Mechanisms underlying impairment of endothelium-dependent relaxation by fetal bovine serum in organ-cultured rat mesenteric artery

European Journal of Pharmacology 668 (2011) 401–406

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Cardiovascular Pharmacology

Mechanisms underlying impairment of endothelium-dependent relaxation by fetal bovine serum in organ-cultured rat mesenteric artery Tomoka Morita, Muneyoshi Okada, Yukio Hara, Hideyuki Yamawaki ⁎ Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Aomori 034-8628, Japan

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Article history: Received 11 May 2011 Received in revised form 21 July 2011 Accepted 30 July 2011 Available online 10 August 2011 Keywords: Endothelium Relaxation Growth factor Nitric oxide Organ culture

a b s t r a c t Organ culture of blood vessels provides a useful technique to investigate long-term effects of drugs because tissue architecture and function are well preserved. Various growth factors are responsible for structural and functional changes during vascular diseases. We investigated long-term effects of fetal bovine serum (FBS) which contains such factors on endothelium-dependent relaxation using organ-culture method. Rat isolated mesenteric arteries with endothelium were cultured for 3 days without or with 10% FBS (FBS). Acetylcholineand bradykinin-induced endothelium-dependent relaxations were significantly impaired in FBS, whereas sodium nitroprusside-induced relaxation of endothelium-removed artery was unchanged. Morphological examination revealed that endothelium was intact in FBS. Acetylcholine-induced nitric oxide (NO) release as detected by 4, 5-diaminofluorescein significantly decreased in FBS, whereas endothelial NO synthase expression was unchanged. A Ca2+ ionophore, A23187-induced relaxation was unchanged in FBS. A phospholipase C activator, m-3M3FBS-induced relaxation of FBS was unchanged in either Ca2+-containing or -free solution. Total expressions of transient receptor potential canonical channels (TRPCs: TRPC-1, -4, -5) were similar in FBS. These data suggest that FBS impairs endothelium-dependent relaxation by inhibiting events upstream of phospholipase C activation including phospholipase C, G-protein, and receptors in endothelium. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Organ culture of blood vessels is a useful technique to investigate long-term direct effects of drugs. This model has several advantages beyond the conventional in vivo or in vitro model such as preservation of a differentiated cell function, easy handling of experimental conditions and dissociation from complicated factors existing in vivo (Kida et al., 2009, 2011; Murata et al., 2001a,b,c, 2004, 2005a,b; Sakamoto et al., 2005; Yamawaki et al., 1999a,b, 2000). In the recent study, we have successfully established an organ-culture technique using rat isolated mesenteric artery (Morita et al., 2010). We demonstrated that 3-day organ-cultured rat mesenteric arteries in a serum-free condition preserved an enough contraction to enable the analysis for long-term effects of drugs. By using this technique, we have found that treatment of endothelium-denuded artery with fetal bovine serum (FBS) for 3 days impairs agonist-induced smooth muscle contractility (Morita et al., 2010).

⁎ Corresponding author at: Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan Tel.: + 81 176 23 4371; fax: + 81 176 24 9456. E-mail address: [email protected] (H. Yamawaki). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.07.040

Vascular endothelium plays important roles on cardiovascular system since it modulates vascular tone, inflammation, permeability, and angiogenesis. Nitric oxide (NO) released from vascular endothelial cells is a most important factor for cardiovascular homeostasis since NO induces vasodilation, and prevents smooth muscle proliferation (Garg and Hassid, 1989), platelet aggregation and lymphocyte adhesion (Gryglewski et al., 1988). In chronic vascular pathological conditions such as hypertension, arteriosclerosis and diabetes in which vascular remodeling occurs, various growth factors are generated in the lesions. They are responsible for the functional and structural changes of blood vessels by chronically affecting on the vessel wall including vascular endothelium to stimulate proliferation (regeneration), migration or inflammatory injury (Ross, 1993; Yamawaki et al., 1999a). In fact, endothelial dysfunction is the most common feature of vascular diseases (Bakris et al., 2010). Organ culture with FBS, as a source of indefinite but various growth factors, may allow us to investigate influences of growth factors on endothelium-dependent relaxation (Yamawaki et al., 1999a). However, the studies on the endothelial function using organ-cultured rat isolated mesenteric arteries are limited (Brum Cde et al., 2005; Chauhan et al., 2007; Jimenez-Altayo et al., 2006; Lu et al., 2007; Luo et al., 2006). The aim of present study was to investigate long-term effects of FBS on endothelium-dependent relaxation using rat mesenteric arterial organ culture model. We have found that FBS may impair agonist-

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mediated endothelium-dependent relaxation by inhibiting events upstream of phospholipase C activation.

industries, kibbutz beit haemek, Israel). The results were analyzed using CS Analyzer 3.0 software (ATTO, Tokyo, Japan).

2. Materials and methods

2.5. Morphological examination

2.1. Tissue preparation and organ culture procedure

Morphological examination was performed as described previously (Yamawaki et al., 1999b, 2000). After the 3-day organ culture, arterial rings were fixed in 10% neutral-buffered formalin for 12 h and embedded in paraffin. The 4-μm-thick sections were stained with hematoxylin and eosin (HE). The slides were examined by a BX-51 light microscope (Olympus, Tokyo, Japan).

Organ culture of isolated blood vessels was performed as described previously (Morita et al., 2010). In brief, male Wistar rats (188–300 g, 6–9-week-old) were anesthetized with urethane (1.5 g/kg, i.p.) and euthanized by exsanguination. The main branch of the superior mesenteric artery was isolated under sterile conditions. After removal of fat and adventitia in sterile Tris-buffered saline, the mesenteric artery was cut into rings (1 mm in diameter) for organ culture and measurement of isometric tension. Arterial rings were then placed in 1 ml Dulbecco's Modified Eagle Medium (DMEM) without (control) or with 10% FBS (FBS) supplemented with 1% penicillin–streptomycin. They were maintained at 37 °C in an atmosphere of 95% air and 5% CO2 for 3 days. Animal care and treatment were conducted in conformity with institutional guidelines of the Kitasato University. 2.2. Measurement of isometric contraction The arterial rings were placed in normal physiological salt solution (PSS), which contained (mM): NaCl 136.9, KCl 5.4, CaCl2 1.5, MgCl2 1.0, NaHCO3 23.8, and glucose 5.5. The Ca2+-free PSS was prepared by adding 2 mM EGTA instead of CaCl2. Smooth muscle contractility was recorded isometrically with a force–displacement transducer (Nihon Kohden, Tokyo, Japan) as described previously (Morita et al., 2010; Mukohda et al., 2010a,b; Yamawaki et al., 2010). Concentration– response curves were obtained by the cumulative application of relaxants during the 30 mM potassium- or 30–40 μM prostaglandin (PG) F2α-induced precontraction. Data was shown as a percent relaxation of the steady-state precontraction. The precontractions induced by KCl (30 mM) and by PGF2α were both unaffected by FBS application. 2.3. Nitric oxide (NO) assay by 4, 5-diaminofluorescein (DAF-2) DAF-2 as a specific NO indicator, selectively traps NO and yields triazolofluorescein, which emits green fluorescence when excited at 490–515 nm (Kojima et al., 1998). After equilibration for 30 min in a 3 ml organ bath, each organ-cultured arterial ring was repeatedly exposed to 72 mM potassium solution to obtain stable response (60–90 min). 30 mM potassium solution and DAF-2 (100 nM) were then simultaneously applied. After 5 min, 200 μl of incubation solution (PSS) in each bath was collected and transferred to a 96well plate as basal NO release control (before acetylcholine stimulation). After 20 min treatment with 30 mM potassium, acetylcholine (100 μM) was applied for 5 min and 200 μl of PSS was also collected. Fluorescence at excitation 485 nm and emission 535 nm was measured using a Tristar LB 941 fluorometer (Berthold technologies, Bad Wildbad, Germany). 2.4. Western blotting Western blotting was performed as described previously (Usui et al., 2010a,b; Yamawaki et al., 2010). Protein lysates were obtained by homogenizing organ-cultured rat mesenteric artery with Triton-based lysis buffer. Equal amounts of proteins (8–15 μg) were separated by SDS–PAGE (7.5%) and transferred to a nitrocellulose membrane (Pall Corporation, Ann Arbor, MI, USA). After being blocked with 0.5% skim milk, membranes were incubated with primary antibody (1:200–1:500 dilution) at 4 °C overnight, and the membrane-bound antibodies were visualized using horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution, 1 h) and the EZ-ECL system (Biological

2.6. Chemicals The chemicals used were as follows: DMEM, bradykinin, sodium nitroprusside (SNP), A23187 (Sigma-Aldrich, St. Louis, MO, USA), FBS and penicillin–streptomycin (Invitrogen/GIBCO, Carlsbad, CA, USA), acetylcholine (Daiichi Pharmaceutical, Tokyo, Japan), DAF-2 (Sekisui medical, Tokyo, Japan), 2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)-benzenesulfonamide (m-3M3FBS) (Calbiochem, San Diego, CA, USA), PGF2α (Cayman, Ann Arbor, MI, USA). Acetylcholine and SNP were dissolved in distilled water. A23187, m-3M3FBS, and PGF2α were dissolved in DMSO. Bradykinin was dissolved in acetic acid (0.1 N). Antibody sources were as follows: total endothelial nitric oxide (NO) synthase (eNOS) (Santa Cruz Biotech, Santa Cruz, CA, USA), transient receptor potential canonical channel (TRPC)-1, TRPC-4, TRPC-5 (Alomone Labs, Jerusalem, Israel), and total actin (Sigma-Aldrich). 2.7. Statistics The results of the experiments are expressed as means ± S.E.M. Statistical evaluation of the data was performed by Student's unpaired t-test. A value of P b 0.05 was taken as statistically significant. 3. Results 3.1. Influence of FBS on acetylcholine- and bradykinin-induced endothelium-dependent relaxations We first examined influences of FBS on agonist-induced endotheliumdependent relaxation. In organ-cultured endothelium-intact arteries under serum-free condition for 3 days (cont), acetylcholine and bradykinin relaxed 30 mM potassium-induced precontraction in a concentration-dependent manner. In organ-cultured endotheliumintact arteries in the presence of FBS for 3 days (FBS), acetylcholineand bradykinin-induced relaxations significantly decreased compared with cont (Fig. 1A, B). 3.2. Influence of FBS on SNP-induced endothelium-independent relaxation We next examined influences of FBS on SNP-induced relaxation in smooth muscle. In cont denuded with endothelium, SNP relaxed 30 mM potassium-induced precontraction in a concentration-dependent manner. In FBS denuded with endothelium, SNP-induced relaxation didn't change compared with cont (Fig. 1C). 3.3. Influence of FBS on endothelial cell morphology To investigate whether endothelium was intact after FBS treatment, we performed morphological analysis in HE-stained cross sections. In cont, endothelial cells were attached along the tunica intima (Fig. 2A). In FBS, endothelial morphology was similar to that in cont (Fig. 2B).

T. Morita et al. / European Journal of Pharmacology 668 (2011) 401–406

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Fig. 2. Light micrographs of hematoxylin and eosin stained sections of endotheliumintact rat mesenteric arteries cultured without (A) or with 10% FBS (B) for 3 days (n = 7 for cont, n = 16 for FBS). The arterial lumen is at the top. Magnification: × 400. Scale bar: 50 μm.

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investigated expression of total eNOS by Western blotting. The expression of eNOS did not change in FBS (Fig. 3B).

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Sodium nitroprusside (log M) Fig. 1. Concentration–response relationships for relaxant effect of acetylcholine (A: 30 nM–300 μM, n = 7 for cont, n = 8 for FBS), bradykinin (B: 0.03–3000 nM, n = 8 for cont, n = 6 for FBS) and sodium nitroprusside (C: 0.1–1000 nM, n = 9 for cont, n = 12 for FBS) on 30 mM potassium-induced precontraction in endothelium-intact (A, B) or -denuded (C) rat mesenteric arteries cultured without (cont, open circle) or with 10% fetal bovine serum (FBS, closed circle) for 3 days. The relaxants were cumulatively applied after the potassium-induced contraction had reached a steady state. Results are expressed as means ± S.E.M. *, **: Significantly different with P b 0.05 and P b 0.01 vs. cont.

3.4. Influence of FBS on acetylcholine-induced NO release We next analyzed whether impairment of endothelium-dependent relaxation in FBS was due to decrease in NO production. In cont, 100 μM acetylcholine increased NO release by 5.2 ± 1.1-fold relative to basal NO-release (Fig. 3A). In FBS, acetylcholine-induced NO release significantly decreased compared with that in cont. 3.5. Influence of FBS on expression of eNOS To determine whether impairment of acetylcholine-induced NO release by FBS was due to down-regulation of eNOS protein, we

To explore mechanisms responsible for the FBS-induced impairment of endothelial NO production, we examined a Ca2+ ionophore, A23187induced relaxation. In cont, A23187 relaxed 30 mM potassium-induced precontraction in a concentration-dependent manner. In FBS, A23187induced relaxation was similar to that in cont (Fig. 4A). 3.7. Influence of FBS on a phospholipase C activator, m-3M3FBS-induced relaxation To further explore the mechanisms, a phospholipase C activator, m-3M3FBS-induced relaxation was examined. In cont, m-3M3FBS relaxed 30 mM potassium precontraction in a concentration-dependent manner. In FBS, the m-3M3FBS-induced relaxation was similar to that in cont (Fig. 4B). In Ca2+-free PSS in the presence of 2 mM EGTA, m-3M3FBS induced a relaxation of PGF2α-precontracted control artery in a concentration-dependent manner. The m-3M3FBS-induced relaxation in the extracellular Ca2+-free condition did not change in FBS (Fig. 4C). 3.8. Influence of FBS on expression of TRPC-1, -4 and -5 In vascular endothelium, TRPC-1, -4, and -5 may mediate agonistinduced Ca 2+ influx (Freichel et al., 2001; Goel et al., 2002; Hofmann et al., 2002; Yoshida et al., 2006). We thus investigated TRPC-1, -4, and -5 expressions by Western blotting. The expression of TRPC-1 in FBS decreased significantly, whereas TRPC-4 expression slightly increased

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Fig. 3. (A) Acetylcholine (100 μM, 5 min)-induced nitric oxide (NO) release was measured by using 4, 5-diaminofluorescein diacetate (DAF-2, 100 nM) in organcultured rat mesenteric arteries in the absence (cont) or presence of 10% FBS for 3 days. Fluorescence at excitation 485 nm and emission 535 nm was measured using a fluorometer. Results are expressed as means ± S.E.M and shown as fold-increase relative to basal NO-release (before acetylcholine stimulation, n = 8). **: Significantly different with P b 0.01 vs. cont. (B) Influence of FBS on endothelial NO synthase (eNOS) expression during a 3-day organ culture. After rat mesenteric arteries were cultured without (cont) or with 10% FBS for 3 days, total protein lysates were harvested. Expression of eNOS was determined by Western blotting using a total eNOS antibody. Equal protein loading was conformed using a total actin antibody. Results are expressed as means ± S.E.M. Expression level of eNOS in each cont was set as 1 and the results were shown as fold-increase relative to cont (n = 7).

although it was not statistically significant (Fig. 5). The expression of TRPC-5 did not change in FBS.

4. Discussion In the present study, we investigated long-term effects of FBS on endothelium-dependent relaxation using rat mesenteric artery in organ culture. The major findings of the present study are as follows; 1) acetylcholine- and bradykinin-mediated endothelium-dependent relaxations were impaired in FBS, 2) SNP-induced relaxation of smooth muscle was unchanged in FBS, 3) endothelium was morphologically intact in FBS, 4) acetylcholine-induced NO release decreased, while eNOS expression was unchanged in FBS, 5) a Ca 2+ ionophore, A23187-induced relaxation was unchanged in FBS, 6) a phospholipase C activator, m-3M3FBS-induced relaxation of FBS was unchanged in either Ca2+-containing or -free solution, and 7) total expressions of TRPCs (TRPC-1 + -4 + -5) was unchanged in FBS. These findings collectively suggest that FBS impairs endothelium-dependent relaxation via suppressing events upstream of phospholipase C activation (Fig. 6).

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m-3M3FBS (log M) Fig. 4. (A) Concentration–response relationship for relaxant effect of a Ca2+ ionophore, A23187 (0.01 nM–3 μM, n= 12 for cont, n= 14 for FBS) on 30 mM potassium-induced precontraction in endothelium-intact rat mesenteric arteries cultured without (cont, open circle) or with 10% FBS (closed circle) for 3 days. (B, C) Concentration–response relationships for relaxant effect of a phospholipase C activator, m-3M3FBS (0.01 nM10 μM) on 30 mM potassium-induced precontraction in normal Ca2+-containing solution (B: n = 4 for cont, n = 10 for FBS) or on 30–40 μM prostaglandin F2α-induced precontraction in Ca2+-free solution (C: n= 6 for cont, n= 7 for FBS). The relaxants were cumulatively applied after the precontraction had reached a steady state. Results are expressed as means± S.E.M.

NO causes smooth muscle relaxation through the cGMP-dependent decreases in cytosolic Ca 2+ concentration and/or Ca 2+ sensitivity of contractile apparatus (Karaki et al., 1988). It was previously shown that sensitivity to NO slightly increased after a 7-day organ culture of rabbit mesenteric arteries in the presence of FBS (Yamawaki et al., 1999a). In this study, an NO donor, SNP-induced relaxation was unchanged in FBS, suggesting that FBS did not affect NO sensitivity in smooth muscle of rat mesenteric arteries during a 3-day organ culture. We then analyzed acetylcholine-induced NO release using a specific fluorescence NO indicator, DAF-2, and found that acetylcholine-induced NO release significantly decreased in FBS. Previously impairment of NO production due to decrease in eNOS mRNA expression was demonstrated after a

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permeable and specific activator of phospholipase C, m-3M3FBS in normal Ca2+-containing or extracellular Ca 2+-free PSS. The m-3M3FBSinduced relaxation of FBS was unchanged in either Ca2+-containing or -free PSS, suggesting that both the extracellular Ca 2+ influx via TRPCs and the IP3-induced Ca 2+ release from sarcoplasmic reticulum may be preserved in FBS. In vascular endothelium, TRPC-4 may mediate receptor agonistinduced Ca 2+ influx since acetylcholine-induced endothelial Ca 2+ entry is reduced in TRPC-4 deficient mice, resulting in a significant decrease in endothelium-dependent NO-mediated vasorelaxation (Freichel et al., 2001). TRPC-5 is also involved since treatment of bovine aortic endothelial cells with small interference RNA against TRPC-5 prevented NO-induced Ca 2+ entry (Yoshida et al., 2006). It was also reported that TRPC-1, -4, and -5 can form functional homoand hetero-tetramers (Goel et al., 2002; Hofmann et al., 2002). In the present study, we observed that total expression level of TRPCs was similar in FBS compared with control. These results well corresponded to the data using m-3M3FBS in which m-3M3FBS-induced relaxation partly caused by TRPCs-related Ca 2+ influx was unchanged in FBS.

TRPC 5

Fig. 5. Influence of FBS on transient potential canonical channel (TRPC)-1, -4 and -5 protein expressions during a 3-day organ culture. After rat mesenteric arteries were cultured without (cont) or with 10% FBS for 3 days, total protein lysates were harvested. Expression of TRPC-1, -4 and -5 was determined by Western blotting. Equal protein loading was conformed using total eNOS antibody. Expression level of TRPC in each cont was set as 1 and the results were shown as fold-increase relative to cont (n = 4). Results are expressed as means ± S.E.M. **: Significantly different with P b 0.01 vs. cont.

7-day organ culture of rabbit mesenteric arteries in the presence of FBS (Yamawaki et al., 1999a). In the present study, eNOS protein expression was unchanged in FBS, suggesting that FBS may affect events upstream of eNOS activation during a 3-day organ culture. Agonist-induced NO producing pathways are as follows; 1) after binding of an agonist to its receptor, G-protein (Gq)-activated phospholipase C-β cleaves phosphatidylinositol 4, 5-bisphosphate into inositol-1, 4, 5 trisphosphate (IP3) and diacylglycerol, 2) an increase in intracellular Ca2+ concentration is caused by an extracellular Ca2+ influx via phospholipase C-activated TRPCs and an IP3-induced Ca2+ release from sarcoplasmic reticulum, 3) after an increase in intracellular Ca2+ concentration, eNOS is activated by a Ca2+–calmodulin complex and produces NO from L-arginine (Braam and Verhaar, 2007; Dietrich et al., 2010). In the present study, a Ca 2+ ionophore-induced relaxation did not change in FBS, suggesting that FBS may affect events upstream of an increase in intracellular Ca2+. We then used a cell-

Fig. 6. Summary of the present results. FBS may impair agonist-mediated endotheliumdependent relaxation by inhibiting events upstream of phospholipase C activation including 1) receptors, 2) G-protein (Gq), and/or 3) phospholipase C.

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