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

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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,

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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

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ACCEPTED MANUSCRIPT ((TensionACh/Tensionprecontraction) ×100) and presented as a relaxation percentage (Brondum et al., 2010).

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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

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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