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Blockade of PKC-beta protects HUVEC from advanced glycation end products induced inflammation
International Immunopharmacology 10 (2010) 1552–1559
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International Immunopharmacology 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 / i n t i m p
Blockade of PKC-beta protects HUVEC from advanced glycation end products induced inflammation Youhua Xu a,1, Shanshan Wang a,1, Liang Feng a, Quan Zhu a,b,⁎, Ping Xiang a, Bao He b a b
Faculty of Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau Institute of Consun Co. for Chinese Medicine in Kidney Diseases, Guangdong Consun Pharmaceutical Group, Dongpeng Road 71, Guangzhou, PR China
a r t i c l e
i n f o
Article history: Received 7 July 2010 Received in revised form 15 August 2010 Accepted 10 September 2010 Keywords: Endothelial cell ICAM-1 Macrophage migration Oxidative stress TGF-beta1 PKC-beta
a b s t r a c t Advanced glycation end products (AGEs) have been recognized as a pivotal inducer in diabetes and kinds of aging-related vasculopathy. Endothelial dysfunction and inflammatory cells adhesion to endothelium have been regarded as important and early factors in the pathogenesis of vascular complications in diabetic patients. Owing to the key role of PKC-beta in AGEs-induced vascular dysfunction, we investigated effects of blocking PKC-beta by LY333531 on macrophage adhesion to HUVEC and the related mechanism. Transwell HUVEC-macrophage co-culture system was established to evaluate macrophage migration and adhesion ability. Immunocytochemistry was applied to examine TGF-beta1, ICAM-1 and RAGE protein expressions by SABC or SABC-AP method; mRNA expression of TGF-beta1, ICAM-1 and RAGE was determined by real-time RT-PCR. SOD and MDA levels in culture supernatant were detected. We found that LY333531 significantly reduced AGEs-induced macrophage adhesion to HUVEC. Blockade of PKC-beta strikingly decreased HUVEC TGF-beta1 and ICAM-1 expression in both protein and mRNA levels, RAGE protein level was also downregulated. Furthermore, the anti-oxidative stress index, SOD/MDA was dramatically elevated on LY333531 application. Therefore we conclude that LY333531 can reduce AGEs-induced macrophage adhesion to endothelial cells and relieve the local inflammation, this was realized by its effect on decreasing inflammatory cytokines' expression and increasing cell anti-oxidative ability. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction Endothelium is the major vector in angiogenesis [1], and endothelial dysfunction is regarded as an important and early factor in the pathogenesis of atherothrombosis [2] and vascular complications [3] in diabetic patients. The complications associated with diabetic vasculopathy are commonly grouped into two categories: micro- and macro-vascular complications. In diabetes, macro-vascular disease is the commonest cause of mortality and morbidity and is responsible for high incidence of vascular diseases such as stroke, myocardial infarction and peripheral vascular diseases [4]. However, the morbidity associated with diabetic micro-vascular disease, including retinopathy, neuropathy, nephropathy, and limb ischemia, is staggering [5]. Infiltration of macrophages in the glomeruli and interstitium is one of the characteristic features of diabetic nephropAbbreviations: ECs, endothelial cells; TGF-beta1, transforming growth factor-beta1; OS, oxidative stress; ICAM-1, intercellular adhesion molecule-1; MCP-1, monocyte chemotactic protein-1. ⁎ Corresponding author. Institute of Consun Co. for Chinese Medicine in Kidney Diseases, Guangdong Consun Pharmaceutical Group, Dongpeng Road 71, Guangzhou 510530, PR China. Tel.: + 86 20 82267589; fax: + 86 20 82017468. E-mail address: [email protected] (Q. Zhu). 1 Youhua Xu and Shanshan Wang contributed equally to the work.
athy, in addition to ECM expansion and interstitial fibrosis [6]. Recruitment of monocyte/macrophage and lymphocytes from the peripheral blood to the endothelial cells (ECs) is an early and central event in vascular dysfunction development [5,7] and experimental diabetes [22], thus inflammation plays a pivotal role in the initiation of this process. Advanced glycation end products (AGEs) have been widely studied in its key role in promoting vascular dysfunction and diabetes development [5]. AGEs impair vessel functions via the receptor for advanced glycation end products (RAGE) [8]. The activation of RAGE may induce a series of inflammatory process, such as up-regulating adhesion molecules' expression, promoting intima proliferation and angiogenesis, and inducing oxidative stress. Cell and animal studies suggest that limiting RAGE expression in vascular cells could modulate expression of various pro-inflammatory mediators and prevent vascular dysfunction development [9]. PKC-beta has been recognized as a key mediator in AGEs-induced micro- and macro-vascular dysfunction [10]. Studies have demonstrated that AGEs can activate PKC-beta and induce a series of pathophysiological changes in cells [10]. In the present study, we demonstrated that selective blockade of PKC-beta with LY333531 can reverse AGEs' effect on macrophage adhesion to HUVEC and reduce AGEs-induced ECs damage.
1567-5769/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2010.09.006
Y. Xu et al. / International Immunopharmacology 10 (2010) 1552–1559
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2. Materials and methods
2.4. Macrophages migration and adhesion assay
2.1. Animals and reagents
Macrophages were collected from mice abdomen according to the reports [13] with minor adjustment. In general, mice were executed by cervical dislocation, and peritoneal exudates were induced by one intraperitoneal injection of 20–25 ml sterile cold phosphate-buffer solution (PBS, pH 7.4). Ten minutes later, abdominal walls were shaved and cleansed with alcohol. Under sterile conditions, the peritoneal exudates were expelled by syringe and centrifuged at room temperature; after red blood cell quassation by 0.83% NH4Cl, the solution was centrifuged at room temperature for two times and the macrophages were re-suspended by DMEM and adjusted to the density of 1 × 106. To study macrophage migration (Fig. 1), HUVECs were firstly seeded on the coverslips in 24-well plates at confluence density of 80– 90%. HUVECs were starved and incubated with BSA (200 μg/ml), AGEs (200 μg/ml) [12], or LY333531 (200 nM) [14] + AGEs (200 μg/ml) for 24 h. Then the 24-well plates were inserted with Transwell inserts (5.0 μm pore size polycarbonate membrane, Corning Costar Corporation, Cambridge, MA); the macrophages were plated into inserts and co-cultured with HUVEC for further 24 h. In this system [15], macrophages were grown on upper compartment of the Transwell microporous membranes. At 48 h, macrophages in the upper compartment were gently wiped with cotton to remove cells that didn't pass through the membrane, then the whole microporous membranes were stained with 10% crystal violet for 10 min; after washing with PBS, the upper side of the microporous membrane and the upper compartment walls were carefully wiped with cotton. Then the membranes were carefully clipped with razor blade (for microscopy observation) or washed with 30% glacial acetic acid (for macrophage counting determination). For macrophage counting determination, the washing solution was transferred to 96-well plate (Corning Costar) and detected at the wavelength of 580 nm.
All animal care and this investigation conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and were approved by the Ethical Review Board of Macau University of Science and Technology and Consun Pharmaceutical Group. Male Swiss mice weighing 20–25 g were supplied by the Shanghai laboratory animal centre; food and water were given ad libitum. The selective PKC-beta inhibitor, LY333531, was purchased from Alexis Biotechnologies (Nottingham, UK). Polyclonal ICAM-1 antibody was obtained from Boster (Wuhan, China); polyclonal TGF-beta1 and monoclonal RAGE antibodies were supplied by Santa Cruz Biotechnology (California, USA). The SOD and MDA detection kits were supplied by Jiancheng (Nanjing, China). All other reagents used were derived from commercial sources. 2.2. Preparation of advanced glycation end products AGEs were prepared as previously described [11,12]. In general, BSA was added into 10 mM/L PBS (pH 7.4, concentration of 5 g/L), incubated with 50 mM/L D-glucose in 95% air/5% CO2 at 37 °C for 12 weeks. Unincorporated glucose was removed by dialysis overnight against 0.01 M PBS. AGEs were stored at −20 °C until use. 2.3. Culture of Human Umbilical Vein Endothelial Cells Human Umbilical Vein Endothelial Cell (HUVEC) line was purchased from ATCC (Manassas, VA, USA). Cells were cultured in DMEM (Gibco) supplemented with 10% FBS (fetal bovine serum), 1% penicillin–streptomycin, at 37 °C in a 95% air/5% CO2 incubator.
A Upper Compartment
Macrophage Lower Compartment
HUVEC 5.0 m Microporous Membrane Coverslip
B Drug incubation end; HUVEC Cell seeding Starving
-24h
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0h
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Fig. 1. Diagram of macrophage migration detection by Transwell. (A) Composition diagram of Transwell. (B) The detection flow of macrophage migration by Transwell. Following different treatment as described in Section 2.4, macrophage migration through microporous membranes was detected by 10% crystal violet staining or 30% glacial acetic acid washing.
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Fig. 2. Macrophage migration assay by crystal violet staining (A) or chromatometry (B). HUVECs at lower compartment were incubated with 200 μg/ml BSA (Aa), 200 μg/ml AGEs (Ab), or 200 nM LY333531 + 200 μg/ml AGEs (Ac) and macrophages migrated through the membrane were stained by 10% crystal violet and observed under microscope (magnification, 200×). For macrophage counting (B), the crystal violet was washed out by 30% glacial acetic acid and the washing solution was detected at wavelength of 580 nm, the data was expressed as means ± SD; ⁎p = 0.073, vs. BSA group. The experiment was repeated for three times, and representative pictures were shown.
HUVECs seeded on the coverslips in the lower compartment were performed with immunocytochemistry detection of TGF-beta1 and macrophage adhesion. 2.5. Immunocytochemistry analysis Immunocytochemistry staining was performed using the StreptAvidin-Biotin-enzyme Complex (SABC) or SABC-alkaline phosphatase (AP) method. Cells were grown on glass coverslips in 24-well plates. Following different treatments as described in Section 2.4, the cells were washed with phosphate-buffer solution (PBS, pH 7.4), fixed with fresh 4% paraformaldehyde for 60 min and blocked with 10% primary antibody-origin serum or 5% BSA (for RAGE detection) for 20 min at room temperature. After being blocked, the cells were incubated at 37 °C with primary antibodies, including ICAM-1 (1:200), TGF-beta1 (1:400) and RAGE (1:200), and then given a sequential 20 min incubation with biotin-conjugated secondary antibody and SABC or SABC-AP at 37 °C. Color was developed by 3,3'-diaminobenzidine (DAB) or BCIP/NBT (for RAGE). Finally, the cells were counterstained with hematoxylin or nuclear fast red (for RAGE) and photographed under microscope. Positive staining was indicated with brown or dark blue (for RAGE) deposits. In negative control, primary antibody was replaced with PBS. 2.6. Real-time RT-PCR analysis HUVECs were plated in flasks and grown to 80–90% confluence. After starvation overnight, cells were incubated with drugs for
24 h. Total RNA was extracted by Trizol (Invitrogen, Carlsbad, CA) and reverse-transcribed using M-MLV RTase (Promega). Quantitative SYBR Green real-time PCR assay was performed to measure expression of mRNAs for TGF-beta1, ICAM-1 and RAGE. Briefly, 1 μl of template cDNA was added to a final volume of 0.4 μM of the primers. Amplification was performed with 40 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, and elongation at 72 °C for 15 s. Sequences of the primers used for real-time PCR were as follows: TGF-beta1 forward 5'GCCCTCCTACCTTTTGC-3' and reverse 5'-AGGCGTCAGCACCAGTAG-3', ICAM-1 forward 5'-GTGGTAGCAGCCGCAGT-3' and reverse 5'-TTCGGTTTCATGGGGGT-3', RAGE forward 5'CTGGTGCTGAAGTGTAAGGG-3' and reverse 5'-GAAGAGGGAGCCGTTGG-3'. During thermal cycling, emission from each sample was recorded and calculated to produce threshold cycle (Ct) values for each sample. The housekeeping gene β-actin was used for internal normalization. The transcript copy number of target gene was determined on the basis of their Ct values.
2.7. Determination of SOD/MDA Levels of SOD and MDA in culture supernatant were determined by SOD and MDA detection kits within 6 h according to the manufacturer's protocol. The final concentrations of SOD and MDA were calculated by the equations supplied by the kits producer.
Fig. 3. Blockade of PKC-beta reduced AGEs-induced TGF-beta1 expression and macrophage adhesion to HUVEC. HUVECs at lower compartment of Transwell system were incubated with 200 μg/ml BSA (Ab), 200 μg/ml AGEs (Ac), or 200 nM LY333531 + 200 μg/ml AGEs (Ad) and TGF-beta1 expression was detected by immunocytochemistry SABC method; HUVECs were counterstained with hematoxylin. Number of macrophages that adhered with HUVECs was counted and quantitated under microscope and compared by the SPSS (B). TGF-beta1 immunoreactivity appears brown as a result of DAB colorimetric reaction, the positive staining of TGF-beta1 was quantitated by Image-Pro Plus software and the difference between groups was compared (C). White arrows show macrophages adhesion with HUVEC; black arrows show positive brown color. In negative control (Aa), primary antibody was replaced with PBS. TGF-beta1 mRNA expression was determined by real-time RT-PCR (D). ⁎⁎p b 0.01, vs. BSA; #p b 0.05, vs. AGEs; ##p b 0.01, vs. AGEs. The experiment was repeated for three times, and representatives were shown (magnification, 200×).
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2.8. Statistical analysis Data were expressed as means ± SD. The significance for the difference among groups was analyzed with SPSS 13.0 by one-way ANOVA. Differences were considered to be statistically significant at p value of b0.05. 3. Results
with LY333531 increased RAGE mRNA to the level even higher than that of AGEs group (p b 0.01, vs. AGEs and BSA groups). This discrepancy with that of protein expression may be attributed to the fact that there is a long process from mRNA to the synthesis of protein at ribosome, other factors' expression that induced by LY333531 may influence RAGE protein or mRNA expression. On the other hand, this increment of mRNA expression may be the negative feedback of cells to the decrement of the receptor protein expression. But the accurate mechanism needs investigation.
3.1. Blockade of PKC-beta reduced macrophages adhesion to HUVEC 4. Discussion As shown in Fig. 2, incubation with AGEs (200 μg/ml) increased macrophage migration through the Transwell microporous membranes (p = 0.073, vs. BSA). Unexpectedly, pretreatment HUVEC with 200 nM of the selective PKC-beta inhibitor, LY333531, cannot reduce AGEs-induced macrophage migration through membranes (p = 0.447, vs. AGEs). To observe if there is more macrophages adhesion to HUVEC in the lower compartment, immunocytochemistry detection was performed. As shown in Fig. 3A and B, AGEs significantly increased macrophage adhesion to HUVEC (Fig. 3Ac), while pretreatment the cells with LY333531 dramatically reduced AGEs' effect on the adhesion (Fig. 3Ad and B). 3.2. LY333531 decreased AGEs-induced TGF-beta1 and ICAM-1 expression in HUVEC TGF-beta1 has been implicated in participating AGEs-induced inflammation [16,17]. In the present study, we observed that AGEs increased TGF-beta1 expression in both protein (Fig. 3A and C) and mRNA (Fig. 3D) levels. Pretreatment HUVEC with LY333531 significantly reduced AGEs-induced TGF-beta1 expression to the normal level (Fig. 3A, C and D). ICAM-1, as a pivotal adhesion molecule that mediates inflammatory cell adhesion, has been recognized as an important factor in the development of vasculitis [18] and AGEs-induced chronic kidney disease [19]. Here we observed that AGEs increased ICAM-1 protein (Fig. 4Ac and B) expression in HUVEC, and blockade of PKC-beta with LY333531 (Fig. 4Ad) reversed its expression to nearly the normal levels as compared with BSA group (Fig. 4B; p b 0.01, vs. AGEs group; p N 0.05, vs. BSA group). Furthermore, the mRNA expression of ICAM-1 after LY333531 incubation was reduced to the level even lower than that of BSA group (Fig. 4C; p b 0.01, vs. BSA group). 3.3. LY333531 pretreatment elevated cell anti-oxidative ability It has been well established that AGEs-induced oxidative stress (OS) plays important role in vascular dysfunction [20]. Here we found that (Fig. 5) the anti-OS index, SOD/MDA was dramatically decreased in HUVEC incubated with AGEs compared with that incubated with BSA (p b 0.01). To assess if blockade of PKC-beta can protect HUVEC from OS damage, cells were pretreated with LY333531 followed by AGEs. As depicted in Fig. 5, blockade of PKC-beta increased HUVEC's anti-OS ability to the normal level (p b 0.05, vs. AGEs; p = 0.229, vs. BSA). 3.4. AGEs induced HUVEC inflammation via RAGE-PKC-beta pathway Receptor for advanced glycation end products (RAGE) is known to mediate AGEs-induced cell damage [8]. Here we observed that AGEs dramatically elevated RAGE protein expression in HUVEC (Fig. 6Ac) and blockade of PKC-beta with LY333531 reversed this effect (Fig. 6Ad). In order to examine if RAGE mRNA expression is in line with the protein expression, real-time RT-PCR was performed. As depicted in Fig. 6B, AGEs incubation significantly increased RAGE mRNA expression (p b 0.01, vs. BSA group); unexpectedly, pretreatment HUVEC
In diabetes, vascular disease is the commonest cause of mortality and morbidity and is responsible for high incidence of stroke, myocardial infarction and peripheral vascular diseases [4]. Advanced glycation end products (AGEs) have been widely studied in its key role in promoting vascular inflammation [5]. Reports have demonstrated that AGEs play a role in kinds of vasculitis such as atherosclerosis and diabetic retinopathy [5]. Macrophages migration and adhesion to endothelial cells (ECs) is a key step in initiating the local inflammation process. In the present study, we demonstrated that blockade of PKC-beta with LY333531 can significantly reduce AGEs-induced macrophage adhesion to HUVEC, in which downregulation of inflammatory cytokines' expression may mediate this process. ECs play in a central role in the pathologic of vascular dysfunction in type 2 diabetic patients [5]. Under inflammatory conditions, ECs secrete a series of inflammatory cytokines, such as ICAM-1 [21], monocyte chemotactic protein-1 (MCP-1) and TNF-alpha [9] et al., and monocyte-derived macrophages migrate to the local site to mediate the inflammatory process. Infiltration of macrophages in the glomeruli and interstitium is one of the characteristic features of diabetic nephropathy, in addition to ECM expansion and interstitial fibrosis [5]. There is a report indicating that leukocyte adherence and accumulation within the vasculature is an early change in experimental diabetes [22]. In the present study we observed that in HUVEC that incubated with 200 μg/ml AGEs, macrophages migration and adhesion were dramatically increased compared with that incubated with BSA; pretreatment HUVEC with the selective PKC-beta inhibitor, LY333531, the macrophage adhesion was dramatically reduced to the normal level. This is in line with previous report. Salonen T et al. have demonstrated that LY333531 dose-dependently alleviate LPS-treated murine J774 macrophages inflammation [23]. But unexpectedly, macrophage migration was not significantly inhibited on application of LY333531 in the present study. The present concentration of LY333531 (200 nM) has been in a high and non-isoform-selective dose [24], thus this unexpected result may be attributed to the highly secretion of inflammatory cytokines that induced by AGEs; however, this hypothesis needs further investigation to demonstrate. ICAM-1 is an important adhesion molecule that controls the influx of inflammatory cells to ECs and its expression is considered as a hallmark in the etiology of atherosclerosis and other vasculitis [18,25], some scholars even regarded it as a powerful independent predictor of type 2 diabetes in initially healthy people [19]. An increase of soluble form of ICAM-1 has been observed in diabetic patients and is associated with an increase risk of developing cardiovascular disease [26]. Chow FY et al. demonstrated that in ICAM-1-deficient db/db mice the development of renal dysfunction was strikingly attenuated [27]. To detect if ICAM-1 participates in LY333531-induced reduction of macrophages adhesion to HUVEC, ICAM-1 expression was detected by immunocytochemistry and real-time RT-PCR. As shown in Fig. 4, blockade of PKC-beta before incubation with AGEs dramatically reduced the elevated ICAM-1 expression in both protein and mRNA levels; furthermore, the mRNA level was even lower than that of BSA group after LY333531 pretreatment. This is consistent with previous report that LY333531 can attenuate macrophage activation by
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Fig. 4. LY333531 decreased ICAM-1 expression in AGEs-activated HUVECs. HUVECs were incubated with 200 μg/ml BSA (Ab), 200 μg/ml AGEs (Ac), or 200 nM LY333531 + 200 μg/ml AGEs (Ad) and ICAM-1 expression was detected by immunocytochemistry SABC method; the cells were counterstained with hematoxylin. In negative control (Aa), primary antibody was replaced with PBS. Immunoreactivity appears brown as a result of DAB colorimetric reaction, and the positive staining of ICAM-1 was quantitated by Image-Pro Plus software and the difference between groups was compared (B). ICAM-1 mRNA expression was determined by real-time RT-PCR (C). ⁎⁎p b 0.01, vs. BSA; ##p b 0.01, vs. AGEs; $$p b 0.01, vs. BSA. The experiment was repeated for at least three times, and representatives were shown (magnification, 200×).
reducing ICAM-1 and MCP-1 protein expression [10]. Thus, we presume that the reduced expression of ICAM-1 may at least in part attribute to the decreased adhesion of macrophage to HUVECs. Oxidative stress (OS) is one of the key factors that participate in AGEs-induced chronic kidney disease and cell damage [28]. AGEsinduced OS has been implicated in both micro- and macro-vascular
disease, and this process is closely related with the protein kinase C activation [5]. Quagliaro L et al. demonstrated that AGEs can stimulate EC production of superoxide [20]. SOD and MDA levels have been applied in evaluating cell anti-OS ability, in which SOD as an anti-OS factor and MDA represent cell OS damage. In the present study, the ratio of SOD and MDA, SOD/MDA, was applied to evaluate cell OS
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SOD/MDA
25
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Fig. 5. LY333531 increased HUVEC anti-oxidative ability. HUVECs were incubated with vehicle, 200 μg/ml BSA, 200 μg/ml AGEs, or 200 nM LY333531 + 200 μg/ml AGEs for 48 h, and the culture supernatants were collected. Levels of SOD and MDA were detected by the detection kits according to the manufacturer's instruction. Ratio of SOD/MDA was obtained to evaluate cell anti-oxidative ability. Data was expressed as means ± SD. The experiment was repeated for three times. ⁎⁎p b 0.01, vs. BSA group. #p b 0.05, vs. AGEs group.
damage in general. Consistent with the previous report [29], we observed the obvious SOD/MDA decrement on AGEs incubation, and LY333531 dramatically increased the index to the normal level. Tumur Z et al. reported that OS was increased under inflammatory conditions, and reduction of OS can decrease ICAM-1 and MCP-1 expression [30]. Therefore, converging effects of LY333531 on decreasing ICAM-1 expression and OS damage may contribute to the reduced macrophage adhesion to HUVEC.
In diabetes, the basement membrane of endothelial cells is thickened and altered in composition, because of the enhanced synthesis of matrix proteins by TGF-beta1 activity [5,31]. Recently, it is recognized that TGF-beta1 can upregulate MCP-1 expression [32] and regulate inflammation through interleukin-1 and NF-kappa B signaling pathways [16,17]. Here we observed blockade of PKCbeta reversed AGEs-induced TGF-beta1 highly expression in both protein and mRNA levels. Converging with previous reports we presume that TGF-beta1 participate AGEs-induced macrophages migration and adhesion process, but this hypothesis needs further study. Receptor for advanced glycation end products (RAGE) is an immunoglobulin protein that mediates AGEs induced cell damage process. RAGE is minimally expressed in normal tissue and vasculature, and is up-regulated when AGE ligands accumulate [8]. The activation of RAGE can induce a series of inflammatory process, such as up-regulating adhesion molecules' expression and inducing OS. Cell and animal studies suggest that limiting RAGE expression in vascular cells could modulate expression of various proinflammatory mediators and prevent atherosclerosis development [9]. In the present study we found that blockade of PKC-beta with LY333531 obviously reduced RAGE protein expression in HUVEC. To detect the receptor mRNA expression, real-time RT-PCR was performed. In line with previous report and the present immunocytochemistry results, RAGE mRNA
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Fig. 6. LY333531 modulated RAGE expression in AGEs-induced HUVEC. HUVECs were incubated with 200 μg/ml BSA (Ab), 200 μg/ml AGEs (Ac), or 200 nM LY333531 + 200 μg/ml AGEs (Ad) for 48 h, and RAGE protein expression was detected by immunocytochemistry SABC-AP method. Immunoreactivity appears dark blue as a result of BCIP/NBT colorimetric reaction; the cells were counterstained with nuclear fast red. Primary antibody was replaced with PBS in negative control (Aa). The picture Ae showed the local magnification of Ac. ICAM-1 mRNA expression was determined by real-time RT-PCR (B). ⁎⁎p b 0.01, vs. BSA; ##p b 0.01, vs. AGEs; $$p b 0.01, vs. BSA. The experiment was repeated for three times and the representatives were shown (magnification, 200× for Aa–d; 400× for Ae).
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was dramatically increased on AGEs application; but unexpectedly, the receptor mRNA was further elevated by LY333531 pretreatment. This discrepancy with the protein expression may be attributed to the fact that there is a long way from mRNA to the protein synthesis at ribosome. On the one hand, other factors' expression that induced by LY333531 may influence RAGE protein or mRNA expression. On the other hand, this increment of mRNA expression may be the negative feedback of cells to the decrement of the receptor protein expression. As well known, the functional protein expression plays more important roles in modulating cell metabolism than that of mRNA expression; therefore, the variation/modulation of the protein expression is more significant than that of mRNA. From this aspect, the RAGE mRNA elevation doesn't counteract the fact that blockade of PKC-beta can down-regulate the receptor protein expression. Researchers have demonstrated that RAGE/ligand interaction plays a key role in the modulation of vascular injury [33,34]. Therefore, we hypothesize that LY333531's effect on reducing macrophage adhesion is through the down-regulation of RAGE protein expression. The exact intracellular mechanism is under further investigation. In conclusion, we demonstrated that the selective PKC-beta inhibitor, LY333531 can reduce AGEs-induced macrophage adhesion to HUVEC; AGEs-induced over-expression of RAGE, ICAM-1, TGFbeta1 and oxidative stress damage in HUVEC were dramatically reversed on application of LY333531. Therefore it is reasonable to conclude that AGEs-induced cell damage was via RAGE-PKC-beta pathways; and ICAM-1, TGF-beta1 and oxidative stress participate in this modulating process. Acknowledgements This work was supported in part by Macau University of Science and Technology Foundation. We would like to thank Lou Chi Han for her kind help during the study performance. References [1] Bakker W, Eringa EC, Sipkema P, van Hinsbergh VW. Endothelial dysfunction and diabetes: roles of hyperglycemia, impaired insulin signaling and obesity. Cell Tissue Res 2009;335:165–89. [2] Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;340:115–26. [3] Stehouwer CD, Lambert J, Donker AJ, van Hinsbergh VW. Endothelial dysfunction and pathogenesis of diabetic angiopathy. Cardiovasc Res 1997;34:55–68. [4] Rahman S, Rahman T, Ismail AA, Rashid AR. Diabetes-associated macrovasculopathy: pathophysiology and pathogenesis. Diabetes Obes Metab 2007;9:767–80. [5] Orasanu G, Plutzky J. The pathologic continuum of diabetic vascular disease. J Am Coll Cardiol 2009;53:S35–42. [6] Okada S, Shikata K, Matsuda M, Ogawa D, Usui H, Kido Y, et al. Intercellular adhesion molecule-1-deficient mice are resistant against renal injury after induction of diabetes. Diabetes 2003;52:2586–93. [7] Osterud B, Bjorklid E. Role of monocytes in atherogenesis. Physiol Rev 2003;83: 1069–112. [8] Goldin A, Beckman JA, Schmidt AM, Creager MA. Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation 2006;114: 597–605. [9] Figarola JL, Shanmugam N, Natarajan R, Rahbar S. Anti-inflammatory effects of the advanced glycation end product inhibitor LR-90 in human monocytes. Diabetes 2007;56:647–55. [10] Geraldes P, King GL. Activation of protein kinase C isoforms and its impact on diabetic complications. Circ Res 2010;106:1319–31. [11] Takata K, Horiuchi S, Araki N, Shiga M, Saitoh M, Morino Y. Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified proteins. J Biol Chem 1988;263:14819–25.
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[12] Ge J, Jia Q, Liang C, Luo Y, Huang D, Sun A, et al. Advanced glycosylation end products might promote atherosclerosis through inducing the immune maturation of dendritic cells. Arterioscler Thromb Vasc Biol 2005;25:2157–63. [13] Ugrinski PS, Kirkpatrick CH. Corneal cellular immunity in the guinea pig. Am J Pathol 1974;74:365–76. [14] Xia L, Wang H, Munk S, Frecker H, Goldberg HJ, Fantus IG, et al. Reactive oxygen species, PKC-beta1, and PKC-zeta mediate high-glucose-induced vascular endothelial growth factor expression in mesangial cells. Am J Physiol Endocrinol Metab 2007;293:E1280–8. [15] Fang X, Neyrinck AP, Matthay MA, Lee JW. Allogeneic human mesenchymal stem cells restore epithelial protein permeability in cultured human alveolar type II cells by secretion of angiopoietin-1. J Biol Chem 2010;285:26211–22. [16] Lu T, Tian L, Han Y, Vogelbaum M, Stark GR. Dose-dependent cross-talk between the transforming growth factor-beta and interleukin-1 signaling pathways. Proc Natl Acad Sci USA 2007;104:4365–70. [17] Yang L, Pang Y, Moses HL. TGF-beta and immune cells: an important regulatory axis in the tumor microenvironment and progression. Trends Immunol 2010;31: 220–7. [18] Xu Y, Li S. Blockade of ICAM-1: a novel way of vasculitis treatment. Biochem Biophys Res Commun 2009;381:459–61. [19] Meigs JB, Hu FB, Rifai N, Manson JE. Biomarkers of endothelial dysfunction and risk of type 2 diabetes mellitus. JAMA 2004;291:1978–86. [20] Quagliaro L, Piconi L, Assaloni R, Da Ros R, Maier A, Zuodar G, et al. Intermittent high glucose enhances ICAM-1, VCAM-1 and E-selectin expression in human umbilical vein endothelial cells in culture: the distinct role of protein kinase C and mitochondrial superoxide production. Atherosclerosis 2005;183:259–67. [21] Zhang R, Xu Y, Fu H, Wang J, Jin L, Li S. Urocortin induced expression of COX-2 and ICAM-1 via corticotrophin-releasing factor type 2 receptor in rat aortic endothelial cells. Br J Pharmacol 2009;158:819–29. [22] Miyamoto K, Khosrof S, Bursell SE, Rohan R, Murata T, Clermont AC, et al. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition. Proc Natl Acad Sci USA 1999;96:10836–41. [23] Salonen T, Sareila O, Jalonen U, Kankaanranta H, Tuominen R, Moilanen E. Inhibition of classical PKC isoenzymes downregulates STAT1 activation and iNOS expression in LPS-treated murine J774 macrophages. Br J Pharmacol 2006;147: 790–9. [24] Cai W, Rook SL, Jiang ZY, Takahara N, Aiello LP. Mechanisms of hepatocyte growth factor-induced retinal endothelial cell migration and growth. Invest Ophthalmol Vis Sci 2000;41:1885–93. [25] Gimbrone Jr MA. Endothelial dysfunction, hemodynamic forces, and atherosclerosis. Thromb Haemost 1999;82:722–6. [26] Becker A, van Hinsbergh VW, Jager A, Kostense PJ, Dekker JM, Nijpels G, et al. Why is soluble intercellular adhesion molecule-1 related to cardiovascular mortality? Eur J Clin Invest 2002;32:1–8. [27] Chow FY, Nikolic-Paterson DJ, Ozols E, Atkins RC, Tesch GH. Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice. J Am Soc Nephrol 2005;16:1711–22. [28] Lal MA, Brismar H, Eklöf AC, Aperia A. Role of oxidative stress in advanced glycation end product-induced mesangial cell activation. Kidney Int 2002;61: 2006–14. [29] Chen J, Song M, Yu S, Gao P, Yu Y, Wang H, et al. Advanced glycation endproducts alter functions and promote apoptosis in endothelial progenitor cells through receptor for advanced glycation endproducts mediate overpression of cell oxidant stress. Mol Cell Biochem 2010;335:137–46. [30] Tumur Z, Shimizu H, Enomoto A, Miyazaki H, Niwa T. Indoxyl sulfate upregulates expression of ICAM-1 and MCP-1 by oxidative stress-induced NF-kappaB activation. Am J Nephrol 2010;31:435–41. [31] Wolf G, Chen S, Ziyadeh FN. From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy. Diabetes 2005;54:1626–34. [32] Qi W, Chen X, Polhill TS, Sumual S, Twigg S, Gilbert RE, et al. TGF-beta1 induces IL-8 and MCP-1 through a connective tissue growth factor-independent pathway. Am J Physiol Renal Physiol 2006;290:F703–9. [33] Zhou Z, Wang K, Penn MS, Marso SP, Lauer MA, Forudi F, et al. Receptor for AGE (RAGE) mediates neointimal formation in response to arterial injury. Circulation 2003;107:2238–43. [34] Sakaguchi T, Yan SF, Yan SD, Belov D, Rong LL, Sousa M, et al. Central role of RAGEdependent neointimal expansion in arterial restenosis. J Clin Invest 2003;111: 959–72.
Contents lists available at ScienceDirect
International Immunopharmacology 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 / i n t i m p
Blockade of PKC-beta protects HUVEC from advanced glycation end products induced inflammation Youhua Xu a,1, Shanshan Wang a,1, Liang Feng a, Quan Zhu a,b,⁎, Ping Xiang a, Bao He b a b
Faculty of Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau Institute of Consun Co. for Chinese Medicine in Kidney Diseases, Guangdong Consun Pharmaceutical Group, Dongpeng Road 71, Guangzhou, PR China
a r t i c l e
i n f o
Article history: Received 7 July 2010 Received in revised form 15 August 2010 Accepted 10 September 2010 Keywords: Endothelial cell ICAM-1 Macrophage migration Oxidative stress TGF-beta1 PKC-beta
a b s t r a c t Advanced glycation end products (AGEs) have been recognized as a pivotal inducer in diabetes and kinds of aging-related vasculopathy. Endothelial dysfunction and inflammatory cells adhesion to endothelium have been regarded as important and early factors in the pathogenesis of vascular complications in diabetic patients. Owing to the key role of PKC-beta in AGEs-induced vascular dysfunction, we investigated effects of blocking PKC-beta by LY333531 on macrophage adhesion to HUVEC and the related mechanism. Transwell HUVEC-macrophage co-culture system was established to evaluate macrophage migration and adhesion ability. Immunocytochemistry was applied to examine TGF-beta1, ICAM-1 and RAGE protein expressions by SABC or SABC-AP method; mRNA expression of TGF-beta1, ICAM-1 and RAGE was determined by real-time RT-PCR. SOD and MDA levels in culture supernatant were detected. We found that LY333531 significantly reduced AGEs-induced macrophage adhesion to HUVEC. Blockade of PKC-beta strikingly decreased HUVEC TGF-beta1 and ICAM-1 expression in both protein and mRNA levels, RAGE protein level was also downregulated. Furthermore, the anti-oxidative stress index, SOD/MDA was dramatically elevated on LY333531 application. Therefore we conclude that LY333531 can reduce AGEs-induced macrophage adhesion to endothelial cells and relieve the local inflammation, this was realized by its effect on decreasing inflammatory cytokines' expression and increasing cell anti-oxidative ability. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction Endothelium is the major vector in angiogenesis [1], and endothelial dysfunction is regarded as an important and early factor in the pathogenesis of atherothrombosis [2] and vascular complications [3] in diabetic patients. The complications associated with diabetic vasculopathy are commonly grouped into two categories: micro- and macro-vascular complications. In diabetes, macro-vascular disease is the commonest cause of mortality and morbidity and is responsible for high incidence of vascular diseases such as stroke, myocardial infarction and peripheral vascular diseases [4]. However, the morbidity associated with diabetic micro-vascular disease, including retinopathy, neuropathy, nephropathy, and limb ischemia, is staggering [5]. Infiltration of macrophages in the glomeruli and interstitium is one of the characteristic features of diabetic nephropAbbreviations: ECs, endothelial cells; TGF-beta1, transforming growth factor-beta1; OS, oxidative stress; ICAM-1, intercellular adhesion molecule-1; MCP-1, monocyte chemotactic protein-1. ⁎ Corresponding author. Institute of Consun Co. for Chinese Medicine in Kidney Diseases, Guangdong Consun Pharmaceutical Group, Dongpeng Road 71, Guangzhou 510530, PR China. Tel.: + 86 20 82267589; fax: + 86 20 82017468. E-mail address: [email protected] (Q. Zhu). 1 Youhua Xu and Shanshan Wang contributed equally to the work.
athy, in addition to ECM expansion and interstitial fibrosis [6]. Recruitment of monocyte/macrophage and lymphocytes from the peripheral blood to the endothelial cells (ECs) is an early and central event in vascular dysfunction development [5,7] and experimental diabetes [22], thus inflammation plays a pivotal role in the initiation of this process. Advanced glycation end products (AGEs) have been widely studied in its key role in promoting vascular dysfunction and diabetes development [5]. AGEs impair vessel functions via the receptor for advanced glycation end products (RAGE) [8]. The activation of RAGE may induce a series of inflammatory process, such as up-regulating adhesion molecules' expression, promoting intima proliferation and angiogenesis, and inducing oxidative stress. Cell and animal studies suggest that limiting RAGE expression in vascular cells could modulate expression of various pro-inflammatory mediators and prevent vascular dysfunction development [9]. PKC-beta has been recognized as a key mediator in AGEs-induced micro- and macro-vascular dysfunction [10]. Studies have demonstrated that AGEs can activate PKC-beta and induce a series of pathophysiological changes in cells [10]. In the present study, we demonstrated that selective blockade of PKC-beta with LY333531 can reverse AGEs' effect on macrophage adhesion to HUVEC and reduce AGEs-induced ECs damage.
1567-5769/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2010.09.006
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2. Materials and methods
2.4. Macrophages migration and adhesion assay
2.1. Animals and reagents
Macrophages were collected from mice abdomen according to the reports [13] with minor adjustment. In general, mice were executed by cervical dislocation, and peritoneal exudates were induced by one intraperitoneal injection of 20–25 ml sterile cold phosphate-buffer solution (PBS, pH 7.4). Ten minutes later, abdominal walls were shaved and cleansed with alcohol. Under sterile conditions, the peritoneal exudates were expelled by syringe and centrifuged at room temperature; after red blood cell quassation by 0.83% NH4Cl, the solution was centrifuged at room temperature for two times and the macrophages were re-suspended by DMEM and adjusted to the density of 1 × 106. To study macrophage migration (Fig. 1), HUVECs were firstly seeded on the coverslips in 24-well plates at confluence density of 80– 90%. HUVECs were starved and incubated with BSA (200 μg/ml), AGEs (200 μg/ml) [12], or LY333531 (200 nM) [14] + AGEs (200 μg/ml) for 24 h. Then the 24-well plates were inserted with Transwell inserts (5.0 μm pore size polycarbonate membrane, Corning Costar Corporation, Cambridge, MA); the macrophages were plated into inserts and co-cultured with HUVEC for further 24 h. In this system [15], macrophages were grown on upper compartment of the Transwell microporous membranes. At 48 h, macrophages in the upper compartment were gently wiped with cotton to remove cells that didn't pass through the membrane, then the whole microporous membranes were stained with 10% crystal violet for 10 min; after washing with PBS, the upper side of the microporous membrane and the upper compartment walls were carefully wiped with cotton. Then the membranes were carefully clipped with razor blade (for microscopy observation) or washed with 30% glacial acetic acid (for macrophage counting determination). For macrophage counting determination, the washing solution was transferred to 96-well plate (Corning Costar) and detected at the wavelength of 580 nm.
All animal care and this investigation conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and were approved by the Ethical Review Board of Macau University of Science and Technology and Consun Pharmaceutical Group. Male Swiss mice weighing 20–25 g were supplied by the Shanghai laboratory animal centre; food and water were given ad libitum. The selective PKC-beta inhibitor, LY333531, was purchased from Alexis Biotechnologies (Nottingham, UK). Polyclonal ICAM-1 antibody was obtained from Boster (Wuhan, China); polyclonal TGF-beta1 and monoclonal RAGE antibodies were supplied by Santa Cruz Biotechnology (California, USA). The SOD and MDA detection kits were supplied by Jiancheng (Nanjing, China). All other reagents used were derived from commercial sources. 2.2. Preparation of advanced glycation end products AGEs were prepared as previously described [11,12]. In general, BSA was added into 10 mM/L PBS (pH 7.4, concentration of 5 g/L), incubated with 50 mM/L D-glucose in 95% air/5% CO2 at 37 °C for 12 weeks. Unincorporated glucose was removed by dialysis overnight against 0.01 M PBS. AGEs were stored at −20 °C until use. 2.3. Culture of Human Umbilical Vein Endothelial Cells Human Umbilical Vein Endothelial Cell (HUVEC) line was purchased from ATCC (Manassas, VA, USA). Cells were cultured in DMEM (Gibco) supplemented with 10% FBS (fetal bovine serum), 1% penicillin–streptomycin, at 37 °C in a 95% air/5% CO2 incubator.
A Upper Compartment
Macrophage Lower Compartment
HUVEC 5.0 m Microporous Membrane Coverslip
B Drug incubation end; HUVEC Cell seeding Starving
-24h
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0h
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Fig. 1. Diagram of macrophage migration detection by Transwell. (A) Composition diagram of Transwell. (B) The detection flow of macrophage migration by Transwell. Following different treatment as described in Section 2.4, macrophage migration through microporous membranes was detected by 10% crystal violet staining or 30% glacial acetic acid washing.
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Fig. 2. Macrophage migration assay by crystal violet staining (A) or chromatometry (B). HUVECs at lower compartment were incubated with 200 μg/ml BSA (Aa), 200 μg/ml AGEs (Ab), or 200 nM LY333531 + 200 μg/ml AGEs (Ac) and macrophages migrated through the membrane were stained by 10% crystal violet and observed under microscope (magnification, 200×). For macrophage counting (B), the crystal violet was washed out by 30% glacial acetic acid and the washing solution was detected at wavelength of 580 nm, the data was expressed as means ± SD; ⁎p = 0.073, vs. BSA group. The experiment was repeated for three times, and representative pictures were shown.
HUVECs seeded on the coverslips in the lower compartment were performed with immunocytochemistry detection of TGF-beta1 and macrophage adhesion. 2.5. Immunocytochemistry analysis Immunocytochemistry staining was performed using the StreptAvidin-Biotin-enzyme Complex (SABC) or SABC-alkaline phosphatase (AP) method. Cells were grown on glass coverslips in 24-well plates. Following different treatments as described in Section 2.4, the cells were washed with phosphate-buffer solution (PBS, pH 7.4), fixed with fresh 4% paraformaldehyde for 60 min and blocked with 10% primary antibody-origin serum or 5% BSA (for RAGE detection) for 20 min at room temperature. After being blocked, the cells were incubated at 37 °C with primary antibodies, including ICAM-1 (1:200), TGF-beta1 (1:400) and RAGE (1:200), and then given a sequential 20 min incubation with biotin-conjugated secondary antibody and SABC or SABC-AP at 37 °C. Color was developed by 3,3'-diaminobenzidine (DAB) or BCIP/NBT (for RAGE). Finally, the cells were counterstained with hematoxylin or nuclear fast red (for RAGE) and photographed under microscope. Positive staining was indicated with brown or dark blue (for RAGE) deposits. In negative control, primary antibody was replaced with PBS. 2.6. Real-time RT-PCR analysis HUVECs were plated in flasks and grown to 80–90% confluence. After starvation overnight, cells were incubated with drugs for
24 h. Total RNA was extracted by Trizol (Invitrogen, Carlsbad, CA) and reverse-transcribed using M-MLV RTase (Promega). Quantitative SYBR Green real-time PCR assay was performed to measure expression of mRNAs for TGF-beta1, ICAM-1 and RAGE. Briefly, 1 μl of template cDNA was added to a final volume of 0.4 μM of the primers. Amplification was performed with 40 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, and elongation at 72 °C for 15 s. Sequences of the primers used for real-time PCR were as follows: TGF-beta1 forward 5'GCCCTCCTACCTTTTGC-3' and reverse 5'-AGGCGTCAGCACCAGTAG-3', ICAM-1 forward 5'-GTGGTAGCAGCCGCAGT-3' and reverse 5'-TTCGGTTTCATGGGGGT-3', RAGE forward 5'CTGGTGCTGAAGTGTAAGGG-3' and reverse 5'-GAAGAGGGAGCCGTTGG-3'. During thermal cycling, emission from each sample was recorded and calculated to produce threshold cycle (Ct) values for each sample. The housekeeping gene β-actin was used for internal normalization. The transcript copy number of target gene was determined on the basis of their Ct values.
2.7. Determination of SOD/MDA Levels of SOD and MDA in culture supernatant were determined by SOD and MDA detection kits within 6 h according to the manufacturer's protocol. The final concentrations of SOD and MDA were calculated by the equations supplied by the kits producer.
Fig. 3. Blockade of PKC-beta reduced AGEs-induced TGF-beta1 expression and macrophage adhesion to HUVEC. HUVECs at lower compartment of Transwell system were incubated with 200 μg/ml BSA (Ab), 200 μg/ml AGEs (Ac), or 200 nM LY333531 + 200 μg/ml AGEs (Ad) and TGF-beta1 expression was detected by immunocytochemistry SABC method; HUVECs were counterstained with hematoxylin. Number of macrophages that adhered with HUVECs was counted and quantitated under microscope and compared by the SPSS (B). TGF-beta1 immunoreactivity appears brown as a result of DAB colorimetric reaction, the positive staining of TGF-beta1 was quantitated by Image-Pro Plus software and the difference between groups was compared (C). White arrows show macrophages adhesion with HUVEC; black arrows show positive brown color. In negative control (Aa), primary antibody was replaced with PBS. TGF-beta1 mRNA expression was determined by real-time RT-PCR (D). ⁎⁎p b 0.01, vs. BSA; #p b 0.05, vs. AGEs; ##p b 0.01, vs. AGEs. The experiment was repeated for three times, and representatives were shown (magnification, 200×).
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2.8. Statistical analysis Data were expressed as means ± SD. The significance for the difference among groups was analyzed with SPSS 13.0 by one-way ANOVA. Differences were considered to be statistically significant at p value of b0.05. 3. Results
with LY333531 increased RAGE mRNA to the level even higher than that of AGEs group (p b 0.01, vs. AGEs and BSA groups). This discrepancy with that of protein expression may be attributed to the fact that there is a long process from mRNA to the synthesis of protein at ribosome, other factors' expression that induced by LY333531 may influence RAGE protein or mRNA expression. On the other hand, this increment of mRNA expression may be the negative feedback of cells to the decrement of the receptor protein expression. But the accurate mechanism needs investigation.
3.1. Blockade of PKC-beta reduced macrophages adhesion to HUVEC 4. Discussion As shown in Fig. 2, incubation with AGEs (200 μg/ml) increased macrophage migration through the Transwell microporous membranes (p = 0.073, vs. BSA). Unexpectedly, pretreatment HUVEC with 200 nM of the selective PKC-beta inhibitor, LY333531, cannot reduce AGEs-induced macrophage migration through membranes (p = 0.447, vs. AGEs). To observe if there is more macrophages adhesion to HUVEC in the lower compartment, immunocytochemistry detection was performed. As shown in Fig. 3A and B, AGEs significantly increased macrophage adhesion to HUVEC (Fig. 3Ac), while pretreatment the cells with LY333531 dramatically reduced AGEs' effect on the adhesion (Fig. 3Ad and B). 3.2. LY333531 decreased AGEs-induced TGF-beta1 and ICAM-1 expression in HUVEC TGF-beta1 has been implicated in participating AGEs-induced inflammation [16,17]. In the present study, we observed that AGEs increased TGF-beta1 expression in both protein (Fig. 3A and C) and mRNA (Fig. 3D) levels. Pretreatment HUVEC with LY333531 significantly reduced AGEs-induced TGF-beta1 expression to the normal level (Fig. 3A, C and D). ICAM-1, as a pivotal adhesion molecule that mediates inflammatory cell adhesion, has been recognized as an important factor in the development of vasculitis [18] and AGEs-induced chronic kidney disease [19]. Here we observed that AGEs increased ICAM-1 protein (Fig. 4Ac and B) expression in HUVEC, and blockade of PKC-beta with LY333531 (Fig. 4Ad) reversed its expression to nearly the normal levels as compared with BSA group (Fig. 4B; p b 0.01, vs. AGEs group; p N 0.05, vs. BSA group). Furthermore, the mRNA expression of ICAM-1 after LY333531 incubation was reduced to the level even lower than that of BSA group (Fig. 4C; p b 0.01, vs. BSA group). 3.3. LY333531 pretreatment elevated cell anti-oxidative ability It has been well established that AGEs-induced oxidative stress (OS) plays important role in vascular dysfunction [20]. Here we found that (Fig. 5) the anti-OS index, SOD/MDA was dramatically decreased in HUVEC incubated with AGEs compared with that incubated with BSA (p b 0.01). To assess if blockade of PKC-beta can protect HUVEC from OS damage, cells were pretreated with LY333531 followed by AGEs. As depicted in Fig. 5, blockade of PKC-beta increased HUVEC's anti-OS ability to the normal level (p b 0.05, vs. AGEs; p = 0.229, vs. BSA). 3.4. AGEs induced HUVEC inflammation via RAGE-PKC-beta pathway Receptor for advanced glycation end products (RAGE) is known to mediate AGEs-induced cell damage [8]. Here we observed that AGEs dramatically elevated RAGE protein expression in HUVEC (Fig. 6Ac) and blockade of PKC-beta with LY333531 reversed this effect (Fig. 6Ad). In order to examine if RAGE mRNA expression is in line with the protein expression, real-time RT-PCR was performed. As depicted in Fig. 6B, AGEs incubation significantly increased RAGE mRNA expression (p b 0.01, vs. BSA group); unexpectedly, pretreatment HUVEC
In diabetes, vascular disease is the commonest cause of mortality and morbidity and is responsible for high incidence of stroke, myocardial infarction and peripheral vascular diseases [4]. Advanced glycation end products (AGEs) have been widely studied in its key role in promoting vascular inflammation [5]. Reports have demonstrated that AGEs play a role in kinds of vasculitis such as atherosclerosis and diabetic retinopathy [5]. Macrophages migration and adhesion to endothelial cells (ECs) is a key step in initiating the local inflammation process. In the present study, we demonstrated that blockade of PKC-beta with LY333531 can significantly reduce AGEs-induced macrophage adhesion to HUVEC, in which downregulation of inflammatory cytokines' expression may mediate this process. ECs play in a central role in the pathologic of vascular dysfunction in type 2 diabetic patients [5]. Under inflammatory conditions, ECs secrete a series of inflammatory cytokines, such as ICAM-1 [21], monocyte chemotactic protein-1 (MCP-1) and TNF-alpha [9] et al., and monocyte-derived macrophages migrate to the local site to mediate the inflammatory process. Infiltration of macrophages in the glomeruli and interstitium is one of the characteristic features of diabetic nephropathy, in addition to ECM expansion and interstitial fibrosis [5]. There is a report indicating that leukocyte adherence and accumulation within the vasculature is an early change in experimental diabetes [22]. In the present study we observed that in HUVEC that incubated with 200 μg/ml AGEs, macrophages migration and adhesion were dramatically increased compared with that incubated with BSA; pretreatment HUVEC with the selective PKC-beta inhibitor, LY333531, the macrophage adhesion was dramatically reduced to the normal level. This is in line with previous report. Salonen T et al. have demonstrated that LY333531 dose-dependently alleviate LPS-treated murine J774 macrophages inflammation [23]. But unexpectedly, macrophage migration was not significantly inhibited on application of LY333531 in the present study. The present concentration of LY333531 (200 nM) has been in a high and non-isoform-selective dose [24], thus this unexpected result may be attributed to the highly secretion of inflammatory cytokines that induced by AGEs; however, this hypothesis needs further investigation to demonstrate. ICAM-1 is an important adhesion molecule that controls the influx of inflammatory cells to ECs and its expression is considered as a hallmark in the etiology of atherosclerosis and other vasculitis [18,25], some scholars even regarded it as a powerful independent predictor of type 2 diabetes in initially healthy people [19]. An increase of soluble form of ICAM-1 has been observed in diabetic patients and is associated with an increase risk of developing cardiovascular disease [26]. Chow FY et al. demonstrated that in ICAM-1-deficient db/db mice the development of renal dysfunction was strikingly attenuated [27]. To detect if ICAM-1 participates in LY333531-induced reduction of macrophages adhesion to HUVEC, ICAM-1 expression was detected by immunocytochemistry and real-time RT-PCR. As shown in Fig. 4, blockade of PKC-beta before incubation with AGEs dramatically reduced the elevated ICAM-1 expression in both protein and mRNA levels; furthermore, the mRNA level was even lower than that of BSA group after LY333531 pretreatment. This is consistent with previous report that LY333531 can attenuate macrophage activation by
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Fig. 4. LY333531 decreased ICAM-1 expression in AGEs-activated HUVECs. HUVECs were incubated with 200 μg/ml BSA (Ab), 200 μg/ml AGEs (Ac), or 200 nM LY333531 + 200 μg/ml AGEs (Ad) and ICAM-1 expression was detected by immunocytochemistry SABC method; the cells were counterstained with hematoxylin. In negative control (Aa), primary antibody was replaced with PBS. Immunoreactivity appears brown as a result of DAB colorimetric reaction, and the positive staining of ICAM-1 was quantitated by Image-Pro Plus software and the difference between groups was compared (B). ICAM-1 mRNA expression was determined by real-time RT-PCR (C). ⁎⁎p b 0.01, vs. BSA; ##p b 0.01, vs. AGEs; $$p b 0.01, vs. BSA. The experiment was repeated for at least three times, and representatives were shown (magnification, 200×).
reducing ICAM-1 and MCP-1 protein expression [10]. Thus, we presume that the reduced expression of ICAM-1 may at least in part attribute to the decreased adhesion of macrophage to HUVECs. Oxidative stress (OS) is one of the key factors that participate in AGEs-induced chronic kidney disease and cell damage [28]. AGEsinduced OS has been implicated in both micro- and macro-vascular
disease, and this process is closely related with the protein kinase C activation [5]. Quagliaro L et al. demonstrated that AGEs can stimulate EC production of superoxide [20]. SOD and MDA levels have been applied in evaluating cell anti-OS ability, in which SOD as an anti-OS factor and MDA represent cell OS damage. In the present study, the ratio of SOD and MDA, SOD/MDA, was applied to evaluate cell OS
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SOD/MDA
25
#
20
**
15 10 5 0
Normal
BSA
AGEs
LY333531+AGEs
Fig. 5. LY333531 increased HUVEC anti-oxidative ability. HUVECs were incubated with vehicle, 200 μg/ml BSA, 200 μg/ml AGEs, or 200 nM LY333531 + 200 μg/ml AGEs for 48 h, and the culture supernatants were collected. Levels of SOD and MDA were detected by the detection kits according to the manufacturer's instruction. Ratio of SOD/MDA was obtained to evaluate cell anti-oxidative ability. Data was expressed as means ± SD. The experiment was repeated for three times. ⁎⁎p b 0.01, vs. BSA group. #p b 0.05, vs. AGEs group.
damage in general. Consistent with the previous report [29], we observed the obvious SOD/MDA decrement on AGEs incubation, and LY333531 dramatically increased the index to the normal level. Tumur Z et al. reported that OS was increased under inflammatory conditions, and reduction of OS can decrease ICAM-1 and MCP-1 expression [30]. Therefore, converging effects of LY333531 on decreasing ICAM-1 expression and OS damage may contribute to the reduced macrophage adhesion to HUVEC.
In diabetes, the basement membrane of endothelial cells is thickened and altered in composition, because of the enhanced synthesis of matrix proteins by TGF-beta1 activity [5,31]. Recently, it is recognized that TGF-beta1 can upregulate MCP-1 expression [32] and regulate inflammation through interleukin-1 and NF-kappa B signaling pathways [16,17]. Here we observed blockade of PKCbeta reversed AGEs-induced TGF-beta1 highly expression in both protein and mRNA levels. Converging with previous reports we presume that TGF-beta1 participate AGEs-induced macrophages migration and adhesion process, but this hypothesis needs further study. Receptor for advanced glycation end products (RAGE) is an immunoglobulin protein that mediates AGEs induced cell damage process. RAGE is minimally expressed in normal tissue and vasculature, and is up-regulated when AGE ligands accumulate [8]. The activation of RAGE can induce a series of inflammatory process, such as up-regulating adhesion molecules' expression and inducing OS. Cell and animal studies suggest that limiting RAGE expression in vascular cells could modulate expression of various proinflammatory mediators and prevent atherosclerosis development [9]. In the present study we found that blockade of PKC-beta with LY333531 obviously reduced RAGE protein expression in HUVEC. To detect the receptor mRNA expression, real-time RT-PCR was performed. In line with previous report and the present immunocytochemistry results, RAGE mRNA
A a
b
d
e
c
RAGE mRNA expression (ratio of BSA)
B 120
##$$
100 80 60 40
**
20 0
BSA
AGEs
LY333531+AGEs
Fig. 6. LY333531 modulated RAGE expression in AGEs-induced HUVEC. HUVECs were incubated with 200 μg/ml BSA (Ab), 200 μg/ml AGEs (Ac), or 200 nM LY333531 + 200 μg/ml AGEs (Ad) for 48 h, and RAGE protein expression was detected by immunocytochemistry SABC-AP method. Immunoreactivity appears dark blue as a result of BCIP/NBT colorimetric reaction; the cells were counterstained with nuclear fast red. Primary antibody was replaced with PBS in negative control (Aa). The picture Ae showed the local magnification of Ac. ICAM-1 mRNA expression was determined by real-time RT-PCR (B). ⁎⁎p b 0.01, vs. BSA; ##p b 0.01, vs. AGEs; $$p b 0.01, vs. BSA. The experiment was repeated for three times and the representatives were shown (magnification, 200× for Aa–d; 400× for Ae).
Y. Xu et al. / International Immunopharmacology 10 (2010) 1552–1559
was dramatically increased on AGEs application; but unexpectedly, the receptor mRNA was further elevated by LY333531 pretreatment. This discrepancy with the protein expression may be attributed to the fact that there is a long way from mRNA to the protein synthesis at ribosome. On the one hand, other factors' expression that induced by LY333531 may influence RAGE protein or mRNA expression. On the other hand, this increment of mRNA expression may be the negative feedback of cells to the decrement of the receptor protein expression. As well known, the functional protein expression plays more important roles in modulating cell metabolism than that of mRNA expression; therefore, the variation/modulation of the protein expression is more significant than that of mRNA. From this aspect, the RAGE mRNA elevation doesn't counteract the fact that blockade of PKC-beta can down-regulate the receptor protein expression. Researchers have demonstrated that RAGE/ligand interaction plays a key role in the modulation of vascular injury [33,34]. Therefore, we hypothesize that LY333531's effect on reducing macrophage adhesion is through the down-regulation of RAGE protein expression. The exact intracellular mechanism is under further investigation. In conclusion, we demonstrated that the selective PKC-beta inhibitor, LY333531 can reduce AGEs-induced macrophage adhesion to HUVEC; AGEs-induced over-expression of RAGE, ICAM-1, TGFbeta1 and oxidative stress damage in HUVEC were dramatically reversed on application of LY333531. Therefore it is reasonable to conclude that AGEs-induced cell damage was via RAGE-PKC-beta pathways; and ICAM-1, TGF-beta1 and oxidative stress participate in this modulating process. Acknowledgements This work was supported in part by Macau University of Science and Technology Foundation. We would like to thank Lou Chi Han for her kind help during the study performance. References [1] Bakker W, Eringa EC, Sipkema P, van Hinsbergh VW. Endothelial dysfunction and diabetes: roles of hyperglycemia, impaired insulin signaling and obesity. Cell Tissue Res 2009;335:165–89. [2] Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;340:115–26. [3] Stehouwer CD, Lambert J, Donker AJ, van Hinsbergh VW. Endothelial dysfunction and pathogenesis of diabetic angiopathy. Cardiovasc Res 1997;34:55–68. [4] Rahman S, Rahman T, Ismail AA, Rashid AR. Diabetes-associated macrovasculopathy: pathophysiology and pathogenesis. Diabetes Obes Metab 2007;9:767–80. [5] Orasanu G, Plutzky J. The pathologic continuum of diabetic vascular disease. J Am Coll Cardiol 2009;53:S35–42. [6] Okada S, Shikata K, Matsuda M, Ogawa D, Usui H, Kido Y, et al. Intercellular adhesion molecule-1-deficient mice are resistant against renal injury after induction of diabetes. Diabetes 2003;52:2586–93. [7] Osterud B, Bjorklid E. Role of monocytes in atherogenesis. Physiol Rev 2003;83: 1069–112. [8] Goldin A, Beckman JA, Schmidt AM, Creager MA. Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation 2006;114: 597–605. [9] Figarola JL, Shanmugam N, Natarajan R, Rahbar S. Anti-inflammatory effects of the advanced glycation end product inhibitor LR-90 in human monocytes. Diabetes 2007;56:647–55. [10] Geraldes P, King GL. Activation of protein kinase C isoforms and its impact on diabetic complications. Circ Res 2010;106:1319–31. [11] Takata K, Horiuchi S, Araki N, Shiga M, Saitoh M, Morino Y. Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified proteins. J Biol Chem 1988;263:14819–25.
1559
[12] Ge J, Jia Q, Liang C, Luo Y, Huang D, Sun A, et al. Advanced glycosylation end products might promote atherosclerosis through inducing the immune maturation of dendritic cells. Arterioscler Thromb Vasc Biol 2005;25:2157–63. [13] Ugrinski PS, Kirkpatrick CH. Corneal cellular immunity in the guinea pig. Am J Pathol 1974;74:365–76. [14] Xia L, Wang H, Munk S, Frecker H, Goldberg HJ, Fantus IG, et al. Reactive oxygen species, PKC-beta1, and PKC-zeta mediate high-glucose-induced vascular endothelial growth factor expression in mesangial cells. Am J Physiol Endocrinol Metab 2007;293:E1280–8. [15] Fang X, Neyrinck AP, Matthay MA, Lee JW. Allogeneic human mesenchymal stem cells restore epithelial protein permeability in cultured human alveolar type II cells by secretion of angiopoietin-1. J Biol Chem 2010;285:26211–22. [16] Lu T, Tian L, Han Y, Vogelbaum M, Stark GR. Dose-dependent cross-talk between the transforming growth factor-beta and interleukin-1 signaling pathways. Proc Natl Acad Sci USA 2007;104:4365–70. [17] Yang L, Pang Y, Moses HL. TGF-beta and immune cells: an important regulatory axis in the tumor microenvironment and progression. Trends Immunol 2010;31: 220–7. [18] Xu Y, Li S. Blockade of ICAM-1: a novel way of vasculitis treatment. Biochem Biophys Res Commun 2009;381:459–61. [19] Meigs JB, Hu FB, Rifai N, Manson JE. Biomarkers of endothelial dysfunction and risk of type 2 diabetes mellitus. JAMA 2004;291:1978–86. [20] Quagliaro L, Piconi L, Assaloni R, Da Ros R, Maier A, Zuodar G, et al. Intermittent high glucose enhances ICAM-1, VCAM-1 and E-selectin expression in human umbilical vein endothelial cells in culture: the distinct role of protein kinase C and mitochondrial superoxide production. Atherosclerosis 2005;183:259–67. [21] Zhang R, Xu Y, Fu H, Wang J, Jin L, Li S. Urocortin induced expression of COX-2 and ICAM-1 via corticotrophin-releasing factor type 2 receptor in rat aortic endothelial cells. Br J Pharmacol 2009;158:819–29. [22] Miyamoto K, Khosrof S, Bursell SE, Rohan R, Murata T, Clermont AC, et al. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition. Proc Natl Acad Sci USA 1999;96:10836–41. [23] Salonen T, Sareila O, Jalonen U, Kankaanranta H, Tuominen R, Moilanen E. Inhibition of classical PKC isoenzymes downregulates STAT1 activation and iNOS expression in LPS-treated murine J774 macrophages. Br J Pharmacol 2006;147: 790–9. [24] Cai W, Rook SL, Jiang ZY, Takahara N, Aiello LP. Mechanisms of hepatocyte growth factor-induced retinal endothelial cell migration and growth. Invest Ophthalmol Vis Sci 2000;41:1885–93. [25] Gimbrone Jr MA. Endothelial dysfunction, hemodynamic forces, and atherosclerosis. Thromb Haemost 1999;82:722–6. [26] Becker A, van Hinsbergh VW, Jager A, Kostense PJ, Dekker JM, Nijpels G, et al. Why is soluble intercellular adhesion molecule-1 related to cardiovascular mortality? Eur J Clin Invest 2002;32:1–8. [27] Chow FY, Nikolic-Paterson DJ, Ozols E, Atkins RC, Tesch GH. Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice. J Am Soc Nephrol 2005;16:1711–22. [28] Lal MA, Brismar H, Eklöf AC, Aperia A. Role of oxidative stress in advanced glycation end product-induced mesangial cell activation. Kidney Int 2002;61: 2006–14. [29] Chen J, Song M, Yu S, Gao P, Yu Y, Wang H, et al. Advanced glycation endproducts alter functions and promote apoptosis in endothelial progenitor cells through receptor for advanced glycation endproducts mediate overpression of cell oxidant stress. Mol Cell Biochem 2010;335:137–46. [30] Tumur Z, Shimizu H, Enomoto A, Miyazaki H, Niwa T. Indoxyl sulfate upregulates expression of ICAM-1 and MCP-1 by oxidative stress-induced NF-kappaB activation. Am J Nephrol 2010;31:435–41. [31] Wolf G, Chen S, Ziyadeh FN. From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy. Diabetes 2005;54:1626–34. [32] Qi W, Chen X, Polhill TS, Sumual S, Twigg S, Gilbert RE, et al. TGF-beta1 induces IL-8 and MCP-1 through a connective tissue growth factor-independent pathway. Am J Physiol Renal Physiol 2006;290:F703–9. [33] Zhou Z, Wang K, Penn MS, Marso SP, Lauer MA, Forudi F, et al. Receptor for AGE (RAGE) mediates neointimal formation in response to arterial injury. Circulation 2003;107:2238–43. [34] Sakaguchi T, Yan SF, Yan SD, Belov D, Rong LL, Sousa M, et al. Central role of RAGEdependent neointimal expansion in arterial restenosis. J Clin Invest 2003;111: 959–72.