cell biochemistry and function Cell Biochem Funct 2015; 33: 226–234. Published online 23 April 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.3108
Chinese medicine Tongxinluo increases tight junction protein levels by inducing KLF5 expression in microvascular endothelial cells Li-Min Li, Bing Zheng, Ruo-Nan Zhang, Li-Shuang Jin, Cui-Ying Zheng, Chang Wang, Pei-Pei Zhou, Zong-Wei Guo, Dong Ma and Jin-Kun Wen* Department of Biochemistry and Molecular Biology, Key Laboratory of Neural and Vascular Biology, China Administration of Education, Hebei Medical University, Shijiazhuang, China
Tongxinluo (TXL) is a compound prescription formulated according to the meridian theory of traditional Chinese medicine. It may play an important role in cardiovascular protection by improving endothelial cell function. The aim of present study was to investigate whether endothelial protection with TXL is related to its regulation of tight junction protein expression. Human cardiac microvascular endothelial cells (HCMECs) were cultured and treated with 10 7 mol l 1 angiotensin II (Ang II) and the different doses of TXL; the expression of tight junction proteins occludin, claudin, VE-cadherin and beta-catenin was determined by Western blotting and real-time PCR. Gain-of-function and loss-of-function of Krüppel-like factor 5 (KLF5) were carried out in HCMEC transfected with either KLF5 adenovirus pAd-KLF5 or siRNA specific for KLF5. Angiotensinogen transgenic mice were treated with TXL by oral administration of TXL of 0.75 g kg 1 day 1, and immunohistochemical staining was performed with antioccludin, anticlaudin, anti-VE-cadherin, antibeta-catenin and anti-KLF5 antibodies. Ang II treatment significantly reduced the expression of tight junction proteins occludin, claudin, VE-cadherin and beta-catenin in cultured HCMECs. TXL pretreatment could abrogate the down-regulation of these tight junction proteins induced by Ang II. Ang II treatment also decreased KLF5 expression at the mRNA and protein levels; TXL pretreatment markedly reversed the inhibitory effect of Ang II on KLF5 expression. Gain-of-function and loss-of-function of KLF5 showed that KLF5 mediated the expression of tight junction proteins in HCMECs. TXL-enhanced expression of the tight junction proteins was mediated by KLF5. In angiotensinogen transgenic mice, TXL also increased the tight junction protein levels by inducing KLF5 expression. Chinese medicine TXL increases tight junction protein levels by inducing KLF5 expression in microvascular endothelial cells. Copyright © 2015 John Wiley & Sons, Ltd. key words—tongxinluo; tight junction protein; KLF5; endothelial cell; angiotensin II
INTRODUCTION Vascular endothelium is the monolayer of endothelial cells lining the lumen of blood vessels and is located between the blood and vascular tissue; it cannot only complete metabolic exchange between blood and tissue fluid but also can synthesize and secrete a variety of biologically active substances. The vascular endothelial dysfunction has been implicated in the development of vascular diseases;1 many high-risk factors are becoming potential risk factors in the development of vascular endothelial dysfunction, such as hypertension, diabetes and harmful social and psychological stresses.2 Recent studies suggest that in the early stage of cardiovascular diseases, endothelial function has been damaged. Thus, repairing vascular endothelial injury and improving endothelial dysfunction are a new direction in the field of cardiovascular diseases.
*Correspondence to: Jin-Kun Wen, Department of Biochemistry and Molecular Biology, Hebei Medical University, No. 361, Zhongshan East Road, Shijiazhuang, 050017, China. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
Tight interendothelial junctions are responsible for the formation and maintenance of the permeability barrier,3 both to the passage of ions and molecules through the paracellular pathway and to the movement of proteins and lipids between the apical and the basolateral domains of the plasma membrane.4 Tight junctions are critical for normal vascular physiologic function.5 Abnormal expression of tight junction proteins can lead to vascular endothelial dysfunction and causes various cardiovascular diseases. It has been known that many transmembrane proteins, such as occludin, claudin, VE-cadherin and beta-catenin, are located at the tight junctions of endothelial cells and are involved in the regulation of endothelial functions including paracellular permeability and intracellular signal transduction. Tongxinluo (TXL) is a traditional Chinese medicine that is extracted, concentrated, freeze-dried and standardized from a mixture of 12 medicinal constituents.6 It is approved in 1996 by the State Food and Drug Administration of China for treatment of angina pectoris and ischemic stroke7 and has been widely used in China to treat patients with cardiovascular8 and cerebrovascular diseases.7 The previous Received 7 January 2015 Revised 18 March 2015 Accepted 23 March 2015
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txl increases tight junction protein levels studies have revealed that treatment with TXL can improve endothelial cell function,9 lower lipid,8 reduce inflammation and apoptosis,7,10 inhibit neointima hyperplasia,10 protect the brain from Blood-brain barrier (BBB) disruption11 and promote angiogenesis.12,13 However, the effects of TXL treatment on the expression of tight junction proteins in the endothelial cells and its action mechanisms are still unclear. Angiotensin II (Ang II), a bioactive peptide regulating vascular tone and promoting vascular smooth muscle cell proliferation, plays a key role in the pathogenesis of cardiovascular diseases.14 As a basic transcription factor, Krueppel-like factor 5 (KLF5) is known to be involved in important biological processes such as cell proliferation and differentiation.15–17 In addition to its pro-proliferation and prodifferentiation roles, KLF5 is also implicated in injury response and the activation of inflammatory pathways following exposure to inflammatory stimuli.18 However, it remains unclear whether protective effects of TXL on Ang II-induced endothelial damage are mediated by KLF5. In this study, we investigated whether and how TXL protects endothelial function by regulating tight junction protein expression.
according to the Chinese Pharmacopoeia 2005. TXL contains 12 medicinal components, which were ground to superfine powder with the diameter ≤10 μm by a micronizer and prepared as capsules. TXL powder was weighed and dissolved in the phosphate-buffered saline. The ultrasound technology was used to promote the melting for about 1 h. The drug was then centrifuged at 1000 × g for 10 min, and the supernatant was put into the microfilter (0.22 μm) to eliminate bacteria, which was then aliquoted and stored at 20 °C before use. Fingerprint chromatography of TXL capsule To reduce the dose variability of TXL capsule among different batches, the species, origin, harvest time, medicinal parts and concocted methods for each component were strictly standardized. Moreover, high-performance liquid chromatography, high-performance capillary electrophoresis and chromatography were applied to quantitate the components of the capsule.7 To assess the dosing reproducibility of TXL capsule, high performance liquid chromatography was applied to record fingerprint chromatograms of the aqueous extracts of the ten different batches for similarity analysis.
MATERIAL AND METHODS
Ethics statement
Components and preparation of TXL
All animal studies were approved by the Ethics Committee of Hebei Medical University, which is fully accredited by the Institutional Animal Care and Use Committee, and all efforts were made to minimize suffering.
TXL powder was provided by the Shijiazhuang Yiling Pharmaceutic (Hebei,China, S-130901). The herbal drugs were authenticated and standardized on marker compounds
Figure 1. Angiotensin II (Ang II) inhibits tight junction protein expression in HCMECs. (A) Human cardiac microvascular endothelial cells (HCMECs) were 7 1 stimulated with Ang II (10 mol l ) for 24 h, and the expression of various tight junction proteins was detected by Western blotting with antibodies against claudin, VE-cadherin, beta-catenin or occludin after the treatment with Ang II (left panel); densitometric scanning (right panel). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with control. (B) HCMECs were grown in six-well plates for 24 h and then were stimulated with Ang II 7 1 (10 mol l ) for 24 h. Total RNA was prepared, and the level of tight junction protein mRNA was examined by Real time Quantitative RT-PCR (qRT-PCR) and presented after normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (means ± SD; n = 3). The symbol ‘*’ means p < 0.05, compared with control Copyright © 2015 John Wiley & Sons, Ltd.
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Animals Adult male C57BL/6 J mice with a mean weight of 25 g were from the Experimental Animal Center of Hebei Medical University. C57BL/6 J mice were randomly assigned to one of the following three groups: control group, NS (saline) pretreatment group and TXL pretreatment group that received oral TXL of 0.75 g kg 1 day 1 (six mice in each group). At the end of the exposure period, we anaesthetized each mouse with pentobarbital injection, removed thoracic aorta and immersed in 4% paraformaldehyde for 24 h. Intravital observation of fluorescein isothiocyanate-labelled albumin leakage in mice Mice were anaesthetized after acute hypoxia, and then, the caudal vein was cannulated with a polyethylene catheter (Atom, Tokyo, Japan) for infusion of fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA). The abdomen was opened via a midline incision, and the ileocaecal portion of the mesentery was gently exposed, and vascular albumin leakage was quantified. Briefly, FITCBSA (25 mg kg 1; Sigma Aldrich Corp., St Louis, MO, USA) was administered intravenous (i.v.) to animals 15 min
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before the start of the experimental procedure. Fluorescence intensity (excitation wavelength, 420–490 nm; emission wavelength, 520 nm) was detected using live cell imaging system (Lecia, DM2600CB). Images were recorded for playback analysis using a videocassette recorder (Figure 2D). Immunohistochemistry The thoracic aorta was dehydrated, cleared and embedded in paraffin wax. The paraffin blocks were cut into 4 μm thick sections. For immunohistochemical staining, sections were deparaffinized and rehydrated in graduated alcohol 19. Then they were treated in a 0.1 mol l 1 sodium citrate buffer and heated for 30 min for antigen retrieval, and then the sections were incubated with anti-VE-cadherin (Abcam, Cambridge, MA, USA; 1:50), antibeta-catenin (ABGENT, San Diego, CA, USA,;1:50), antioccludin (Epitomics, Burlingame, CA, USA; 1:50), anticlaudin1 (Abcam, Cambridge, MA, USA; 1:50) and antiKLF5 (Epitomics, Burlingame, CA, USA; 1:50) antibodies. After overnight incubation, the sections were incubated with the secondary antibodies (Abcam, Cambridge, MA, USA; 1:1000) and visualized with 3, 3-diaminobenzidine and counterstained using haematoxylin. Brown and yellow colours indicate positive results.
Figure 2. Tongxinluo (TXL) abrogates the down-regulation of tight junction proteins induced by angiotensin II (Ang II). (A) Human cardiac microvascular endothelial cells were treated with TXL at different doses for 24 h, and then cells were stimulated with Ang IIfor 24 h, and expression of the tight junction proteins was determined by Western blotting with antibodies against claudin, VE-cadherin, occludin or beta-catenin. (B) Densitometric scanning. Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with control. (C) Immunohistochemical staining on sections of the thoracic artery with antibodies against claudin, VE-cadherin, occludin or beta-catenin (×200). (D) Images showing TXL-reduced permeability of FITC-BSA (green), lower panel Copyright © 2015 John Wiley & Sons, Ltd.
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Western blotting Human cardiac microvascular endothelial cells (HCMECs) were cultured in endothelial cell medium with 10% fetal bovine serum and maintained in 5% CO2 at 37 °C in a humidified atmosphere. Cells were treated with the different doses of TXL (200, 400 and 600 μg ml 1), Ang II (10 7 mol l 1), pAd-GFP and pAd-KLF5, respectively, for Copyright © 2015 John Wiley & Sons, Ltd.
24 h, then harvested and lysed with lysis buffer containing 1% NP-40,150 mM NaCl,50 mM Tris-HCl,pH 7.5, 10% glycerin, 1 mM Na3VO4, 1 mM PMSF (phenylmethyl sulfonylfluoride) and 1 mM DTT. Proteins were isolated from HCMECs, and then separated on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride membrane.20 Membranes were blocked with 5% milk in Tris-HCl tween Cell Biochem Funct 2015; 33: 226–234.
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buffer solution for 2 h at 37 °C and incubated overnight at 4 °C with specific VE-cadherin (1:1000), beta-catenin (1:1000), occludin (1:1000), claudin1 (1:500) and KLF5 (1:1000) antibodies. After incubation with appropriate secondary antibody, the membranes were developed with the Chemiluminescence Plus Western blot analysis kit (Millipore). SiRNA transfection siRNA specific for KLF5 (si-KLF5) was synthesized by Sigma-Aldrich. Nonspecific siRNA (si-Con) was used as the negative control. Transfections were done using Lipofectamine Reagent (Invitrogen, San Diego, CA, USA) following manufacturer’s instructions.21 Twenty hours after transfection, HCMECs were treated with or without TXL. Then cells were harvested and lysed for Western blotting. Statistical analysis All results are expressed as means ± SD from three or more independent experiments. Statistical significance was assessed by one-way ANOVA for comparison of different time points within a group. A p-value below 0.05 was considered statistically significant.
RESULTS Ang II inhibits tight junction protein expression in HCMECs Because Ang II is known to cause vascular endothelial injury, we sought to examine whether Ang II affected tight junction protein expression. To do this, HCMECs were treated with Ang II and some tight junction proteins were detected by Western blotting. As shown in Figure 1A, Ang II significantly reduced the expression of tight junction proteins, VE-cadherin, beta-catenin and occludin, except for claudin. Real-time PCR analysis got the same results (Figure 1B). TXL abrogates the down-regulation of tight junction proteins induced by Ang II To examine whether TXL influenced tight junction protein expression,HCMEC was preincubated with the different doses of TXL for 24 h and then treated with Ang II. As shown in Figure 2A and B, TXL preincubation abrogated the down-regulation of these tight junction proteins (Figure 2A, lane 2 versus lane 5) induced by Ang II, with a dose-dependent manner, especially when 600 μg ml 1 TXL were used (lane 5). To further investigate the effect of TXL on the expression of vascular endothelial tight junction proteins in vivo, angiotensinogen transgenic mice were used to examine endothelial integrity by immunohistochemical staining. As shown in Figure 2C, the expression of tight junction proteins was markedly attenuated in the thoracic artery of transgenic mice but markedly increased upon TXL treatment. These results indicated that TXL can abrogate the down-regulation of tight junction proteins induced by Ang II. Paralleling these results, TXL treatment Copyright © 2015 John Wiley & Sons, Ltd.
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markedly reduced the leakage of FITC-BSA across mesenteric venules-induced acute hypoxia compared with the control (Figure 2D). These results indicated that TXL can improve the endothelial function by increasing the expression of tight junction proteins. KLF5 is involved in the Ang II-suppressed and TXL-induced expression of tight junction proteins Because KLF5 is an essential transcription factor in cardiovascular remodelling,22 next we sought to detect whether Ang II affected the expression of KLF5 in HCMECs. As shown in Figure 3A, Ang II decreased the expression of KLF5 protein to some extent (left panel), especially in mRNA level (right panel), suggesting that KLF5 might be responsible for the expressions of these tight junction proteins. Next, we detected the effect of TXL on the expression of KLF5 in vitro and in vivo As shown in Figure 3B, TXL significantly reversed the inhibitory effect of Ang II on the expression of KLF5, consistent with the expression of tight junction proteins induced by Ang II in HCMEC (Figure 2). Furthermore, using immunohistochemical staining, we further detected the expression of KLF5 in angiotensinogen transgenic mice. As shown in Figure 3C, the expression of KLF5 was reduced in the thoracic artery of transgenic mice but markedly increased upon TXL treatment. These results indicated that TXL can abrogate the down-regulation of KLF5 induced by Ang II in vivo, which is consistent with the results in vitro (Figure 3B). These results further promoted us to believe that KLF5 might be involved in Ang II-suppressed and TXLpromoted expression of tight junction proteins. KLF5 mediates the expression of tight junction proteins in HCMECs To further address the earlier hypotheses, gain-of-function and loss-of-function experiments were carried out in HCMECs that were transfected with either KLF5 adenovirus pAd-KLF5 or siRNA si-KLF5 for 24 h. As shown in Figure 4A, KLF5 overexpression increased the expression of VE-cadherin, claudin and occludin, but had no effect on beta-catenin expression. Knockdown of KLF5 using si-KLF5 in HCMECs markedly decreased the expression of tight junction proteins (Figure 4B). Real-time PCR analysis also got the similar results (Figure 4C and D). These results suggest that KLF5 mediates the expression of VE-cadherin, claudin and occludin in HCMECs. KLF5 mediates TXL-enhanced expression of the tight junction proteins To clarify whether KLF5 mediated TXL-induced tight junction protein expression, HCMECs were preincubated with TXL for 24 h and then transfected with si-KLF5. As shown in Figure 5A and B, TXL could partly upregulate Ang IIsuppressed expression of claudin and occludin but had little effect on the expression of VE-cadherin and beta-catenin, indicating that KLF5 mediates the stimulatory effect of TXL on the expression of part tight junction proteins in HCMECs. Cell Biochem Funct 2015; 33: 226–234.
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Figure 3. Krueppel-like factor 5 (KLF5) is involved in the angiotensin II (Ang II)-suppressed and tongxinluo (TXL)-enhanced expression of tight junction proteins. (A) The expression of KLF5 after the treatment with Ang II was detected by Western blotting with antibodies against KLF5 (left panel). Densitometric scanning (middle). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with control; the level of KLF5 mRNA was detected by qRT-PCR after the treatment with Ang II (right panel). (B) TXL promotes the expression of tight junction proteins after the treatment with different doses of Ang II for 24 h (left panel). Densitometric scanning (right panel). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with control. (C) Immunohistochemical staining on sections of the thoracic artery with antibody against KLF5 (×200)
DISCUSSION TXL in capsule form is a compound prescription formulated according to the meridian theory of traditional Chinese medicine including ginseng, Radix Paeoniae Rubra and borneol and spiny jujube seed and has been used to treat patients with angina pectoris for more than one decade in Copyright © 2015 John Wiley & Sons, Ltd.
clinical practice.23 Ginseng is the major ingredient of TXL and contains a group of triterpene glycosides called ginsenosides.7 It has been reported that ginsenoside Rb1 effectively blocks homocysteine-induced endothelial dysfunction, superoxide anion production and endothelial nitric oxide synthesis down-regulation in porcine coronary arteries.24 Several studies found that paeonia has antioxidative, vasodilatory, Cell Biochem Funct 2015; 33: 226–234.
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Figure 4. Overexpression of Krueppel-like factor 5 (KLF5) promotes the expression of tight junction proteins. (A) Human cardiac microvascular endothelial cells (HCMECs) were transfected with adenovirus to overexpress KLF5, and Western blotting was used to detect tight junction proteins (left panel). Densitometric scanning (right panel). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with pAd. (B) HCMECs were transfected with siRNA targeting KLF5 (si-KLF5) or control siRNA (si-Con) for 24 h. The expression of tight junction proteins was detected by Western blotting with antibodies against VE-cadherin, beta-catenin, occludin, claudin or KLF5 (left panel). Densitometric scanning (right panel). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with si-Con. (C) HCMECs were grown in six-well plates for 24 h, and then were transfected with pAd-KLF5 for 6 h. Total RNA was prepared, and the level of KLF5 mRNA was examined by qRT-PCR (means ± SD; n = 3). The symbol ‘*’ means p < 0.05, compared with pAd. (D) HCMECs were grown in six-well plates for 24 h and then were transfected with si-KLF5 or si-Con for 6 h, and the level of KLF5 mRNA was detected by qRT-PCR (means ± SD; n = 3). The symbol ‘*’ means p < 0.05, compared with si-Con
antiplatelet, lipid-lowering and anti-inflammatory capacities.25 Spiny jujube seed showed potent immunological adjuvant activity. All these pharmacological effects may contribute to the antianginal efficacy of TXL in clinical practice. Ang II, an endogenous peptide hormone, plays a critical role in the pathophysiological modulation of cardiovascular functions. Ang II is the principal effector of the reninangiotensin system for maintaining homeostasis in the Copyright © 2015 John Wiley & Sons, Ltd.
cardiovascular system. Many pieces of evidence indicate that Ang II not only induces hypertension, but also directly contributes to vascular thickening and atherosclerosis by injuring the vascular endothelium.26–28 In this study, we found that KLF5 was responsible for the expression of tight junction proteins induced by TXL. Our findings suggest that KLF5 may represent a new target molecule for treating cardiovascular diseases with TXL. Cell Biochem Funct 2015; 33: 226–234.
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Figure 5. Knockdown of Krueppel-like factor 5 (KLF5) abolishes the up-regulation of tight junction proteins induced by tongxinluo (TXL). (A) Human cardiac microvascular endothelial cells were incubated with TXL for 24 h and then were transfected with si-KLF5, and Western blot analysis was performed using antibodies against VE-cadherin, beta-catenin, occludin, claudin or KLF5 (left panel). (B) Densitometric scanning (right panel). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with si-Con
We first investigated the effects of Ang II on the expression of tight junction proteins in microvascular endothelial cells and explored the protective effect of TXL on the endothelial function. We found that Ang II significantly reduced the expression of the tight junction proteins in HCMECs. Importantly, TXL treatment reversed Ang II-suppressed expression of the tight junction proteins, indicating that TXL may protect the vascular endothelial cells against Ang II-induced injury. Further experiments confirmed that KLF5 mediated TXL-promoted expression of the tight junction proteins. However, TXL-mediated cardiovascular protection is not only via improvement of endothelial function but also via anti-inflammatory effect on macrophages and antiproliferation effect on vascular smooth muscle cells.10 In conclusion, our results indicated that the traditional Chinese medical compound TXL can up-regulate tight junction protein expression to a KLF5-dependent manner, thus improving the vascular endothelial function. This finding not only provides a theoretical basis for TXL in treating cardiovascular and cerebrovascular diseases but also provides a new therapeutic approach to target vascular tight interendothelial junctions. CONFLICT OF INTEREST The authors have declared that there is no conflict of interest. ACKNOWLEDGEMENTS This study was supported by the National Basic Research Program of China (No. 2012CB518601), the National Natural Science Foundation of China (No. 31271396, 31271224, 31301136) and the Fok Ying Tung Education Foundation (131037).
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2. Hoefer IE, den Adel B, Daemen MJ. Biomechanical factors as triggers of vascular growth. Cardiovasc Res 2013; 99: 276–283. 3. Cristante E, McArthur S, Mauro C, et al. Identification of an essential endogenous regulator of blood-brain barrier integrity, and its pathological and therapeutic implications. Proc Natl Acad Sci U S A 2013; 110: 832–841. 4. Farquhar MG, Palade GE. Junctional complexes in various epithelia. J Cell Biol 1963; 17: 375–412. 5. Beaufort N, Corvazier E, Mlanaoindrou S, de Bentzmann S, Pidard D. Disruption of the endothelial barrier by proteases from the bacterial pathogen Pseudomonas aeruginosa: implication of matrilysis and receptor cleavage. PLoS One 2013; 8: e75708. 6. Cheng YT, Yang YJ, Zhang HT, et al. Pretreatment with Tongxinluo protects porcine myocardium from ischaemia/reperfusion injury through a nitric oxide related mechanism. Chin Med J (Engl) 2009; 122: 1529–1538. 7. Zhang L, Liu Y, Lu XT, et al. Traditional Chinese medication Tongxinluo dose-dependently enhances stability of vulnerable plaques: a comparison with a high-dose simvastatin therapy. Am J Physiol Heart Circ Physiol 2009; 297: H2004–H2014. 8. Chen WQ, Zhong L, Zhang L, et al. Chinese medicine tongxinluo significantly lowers serum lipid levels and stabilizes vulnerable plaques in a rabbit model. J Ethnopharmacol 2009; 124: 103–110. 9. Liang JQ, Wu K, Jia ZH, et al. Chinese medicine Tongxinluo modulates vascular endothelial function by inducing eNOS expression via the PI-3K/Akt/HIF-dependent signaling pathway. J Ethnopharmacol 2011; 133: 517–523. 10. Zhang RN, Zheng B, Li LM, et al. Tongxinluo inhibits vascular inflammation and neointimal hyperplasia through blocking positive feedback loop between miR-155 and TNF-α. Am J Physiol Heart Circ Physiol 2014; 307: H552–H562. 11. Liu Y, Tang GH, Sun YH, et al. The protective role of Tongxinluo on blood-brain barrier after ischemia-reperfusion brain injury. J Ethnopharmacol 2013; 148: 632–639. 12. Chen L, Wang X, Chen X, et al. Tongxinluo attenuates neuronal loss and enhances neurogenesis and angiogenesis in the ipsilateral thalamus and improves neurological outcome after focal cortical infarction in hypertensive rats. Restor Neurol Neurosci 2014; 32: 533–546. 13. Wang B, Yang Q, Bai WW, et al. Tongxinluo protects against pressure overload-induced heart failure in mice involving VEGF/Akt/eNOS pathway activation. PLoS One 2014; 9: e98047. 14. Shindo T, Manabe I, Fukushima Y, et al. Krüppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling. Nat Med 2002; 8: 856–863. 15. Diakiw SM, D’Andrea RJ, Brown AL. The double life of KLF5: opposing roles in regulation of gene-expression, cellular function, and transformation. IUBMB Life 2013; 65: 999–1011. Cell Biochem Funct 2015; 33: 226–234.
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Chinese medicine Tongxinluo increases tight junction protein levels by inducing KLF5 expression in microvascular endothelial cells Li-Min Li, Bing Zheng, Ruo-Nan Zhang, Li-Shuang Jin, Cui-Ying Zheng, Chang Wang, Pei-Pei Zhou, Zong-Wei Guo, Dong Ma and Jin-Kun Wen* Department of Biochemistry and Molecular Biology, Key Laboratory of Neural and Vascular Biology, China Administration of Education, Hebei Medical University, Shijiazhuang, China
Tongxinluo (TXL) is a compound prescription formulated according to the meridian theory of traditional Chinese medicine. It may play an important role in cardiovascular protection by improving endothelial cell function. The aim of present study was to investigate whether endothelial protection with TXL is related to its regulation of tight junction protein expression. Human cardiac microvascular endothelial cells (HCMECs) were cultured and treated with 10 7 mol l 1 angiotensin II (Ang II) and the different doses of TXL; the expression of tight junction proteins occludin, claudin, VE-cadherin and beta-catenin was determined by Western blotting and real-time PCR. Gain-of-function and loss-of-function of Krüppel-like factor 5 (KLF5) were carried out in HCMEC transfected with either KLF5 adenovirus pAd-KLF5 or siRNA specific for KLF5. Angiotensinogen transgenic mice were treated with TXL by oral administration of TXL of 0.75 g kg 1 day 1, and immunohistochemical staining was performed with antioccludin, anticlaudin, anti-VE-cadherin, antibeta-catenin and anti-KLF5 antibodies. Ang II treatment significantly reduced the expression of tight junction proteins occludin, claudin, VE-cadherin and beta-catenin in cultured HCMECs. TXL pretreatment could abrogate the down-regulation of these tight junction proteins induced by Ang II. Ang II treatment also decreased KLF5 expression at the mRNA and protein levels; TXL pretreatment markedly reversed the inhibitory effect of Ang II on KLF5 expression. Gain-of-function and loss-of-function of KLF5 showed that KLF5 mediated the expression of tight junction proteins in HCMECs. TXL-enhanced expression of the tight junction proteins was mediated by KLF5. In angiotensinogen transgenic mice, TXL also increased the tight junction protein levels by inducing KLF5 expression. Chinese medicine TXL increases tight junction protein levels by inducing KLF5 expression in microvascular endothelial cells. Copyright © 2015 John Wiley & Sons, Ltd. key words—tongxinluo; tight junction protein; KLF5; endothelial cell; angiotensin II
INTRODUCTION Vascular endothelium is the monolayer of endothelial cells lining the lumen of blood vessels and is located between the blood and vascular tissue; it cannot only complete metabolic exchange between blood and tissue fluid but also can synthesize and secrete a variety of biologically active substances. The vascular endothelial dysfunction has been implicated in the development of vascular diseases;1 many high-risk factors are becoming potential risk factors in the development of vascular endothelial dysfunction, such as hypertension, diabetes and harmful social and psychological stresses.2 Recent studies suggest that in the early stage of cardiovascular diseases, endothelial function has been damaged. Thus, repairing vascular endothelial injury and improving endothelial dysfunction are a new direction in the field of cardiovascular diseases.
*Correspondence to: Jin-Kun Wen, Department of Biochemistry and Molecular Biology, Hebei Medical University, No. 361, Zhongshan East Road, Shijiazhuang, 050017, China. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
Tight interendothelial junctions are responsible for the formation and maintenance of the permeability barrier,3 both to the passage of ions and molecules through the paracellular pathway and to the movement of proteins and lipids between the apical and the basolateral domains of the plasma membrane.4 Tight junctions are critical for normal vascular physiologic function.5 Abnormal expression of tight junction proteins can lead to vascular endothelial dysfunction and causes various cardiovascular diseases. It has been known that many transmembrane proteins, such as occludin, claudin, VE-cadherin and beta-catenin, are located at the tight junctions of endothelial cells and are involved in the regulation of endothelial functions including paracellular permeability and intracellular signal transduction. Tongxinluo (TXL) is a traditional Chinese medicine that is extracted, concentrated, freeze-dried and standardized from a mixture of 12 medicinal constituents.6 It is approved in 1996 by the State Food and Drug Administration of China for treatment of angina pectoris and ischemic stroke7 and has been widely used in China to treat patients with cardiovascular8 and cerebrovascular diseases.7 The previous Received 7 January 2015 Revised 18 March 2015 Accepted 23 March 2015
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txl increases tight junction protein levels studies have revealed that treatment with TXL can improve endothelial cell function,9 lower lipid,8 reduce inflammation and apoptosis,7,10 inhibit neointima hyperplasia,10 protect the brain from Blood-brain barrier (BBB) disruption11 and promote angiogenesis.12,13 However, the effects of TXL treatment on the expression of tight junction proteins in the endothelial cells and its action mechanisms are still unclear. Angiotensin II (Ang II), a bioactive peptide regulating vascular tone and promoting vascular smooth muscle cell proliferation, plays a key role in the pathogenesis of cardiovascular diseases.14 As a basic transcription factor, Krueppel-like factor 5 (KLF5) is known to be involved in important biological processes such as cell proliferation and differentiation.15–17 In addition to its pro-proliferation and prodifferentiation roles, KLF5 is also implicated in injury response and the activation of inflammatory pathways following exposure to inflammatory stimuli.18 However, it remains unclear whether protective effects of TXL on Ang II-induced endothelial damage are mediated by KLF5. In this study, we investigated whether and how TXL protects endothelial function by regulating tight junction protein expression.
according to the Chinese Pharmacopoeia 2005. TXL contains 12 medicinal components, which were ground to superfine powder with the diameter ≤10 μm by a micronizer and prepared as capsules. TXL powder was weighed and dissolved in the phosphate-buffered saline. The ultrasound technology was used to promote the melting for about 1 h. The drug was then centrifuged at 1000 × g for 10 min, and the supernatant was put into the microfilter (0.22 μm) to eliminate bacteria, which was then aliquoted and stored at 20 °C before use. Fingerprint chromatography of TXL capsule To reduce the dose variability of TXL capsule among different batches, the species, origin, harvest time, medicinal parts and concocted methods for each component were strictly standardized. Moreover, high-performance liquid chromatography, high-performance capillary electrophoresis and chromatography were applied to quantitate the components of the capsule.7 To assess the dosing reproducibility of TXL capsule, high performance liquid chromatography was applied to record fingerprint chromatograms of the aqueous extracts of the ten different batches for similarity analysis.
MATERIAL AND METHODS
Ethics statement
Components and preparation of TXL
All animal studies were approved by the Ethics Committee of Hebei Medical University, which is fully accredited by the Institutional Animal Care and Use Committee, and all efforts were made to minimize suffering.
TXL powder was provided by the Shijiazhuang Yiling Pharmaceutic (Hebei,China, S-130901). The herbal drugs were authenticated and standardized on marker compounds
Figure 1. Angiotensin II (Ang II) inhibits tight junction protein expression in HCMECs. (A) Human cardiac microvascular endothelial cells (HCMECs) were 7 1 stimulated with Ang II (10 mol l ) for 24 h, and the expression of various tight junction proteins was detected by Western blotting with antibodies against claudin, VE-cadherin, beta-catenin or occludin after the treatment with Ang II (left panel); densitometric scanning (right panel). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with control. (B) HCMECs were grown in six-well plates for 24 h and then were stimulated with Ang II 7 1 (10 mol l ) for 24 h. Total RNA was prepared, and the level of tight junction protein mRNA was examined by Real time Quantitative RT-PCR (qRT-PCR) and presented after normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (means ± SD; n = 3). The symbol ‘*’ means p < 0.05, compared with control Copyright © 2015 John Wiley & Sons, Ltd.
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Animals Adult male C57BL/6 J mice with a mean weight of 25 g were from the Experimental Animal Center of Hebei Medical University. C57BL/6 J mice were randomly assigned to one of the following three groups: control group, NS (saline) pretreatment group and TXL pretreatment group that received oral TXL of 0.75 g kg 1 day 1 (six mice in each group). At the end of the exposure period, we anaesthetized each mouse with pentobarbital injection, removed thoracic aorta and immersed in 4% paraformaldehyde for 24 h. Intravital observation of fluorescein isothiocyanate-labelled albumin leakage in mice Mice were anaesthetized after acute hypoxia, and then, the caudal vein was cannulated with a polyethylene catheter (Atom, Tokyo, Japan) for infusion of fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA). The abdomen was opened via a midline incision, and the ileocaecal portion of the mesentery was gently exposed, and vascular albumin leakage was quantified. Briefly, FITCBSA (25 mg kg 1; Sigma Aldrich Corp., St Louis, MO, USA) was administered intravenous (i.v.) to animals 15 min
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before the start of the experimental procedure. Fluorescence intensity (excitation wavelength, 420–490 nm; emission wavelength, 520 nm) was detected using live cell imaging system (Lecia, DM2600CB). Images were recorded for playback analysis using a videocassette recorder (Figure 2D). Immunohistochemistry The thoracic aorta was dehydrated, cleared and embedded in paraffin wax. The paraffin blocks were cut into 4 μm thick sections. For immunohistochemical staining, sections were deparaffinized and rehydrated in graduated alcohol 19. Then they were treated in a 0.1 mol l 1 sodium citrate buffer and heated for 30 min for antigen retrieval, and then the sections were incubated with anti-VE-cadherin (Abcam, Cambridge, MA, USA; 1:50), antibeta-catenin (ABGENT, San Diego, CA, USA,;1:50), antioccludin (Epitomics, Burlingame, CA, USA; 1:50), anticlaudin1 (Abcam, Cambridge, MA, USA; 1:50) and antiKLF5 (Epitomics, Burlingame, CA, USA; 1:50) antibodies. After overnight incubation, the sections were incubated with the secondary antibodies (Abcam, Cambridge, MA, USA; 1:1000) and visualized with 3, 3-diaminobenzidine and counterstained using haematoxylin. Brown and yellow colours indicate positive results.
Figure 2. Tongxinluo (TXL) abrogates the down-regulation of tight junction proteins induced by angiotensin II (Ang II). (A) Human cardiac microvascular endothelial cells were treated with TXL at different doses for 24 h, and then cells were stimulated with Ang IIfor 24 h, and expression of the tight junction proteins was determined by Western blotting with antibodies against claudin, VE-cadherin, occludin or beta-catenin. (B) Densitometric scanning. Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with control. (C) Immunohistochemical staining on sections of the thoracic artery with antibodies against claudin, VE-cadherin, occludin or beta-catenin (×200). (D) Images showing TXL-reduced permeability of FITC-BSA (green), lower panel Copyright © 2015 John Wiley & Sons, Ltd.
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Figure 2.
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Western blotting Human cardiac microvascular endothelial cells (HCMECs) were cultured in endothelial cell medium with 10% fetal bovine serum and maintained in 5% CO2 at 37 °C in a humidified atmosphere. Cells were treated with the different doses of TXL (200, 400 and 600 μg ml 1), Ang II (10 7 mol l 1), pAd-GFP and pAd-KLF5, respectively, for Copyright © 2015 John Wiley & Sons, Ltd.
24 h, then harvested and lysed with lysis buffer containing 1% NP-40,150 mM NaCl,50 mM Tris-HCl,pH 7.5, 10% glycerin, 1 mM Na3VO4, 1 mM PMSF (phenylmethyl sulfonylfluoride) and 1 mM DTT. Proteins were isolated from HCMECs, and then separated on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride membrane.20 Membranes were blocked with 5% milk in Tris-HCl tween Cell Biochem Funct 2015; 33: 226–234.
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buffer solution for 2 h at 37 °C and incubated overnight at 4 °C with specific VE-cadherin (1:1000), beta-catenin (1:1000), occludin (1:1000), claudin1 (1:500) and KLF5 (1:1000) antibodies. After incubation with appropriate secondary antibody, the membranes were developed with the Chemiluminescence Plus Western blot analysis kit (Millipore). SiRNA transfection siRNA specific for KLF5 (si-KLF5) was synthesized by Sigma-Aldrich. Nonspecific siRNA (si-Con) was used as the negative control. Transfections were done using Lipofectamine Reagent (Invitrogen, San Diego, CA, USA) following manufacturer’s instructions.21 Twenty hours after transfection, HCMECs were treated with or without TXL. Then cells were harvested and lysed for Western blotting. Statistical analysis All results are expressed as means ± SD from three or more independent experiments. Statistical significance was assessed by one-way ANOVA for comparison of different time points within a group. A p-value below 0.05 was considered statistically significant.
RESULTS Ang II inhibits tight junction protein expression in HCMECs Because Ang II is known to cause vascular endothelial injury, we sought to examine whether Ang II affected tight junction protein expression. To do this, HCMECs were treated with Ang II and some tight junction proteins were detected by Western blotting. As shown in Figure 1A, Ang II significantly reduced the expression of tight junction proteins, VE-cadherin, beta-catenin and occludin, except for claudin. Real-time PCR analysis got the same results (Figure 1B). TXL abrogates the down-regulation of tight junction proteins induced by Ang II To examine whether TXL influenced tight junction protein expression,HCMEC was preincubated with the different doses of TXL for 24 h and then treated with Ang II. As shown in Figure 2A and B, TXL preincubation abrogated the down-regulation of these tight junction proteins (Figure 2A, lane 2 versus lane 5) induced by Ang II, with a dose-dependent manner, especially when 600 μg ml 1 TXL were used (lane 5). To further investigate the effect of TXL on the expression of vascular endothelial tight junction proteins in vivo, angiotensinogen transgenic mice were used to examine endothelial integrity by immunohistochemical staining. As shown in Figure 2C, the expression of tight junction proteins was markedly attenuated in the thoracic artery of transgenic mice but markedly increased upon TXL treatment. These results indicated that TXL can abrogate the down-regulation of tight junction proteins induced by Ang II. Paralleling these results, TXL treatment Copyright © 2015 John Wiley & Sons, Ltd.
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markedly reduced the leakage of FITC-BSA across mesenteric venules-induced acute hypoxia compared with the control (Figure 2D). These results indicated that TXL can improve the endothelial function by increasing the expression of tight junction proteins. KLF5 is involved in the Ang II-suppressed and TXL-induced expression of tight junction proteins Because KLF5 is an essential transcription factor in cardiovascular remodelling,22 next we sought to detect whether Ang II affected the expression of KLF5 in HCMECs. As shown in Figure 3A, Ang II decreased the expression of KLF5 protein to some extent (left panel), especially in mRNA level (right panel), suggesting that KLF5 might be responsible for the expressions of these tight junction proteins. Next, we detected the effect of TXL on the expression of KLF5 in vitro and in vivo As shown in Figure 3B, TXL significantly reversed the inhibitory effect of Ang II on the expression of KLF5, consistent with the expression of tight junction proteins induced by Ang II in HCMEC (Figure 2). Furthermore, using immunohistochemical staining, we further detected the expression of KLF5 in angiotensinogen transgenic mice. As shown in Figure 3C, the expression of KLF5 was reduced in the thoracic artery of transgenic mice but markedly increased upon TXL treatment. These results indicated that TXL can abrogate the down-regulation of KLF5 induced by Ang II in vivo, which is consistent with the results in vitro (Figure 3B). These results further promoted us to believe that KLF5 might be involved in Ang II-suppressed and TXLpromoted expression of tight junction proteins. KLF5 mediates the expression of tight junction proteins in HCMECs To further address the earlier hypotheses, gain-of-function and loss-of-function experiments were carried out in HCMECs that were transfected with either KLF5 adenovirus pAd-KLF5 or siRNA si-KLF5 for 24 h. As shown in Figure 4A, KLF5 overexpression increased the expression of VE-cadherin, claudin and occludin, but had no effect on beta-catenin expression. Knockdown of KLF5 using si-KLF5 in HCMECs markedly decreased the expression of tight junction proteins (Figure 4B). Real-time PCR analysis also got the similar results (Figure 4C and D). These results suggest that KLF5 mediates the expression of VE-cadherin, claudin and occludin in HCMECs. KLF5 mediates TXL-enhanced expression of the tight junction proteins To clarify whether KLF5 mediated TXL-induced tight junction protein expression, HCMECs were preincubated with TXL for 24 h and then transfected with si-KLF5. As shown in Figure 5A and B, TXL could partly upregulate Ang IIsuppressed expression of claudin and occludin but had little effect on the expression of VE-cadherin and beta-catenin, indicating that KLF5 mediates the stimulatory effect of TXL on the expression of part tight junction proteins in HCMECs. Cell Biochem Funct 2015; 33: 226–234.
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Figure 3. Krueppel-like factor 5 (KLF5) is involved in the angiotensin II (Ang II)-suppressed and tongxinluo (TXL)-enhanced expression of tight junction proteins. (A) The expression of KLF5 after the treatment with Ang II was detected by Western blotting with antibodies against KLF5 (left panel). Densitometric scanning (middle). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with control; the level of KLF5 mRNA was detected by qRT-PCR after the treatment with Ang II (right panel). (B) TXL promotes the expression of tight junction proteins after the treatment with different doses of Ang II for 24 h (left panel). Densitometric scanning (right panel). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with control. (C) Immunohistochemical staining on sections of the thoracic artery with antibody against KLF5 (×200)
DISCUSSION TXL in capsule form is a compound prescription formulated according to the meridian theory of traditional Chinese medicine including ginseng, Radix Paeoniae Rubra and borneol and spiny jujube seed and has been used to treat patients with angina pectoris for more than one decade in Copyright © 2015 John Wiley & Sons, Ltd.
clinical practice.23 Ginseng is the major ingredient of TXL and contains a group of triterpene glycosides called ginsenosides.7 It has been reported that ginsenoside Rb1 effectively blocks homocysteine-induced endothelial dysfunction, superoxide anion production and endothelial nitric oxide synthesis down-regulation in porcine coronary arteries.24 Several studies found that paeonia has antioxidative, vasodilatory, Cell Biochem Funct 2015; 33: 226–234.
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Figure 4. Overexpression of Krueppel-like factor 5 (KLF5) promotes the expression of tight junction proteins. (A) Human cardiac microvascular endothelial cells (HCMECs) were transfected with adenovirus to overexpress KLF5, and Western blotting was used to detect tight junction proteins (left panel). Densitometric scanning (right panel). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with pAd. (B) HCMECs were transfected with siRNA targeting KLF5 (si-KLF5) or control siRNA (si-Con) for 24 h. The expression of tight junction proteins was detected by Western blotting with antibodies against VE-cadherin, beta-catenin, occludin, claudin or KLF5 (left panel). Densitometric scanning (right panel). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with si-Con. (C) HCMECs were grown in six-well plates for 24 h, and then were transfected with pAd-KLF5 for 6 h. Total RNA was prepared, and the level of KLF5 mRNA was examined by qRT-PCR (means ± SD; n = 3). The symbol ‘*’ means p < 0.05, compared with pAd. (D) HCMECs were grown in six-well plates for 24 h and then were transfected with si-KLF5 or si-Con for 6 h, and the level of KLF5 mRNA was detected by qRT-PCR (means ± SD; n = 3). The symbol ‘*’ means p < 0.05, compared with si-Con
antiplatelet, lipid-lowering and anti-inflammatory capacities.25 Spiny jujube seed showed potent immunological adjuvant activity. All these pharmacological effects may contribute to the antianginal efficacy of TXL in clinical practice. Ang II, an endogenous peptide hormone, plays a critical role in the pathophysiological modulation of cardiovascular functions. Ang II is the principal effector of the reninangiotensin system for maintaining homeostasis in the Copyright © 2015 John Wiley & Sons, Ltd.
cardiovascular system. Many pieces of evidence indicate that Ang II not only induces hypertension, but also directly contributes to vascular thickening and atherosclerosis by injuring the vascular endothelium.26–28 In this study, we found that KLF5 was responsible for the expression of tight junction proteins induced by TXL. Our findings suggest that KLF5 may represent a new target molecule for treating cardiovascular diseases with TXL. Cell Biochem Funct 2015; 33: 226–234.
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Figure 5. Knockdown of Krueppel-like factor 5 (KLF5) abolishes the up-regulation of tight junction proteins induced by tongxinluo (TXL). (A) Human cardiac microvascular endothelial cells were incubated with TXL for 24 h and then were transfected with si-KLF5, and Western blot analysis was performed using antibodies against VE-cadherin, beta-catenin, occludin, claudin or KLF5 (left panel). (B) Densitometric scanning (right panel). Values are the means ± SD from three independent experiments. The symbol ‘*’ means p < 0.05, compared with si-Con
We first investigated the effects of Ang II on the expression of tight junction proteins in microvascular endothelial cells and explored the protective effect of TXL on the endothelial function. We found that Ang II significantly reduced the expression of the tight junction proteins in HCMECs. Importantly, TXL treatment reversed Ang II-suppressed expression of the tight junction proteins, indicating that TXL may protect the vascular endothelial cells against Ang II-induced injury. Further experiments confirmed that KLF5 mediated TXL-promoted expression of the tight junction proteins. However, TXL-mediated cardiovascular protection is not only via improvement of endothelial function but also via anti-inflammatory effect on macrophages and antiproliferation effect on vascular smooth muscle cells.10 In conclusion, our results indicated that the traditional Chinese medical compound TXL can up-regulate tight junction protein expression to a KLF5-dependent manner, thus improving the vascular endothelial function. This finding not only provides a theoretical basis for TXL in treating cardiovascular and cerebrovascular diseases but also provides a new therapeutic approach to target vascular tight interendothelial junctions. CONFLICT OF INTEREST The authors have declared that there is no conflict of interest. ACKNOWLEDGEMENTS This study was supported by the National Basic Research Program of China (No. 2012CB518601), the National Natural Science Foundation of China (No. 31271396, 31271224, 31301136) and the Fok Ying Tung Education Foundation (131037).
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