 1-matrix metalloproteinase expression in endothelium. J Biol Chem 2002;277:34808–14. [36] Parmar KM, Larman HB, Dai G, Zhang Y, Wang ET, Moorthy SN, et al. Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2. J Clin Invest 2006;116:49–58. [37] Atochin DN, Huang PL. Endothelial nitric oxide synthase transgenic models of endothelial dysfunction. Pflugers Arch 2010;460:965–74. [38] Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 1999;399:601–5. [39] Uematsu M, Ohara Y, Navas JP, Nishida K, Murphy TJ, Alexander RW, et al. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress. Am J Physiol 1995;269:C1371–8. [40] Moncada S, Higgs EA. Nitric oxide and the vascular endothelium. Handb Exp Pharmacol 2006:213254. [41] Wilcox JN, Subramanian RR, Sundell CL, Tracey WR, Pollock JS, Harrison DG, et al. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol 1997;17:2479–88. [42] Ozawa N, Shichiri M, Iwashina M, Fukai N, Yoshimoto T, Hirata Y. Laminar shear stress up-regulates inducible nitric oxide synthase in the endothelium. Hypertens Res 2004;27:93–9. [43] Leffler CW, Parfenova H, Jaggar JH. Carbon monoxide as an endogenous vascular modulator. Am J Physiol Heart Circ Physiol 2011;301:H1-11. [44] De Keulenaer GW, Chappell DC, Ishizaka N, Nerem RM, Alexander RW, Griendling KK. Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: role of a superoxide-producing NADH oxidase. Circ Res 1998;82:1094–101. [45] Chen XL, Varner SE, Rao AS, Grey JY, Thomas S, Cook CK, et al. Laminar flow induction of antioxidant response element-mediated genes in endothelial cells. A novel anti-inflammatory mechanism. J Biol Chem 2003;278:703–11. [46] Vandiver M, Snyder SH. Hydrogen sulfide: a gasotransmitter of clinical relevance. J Mol Med (Berl) 2012;90:255–63. [47] Go YM, Lee HR, Park H. H2S inhibits oscillatory shear stress-induced monocyte binding to endothelial cells via nitric oxide production. Mol Cells 2012;34:449–55. [48] Yet SF, Layne MD, Liu X, Chen YH, Ith B, Sibinga NE, et al. Absence of heme oxygenase-1 exacerbates atherosclerotic lesion formation and vascular remodeling. FASEB J 2003;17:1759–61. [49] Zhang D, Jiang X, Fang P, Yan Y, Song J, Gupta S, et al. Hyperhomocysteinemia promotes inflammatory monocyte generation and accelerates atherosclerosis in transgenic cystathionine beta-synthase-deficient mice. Circulation 2009;120:1893–902. [50] Mani S, Li H, Untereiner A, Wu L, Yang G, Austin RC, et al. Decreased endogenous production of hydrogen sulfide accelerates atherosclerosis. Circulation 2013;127:2523–34. [51] Laufs U, Gertz K, Dirnagl U, Bohm M, Nickenig G, Endres M. Rosuvastatin, a new HMG-CoA reductase inhibitor, upregulates endothelial nitric oxide synthase and protects from ischemic stroke in mice. Brain Res 2002;942:23–30. [52] Wenzel P, Daiber A, Oelze M, Brandt M, Closs E, Xu J, et al. Mechanisms underlying recoupling of eNOS by HMG-CoA reductase inhibition in a rat model of streptozotocin-induced diabetes mellitus. Atherosclerosis 2008;198:65–76. [53] Schlimmer N, Kratz M, Bohm M, Baumhakel M. Telmisartan, ramipril and their combination improve endothelial function in different tissues in a murine model of cholesterol-induced atherosclerosis. Br J Pharmacol 2011;163:804–14. [54] Frohlich ED. Local hemodynamic changes in hypertension: insights for therapeutic preservation of target organs. Hypertension 2001;38:1388–94.
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