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Salvianolic acid A inhibits endothelial dysfunction and vascular remodeling in spontaneously hypertensive rats
Salvianolic acid A inhibits endothelial dysfunction and vascular remodeling in spontaneously hypertensive rats Fukang Teng, Ying Yin, Yajun Cui, Yanping Deng, Defang Li, Kenka Cho, Ge Zhang, Aiping Lu, Wanying Wu, Min Yang, Xuan Liu, De-an Guo, Jun Yin, Baohong Jiang PII: DOI: Reference:
S0024-3205(15)00333-1 doi: 10.1016/j.lfs.2015.06.010 LFS 14417
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
3 February 2015 11 June 2015 14 June 2015
Please cite this article as: Teng Fukang, Yin Ying, Cui Yajun, Deng Yanping, Li Defang, Cho Kenka, Zhang Ge, Lu Aiping, Wu Wanying, Yang Min, Liu Xuan, Guo De-an, Yin Jun, Jiang Baohong, Salvianolic acid A inhibits endothelial dysfunction and vascular remodeling in spontaneously hypertensive rats, Life Sciences (2015), doi: 10.1016/j.lfs.2015.06.010
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Salvianolic acid A inhibits endothelial dysfunction and vascular remodeling in
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spontaneously hypertensive rats Fukang Tenga, b, 1, Ying Yinb, Yajun Cuic,Yanping Dengb, Defang Lie, Kenka Chod,
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Ge Zhange, Aiping Lue, Wanying Wub, Min Yangb, Xuan Liub, De-an Guob, Jun Yina*,
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Baohong Jiangb*
Shenyang Pharmaceutical University, Wenhua Road #103, Shenyang 110016, China
b
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
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Affiliation
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201203, China.
Shanghai University of Traditional Chinese Medicine, Cailun Road #1200, Shanghai
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201203, China
Takarazuka University of Medical and Health Care, Hanayashiki-Midorigaoka,
e
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Takarazuka City 6660162, Japan Institute for Advancing Translational Medicine in Bone and Joint Diseases, School of
Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China.
*
Corresponding author
Pro. Jun Yin, E-mail: [email protected], 86-24-23986491 Pro. Baohong Jiang, E-mail: [email protected], 86-21-50272223
Conflicts of Interest 1
ACCEPTED MANUSCRIPT The authors declare that there are no conflicts of interest.
Abstract
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Aims: Despite the numerous pharmacological agents available for hypertension
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therapy, hypertension-related microvascular remodeling is not resolved, eventually leading to end-organ damage. The aim of the present study was to investigate the protection of salvianolic acid A (SalA) against microvascular remodeling in vitro and
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in vivo.
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Main methods: Spontaneously hypertensive rats (SHR) were administered 2.5, 5 or 10 mg/kg SalA via intraperitoneal injection once a day for 4 weeks. The tail-cuff method
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was applied to monitor blood pressure; the microvascular structure of retina was detected by hematoxylin-eosin and immunohistochemical staining; the function of
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mesenteric arteries was measured by DMT wire myography; endothelial cell proliferation was estimated using Cell Counting Kit 8; endothelial cell migration was
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evaluated by wound healing and transwell assay; and endothelial cell integrity was detected by transendothelial electrical resistance and permeability assays. Key findings: Although no antihypertensive effects of SalA were observed, SalA attenuated the microvascular inward remodeling of the retina and improved microvascular function in the mesenteries in vivo. Further cell experiments confirmed the beneficial effects of SalA on the integrity of the endothelial monolayer in vitro. Significance: Salvianolic acid A inhibited endothelial dysfunction and vascular remodeling in spontaneously hypertensive rats. Therefore, salvianolic acid A could be 2
ACCEPTED MANUSCRIPT a potential drug therapy to prevent further targeted organ damage induced by vascular remodeling. salvianolic
acid
A;
hypertension;
microvascular
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spontaneously hypertensive rats; vascular endothelial function
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Keywords:
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remodeling;
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Introduction Hypertension is the most common cause of damage to the microvascular system in
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humans, potentially leading to microvascular remodeling and ultimately resulting in
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severe complications, such as retinopathy, renal failure and heart failure (Feihl et al., 2008). Although antihypertensive drugs, such as angiotensin-converting enzyme
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(ACE) inhibitors, calcium-channel blockers and angiotensin II receptor blockers, are extensively applied for clinical therapy, the hypertension-induced impairment of the
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microvascular system is not resolved (Higashi et al., 2000). Hence, new drugs and therapeutic strategies for hypertension-related microvascular remodeling are urgently
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needed.
Salvia miltiorrhiza (Danshen), a traditional Chinese medicine, is broadly used as a
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clinical drug to treat cardiovascular diseases. Salvianolic acid A (SalA), one of the most bioactive compounds in Salvia miltiorrhiza, has many pharmacological activities,
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including anti-oxidation, myocardial protection, antithrombosis, antifibrosis, the prevention of diabetes complications (Ho and Hong, 2011). Recently, we observed that SalA is a novel matrix metalloproteinase-9 inhibitor that prevents cardiac remodeling in spontaneously hypertensive rats (Jiang et al., 2013). Microvascular remodeling is an important step for end-organ damage. However, whether SalA prevents microvascular remodeling induced by hypertension remains unknown. To elucidate the effects of SalA on microvascular remodeling, we estimated the
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ACCEPTED MANUSCRIPT protective effects of SalA on microvascular structure and function in representative organs prone to damage under hypertension in vivo. Based on the results of in vivo
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detection, the direct protection of microvascular endothelial cells by SalA was
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confirmed in vitro.
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Materials and Methods Animals
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Eight-week-old male spontaneously hypertensive rats (SHR, 280–300 g) and Wistar
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Kyoto rats (WKY, 280–300 g) were obtained from the Shanghai Center of Experimental Animals and acclimatized for 1 week. WKY rats fed ordinary laboratory
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chow and administered with saline via intraperitoneal injection were presented as normal controls (WKY, n=10). SHR were randomly divided into four groups of ten
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rats each and fed with 8.0 % high salt chow throughout the whole experiment. SHR treated with saline were regarded as a hypertensive model (SHR, n=10); SHR in the
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other three groups were administered with 2.5 mg/kg SalA (SHR-SalA(2.5), n=10), 5 mg/kg SalA (SHR-SalA(5), n=10) or 10 mg/kg SalA (SHR-SalA(10), n=10).
SalA
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dissolved in saline was administered once a day via intraperitoneal injection. All rats were maintained in a room with a 12/12 h light/dark cycle at constant temperature
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(22–23 °C) and free access to food and water. This study was approved by the Animal Care and Use Committee (SIMM-AE-GDA-2010-05) in accordance with the guidelines for the ethical care of experimental animals. The National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals was followed throughout the entire experiment.
SalA purity assay SalA was purchased from Shanghai Yousi Bio-Tech Co., Ltd (Yousi, Shanghai
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ACCEPTED MANUSCRIPT Yousi Bio-Tech Co., China). The high performance liquid chromatography (HPLC) gradient elution method was used to determine the purity of SalA. Briefly, SalA was
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dissolved in acetonitrile, passed through a 0.45 μm filter membrane and detected
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using the Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA) with a Zorbax Extend SB-C18 column (5 μm, 250 mm×4.6 mm). The loading volume
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of the sample was 10 μl, the wavelength of detection was set at 280 nm, the flow rate of mobile phase was 0.8 mL/min and the column temperature was maintained at
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20 °C. The composition of the mobile phase was 0.05 % aqueous trifluoroacetic acid (V/V), 2 %-10 % acetonitrile at 0-7 min, 10 %-30 % acetonitrile at 7-20 min,
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23 %-27 % acetonitrile at 20-35 min, and 27 %-60 % acetonitrile at 35-50 min (Jiang et al., 2008).
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Body weight and blood pressure monitoring Body weight and blood pressure were monitored at the indicated times (Fig. S1).
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Blood pressure was indirectly measured in a conscious rat using the tail cuff method as previously described. Briefly, the conscious animals were conditioned to restraint on a heating pad. After stabilization for 10–20 min on the heating pad at 37 °C, the blood pressure was continuously recorded using a tail-cuff apparatus (ALC-NIBP, Shanghai Alcott Biotech Co., China) (Zhang et al., 2014, Chen et al., 2012).
Hematoxylin-eosin staining At the end of the experiment, the rats were euthanized with an overdose of chloral
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ACCEPTED MANUSCRIPT hydrate. The rat eyes were harvested and fixed in 4 % formaldehyde solution for at least 24 h and embedded in paraffin. The paraffin-embedded specimens were cut into
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sections (3-µm-thick) and stained with hematoxylin-eosin. The images were digitally
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captured (magnification ×400) using an Olympus BX51 microscope equipped with an Olympus DP71 CCD camera (Olympus Corporation, Japan).
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Immunohistochemical detection of the retinal vessels After euthanasia, the rat eyes were removed and fixed in a 4 % formaldehyde
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solution for 1 day. The retinas were dissected and flattened onto slides, washed 3 times with PBS, and subsequently incubated with PBS containing 0.5 % Triton X-100
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and 10 % normal goat serum for 1 h at room temperature. After washing 3 times with PBS, the retinas were incubated with α-SMA-Cy3 (red) (1:400, Sigma-Aldrich,
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Germany) and lectin-FITC (green) (1:200, Sigma-Aldrich, Germany) at 37 °C for 1.5 h, followed by washing 3 times with PBS. The images were digitally captured
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(magnification ×100) using an Olympus BX51 fluorescence microscope equipped with an Olympus DP71 CCD camera (Olympus Corporation, Japan), and the retinal vascular diameter was directly measured at a distance of 0.4 mm from the optical disc using Image-Pro Plus 6.0 image software (IPP 6.0, Media Cybemetics, USA) (Licht et al., 2010).
Microvascular function measurements Microvascular function was measured using DMT wire myography (Model 620M,
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ACCEPTED MANUSCRIPT Danish Myo Technology, Aarhus, Denmark). After euthanasia, the rat mesenteries were removed and placed in 1X cold physiological salt solution (10X PSS
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composition in (g/l): NaCl 69.54, KCl 3.5, CaCl2 2.78, MgSO4 1.4112, KH2PO4 1.61,
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EDTA 0.1, NaHCO3 21 and glucose 10.91). The mesenteries were maintained in oxygenated (5 % CO2, 95 % O2 mixture) PSS at 4 °C until dissection. Mesenteric
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small arteries (2 mm long) were isolated from adipose or connective tissue under a dissecting microscope. After passage through two stainless steel wires (40 μm in
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diameter), the dissected mesenteric small arteries were mounted as ring-shaped preparations on a quadruple wire to measure microvascular function. The bath of each
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myograph contained 5 mL oxygenated (5 % CO2, 95 % O2 mixture) PSS at 37 °C to keep pH 7.4. Normalization was performed to set the pre-tension of the mesenteric
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small arteries. Briefly, after stabilizing for 30 min in PSS, the mesenteric small arteries were contracted with DL-phenylephrine hydrochloride (PE) (Adamas Reagent
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Co., Ltd) (1×10-2 M) dissolved in PSS with 125 mM K+, washed to determine viability and stabilized for 30 min. After preconstruction with PE (3×10-5 M), the mesenteric
small
arterial
contraction
was
stabilized.
Subsequently,
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endothelium-dependent vasodilator acetylcholine chloride (ACh) (Sinopharm Chemical Reagent Co., Ltd, China) (10 pmol/L to 10 μmol/L) was added in a cumulative manner, and concentration–response curves were obtained. The relaxation induced
by
ACh
was
normalized
to
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the
contraction
induced
by
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Cell line and cell culture
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The human umbilical vein endothelial cell line (HUVEC) was purchased from the Beijing North Albert Development Co. Ltd. HUVECs were cultured in 75-cm2 flasks
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containing RPMI-1640 medium (Hyclone, Thermo Fisher Scientific, USA) supplemented with 10 % fetal bovine serum (GIBCO, Invitrogen Inc., USA), 100
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U/mL penicillin and 100 mg/L streptomycin (Sigma, Sigma Inc., USA) at 37 °C in a 5 % CO2 humidified atmosphere. The HUVECs were subcultured until reaching 80 %
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- 90 % confluence. After 4-9 passages, the HUVECs were collected for in vitro experiments. LPS was purchased from Sigma-Aldrich (L9641; St. Louis, MO, USA).
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Cell proliferation assay
The HUVEC suspension (100 μl per well) was seeded at a density of 5×10 4
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cells/mL onto 96-well culture plates for 24 h before treatment. SalA was added at various final concentrations (0.01, 0.1, 1 and 10 μmol/L) with or without 10 μg/mL LPS (n=6) (Fu et al., 2011). After treatment with SalA for 24 h, HUVEC proliferation was estimated using the Cell Counting Kit 8 (CCK-8; Dojindo Laboratories, Japan), according to the manufacturer’s instructions. The optical density at 450 nm was measured using a microplate reader (Tecan, Männedorf, Switzerland) with XFluor™ software.
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ACCEPTED MANUSCRIPT Wound-healing assay A HUVEC suspension at 1×105 cells/mL was seeded onto 6-well culture plates (2
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mL per well) until 90 % confluence was reached. After scratching with a sterile 1 mL
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pipette tip, the monolayer of HUVECs was washed three times with PBS to remove the detached cells, and fresh culture medium containing either LPS or LPS with SalA
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was added. The wound closure was digitally captured at 0 and 24 h (magnification ×40) using an Olympus CKX41 microscope with Olympus DP71 CCD camera
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(Olympus Corporation, Japan). The migrated area was quantified using Image-Pro Plus 6.0 image software (IPP 6.0, Media Cybemetics, Georgia, USA). The cells that
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migrated into the wound site were quantified and presented as relative would closure
al., 2012).
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compared with the control. The magnification of each picture was 40× (Korybalska et
Cell migration assay
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For the cell migration assay, 24-well transwell chambers with 8 µm pore size membranes (Millipore. Life Sciences, The Netherlands) were used. HUVECs (200 µL per chamber) were re-suspended in serum-free RPMI-1640 medium containing LPS or LPS with SalA at indicated concentration and seeded at a density of 5×104 cells/mL into the upper chambers. A total of 600 μL RPMI-1640 medium containing 10 % FBS was added to the lower chambers. After a 24-h incubation, HUVECs that adhered to the upper side of the membrane were removed with a cotton swab. The HUVECs that
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ACCEPTED MANUSCRIPT migrated to the lower side of membrane were washed three times with PBS, fixed with 95 % ethanol for 30 min and stained with 0.2 % crystal violet at room
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temperature. After washing, the membranes were digitally captured (magnification
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×200) using an Olympus BX51 microscope equipped with an Olympus DP71 CCD camera (Olympus Corporation, Japan). The number of migrated cells was quantified
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using Image-Pro Plus 6.0 image software (IPP 6.0, Media Cybemetics, Georgia, USA) (Giordano et al., 2014).
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Transendothelial electrical resistance assay The transendothelial electrical resistance (TEER) across HUVEC monolayers,
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reflecting the endothelial barrier integrity, was measured using a Millicell-ERS ohmmeter (Millipore, Bedford, MA, USA) according to the manufacturer’s
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instructions. Briefly, 200 μL HUVECs at 5×104 cells/mL were cultured in the upper chambers of the transwells (24-well type, Millipore, USA) and 1250 μL of culture
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medium were added to the lower chambers. The culture medium was exchanged every 2 days. LPS with or without SalA was administered at the indicated concentrations until the HUVECs formed a tight monolayer. After an additional 24 h of culture, the barrier function of the HUVEC monolayer was monitored using a Millicell-ERS ohmmeter (Kaneda et al., 2006).
Permeability assay HUVECs were seeded onto 24-well transwells with 8 μm pore-size membranes and
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ACCEPTED MANUSCRIPT cultured to form a restrictive endothelial monolayer. After the cells were treated with LPS with or without SalA as described above, the HUVEC permeability was
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evaluated after measuring the optical density of fluorescein isothiocyanate dextran
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4000Da (FD4, Sigma Inc., USA). Briefly, FD4 was added to the upper chambers at final concentration of 100 µg/mL, and then 100-µL samples were collected from
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lower chambers at 0, 0.5, 1, 2, 3, and 4 h. Each sample was transferred to a 96-well plate (100 μL per well), and the optical density was detected using a Tecan GENios
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FL Microplate Reader (Tecan, Männedorf, Switzerland) with XFluor™ software at an excitation wavelength of 485 nm and an emission wavelength of 535 nm (Kaneda et
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al., 2006).
Statistical analysis
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All quantitative values are expressed as the mean ± S.E.M. Nonlinear regression analysis of each dose-response curve was analyzed using GraphPad Prism 5.0
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software (San Diego, CA, USA). The data were calculated using one-way analysis of variance, and statistical calculations were analyzed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). The data from different groups were compared using one-way ANOVA. When equal variance was assumed, the least-significant difference (LSD) was used to compare the significance between the groups. When equal variance was not assumed, Dunnett’s T3 was used to compare the significance between the groups. P < 0.05 was considered statistically significant.
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Results Structure and Purity of SalA
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The molecular structure of SalA is displayed in Fig. 1A. The purity of SalA was
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purity of SalA was confirmed as 99.471 %.
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detected by HPLC, and the representative chromatograms are shown in Fig. 1B. The
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Fig. 1. Structure and purity of SalA. (A) The structure of SalA. (B) Purity of SalA was detected using high-performance liquid chromatography.
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Effect of SalA on body weight and blood pressure of SHR All animals survived to the predetermined endpoint. The body weight was monitored during the every two weeks for the entire experimental period, as shown in Fig. S1. Initially, there was no difference in body weight among the five groups. At the 2nd week, the body weight of the SHR group was lower than that of the WKY group (317.8±7.4 g versus 343.4±5.0 g, P < 0.01), while SalA treatment did not show any effects on the body weight compared with the SHR group. The value of body
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ACCEPTED MANUSCRIPT weight was 327.8±5.2 g, 329.2±4.3 g, and 321.5±6.9 g for SHR-SalA(2.5), SHR-SalA(5), and SHR-SalA(10), respectively. At the 4th week, no influence of SalA
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on body weight was observed compared with SHR group (Fig. 2A).
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Blood pressure was monitored every two weeks at the indicated time. At the beginning of the experiment, the blood pressure of the WKY group was lower than
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that of the SHR group (151.5±2.8 mmHg versus 182.2±3.6 mmHg, P < 0.001). During the entire experiment, SalA treatment showed no effect on blood pressure
mmHg, 199±2.7
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compared with the SHR group. At the 4th week, the blood pressure was 199.8±1.9 mmHg, 194.5±2.2
mmHg,
199.6±1.6
mmHg for SHR,
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SHR-SalA(2.5), SHR-SalA(5),and SHR-SalA(10), respectively (Fig. 2B). The influence of SalA on the heart rate of SHR was detected in the present study. The
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results showed no influence of SalA on the heart rate of SHR (data not shown).
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Fig. 2. The effect of SalA on body weight and blood pressure. (A) Body weight. (B) Blood pressure. The data are expressed as the mean ± S.E.M; #P < 0.5, ##P < 0.01, ###P < 0.001 compared with WKY.
Effect of SalA on retinal microvascular remodeling Previous studies have suggested that an assessment of retinal vascular changes might provide meaningful information concerning organ damage in persons with hypertension (Kim et al., 2010, Triantafyllou et al., 2014). In the present study, the 16
ACCEPTED MANUSCRIPT protection of SalA against retinal microvascular remodeling was evaluated using hematoxylin-eosin
staining
and
immunohistochemical
analysis.
For
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hematoxylin-eosin staining, the retinal endothelial cells in the SHR group were
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loosely arranged, while the retinal endothelial cells showed a closer and more regular arrangement in the SalA-treated SHR groups (Fig. 3A).
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The retinal vascular diameter is an important index to evaluate vascular remodeling. The retinal microvessels were further investigated using an immunohistochemical
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assay. The retinal veins were stained with FITC-coupled lectin (green), and the retinal arteries were more pronounced with α-SMA staining (red) (Fig. 3B). After staining,
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the retinal vascular diameter was directly quantified at a distance of 0.4 mm from the optical disc. The retinal arterial diameter of the SHR group was less than that of the
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WKY group (18.41±0.63 µm versus 24.83±0.82 µm, P < 0.001). SalA treatment inhibited this inward remodeling compared with the SHR group. The retinal arterial
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diameter was significantly restored in the SHR-SalA(5) (23.64±0.63 µm, P < 0.001) and SHR-SalA(10) (21.42±0.41 µm, P<0.001) groups compared with the SHR group (Fig. 3C). In addition, the diameter of the retinal vein was also significantly decreased in the SHR group compared with the WKY group (23.50±0.52 µm versus 38.03±1.63 µm, P < 0.001). The inward remodeling of the retinal vein was inhibited after treatment with SalA at 2.5, 5 and 10 mg/kg (Fig. 3D).
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Fig. 3. Effect of SalA on retinal perivascular cells and the retinal microvascular diameter of SHR. (A) Retinal vessels stained with hematoxylin-eosin. The arrows indicate retinal endothelial cells. Scale bar: 50 μm. (B) Retinal vessels stained with FITC-coupled lectin (green) and α-SMA antibody (red), the top graph shows lectin-positive veins, the middle graph shows α-SMA-positive arteries, and the bottom graph is a merged picture. Scale bar: 200 μm. (C) Quantitative data of lumen diameter
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ACCEPTED MANUSCRIPT for the retinal arteries. (D) Quantitative data of the lumen diameter of the retinal veins. The data are expressed as the mean ± S.E.M; ###P < 0.001 compared with WKY; *P <
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0.05, **P < 0.01, ***P < 0.001 compared with SHR.
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Effect of SalA on mesenteric vascular function
The mesenteric artery is the candidate artery for the evaluation of microvascular
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function. Because SalA inhibited the inward remodeling of retinal microvessels, we assessed the effect of SalA on microvascular function in SHR using DMT wire
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myography (Fig. 4). Acetylcholine (Ach) induced concentration-dependent relaxation in the mesenteric arteries with functional endothelium after pre-contraction with
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DL-phenylephrine hydrochloride. The relaxation of the arteries in the SHR group, elicited by treatment with 10 μM ACh, was reduced compared with the arteries from
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the WKY group (48.82±7.23 % versus 87.81±3.51 %, P < 0.001). SalA treatment improved endothelial function, and the values of relaxation induced by 10 μM ACh
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were 70.83±3.61 %, 63.91±12.82 %, and 72.93±4.84 % for SHR-SalA(2.5), SHR-SalA(5), and SHR-SalA(10), respectively.
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Fig. 4. Effect of SalA on the microvascular endothelial diastolic function of SHR
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measurement using DMT wire myography. Relative relaxation of small mesenteric arteries from WKY and SHR groups in response to acetylcholine (ACh). The data are ###
P < 0.001 compared with WKY; *P <
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expressed as the mean ± S.E.M; #P < 0.05, 0.05, **P < 0.01 compared with SHR.
The
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Effect of SalA on HUVEC proliferation results
of
the
present
study
showed
that
SalA
improved
the
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endothelial-dependent vasodilatation of mesenteric vessels for SHR in vivo. Subsequently, the direct effects of SalA on the function of endothelial cells were detected in vitro. There was no obvious impact on HUVEC proliferation at the detected concentrations of SalA (Fig. 5A). Treatment with 10 μg/mL of LPS induced endothelial cell dysfunction, and the cell viability was significantly decreased after LPS treatment compared with the control group (0.26±0.02 versus 0.30±0.01, P < 0.01). No effect of SalA on cell proliferation was observed at the detected dose used
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ACCEPTED MANUSCRIPT for LPS stimulation (Fig. 5B). Treatment with1 and 10 μM SalA was used in the
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following studies.
Fig. 5. Effect of SalA on HUVEC proliferation. (A) HUVECs were treated with SalA at concentrations of 0, 0.01, 0.1, 1 and 10 μmol/L for 24 h. The effect of SalA on the proliferation of HUVECs was determined using the Cell Counting Kit-8. (B) HUVECs were simultaneously treated with LPS (10 μg/mL) and increasing concentrations of 0, 0.01, 0.1, 1 and 10 μmol/L SalA for 24 h. The viability of HUVECs was evaluated using the Cell Counting Kit-8. The data are expressed as the
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ACCEPTED MANUSCRIPT mean ± S.E.M; #P < 0.05, ##P < 0.001 compared with Control.
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Effect of SalA on HUVEC wound healing and migration
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The direct protection effect of SalA was further evaluated in the migration of HUVECs. As shown in Fig. 6A and 6C, SalA did not affect the motility of HUVECs
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in the wound-healing assay. The migration was further evaluated using a transwell assay. As shown in Fig. 6B and 6D, the considerable enhancement of LPS on
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HUVECs migration was observed (55.1±15.3 versus 28.6±9.0 cells per field), and SalA significantly inhibited this effect (26.0±7.7 versus 55.1±15.3 cells per field for 1
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μM SalA and 20.8±6.4 versus 55.1±15.3 cells per field for 10 μM SalA).
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Fig. 6. Effect of SalA on HUVEC wound healing and migration. (A) Wound closure
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at 0 and 24 h. Scale bar: 200 μm (B) Representative photomicrographs of cell migration across the membrane of the chambers. Scale bar: 100 μm. (C) The quantitative data for wound closure. The data are expressed as the mean ± S.E.M. (D) The quantitative data for migration cell. The data are expressed as the mean ± S.E.M; ##
P < 0.01 compared with control; ** P < 0.01 compared with LPS.
Effect of SalA on HUVEC TEER and the permeability assay The integrity of the endothelial monolayer has important physiological significance
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ACCEPTED MANUSCRIPT in the microvascular system. Two methods, TEER and the permeability assay, were used to assess the integrity of the endothelial monolayer on HUVECs. After
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incubation with LPS with or without SalA for 24 h, TEER was determined using a
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Millicell-ERS ohmmeter. The value of TEER was significantly decreased in the HUVEC monolayer stimulated with LPS compared with the control group (62.1±2.5
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Ω·cm2 versus 80.8±2.3 Ω·cm2, P < 0.01). Compared with the LPS group, TEER significantly increased the HUVEC monolayer treated with SalA (P < 0.01) (Fig. 7A).
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The values of TEER were 79.2±2.5 Ω·cm2 and 83.9±5.4 Ω·cm2 for LPS-SalA(1) and LPS-SalA(10), respectively.
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In addition to the TEER assay, a permeability assay was also used to evaluate the integrity of the endothelial monolayer. The optical density of fluorescein
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isothiocyanate dextran 4000Da was used to evaluate the permeability of the HUVEC monolayer. Fluorescein isothiocyanate dextran 4000Da infiltrated the endothelial
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monolayer with increasing time. After 4 h, LPS treatment significantly increased the optical density compared with the control (40891 versus 25905 P<0.01), and 10 μM SalA exhibited an obvious protective effect on the HUVEC monolayer compared with the LPS group (20078 versus 40891 P < 0.01) (Fig. 7B).
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Fig. 7. Effect of SalA on HUVEC TEER and solute permeability. (A) The TEER of
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confluent HUVECs was monitored using a Millicell-ERS ohmmeter. The quantitative data are expressed as the mean ± S.E.M. (B) The cell permeability was assessed after
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adding fluoresceinated dextran-4000 to the upper chambers. Fluorescence in the lower chamber was measured at 0, 0.5, 1, 2, 3 and 4 h. The quantitative data are expressed as the mean ± S.E.M; ##P < 0.01 compared with control; **P < 0.01 compared with LPS.
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Discussion In the present study, SalA showed obvious protection against microvascular
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remodeling in vivo and directly protected endothelial cells in vitro.
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After SalA administration for 4 weeks, there was no impact on body weight and blood pressure in the SHR groups. SalA showed significant protection against
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remodeling for both microvascular structure and function, suggesting that the beneficial effect of SalA was not associated with the effect of this compound on blood
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pressure. Microvascular remodeling is a key pathological characteristic, associated with the incidence of end-organ damage induced by hypertension. Because the retinal
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microvessels are similar in anatomical features and physiological properties to those of the kidney, the retinal microvessels are considered to be a predictor to examine
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kidney damage. Grunwald et al. revealed that the pathology of retinal microvessels reflected renal microvascular pathology (Grunwald et al., 2012a; Grunwald et al.,
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2012b). Baki et al. also had reported that the change of retinal microvessels was closely associated with that of kidney impairment (Baki et al., 2014). In previous studies, we also reported that vascular remodeling eventually caused targeted organ damage, such as cardiomyocyte hypertrophy, cardio-fibrosis, and glomerular hyalinization in spontaneously hypertensive rats (Chen et al., 2012). In the present study, the protective effect of SalA against retinal vascular remodeling was confirmed, indicating the potential protection against end-organ damage induced by hypertension,
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ACCEPTED MANUSCRIPT such as renal damage. The vascular endothelium plays a key role in maintaining vascular homeostasis, such as barrier function, regulating local cellular growth and
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secreting cytokines. Almost all patients with hypertension have vascular endothelial
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injuries. Karaca et al. reported that endothelial dysfunction in vascular lesions might lead to end-organ damage under hypertension (Karaca et al., 2014). Zhai et al.
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demonstrated that, in the absence of VEGF signaling, glomerular endothelial cells could not maintain the integrity of the glomerular filtration barrier, resulting in
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proteinuria. The dysfunction of endothelium-dependent relaxation is serves as a pathological characteristic of hypertension (Zhai et al., 2014). ACh activates M
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subtype vascular endothelial cells, which release nitric oxide and eventually induce vascular vasodilator, as a tool to evaluate vascular endothelial function (Wassmann et
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al., 2002). In the present study, endothelial function was evaluated by DMT wire myography in vivo, and SalA could improve ACh-mediated and endothelial
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cell-dependent microvascular diastolic function in SHR. Increasing nitric oxide also could decrease the mean arterial pressure, heart rate and sympathetic activity in rats by the activation of adenosine A2A receptors and reduction of ACh and M1 receptor levels in the rostral ventrolateral medulla (Jiang et al., 2011). In addition to confirming the beneficial effects of SalA on microvascular function in vivo, the direct protection of endothelial cells by SalA was further evaluated in vitro using HUVECs. The mobility of endothelial cells was assessed using wound healing and transwell
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ACCEPTED MANUSCRIPT assays, and the integrity of endothelial cell was assessed using TEER and permeability assays. HUVECs exposed to LPS secreted large amounts of MMP-9.
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MMP-9 causes the degradation of the extracellular matrix and damages cell junctions,
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ultimately enhancing the capability of cell migration and opening of intercellular gaps to increase permeability in HUVECs (Lappas, 2012, Jang et al., 2014). In a previous
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study, we showed that SalA is a novel matrix metalloproteinase-9 inhibitor for the suppression of vascular remodeling (Zhang et al., 2014). Southgate et al. reported that
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MMP-9 might be involved in the early vascular remodeling associated with hypertension (Southgate et al., 1999). Therefore, we proposed that SalA would inhibit
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the expression of MMP-9, which might reduce the damage to vascular endothelial cells, maintain the permeability and integrity of vascular intima and improve the
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vascular diastolic function in SHR rats. The results showed that SalA not only maintained the integrity of the endothelial
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monolayer but also inhibited endothelial mobility, consistent with the findings in SHR. In the present study, we showed that SalA could be a potential drug candidate to prevent targeted organ damage induced by vascular remodeling.
Conclusion In summary, SalA inhibited the remodeling of microvessels and normalized endothelial dysfunction, suggesting that SalA has potential beneficial effects on the microvascular system and against microvascular complications induced by
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hypertension.
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Acknowledgments This work was financially supported by grants from the National Natural Science
for
“Key
New
Drug
Creation
and
Manufacturing
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Project
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Foundation of China Grants (81173587), National Science and Technology Major Program”
(2013ZX09103002-024), and the Shanghai Science and Technology Development
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Foundation (14401900900). This work was also partially supported by the Croucher
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Foundation (CAS14201).
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Figure captions Fig. 1. Structure and purity of SalA. (A) The structure of SalA. (B) Purity of SalA was
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detected using high-performance liquid chromatography.
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Fig. 2. The effect of SalA on body weight and blood pressure. (A) Body weight. (B) Blood pressure. The data are expressed as the mean ± S.E.M; #P < 0.5, ##P < 0.01, ###P
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< 0.001 compared with WKY.
Fig. 3. Effect of SalA on retinal perivascular cells and the retinal microvascular
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diameter of SHR. (A) Retinal vessels stained with hematoxylin-eosin. The arrows indicate retinal endothelial cells. Scale bar: 50 μm. (B) Retinal vessels stained with
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FITC-coupled lectin (green) and α-SMA antibody (red), the top graph shows lectin-positive veins, the middle graph shows α-SMA-positive arteries, and the bottom
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graph is a merged picture. Scale bar: 200 μm. (C) Quantitative data of lumen diameter for the retinal arteries. (D) Quantitative data of the lumen diameter of the retinal veins.
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The data are expressed as the mean ± S.E.M; ###P < 0.001 compared with WKY; *P < 0.05, **P < 0.01, ***P < 0.001 compared with SHR. Fig. 4. Effect of SalA on the microvascular endothelial diastolic function of SHR measurement using DMT wire myography. Relative relaxation of small mesenteric arteries from WKY and SHR groups in response to acetylcholine (ACh). The data are expressed as the mean ± S.E.M; #P < 0.05, 0.05, **P < 0.01 compared with SHR.
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###
P < 0.001 compared with WKY; *P <
ACCEPTED MANUSCRIPT Fig. 5. Effect of SalA on HUVEC proliferation. (A) HUVECs were treated with SalA at concentrations of 0, 0.01, 0.1, 1 and 10 μmol/L for 24 h. The effect of SalA on the
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proliferation of HUVECs was determined using the Cell Counting Kit-8. (B)
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HUVECs were simultaneously treated with LPS (10 μg/mL) and increasing concentrations of 0, 0.01, 0.1, 1 and 10 μmol/L SalA for 24 h. The viability of
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HUVECs was evaluated using the Cell Counting Kit-8. The data are expressed as the mean ± S.E.M; #P < 0.05, ##P < 0.001 compared with Control.
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Fig. 6. Effect of SalA on HUVEC wound healing and migration. (A) Wound closure at 0 and 24 h. Scale bar: 200 μm (B) Representative photomicrographs of cell
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migration across the membrane of the chambers. Scale bar: 100 μm. (C) The quantitative data for wound closure. The data are expressed as the mean ± S.E.M. (D)
##
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The quantitative data for migration cell. The data are expressed as the mean ± S.E.M; P < 0.01 compared with control; ** P < 0.01 compared with LPS.
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Fig. 7. Effect of SalA on HUVEC TEER and solute permeability. (A) The TEER of confluent HUVECs was monitored using a Millicell-ERS ohmmeter. The quantitative data are expressed as the mean ± S.E.M. (B) The cell permeability was assessed after adding fluoresceinated dextran-4000 to the upper chambers. Fluorescence in the lower chamber was measured at 0, 0.5, 1, 2, 3 and 4 h. The quantitative data are expressed as the mean ± S.E.M;
##
P < 0.01 compared with control;
LPS.
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**
P < 0.01 compared with
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Baik, S.Y., Lim, Y.A., Kang, S.J., Ahn, S.H., Lee, W.G., Kim, C.H., 2014. Effects of platelet lysate preparations on the proliferation of HaCaT cells. Annals of laboratory medicine 34, 43-50. Baki, A.H., Soliman, Y., Seif, E.I., 2014. Histopathological Association between Vascular Hypertensive Changes and Different Types of Glomerulopathies. Arab journal of nephrology and transplantation 7, 21-26. Brondum, E., Kold-Petersen, H., Simonsen, U., Aalkjaer, C., 2010. NS309 restores EDHF-type relaxation in mesenteric small arteries from type 2 diabetic ZDF rats. British journal of pharmacology 159, 154-165. Chen, H., Yin, J., Deng, Y., Yang, M., Xu, L., Teng, F., Li, D., Cheng, Y., Liu, S., Wang, D., Zhang, T., Wu, W., Liu, X., Guan, S., Jiang, B., Guo, D., 2012. The protective effects of ginsenoside Rg1 against hypertension target-organ damage in spontaneously hypertensive rats. BMC complementary and alternative medicine 12, 53. Feihl, F., Liaudet, L., Levy, B.I., Waeber, B., 2008. Hypertension and microvascular remodelling. Cardiovascular research 78, 274-285. Fu R, Chen Z, Wang Q, Guo Q, Xu J, Wu X. XJP-1, a novel ACEI, with anti-inflammatory properties in HUVECs. Atherosclerosis. 2011;219:40-8 Giordano, A., D'Angelillo, A., Romano, S., D'Arrigo, P., Corcione, N., Bisogni, R., Messina, S., Polimeno, M., Pepino, P., Ferraro, P., Romano, M.F., 2014. Tirofiban induces VEGF production and stimulates migration and proliferation of endothelial cells. Vascul Pharmacol 61, 63-71. Grunwald, J.E., Alexander, J., Ying, G.S., Maguire, M., Daniel, E., Whittock-Martin, R., Parker, C., McWilliams, K., Lo, J.C., Go, A., Townsend, R., Gadegbeku, C.A., Lash, J.P., Fink, J.C., Rahman, M., Feldman, H., Kusek, J.W., Xie, D., Jaar, B.G., Group, C.S., 2012a. Retinopathy and chronic kidney disease in the Chronic Renal Insufficiency Cohort (CRIC) study. Arch Ophthalmol 130, 1136-1144. Grunwald, J.E., Ying, G.S., Maguire, M., Pistilli, M., Daniel, E., Alexander, J., Whittock-Martin, R., Parker, C., Mohler, E., Lo, J.C.M., Townsend, R., Gadegbeku, C.A., Lash, J.P., Fink, J.C., Rahman, M., Feldman, H., Kusek, J.W., Xie, D.W., Coleman, M., Keane, M.G., Cohort, C.R.I., 2012b. Association Between Retinopathy and Cardiovascular Disease in Patients With Chronic Kidney Disease (from the Chronic Renal Insufficiency Cohort [CRIC] Study). Am J Cardiol 110, 246-253. Higashi, Y., Sasaki, S., Nakagawa, K., Ueda, T., Yoshimizu, A., Kurisu, S., Matsuura, H., Kajiyama, G., Oshima, T., 2000. A comparison of angiotensin-converting enzyme inhibitors, calcium antagonists, beta-blockers and diuretic agents on reactive hyperemia in patients with essential hypertension: a multicenter study. Journal of the American College of Cardiology 35, 284-291. 34
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Ho, J.H., Hong, C.Y., 2011. Salvianolic acids: small compounds with multiple mechanisms for cardiovascular protection. Journal of biomedical science 18, 30. Jang CH, Cho YB, Choi CH, Jang YS, Jang SJ, Jung WK, et al. ALH-L1005 attenuates endotoxin induced inner ear damage. International journal of pediatric otorhinolaryngology. 2014;78:465-70. Jiang, B., Li, D., Deng, Y., Teng, F., Chen, J., Xue, S., Kong, X., Luo, C., Shen, X., Jiang, H., Xu, F., Yang, W., Yin, J., Wang, Y., Chen, H., Wu, W., Liu, X., Guo, D.A., 2013. Salvianolic acid A, a novel matrix metalloproteinase-9 inhibitor, prevents cardiac remodeling in spontaneously hypertensive rats. PloS one 8, e59621. Jiang, B., Zhang, L., Li, M., Wu, W., Yang, M., Wang, J., Guo, D.A., 2008. Salvianolic acids prevent acute doxorubicin cardiotoxicity in mice through suppression of oxidative stress. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 46, 1510-1515. Jiang MY, Chen J, Wang J, Xiao F, Zhang HH, Zhang CR, et al. Nitric oxide modulates cardiovascular function in the rat by activating adenosine A2A receptors and inhibiting acetylcholine release in the rostral ventrolateral medulla. Clinical and experimental pharmacology & physiology. 2011;38:380-6 Kaneda, K., Miyamoto, K., Nomura, S., Horiuchi, T., 2006. Intercellular localization of occludins and ZO-1 as a solute transport barrier of the mesothelial monolayer. Journal of artificial organs : the official journal of the Japanese Society for Artificial Organs 9, 241-250. Karaca, M., Coban, E., Ozdem, S., Unal, M., Salim, O., OrhanYucel, 2014. The association between endothelial dysfunction and hypertensive retinopathy in essential hypertension. Med Sci Monitor 20, 78-82. Kim GH, Youn HJ, Kang S, Choi YS, Moon JI. Relation between grade II hypertensive retinopathy and coronary artery disease in treated essential hypertensives. Clin Exp Hypertens. 2010;32:469-73. Korybalska, K., Kawka, E., Breborowicz, A., Witowski, J., 2012. Atorvastatin does not impair endothelial cell wound healing in an in vitro model of vascular injury. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society 63, 389-395. Lappas M. Anti-inflammatory properties of sirtuin 6 in human umbilical vein endothelial cells. Mediators of inflammation. 2012;2012:597514. Licht, A.H., Nubel, T., Feldner, A., Jurisch-Yaksi, N., Marcello, M., Demicheva, E., Hu, J.H., Hartenstein, B., Augustin, H.G., Hecker, M., Angel, P., Korff, T., Schorpp-Kistner, M., 2010. Junb regulates arterial contraction capacity, cellular contractility, and motility via its target Myl9 in mice. The Journal of clinical investigation 120, 2307-2318. Southgate KM, Mehta D, Izzat MB, Newby AC, Angelini GD. Increased secretion of 35
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basement membrane-degrading metalloproteinases in pig saphenous vein into carotid artery interposition grafts. Arteriosclerosis, thrombosis, and vascular biology. 1999;19:1640-9. Triantafyllou A, Anyfanti P, Zabulis X, Gavriilaki E, Karamaounas P, Gkaliagkousi E, et al. Accumulation of microvascular target organ damage in newly diagnosed hypertensive patients. Journal of the American Society of Hypertension : JASH. 2014;8:542-9. Wassmann, S., Laufs, U., Stamenkovic, D., Linz, W., Stasch, J.P., Ahlbory, K., Rosen, R., Bohm, M., Nickenig, G., 2002. Raloxifene improves endothelial dysfunction in hypertension by reduced oxidative stress and enhanced nitric oxide production. Circulation 105, 2083-2091. Zhai, Y.L., Zhu, L., Shi, S.F., Liu, L.J., Lv, J.C., Zhang, H., 2014. Elevated Soluble VEGF Receptor sFlt-1 Correlates with Endothelial Injury in IgA Nephropathy. PloS one 9. Zhang T, Xu J, Li D, Chen J, Shen X, Xu F, et al. Salvianolic acid A, a matrix metalloproteinase-9 inhibitor of Salvia miltiorrhiza, attenuates aortic aneurysm formation in apolipoprotein E-deficient mice. Phytomedicine : international journal of phytotherapy and phytopharmacology. 2014;21:1137-45.
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Graphical Abstract
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S0024-3205(15)00333-1 doi: 10.1016/j.lfs.2015.06.010 LFS 14417
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
3 February 2015 11 June 2015 14 June 2015
Please cite this article as: Teng Fukang, Yin Ying, Cui Yajun, Deng Yanping, Li Defang, Cho Kenka, Zhang Ge, Lu Aiping, Wu Wanying, Yang Min, Liu Xuan, Guo De-an, Yin Jun, Jiang Baohong, Salvianolic acid A inhibits endothelial dysfunction and vascular remodeling in spontaneously hypertensive rats, Life Sciences (2015), doi: 10.1016/j.lfs.2015.06.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Salvianolic acid A inhibits endothelial dysfunction and vascular remodeling in
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spontaneously hypertensive rats Fukang Tenga, b, 1, Ying Yinb, Yajun Cuic,Yanping Dengb, Defang Lie, Kenka Chod,
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Ge Zhange, Aiping Lue, Wanying Wub, Min Yangb, Xuan Liub, De-an Guob, Jun Yina*,
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Baohong Jiangb*
Shenyang Pharmaceutical University, Wenhua Road #103, Shenyang 110016, China
b
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
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a
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Affiliation
c
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201203, China.
Shanghai University of Traditional Chinese Medicine, Cailun Road #1200, Shanghai
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201203, China
Takarazuka University of Medical and Health Care, Hanayashiki-Midorigaoka,
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Takarazuka City 6660162, Japan Institute for Advancing Translational Medicine in Bone and Joint Diseases, School of
Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China.
*
Corresponding author
Pro. Jun Yin, E-mail: [email protected], 86-24-23986491 Pro. Baohong Jiang, E-mail: [email protected], 86-21-50272223
Conflicts of Interest 1
ACCEPTED MANUSCRIPT The authors declare that there are no conflicts of interest.
Abstract
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Aims: Despite the numerous pharmacological agents available for hypertension
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therapy, hypertension-related microvascular remodeling is not resolved, eventually leading to end-organ damage. The aim of the present study was to investigate the protection of salvianolic acid A (SalA) against microvascular remodeling in vitro and
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in vivo.
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Main methods: Spontaneously hypertensive rats (SHR) were administered 2.5, 5 or 10 mg/kg SalA via intraperitoneal injection once a day for 4 weeks. The tail-cuff method
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was applied to monitor blood pressure; the microvascular structure of retina was detected by hematoxylin-eosin and immunohistochemical staining; the function of
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mesenteric arteries was measured by DMT wire myography; endothelial cell proliferation was estimated using Cell Counting Kit 8; endothelial cell migration was
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evaluated by wound healing and transwell assay; and endothelial cell integrity was detected by transendothelial electrical resistance and permeability assays. Key findings: Although no antihypertensive effects of SalA were observed, SalA attenuated the microvascular inward remodeling of the retina and improved microvascular function in the mesenteries in vivo. Further cell experiments confirmed the beneficial effects of SalA on the integrity of the endothelial monolayer in vitro. Significance: Salvianolic acid A inhibited endothelial dysfunction and vascular remodeling in spontaneously hypertensive rats. Therefore, salvianolic acid A could be 2
ACCEPTED MANUSCRIPT a potential drug therapy to prevent further targeted organ damage induced by vascular remodeling. salvianolic
acid
A;
hypertension;
microvascular
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spontaneously hypertensive rats; vascular endothelial function
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Keywords:
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remodeling;
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Introduction Hypertension is the most common cause of damage to the microvascular system in
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humans, potentially leading to microvascular remodeling and ultimately resulting in
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severe complications, such as retinopathy, renal failure and heart failure (Feihl et al., 2008). Although antihypertensive drugs, such as angiotensin-converting enzyme
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(ACE) inhibitors, calcium-channel blockers and angiotensin II receptor blockers, are extensively applied for clinical therapy, the hypertension-induced impairment of the
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microvascular system is not resolved (Higashi et al., 2000). Hence, new drugs and therapeutic strategies for hypertension-related microvascular remodeling are urgently
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needed.
Salvia miltiorrhiza (Danshen), a traditional Chinese medicine, is broadly used as a
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clinical drug to treat cardiovascular diseases. Salvianolic acid A (SalA), one of the most bioactive compounds in Salvia miltiorrhiza, has many pharmacological activities,
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including anti-oxidation, myocardial protection, antithrombosis, antifibrosis, the prevention of diabetes complications (Ho and Hong, 2011). Recently, we observed that SalA is a novel matrix metalloproteinase-9 inhibitor that prevents cardiac remodeling in spontaneously hypertensive rats (Jiang et al., 2013). Microvascular remodeling is an important step for end-organ damage. However, whether SalA prevents microvascular remodeling induced by hypertension remains unknown. To elucidate the effects of SalA on microvascular remodeling, we estimated the
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ACCEPTED MANUSCRIPT protective effects of SalA on microvascular structure and function in representative organs prone to damage under hypertension in vivo. Based on the results of in vivo
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detection, the direct protection of microvascular endothelial cells by SalA was
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confirmed in vitro.
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Materials and Methods Animals
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Eight-week-old male spontaneously hypertensive rats (SHR, 280–300 g) and Wistar
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Kyoto rats (WKY, 280–300 g) were obtained from the Shanghai Center of Experimental Animals and acclimatized for 1 week. WKY rats fed ordinary laboratory
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chow and administered with saline via intraperitoneal injection were presented as normal controls (WKY, n=10). SHR were randomly divided into four groups of ten
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rats each and fed with 8.0 % high salt chow throughout the whole experiment. SHR treated with saline were regarded as a hypertensive model (SHR, n=10); SHR in the
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other three groups were administered with 2.5 mg/kg SalA (SHR-SalA(2.5), n=10), 5 mg/kg SalA (SHR-SalA(5), n=10) or 10 mg/kg SalA (SHR-SalA(10), n=10).
SalA
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dissolved in saline was administered once a day via intraperitoneal injection. All rats were maintained in a room with a 12/12 h light/dark cycle at constant temperature
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(22–23 °C) and free access to food and water. This study was approved by the Animal Care and Use Committee (SIMM-AE-GDA-2010-05) in accordance with the guidelines for the ethical care of experimental animals. The National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals was followed throughout the entire experiment.
SalA purity assay SalA was purchased from Shanghai Yousi Bio-Tech Co., Ltd (Yousi, Shanghai
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ACCEPTED MANUSCRIPT Yousi Bio-Tech Co., China). The high performance liquid chromatography (HPLC) gradient elution method was used to determine the purity of SalA. Briefly, SalA was
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dissolved in acetonitrile, passed through a 0.45 μm filter membrane and detected
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using the Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA) with a Zorbax Extend SB-C18 column (5 μm, 250 mm×4.6 mm). The loading volume
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of the sample was 10 μl, the wavelength of detection was set at 280 nm, the flow rate of mobile phase was 0.8 mL/min and the column temperature was maintained at
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20 °C. The composition of the mobile phase was 0.05 % aqueous trifluoroacetic acid (V/V), 2 %-10 % acetonitrile at 0-7 min, 10 %-30 % acetonitrile at 7-20 min,
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23 %-27 % acetonitrile at 20-35 min, and 27 %-60 % acetonitrile at 35-50 min (Jiang et al., 2008).
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Body weight and blood pressure monitoring Body weight and blood pressure were monitored at the indicated times (Fig. S1).
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Blood pressure was indirectly measured in a conscious rat using the tail cuff method as previously described. Briefly, the conscious animals were conditioned to restraint on a heating pad. After stabilization for 10–20 min on the heating pad at 37 °C, the blood pressure was continuously recorded using a tail-cuff apparatus (ALC-NIBP, Shanghai Alcott Biotech Co., China) (Zhang et al., 2014, Chen et al., 2012).
Hematoxylin-eosin staining At the end of the experiment, the rats were euthanized with an overdose of chloral
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sections (3-µm-thick) and stained with hematoxylin-eosin. The images were digitally
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captured (magnification ×400) using an Olympus BX51 microscope equipped with an Olympus DP71 CCD camera (Olympus Corporation, Japan).
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Immunohistochemical detection of the retinal vessels After euthanasia, the rat eyes were removed and fixed in a 4 % formaldehyde
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solution for 1 day. The retinas were dissected and flattened onto slides, washed 3 times with PBS, and subsequently incubated with PBS containing 0.5 % Triton X-100
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and 10 % normal goat serum for 1 h at room temperature. After washing 3 times with PBS, the retinas were incubated with α-SMA-Cy3 (red) (1:400, Sigma-Aldrich,
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Germany) and lectin-FITC (green) (1:200, Sigma-Aldrich, Germany) at 37 °C for 1.5 h, followed by washing 3 times with PBS. The images were digitally captured
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(magnification ×100) using an Olympus BX51 fluorescence microscope equipped with an Olympus DP71 CCD camera (Olympus Corporation, Japan), and the retinal vascular diameter was directly measured at a distance of 0.4 mm from the optical disc using Image-Pro Plus 6.0 image software (IPP 6.0, Media Cybemetics, USA) (Licht et al., 2010).
Microvascular function measurements Microvascular function was measured using DMT wire myography (Model 620M,
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composition in (g/l): NaCl 69.54, KCl 3.5, CaCl2 2.78, MgSO4 1.4112, KH2PO4 1.61,
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EDTA 0.1, NaHCO3 21 and glucose 10.91). The mesenteries were maintained in oxygenated (5 % CO2, 95 % O2 mixture) PSS at 4 °C until dissection. Mesenteric
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small arteries (2 mm long) were isolated from adipose or connective tissue under a dissecting microscope. After passage through two stainless steel wires (40 μm in
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diameter), the dissected mesenteric small arteries were mounted as ring-shaped preparations on a quadruple wire to measure microvascular function. The bath of each
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myograph contained 5 mL oxygenated (5 % CO2, 95 % O2 mixture) PSS at 37 °C to keep pH 7.4. Normalization was performed to set the pre-tension of the mesenteric
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small arteries. Briefly, after stabilizing for 30 min in PSS, the mesenteric small arteries were contracted with DL-phenylephrine hydrochloride (PE) (Adamas Reagent
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Co., Ltd) (1×10-2 M) dissolved in PSS with 125 mM K+, washed to determine viability and stabilized for 30 min. After preconstruction with PE (3×10-5 M), the mesenteric
small
arterial
contraction
was
stabilized.
Subsequently,
the
endothelium-dependent vasodilator acetylcholine chloride (ACh) (Sinopharm Chemical Reagent Co., Ltd, China) (10 pmol/L to 10 μmol/L) was added in a cumulative manner, and concentration–response curves were obtained. The relaxation induced
by
ACh
was
normalized
to
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the
contraction
induced
by
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Cell line and cell culture
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The human umbilical vein endothelial cell line (HUVEC) was purchased from the Beijing North Albert Development Co. Ltd. HUVECs were cultured in 75-cm2 flasks
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containing RPMI-1640 medium (Hyclone, Thermo Fisher Scientific, USA) supplemented with 10 % fetal bovine serum (GIBCO, Invitrogen Inc., USA), 100
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U/mL penicillin and 100 mg/L streptomycin (Sigma, Sigma Inc., USA) at 37 °C in a 5 % CO2 humidified atmosphere. The HUVECs were subcultured until reaching 80 %
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- 90 % confluence. After 4-9 passages, the HUVECs were collected for in vitro experiments. LPS was purchased from Sigma-Aldrich (L9641; St. Louis, MO, USA).
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Cell proliferation assay
The HUVEC suspension (100 μl per well) was seeded at a density of 5×10 4
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cells/mL onto 96-well culture plates for 24 h before treatment. SalA was added at various final concentrations (0.01, 0.1, 1 and 10 μmol/L) with or without 10 μg/mL LPS (n=6) (Fu et al., 2011). After treatment with SalA for 24 h, HUVEC proliferation was estimated using the Cell Counting Kit 8 (CCK-8; Dojindo Laboratories, Japan), according to the manufacturer’s instructions. The optical density at 450 nm was measured using a microplate reader (Tecan, Männedorf, Switzerland) with XFluor™ software.
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ACCEPTED MANUSCRIPT Wound-healing assay A HUVEC suspension at 1×105 cells/mL was seeded onto 6-well culture plates (2
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mL per well) until 90 % confluence was reached. After scratching with a sterile 1 mL
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pipette tip, the monolayer of HUVECs was washed three times with PBS to remove the detached cells, and fresh culture medium containing either LPS or LPS with SalA
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was added. The wound closure was digitally captured at 0 and 24 h (magnification ×40) using an Olympus CKX41 microscope with Olympus DP71 CCD camera
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(Olympus Corporation, Japan). The migrated area was quantified using Image-Pro Plus 6.0 image software (IPP 6.0, Media Cybemetics, Georgia, USA). The cells that
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migrated into the wound site were quantified and presented as relative would closure
al., 2012).
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compared with the control. The magnification of each picture was 40× (Korybalska et
Cell migration assay
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For the cell migration assay, 24-well transwell chambers with 8 µm pore size membranes (Millipore. Life Sciences, The Netherlands) were used. HUVECs (200 µL per chamber) were re-suspended in serum-free RPMI-1640 medium containing LPS or LPS with SalA at indicated concentration and seeded at a density of 5×104 cells/mL into the upper chambers. A total of 600 μL RPMI-1640 medium containing 10 % FBS was added to the lower chambers. After a 24-h incubation, HUVECs that adhered to the upper side of the membrane were removed with a cotton swab. The HUVECs that
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temperature. After washing, the membranes were digitally captured (magnification
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×200) using an Olympus BX51 microscope equipped with an Olympus DP71 CCD camera (Olympus Corporation, Japan). The number of migrated cells was quantified
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using Image-Pro Plus 6.0 image software (IPP 6.0, Media Cybemetics, Georgia, USA) (Giordano et al., 2014).
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Transendothelial electrical resistance assay The transendothelial electrical resistance (TEER) across HUVEC monolayers,
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reflecting the endothelial barrier integrity, was measured using a Millicell-ERS ohmmeter (Millipore, Bedford, MA, USA) according to the manufacturer’s
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instructions. Briefly, 200 μL HUVECs at 5×104 cells/mL were cultured in the upper chambers of the transwells (24-well type, Millipore, USA) and 1250 μL of culture
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medium were added to the lower chambers. The culture medium was exchanged every 2 days. LPS with or without SalA was administered at the indicated concentrations until the HUVECs formed a tight monolayer. After an additional 24 h of culture, the barrier function of the HUVEC monolayer was monitored using a Millicell-ERS ohmmeter (Kaneda et al., 2006).
Permeability assay HUVECs were seeded onto 24-well transwells with 8 μm pore-size membranes and
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evaluated after measuring the optical density of fluorescein isothiocyanate dextran
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4000Da (FD4, Sigma Inc., USA). Briefly, FD4 was added to the upper chambers at final concentration of 100 µg/mL, and then 100-µL samples were collected from
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lower chambers at 0, 0.5, 1, 2, 3, and 4 h. Each sample was transferred to a 96-well plate (100 μL per well), and the optical density was detected using a Tecan GENios
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FL Microplate Reader (Tecan, Männedorf, Switzerland) with XFluor™ software at an excitation wavelength of 485 nm and an emission wavelength of 535 nm (Kaneda et
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Statistical analysis
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All quantitative values are expressed as the mean ± S.E.M. Nonlinear regression analysis of each dose-response curve was analyzed using GraphPad Prism 5.0
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software (San Diego, CA, USA). The data were calculated using one-way analysis of variance, and statistical calculations were analyzed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). The data from different groups were compared using one-way ANOVA. When equal variance was assumed, the least-significant difference (LSD) was used to compare the significance between the groups. When equal variance was not assumed, Dunnett’s T3 was used to compare the significance between the groups. P < 0.05 was considered statistically significant.
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Results Structure and Purity of SalA
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The molecular structure of SalA is displayed in Fig. 1A. The purity of SalA was
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purity of SalA was confirmed as 99.471 %.
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detected by HPLC, and the representative chromatograms are shown in Fig. 1B. The
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Fig. 1. Structure and purity of SalA. (A) The structure of SalA. (B) Purity of SalA was detected using high-performance liquid chromatography.
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Effect of SalA on body weight and blood pressure of SHR All animals survived to the predetermined endpoint. The body weight was monitored during the every two weeks for the entire experimental period, as shown in Fig. S1. Initially, there was no difference in body weight among the five groups. At the 2nd week, the body weight of the SHR group was lower than that of the WKY group (317.8±7.4 g versus 343.4±5.0 g, P < 0.01), while SalA treatment did not show any effects on the body weight compared with the SHR group. The value of body
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on body weight was observed compared with SHR group (Fig. 2A).
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Blood pressure was monitored every two weeks at the indicated time. At the beginning of the experiment, the blood pressure of the WKY group was lower than
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that of the SHR group (151.5±2.8 mmHg versus 182.2±3.6 mmHg, P < 0.001). During the entire experiment, SalA treatment showed no effect on blood pressure
mmHg, 199±2.7
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compared with the SHR group. At the 4th week, the blood pressure was 199.8±1.9 mmHg, 194.5±2.2
mmHg,
199.6±1.6
mmHg for SHR,
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SHR-SalA(2.5), SHR-SalA(5),and SHR-SalA(10), respectively (Fig. 2B). The influence of SalA on the heart rate of SHR was detected in the present study. The
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results showed no influence of SalA on the heart rate of SHR (data not shown).
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Fig. 2. The effect of SalA on body weight and blood pressure. (A) Body weight. (B) Blood pressure. The data are expressed as the mean ± S.E.M; #P < 0.5, ##P < 0.01, ###P < 0.001 compared with WKY.
Effect of SalA on retinal microvascular remodeling Previous studies have suggested that an assessment of retinal vascular changes might provide meaningful information concerning organ damage in persons with hypertension (Kim et al., 2010, Triantafyllou et al., 2014). In the present study, the 16
ACCEPTED MANUSCRIPT protection of SalA against retinal microvascular remodeling was evaluated using hematoxylin-eosin
staining
and
immunohistochemical
analysis.
For
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hematoxylin-eosin staining, the retinal endothelial cells in the SHR group were
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loosely arranged, while the retinal endothelial cells showed a closer and more regular arrangement in the SalA-treated SHR groups (Fig. 3A).
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The retinal vascular diameter is an important index to evaluate vascular remodeling. The retinal microvessels were further investigated using an immunohistochemical
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assay. The retinal veins were stained with FITC-coupled lectin (green), and the retinal arteries were more pronounced with α-SMA staining (red) (Fig. 3B). After staining,
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the retinal vascular diameter was directly quantified at a distance of 0.4 mm from the optical disc. The retinal arterial diameter of the SHR group was less than that of the
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WKY group (18.41±0.63 µm versus 24.83±0.82 µm, P < 0.001). SalA treatment inhibited this inward remodeling compared with the SHR group. The retinal arterial
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diameter was significantly restored in the SHR-SalA(5) (23.64±0.63 µm, P < 0.001) and SHR-SalA(10) (21.42±0.41 µm, P<0.001) groups compared with the SHR group (Fig. 3C). In addition, the diameter of the retinal vein was also significantly decreased in the SHR group compared with the WKY group (23.50±0.52 µm versus 38.03±1.63 µm, P < 0.001). The inward remodeling of the retinal vein was inhibited after treatment with SalA at 2.5, 5 and 10 mg/kg (Fig. 3D).
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Fig. 3. Effect of SalA on retinal perivascular cells and the retinal microvascular diameter of SHR. (A) Retinal vessels stained with hematoxylin-eosin. The arrows indicate retinal endothelial cells. Scale bar: 50 μm. (B) Retinal vessels stained with FITC-coupled lectin (green) and α-SMA antibody (red), the top graph shows lectin-positive veins, the middle graph shows α-SMA-positive arteries, and the bottom graph is a merged picture. Scale bar: 200 μm. (C) Quantitative data of lumen diameter
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ACCEPTED MANUSCRIPT for the retinal arteries. (D) Quantitative data of the lumen diameter of the retinal veins. The data are expressed as the mean ± S.E.M; ###P < 0.001 compared with WKY; *P <
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0.05, **P < 0.01, ***P < 0.001 compared with SHR.
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Effect of SalA on mesenteric vascular function
The mesenteric artery is the candidate artery for the evaluation of microvascular
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function. Because SalA inhibited the inward remodeling of retinal microvessels, we assessed the effect of SalA on microvascular function in SHR using DMT wire
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myography (Fig. 4). Acetylcholine (Ach) induced concentration-dependent relaxation in the mesenteric arteries with functional endothelium after pre-contraction with
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DL-phenylephrine hydrochloride. The relaxation of the arteries in the SHR group, elicited by treatment with 10 μM ACh, was reduced compared with the arteries from
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the WKY group (48.82±7.23 % versus 87.81±3.51 %, P < 0.001). SalA treatment improved endothelial function, and the values of relaxation induced by 10 μM ACh
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were 70.83±3.61 %, 63.91±12.82 %, and 72.93±4.84 % for SHR-SalA(2.5), SHR-SalA(5), and SHR-SalA(10), respectively.
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Fig. 4. Effect of SalA on the microvascular endothelial diastolic function of SHR
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measurement using DMT wire myography. Relative relaxation of small mesenteric arteries from WKY and SHR groups in response to acetylcholine (ACh). The data are ###
P < 0.001 compared with WKY; *P <
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expressed as the mean ± S.E.M; #P < 0.05, 0.05, **P < 0.01 compared with SHR.
The
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Effect of SalA on HUVEC proliferation results
of
the
present
study
showed
that
SalA
improved
the
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endothelial-dependent vasodilatation of mesenteric vessels for SHR in vivo. Subsequently, the direct effects of SalA on the function of endothelial cells were detected in vitro. There was no obvious impact on HUVEC proliferation at the detected concentrations of SalA (Fig. 5A). Treatment with 10 μg/mL of LPS induced endothelial cell dysfunction, and the cell viability was significantly decreased after LPS treatment compared with the control group (0.26±0.02 versus 0.30±0.01, P < 0.01). No effect of SalA on cell proliferation was observed at the detected dose used
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ACCEPTED MANUSCRIPT for LPS stimulation (Fig. 5B). Treatment with1 and 10 μM SalA was used in the
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following studies.
Fig. 5. Effect of SalA on HUVEC proliferation. (A) HUVECs were treated with SalA at concentrations of 0, 0.01, 0.1, 1 and 10 μmol/L for 24 h. The effect of SalA on the proliferation of HUVECs was determined using the Cell Counting Kit-8. (B) HUVECs were simultaneously treated with LPS (10 μg/mL) and increasing concentrations of 0, 0.01, 0.1, 1 and 10 μmol/L SalA for 24 h. The viability of HUVECs was evaluated using the Cell Counting Kit-8. The data are expressed as the
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ACCEPTED MANUSCRIPT mean ± S.E.M; #P < 0.05, ##P < 0.001 compared with Control.
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Effect of SalA on HUVEC wound healing and migration
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The direct protection effect of SalA was further evaluated in the migration of HUVECs. As shown in Fig. 6A and 6C, SalA did not affect the motility of HUVECs
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in the wound-healing assay. The migration was further evaluated using a transwell assay. As shown in Fig. 6B and 6D, the considerable enhancement of LPS on
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HUVECs migration was observed (55.1±15.3 versus 28.6±9.0 cells per field), and SalA significantly inhibited this effect (26.0±7.7 versus 55.1±15.3 cells per field for 1
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μM SalA and 20.8±6.4 versus 55.1±15.3 cells per field for 10 μM SalA).
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Fig. 6. Effect of SalA on HUVEC wound healing and migration. (A) Wound closure
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at 0 and 24 h. Scale bar: 200 μm (B) Representative photomicrographs of cell migration across the membrane of the chambers. Scale bar: 100 μm. (C) The quantitative data for wound closure. The data are expressed as the mean ± S.E.M. (D) The quantitative data for migration cell. The data are expressed as the mean ± S.E.M; ##
P < 0.01 compared with control; ** P < 0.01 compared with LPS.
Effect of SalA on HUVEC TEER and the permeability assay The integrity of the endothelial monolayer has important physiological significance
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ACCEPTED MANUSCRIPT in the microvascular system. Two methods, TEER and the permeability assay, were used to assess the integrity of the endothelial monolayer on HUVECs. After
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incubation with LPS with or without SalA for 24 h, TEER was determined using a
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Millicell-ERS ohmmeter. The value of TEER was significantly decreased in the HUVEC monolayer stimulated with LPS compared with the control group (62.1±2.5
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Ω·cm2 versus 80.8±2.3 Ω·cm2, P < 0.01). Compared with the LPS group, TEER significantly increased the HUVEC monolayer treated with SalA (P < 0.01) (Fig. 7A).
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The values of TEER were 79.2±2.5 Ω·cm2 and 83.9±5.4 Ω·cm2 for LPS-SalA(1) and LPS-SalA(10), respectively.
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In addition to the TEER assay, a permeability assay was also used to evaluate the integrity of the endothelial monolayer. The optical density of fluorescein
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isothiocyanate dextran 4000Da was used to evaluate the permeability of the HUVEC monolayer. Fluorescein isothiocyanate dextran 4000Da infiltrated the endothelial
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monolayer with increasing time. After 4 h, LPS treatment significantly increased the optical density compared with the control (40891 versus 25905 P<0.01), and 10 μM SalA exhibited an obvious protective effect on the HUVEC monolayer compared with the LPS group (20078 versus 40891 P < 0.01) (Fig. 7B).
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Fig. 7. Effect of SalA on HUVEC TEER and solute permeability. (A) The TEER of
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confluent HUVECs was monitored using a Millicell-ERS ohmmeter. The quantitative data are expressed as the mean ± S.E.M. (B) The cell permeability was assessed after
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adding fluoresceinated dextran-4000 to the upper chambers. Fluorescence in the lower chamber was measured at 0, 0.5, 1, 2, 3 and 4 h. The quantitative data are expressed as the mean ± S.E.M; ##P < 0.01 compared with control; **P < 0.01 compared with LPS.
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Discussion In the present study, SalA showed obvious protection against microvascular
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remodeling in vivo and directly protected endothelial cells in vitro.
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After SalA administration for 4 weeks, there was no impact on body weight and blood pressure in the SHR groups. SalA showed significant protection against
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remodeling for both microvascular structure and function, suggesting that the beneficial effect of SalA was not associated with the effect of this compound on blood
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pressure. Microvascular remodeling is a key pathological characteristic, associated with the incidence of end-organ damage induced by hypertension. Because the retinal
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microvessels are similar in anatomical features and physiological properties to those of the kidney, the retinal microvessels are considered to be a predictor to examine
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kidney damage. Grunwald et al. revealed that the pathology of retinal microvessels reflected renal microvascular pathology (Grunwald et al., 2012a; Grunwald et al.,
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2012b). Baki et al. also had reported that the change of retinal microvessels was closely associated with that of kidney impairment (Baki et al., 2014). In previous studies, we also reported that vascular remodeling eventually caused targeted organ damage, such as cardiomyocyte hypertrophy, cardio-fibrosis, and glomerular hyalinization in spontaneously hypertensive rats (Chen et al., 2012). In the present study, the protective effect of SalA against retinal vascular remodeling was confirmed, indicating the potential protection against end-organ damage induced by hypertension,
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ACCEPTED MANUSCRIPT such as renal damage. The vascular endothelium plays a key role in maintaining vascular homeostasis, such as barrier function, regulating local cellular growth and
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secreting cytokines. Almost all patients with hypertension have vascular endothelial
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injuries. Karaca et al. reported that endothelial dysfunction in vascular lesions might lead to end-organ damage under hypertension (Karaca et al., 2014). Zhai et al.
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demonstrated that, in the absence of VEGF signaling, glomerular endothelial cells could not maintain the integrity of the glomerular filtration barrier, resulting in
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proteinuria. The dysfunction of endothelium-dependent relaxation is serves as a pathological characteristic of hypertension (Zhai et al., 2014). ACh activates M
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subtype vascular endothelial cells, which release nitric oxide and eventually induce vascular vasodilator, as a tool to evaluate vascular endothelial function (Wassmann et
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al., 2002). In the present study, endothelial function was evaluated by DMT wire myography in vivo, and SalA could improve ACh-mediated and endothelial
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cell-dependent microvascular diastolic function in SHR. Increasing nitric oxide also could decrease the mean arterial pressure, heart rate and sympathetic activity in rats by the activation of adenosine A2A receptors and reduction of ACh and M1 receptor levels in the rostral ventrolateral medulla (Jiang et al., 2011). In addition to confirming the beneficial effects of SalA on microvascular function in vivo, the direct protection of endothelial cells by SalA was further evaluated in vitro using HUVECs. The mobility of endothelial cells was assessed using wound healing and transwell
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ACCEPTED MANUSCRIPT assays, and the integrity of endothelial cell was assessed using TEER and permeability assays. HUVECs exposed to LPS secreted large amounts of MMP-9.
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MMP-9 causes the degradation of the extracellular matrix and damages cell junctions,
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ultimately enhancing the capability of cell migration and opening of intercellular gaps to increase permeability in HUVECs (Lappas, 2012, Jang et al., 2014). In a previous
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study, we showed that SalA is a novel matrix metalloproteinase-9 inhibitor for the suppression of vascular remodeling (Zhang et al., 2014). Southgate et al. reported that
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MMP-9 might be involved in the early vascular remodeling associated with hypertension (Southgate et al., 1999). Therefore, we proposed that SalA would inhibit
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the expression of MMP-9, which might reduce the damage to vascular endothelial cells, maintain the permeability and integrity of vascular intima and improve the
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vascular diastolic function in SHR rats. The results showed that SalA not only maintained the integrity of the endothelial
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monolayer but also inhibited endothelial mobility, consistent with the findings in SHR. In the present study, we showed that SalA could be a potential drug candidate to prevent targeted organ damage induced by vascular remodeling.
Conclusion In summary, SalA inhibited the remodeling of microvessels and normalized endothelial dysfunction, suggesting that SalA has potential beneficial effects on the microvascular system and against microvascular complications induced by
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hypertension.
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Acknowledgments This work was financially supported by grants from the National Natural Science
for
“Key
New
Drug
Creation
and
Manufacturing
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Project
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Foundation of China Grants (81173587), National Science and Technology Major Program”
(2013ZX09103002-024), and the Shanghai Science and Technology Development
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Foundation (14401900900). This work was also partially supported by the Croucher
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Foundation (CAS14201).
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Figure captions Fig. 1. Structure and purity of SalA. (A) The structure of SalA. (B) Purity of SalA was
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detected using high-performance liquid chromatography.
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Fig. 2. The effect of SalA on body weight and blood pressure. (A) Body weight. (B) Blood pressure. The data are expressed as the mean ± S.E.M; #P < 0.5, ##P < 0.01, ###P
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< 0.001 compared with WKY.
Fig. 3. Effect of SalA on retinal perivascular cells and the retinal microvascular
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diameter of SHR. (A) Retinal vessels stained with hematoxylin-eosin. The arrows indicate retinal endothelial cells. Scale bar: 50 μm. (B) Retinal vessels stained with
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FITC-coupled lectin (green) and α-SMA antibody (red), the top graph shows lectin-positive veins, the middle graph shows α-SMA-positive arteries, and the bottom
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graph is a merged picture. Scale bar: 200 μm. (C) Quantitative data of lumen diameter for the retinal arteries. (D) Quantitative data of the lumen diameter of the retinal veins.
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The data are expressed as the mean ± S.E.M; ###P < 0.001 compared with WKY; *P < 0.05, **P < 0.01, ***P < 0.001 compared with SHR. Fig. 4. Effect of SalA on the microvascular endothelial diastolic function of SHR measurement using DMT wire myography. Relative relaxation of small mesenteric arteries from WKY and SHR groups in response to acetylcholine (ACh). The data are expressed as the mean ± S.E.M; #P < 0.05, 0.05, **P < 0.01 compared with SHR.
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###
P < 0.001 compared with WKY; *P <
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proliferation of HUVECs was determined using the Cell Counting Kit-8. (B)
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HUVECs were simultaneously treated with LPS (10 μg/mL) and increasing concentrations of 0, 0.01, 0.1, 1 and 10 μmol/L SalA for 24 h. The viability of
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HUVECs was evaluated using the Cell Counting Kit-8. The data are expressed as the mean ± S.E.M; #P < 0.05, ##P < 0.001 compared with Control.
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Fig. 6. Effect of SalA on HUVEC wound healing and migration. (A) Wound closure at 0 and 24 h. Scale bar: 200 μm (B) Representative photomicrographs of cell
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migration across the membrane of the chambers. Scale bar: 100 μm. (C) The quantitative data for wound closure. The data are expressed as the mean ± S.E.M. (D)
##
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The quantitative data for migration cell. The data are expressed as the mean ± S.E.M; P < 0.01 compared with control; ** P < 0.01 compared with LPS.
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Fig. 7. Effect of SalA on HUVEC TEER and solute permeability. (A) The TEER of confluent HUVECs was monitored using a Millicell-ERS ohmmeter. The quantitative data are expressed as the mean ± S.E.M. (B) The cell permeability was assessed after adding fluoresceinated dextran-4000 to the upper chambers. Fluorescence in the lower chamber was measured at 0, 0.5, 1, 2, 3 and 4 h. The quantitative data are expressed as the mean ± S.E.M;
##
P < 0.01 compared with control;
LPS.
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**
P < 0.01 compared with
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Baik, S.Y., Lim, Y.A., Kang, S.J., Ahn, S.H., Lee, W.G., Kim, C.H., 2014. Effects of platelet lysate preparations on the proliferation of HaCaT cells. Annals of laboratory medicine 34, 43-50. Baki, A.H., Soliman, Y., Seif, E.I., 2014. Histopathological Association between Vascular Hypertensive Changes and Different Types of Glomerulopathies. Arab journal of nephrology and transplantation 7, 21-26. Brondum, E., Kold-Petersen, H., Simonsen, U., Aalkjaer, C., 2010. NS309 restores EDHF-type relaxation in mesenteric small arteries from type 2 diabetic ZDF rats. British journal of pharmacology 159, 154-165. Chen, H., Yin, J., Deng, Y., Yang, M., Xu, L., Teng, F., Li, D., Cheng, Y., Liu, S., Wang, D., Zhang, T., Wu, W., Liu, X., Guan, S., Jiang, B., Guo, D., 2012. The protective effects of ginsenoside Rg1 against hypertension target-organ damage in spontaneously hypertensive rats. BMC complementary and alternative medicine 12, 53. Feihl, F., Liaudet, L., Levy, B.I., Waeber, B., 2008. Hypertension and microvascular remodelling. Cardiovascular research 78, 274-285. Fu R, Chen Z, Wang Q, Guo Q, Xu J, Wu X. XJP-1, a novel ACEI, with anti-inflammatory properties in HUVECs. Atherosclerosis. 2011;219:40-8 Giordano, A., D'Angelillo, A., Romano, S., D'Arrigo, P., Corcione, N., Bisogni, R., Messina, S., Polimeno, M., Pepino, P., Ferraro, P., Romano, M.F., 2014. Tirofiban induces VEGF production and stimulates migration and proliferation of endothelial cells. Vascul Pharmacol 61, 63-71. Grunwald, J.E., Alexander, J., Ying, G.S., Maguire, M., Daniel, E., Whittock-Martin, R., Parker, C., McWilliams, K., Lo, J.C., Go, A., Townsend, R., Gadegbeku, C.A., Lash, J.P., Fink, J.C., Rahman, M., Feldman, H., Kusek, J.W., Xie, D., Jaar, B.G., Group, C.S., 2012a. Retinopathy and chronic kidney disease in the Chronic Renal Insufficiency Cohort (CRIC) study. Arch Ophthalmol 130, 1136-1144. Grunwald, J.E., Ying, G.S., Maguire, M., Pistilli, M., Daniel, E., Alexander, J., Whittock-Martin, R., Parker, C., Mohler, E., Lo, J.C.M., Townsend, R., Gadegbeku, C.A., Lash, J.P., Fink, J.C., Rahman, M., Feldman, H., Kusek, J.W., Xie, D.W., Coleman, M., Keane, M.G., Cohort, C.R.I., 2012b. Association Between Retinopathy and Cardiovascular Disease in Patients With Chronic Kidney Disease (from the Chronic Renal Insufficiency Cohort [CRIC] Study). Am J Cardiol 110, 246-253. Higashi, Y., Sasaki, S., Nakagawa, K., Ueda, T., Yoshimizu, A., Kurisu, S., Matsuura, H., Kajiyama, G., Oshima, T., 2000. A comparison of angiotensin-converting enzyme inhibitors, calcium antagonists, beta-blockers and diuretic agents on reactive hyperemia in patients with essential hypertension: a multicenter study. Journal of the American College of Cardiology 35, 284-291. 34
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