Respiratory Physiology & Neurobiology 205 (2015) 120–128

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Tanshinone IIA attenuates hypoxic pulmonary hypertension via modulating KV currents Lianhe Zheng a,1 , Manling Liu b,c,1 , Min Wei b,c,1 , Yi Liu b,c , Mingqing Dong b,c , Ying Luo b,c , Pengtao Zhao b,c , Haiying Dong b,c , Wen Niu b,c , Zhiqiang Yan d , Zhichao Li b,∗ a

Center of Orthopedic Surgery, Orthopedics Oncology Institute of Chinese PLA, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, PR China Department of Pathology and Pathophysiology, Fourth Military Medical University, Xi’an 710032, PR China c Lung Injury and Repair Center, Fourth Military Medical University, Xi’an 710032, PR China d Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xi’an 710038, PR China b

a r t i c l e

i n f o

Article history: Accepted 30 September 2014 Available online 7 October 2014 Keywords: Tanshinone IIA (TIIA) Chronic hypoxia pulmonary hypertension (CH-PH) The voltage-gated potassium ion (KV ) channels

a b s t r a c t The voltage-gated K+ (KV ) channels play an essential role in the etiology of chronic hypoxic pulmonary hypertension (CH-PH).Tanshinone IIA (TIIA), a major active component of Salvia miltiorrhiza Bunge (S. miltiorrhiza), has many biological protective effects. In the present study, we investigated whether KV channels were responsible for the protective effect of TIIA on CH-PH. In acute hypoxia experiments, the IKV currents of pulmonary artery smooth muscle cells (PASMCs) isolated from healthy rats were determined in the absence or presence of TIIA (5 ␮g/ml or 25 ␮g/ml) or 4-AP (1 mM). In chronic hypoxia experiments, rats were challenged by intermittent hypoxia or sustained hypoxia exposure for 4 weeks with or without TIIA (10 mg/kg) treatment. Subsequently, the hemodynamic data and the pathomorphological changes of pulmonary arteries were gathered. The expressions of KV 2.1 and KV 1.5 in pulmonary arteries were tested by Western blotting and RT-PCR, respectively. PASMCs were detached from intermittent hypoxia or sustained hypoxia exposure rats to evaluate the IKV currents. Results showed that TIIA markedly recovered acute hypoxia-induced the down-regulation of IKV currents in PASMCs. Moreover, TIIA significantly restrained chronic intermittent hypoxia or sustained hypoxia-induced pulmonary artery wall remodeling, accompanied with modulating the expressions of KV 2.1 and KV 1.5, and reversing the down-regulation of IKV currents. TIIA is thus an attractive potential therapy for CH-PH. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Pulmonary hypertension (PH) is defined as a multifactorial progressive disease, which may ultimately lead to severe pulmonary hypertension, right ventricular hypertrophy and right heart failure with a high morbidity and mortality (Chazova et al., 1995; Farber and Loscalzo, 2004). Chronic hypoxia-induced PH (CH-PH) is a common type of PH, mainly secondary to disorders of the respiratory system, such as chronic obstructive pulmonary disease (COPD), obstructive sleep apnea, interstitial lung disease, or chronic mountain sickness in plateau residents. Hypoxia is regarded as a pivotal factor in the etiology of CH-PH. Hypoxia provokes hypoxic pulmonary vasoconstriction (HPV), a unique physiological

∗ Corresponding author. Tel.: +86 29 84774548; fax: +86 29 84774548. E-mail address: [email protected] (Z. Li). 1 These authors contributed equally to this paper. http://dx.doi.org/10.1016/j.resp.2014.09.025 1569-9048/© 2014 Elsevier B.V. All rights reserved.

mechanism observed in pulmonary arterioles (Moudgil et al., 2005). Chronic exposure to hypoxia or sustained HPV leads to gradual increased pulmonary vascular remodeling (PVR) (Girgis and Mathai, 2007; Howell et al., 2003). The pathological changes of PVR include vasoconstriction, endothelial injury, varying degrees of cellular proliferation, and hypertrophy in intimae and media, as well as distal pulmonary arterioles muscularization (Tucker and Rhodes, 2001). Despite abundant therapies focused on the improvement of the pulmonary vascular dysfunction and vasoconstriction, effective preventions and newer treatments for CH-PH are inadequate (Farber and Loscalzo, 2004; Runo and Loyd, 2003). It is well known that there are five functionally-distinguishable potassium ion (K+ ) channels identified in pulmonary artery smooth muscle cells (PASMCs), namely, (1) voltage-gated K+ (KV ) channels, (2) Ca2+ -activated K+ (KCa ) channels, (3) ATP-sensitive K+ (KATP ) channels, (4) inward rectifier K+ (KIR ) channels, and (5) tandem pore domain K+ (KT ) channels (Gurney et al., 2003; Peng et al., 1999; Post et al., 1995; Yuan, 1995). Among the five types of K+ channels,

L. Zheng et al. / Respiratory Physiology & Neurobiology 205 (2015) 120–128

KV channels have been demonstrated to contribute to regulating the resting membrane potential in PASMCs and determining pulmonary vascular tone (Nelson and Quayle, 1995). Studies have revealed that KV channels in PASMCs play an important role in the development of HPV and PVR, in which acute hypoxia inhibits KV channel function and triggers contraction, whereas chronic hypoxia down-regulates KV channel gene expression, which in turn stimulates proliferation and inhibits apoptosis in PASMCs (Wang et al., 1997; Wang et al., 2005). Tanshinone IIA (TIIA), one of the major active component of S. miltiorrhiza Bunge (S. miltiorrhiza) (Jang et al., 2003), exhibits many vascular biological activities and has been commonly used in traditional oriental herbal medicine for cardiovascular diseases. Some studies have showed that TIIA attenuates cardiomyocyte hypertrophy (Tan et al., 2011), induces coronary artery vasodilatation (Wu et al., 2009), protects against ischemia-reperfusion injury and has anti-arrhythmic effects (Shan et al., 2009; Xu et al., 2009a,b). Our previous studies have also showed that pharmacological treatment with TIIA has exerted protective effects on CH-PH in vivo and stimulated KV 2.1 expression in vitro (Huang et al., 2009). Moreover, TIIA inhibited hypoxia-induce PASMCs proliferation, which may be due to arresting the cells in G1/G0-phase by slowing down the hypoxia-induced degradation of p27 via Akt/Skp2-associated pathway (Luo et al., 2013). We also found that TIIA eliminates the hypoxia-induced initial contraction and potentiates the following vasorelaxation on isolated pulmonary arteries (Wang et al., 2010). But, TIIA ameliorated the hypoxia induced vasoconstriction and remodeling of pulmonary artery is not full defined. Therefore, in the current study, we investigated whether the protective effects of TIIA against CH-PH were correlated with modulating KV currents under acute and chronic hypoxia exposure.

2. Methods 2.1. Chemicals and animal Tanshinone IIA (TIIA, sulfonate, purity is 99%) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, China) and the chemical structure of TIIA is shown in Fig. 1. Polyclonal KV 1.5, KV 2.1 and monoclonal ␤-actin antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Sigma-Aldrich Inc. (St. Louis, MO, USA), respectively. Tetraethylammonium (TEA) and 4aminopyridine (4-AP) were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). And all the other reagents were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). Adult male Sprague-Dawley rats (7–8 weeks old, and 200–250 g weight) were obtained from the animal center (Fourth Military Medical University, Xi’an, P R China). Rats were kept in a temperature-controlled house with 12-h light-dark cycles. All experiments were approved by Animal Care and Use Committee at Fourth Military Medical University and were in accordance with the

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Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication no. 85-23, revised 1985). 2.2. Animal model and grouping Rats were randomly divided into various groups, i.e., (1) normoxia group and (2) normoxia/TIIA group: rats were housed continuously in room air and ambient barometric pressure (−718 mmHg, 21% oxygen) for 4 weeks with or without TIIA (10 mg/kg) via intraperitoneal injection; (3) intermittent hypoxia group: rats were housed intermittently in a hypobaric hypoxia chamber depressurized to 380 mmHg (oxygen concentration reduced to only 10%) for 8 h. After 8 h hypoxia exposure, rats were placed in room air (21% oxygen) again (Ostadal et al., 1981; Thomas and Wanstall, 2003). These steps were repeated daily for a total of 4 weeks; (4) intermittent hypoxia/TIIA group: rats also were exposed to intermittent hypobaric hypoxia in the same way. After 8 h hypoxia exposure, rats were injected TIIA (10 mg/kg) via intraperitoneal daily for 4 weeks; (5) sustained hypoxia group: rats were housed sustainedly in a hypobaric hypoxia chamber depressurized to 380 mmHg (oxygen concentration reduced to only 10%) at all times for 4 weeks(Reeves et al., 2003); (6) sustained hypoxia/TIIA group: rats were housed sustainedly in a hypobaric hypoxia chamber depressurized to 380 mmHg (oxygen concentration reduced to only 10%) for 4 weeks, and then they were injected TIIA (10 mg/kg) via intraperitoneal daily for another 2 weeks. The current dose of TIIA was based on our previous experiments. (Huang et al., 2009; Xu et al., 2011; Xu et al., 2009a,b; Zhang et al., 2011). 2.3. Pulmonary hypertension and PVR assessment Rats were anesthetized with sodium pentobarbital (30 mg/kg, i.p.). Two heparin-filled blunt-ended polyethylene catheters connected to pressure transducers (AD Instruments, Colorado Springs, CO, Australia) were inserted into the right ventricle and the left carotid artery, respectively, and then the right ventricular systolic pressure (RVSP) and the mean carotid arterial pressure (mCAP) were recorded. Next, the hearts were dissected out, divided into right ventricle (RV), left ventricle (LV) and septum (S), blotted and weighed, respectively, to determine the RV/(LV + S) % as an index of RV hypertrophy. At the end of the experiments, right lung sagittal sections were placed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5 ␮m thick, and then were stained with hematoxylineosin. Microscopic evaluation was performed to characterize structure remodeling of the pulmonary arteries. The pulmonary arteries (external diameters of 50–200 ␮m) were chosen randomly at a magnification of 400× and were analyzed using an image-processing program (Image-Pro Plus, Version 5.1, Media Cybernetics, USA). The outside diameter and inside diameter of pulmonary arterioles were measured, and the medial wall thickness, the cross sectional area of medial wall, and the total cross sectional vessel area were obtained, following which the two indexes were calculated: the ratio of medial wall thickness (WT%) = 100 × (medial wall thickness)/(vessel semidiameter); the ratio of medial wall area (WA%) = 100 × (cross-sectional medial wall area)/(total cross-sectional vessel area). All the morphological analysis was conducted in a double-blind method. 2.4. RT-PCR analysis and Western blotting

Fig. 1. The chemical structure of tanshinone IIA (TIIA, sulfonate, purity is 99%).

The lungs were dissected and immediately placed on ice. The intrapulmonary arteries (IPAs, 2–3th divisions) were carefully isolated, and the adventitial tissues were removed. Total RNA was isolated from PA tissue homogenate using TRIzol. Samples with

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a 260:280 nm absorbance ratio of 1.9 or greater were used for subsequent reverse transcription. The primer pairs were designed by primer premier 5 (PREMIER Biosoft International, Palo Alto CA, USA), and original information of cDNA were aligned in the GeneBank. The primer pairs for KV 2.1, KV 1.5, and ␤-actin were following: (1) KV 2.1 [Genebank number NM013186] forward: 5 AGG CCG AAC TGT GTC TAC TC-3 ; reverse: 5 -GTC CTC TGC ACC CTC CTA AC-3 , 557 bp. (2) KV 1.5 [Genebank number M27158] forward: 5 -ATG CAG GGT CAC TCC ATC–‘; reverse: 5 -GGC TTC TCC TCT TCC TTG-3 , 340 bp. (3) ␤-actin [Genebank number NM031144] forward: 5 -CAT CTC TTG CTC GAA GTC CA-3 ; reverse: 5 -ATC ATG TTT GAG ACC TTC AAC A-3 , 318 bp. (4) ␤-actin [Genebank number NM031144] forward: 5 -TAA AGA CCT CTA TGC CAA CAC AGT-3 ; reverse: 5 -CAC GAT GGA GGG CCG GAC TCA TC-3 ; 240 bp. PCR reaction conditions for KV 2.1 were started by a 2 min denaturation procedure at 94 ◦ C, followed by 35 cycles of 94 ◦ C for 45 s, 62 ◦ C for 45 s and 72 ◦ C for 1 min, and a final extension at 72 ◦ C for 8 min; for KV 1.5 was initiated by a 5 min denaturation step at 94 ◦ C, followed by 35 cycles of 94 ◦ C for 1 min, 54 ◦ C for 1 min and 72 ◦ C for 1 min, and a final extension at 72 ◦ C for 10 min. PCR products were analyzed by agarose gel electrophoresis. An invariant mRNA quantity of ␤-actin was used as an internal control to quantify PCR products. Total proteins were extracted as well. Protein concentrations were determined by coomassie brilliant blue assay. Western blot analysis was performed by using primary antibodies for KV 1.5 (1:200), KV 2.1 (1:200), and ␤-actin (1:5000). Immunoreactivity was visualized with the corresponding peroxidase-conjugated secondary antibodies; the relative content of target proteins was detected by chemiluminescence.

2.5. PASMCs isolation and electrophysiology record PASMCs were dispersed from rats’ IPA branches (3th division) as previously described (Shimoda et al., 2000). Briefly, IPA branches were dissected in Ca2+ -free physiological saline solution (PSS) containing (mM): NaCl 118.3, KCl 4.7, KH2 PO4 1.2, MgSO4 1.2, NaHCO3 25, EDTA 0.026, and glucose 7.0, bubbled with 95% O2 and 5% CO2 at 4 ◦ C with pH adjusted to 7.4. After endothelium was removed with the wooden end of a cotton swab, IPA branches were cut into small pieces (1 mm3 ) in Ca2+ -free PSS and recovered for 20 min at 37 ◦ C. Then, the small pieces were transferred to Ca2+ -free PSS

containing: collagenase (1 mg/ml), papain (1 mg/ml), bovine serum albumin (2 mg/ml) and dithiothreitol (1.25 mg/ml) for 45 min at 37 ◦ C. After enzyme treatment, the small pieces were washed 3 times by Ca2+ -free PSS. Thus, single cells were released by gentle trituration through a Pasteur pipette. Isolated PASMCs were placed in recording chamber for whole-cell patch-clamping study. After a brief period to allow partial adherence to the bottom of the recording chamber, cells were continuously perfused (2 ml/min) with a solution composed of (in mM): NaCl 115, KCl 5.4, MgCl2 1, EGTA 1, NaHCO3 25, HEPES 10, glucose 10, and tetraethylammonium (TEA, a BKCa blocker) 5 (pH 7.3 with NaOH), heated to 37 ◦ C. For acute hypoxia experiment, hypoxic solutions (pO2 , about 15 mmHg) were bubbled with 95% N2 and 5% CO2 for at least 30 min, whereas normoxic solutions (pO2 , about 150 mmHg) were bubbled with 95% O2 and 5% CO2 . The IKV currents of PASMC from normal rats’ IPA branches (3th division) were determined under normoxic or hypoxic conditions in the absence or presence of TIIA (5 ␮g/ml or 25 ␮g/ml) or 4-AP (1 mM). For chronic hypoxia experiment, the IKV currents of PASMC from CH-PH rats’ or CH-PH/TIIA rats’ IPA branches (3th division) were determined by using normoxic solutions. pO2 were measured with an O2 microelectrode (World Precision Instrument, USA) placed near the recording site. Whole-cell recordings were made with borosilicate glass pipettes of 3–6 M resistance containing (in mM): KCl 125, MgCl2 4, Na2 ATP 2, EGTA10 and HEPES 10 (pH 7.3; osmolarity 285–290). Recorded electrical signals were amplified with an Axopatch700B amplifier (Molecular Devices Corporation, USA), filtered at 3 kHz. Data were acquired using a computer with the digidata 1440A acquisition system (Molecular Devices Corporation, USA) and analyzed using pCLAMP software (version 10.2). Cells were voltage-clamped at −70 mV and currents were evoked in 20 mV steps from −70 mV to +70 mV using 400 ms pulses. Whole-cell currents were normalized by cell capacitance and expressed as current density. 2.6. Statistical analysis Data were expressed as mean ± S.EM. Statistical analysis was performed with analysis of variance (one way-ANOVA), followed by a LSD-t test for multiple comparisons. Statistical significance was accepted as P < 0.05.

Fig. 2. TIIA reversed acute hypoxia-induced IKV current down-regulation in PASMCs. The IKV currents of freshly isolated PASMCs from healthy rats were determined under acute hypoxia exposure (A–D) in the absence or presence of TIIA (5 ␮g/ml or 25 ␮g/ml). BKCa channels were inhibited by 5 mM TEA. Cells were voltage-clamped at −70 mV and currents were evoked in 20 mV steps from −70 mV to +70 mV using 400 ms pulses. TIIA (25 ␮g/ml) did not affect IKV currents under normoxia exposure (A and B). The IKV currents were suppressed by acute hypoxia (C), which was not changed in the presence of 5 ␮g/ml TIIA (D), but partially reversed in the presence of 25 ␮g/ml TIIA (E). Moreover, treated with acute hypoxia and 4-AP (1 mM), the IKV currents was extremely suppressed (F), and it was not altered in the presence of 25 ␮g/ml TAII (G).The current–voltage (I–V) curve was recorded in PASMCs (H). Data are expressed as means ± S. EM. n = 6 replicates per group. ** P < 0.01 vs. normoxia protocol, # P < 0.05 vs. hypoxia protocol, and + P < 0.05 vs. hypoxia + TIIA (5 ␮g/ml) protocol.

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3. Results 3.1. TIIA reversed acute hypoxia-induced IKV current down-regulation in PASMC TIIA (25 ␮g/ml) did not affect the IKV currents under normoxia exposure (Fig. 2A, B and H). Acute hypoxia markedly downregulated the IKV currents (P < 0.01, Fig. 2C and H), which was not changed in the presence of 5 ␮g/ml TIIA (P > 0.05, Fig. 2D and H), but partially reversed in the presence of 25 ␮g/ml TIIA (P < 0.05, Fig. 2E and H). Moreover, treated with hypoxia and 4-AP, a KV channels inhibitor, the IKV currents was extremely suppressed (Fig. 2F and H), which was not altered in the presence of 25 ␮g/ml TAII (P > 0.05, Fig. 2G and H). 3.2. TIIA improved chronic intermittent hypoxia and sustained hypoxia-induced PH and pulmonary artery remodeling in rats TIIA did not affect mCAP and RVSP under normoxia exposure (Figs. 3A–B and 4A–B). Rats subjected to intermittent hypoxia for 4 weeks were found suffering from pulmonary hypertension, as evidenced by a significant increase in RVSP compared with that in normoxia group (Fig. 3B, P < 0.01). However, this elevated RVSP strikingly decreased in intermittent hypoxia/TIIA group (P < 0.05). The ratio of RV/(LV + S) % as an index of RV hypertrophy is shown in Fig. 3C. No significant difference was found between normoxia group and normoxia/TIIA group. In intermittent hypoxia group, (RV/LV + S) % significantly increased (P < 0.01), but TIIA markedly suppressed the chronic hypoxia-induced RV hypertrophy in the treatment protocol (P < 0.05). In terms of pulmonary arteries histological changes, the structure of pulmonary arteries in normoxia group and normoxia/TIIA group were normal (Fig. 3D, a and b). Chronic intermittent hypoxia exposure caused a marked pulmonary artery remodeling, characterized by thickened small pulmonary artery vessel walls, deflated vessel lumina (Fig. 3D, c). However, after TIIA treatment (Fig. 3D, d), these changes were less pronounced compared with those in hypoxia group. Furthermore, the medial wall thickness (WT) % (Fig. 4E) and the medial wall area (WA) % (Fig. 3F) of the arteries, both indexes of pulmonary artery remodeling, were dramatically elevated after chronic hypoxia exposure compared with those in normoxia group (P < 0.01, respectively). In contrast, TIIA treatment significantly diminished these changes (P < 0.05, respectively). But no significant difference in both (WT) % and (WA) % was observed between normoxia group and normoxia/TIIA group. Additionally, sustained hypoxia also induced serious PH and pulmonary artery remodeling in rats. As shown in Fig. 4, rats subjected to sustained hypoxia for 4 weeks were found significant increases in RVSP and (RV/LV + S) % compared with those in normoxia group (Fig. 4B and C, P < 0.01, respectively). Moreover, sustained hypoxia exposure also thickened the small pulmonary artery vessel walls, deflated vessel lumina (Fig. 4D, c), and increased the WT% and WA% (Fig. 4E and F, P < 0.01, respectively). However, these elevated parameters strikingly decreased by TIIA treatment (Fig. 4B–F, P < 0.05, respectively). 3.3. TIIA stabilized chronic intermittent hypoxia and sustained hypoxia-induced KV 2.1 and KV 1.5 expression in pulmonary arteries. For the results of intermittent hypoxia experiments, both KV 2.1 and KV 1.5 mRNA levels observably decreased after chronic intermittent hypoxia exposure (P < 0.01, respectively). TIIA treatment partially recovered the down-regulation of KV 1.5 and KV 2.1 (P < 0.05, respectively), but TIIA had no effect on the mRNA levels of KV 1.5 and KV 2.1 in normoxia group and normoxia/TIIA

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group (Fig. 5A–D). Meanwhile, the protein expressions of KV 1.5 and KV 2.1 in pulmonary arteries from intermittent hypoxia group rats decreased compared with those in normoxia group (P < 0.01, respectively). However, these changes were partially reversed by TIIA treatment (P < 0.05, respectively), but TIIA had no effect on the protein expressions of KV 1.5 and KV 2.1 in normoxia group and normoxia/TIIA group either (Fig. 5E–G). Similarly, the results of sustained hypoxia experiments shown that sustained hypoxia markedly degraded both the mRNA and protein expression of KV 2.1 and KV 1.5 (Fig. 6A–G, P < 0.01, respectively), which were dramatically elevated after TIIA treatment (P < 0.05, respectively). 3.4. TIIA ameliorated chronic intermittent hypoxia and sustained hypoxia-induced IKV current down-regulation in PASMC. TIIA treatment did not affect the mean IKV currents in PASMCs from the normoxia group and normoxia/TIIA group rats (Fig. 7A, B, F and G). Chronic intermittent hypoxia led to a dramatical decrease in the IKV currents compared with the normoxia group (Fig. 7C), TIIA treatment protocols evidently reversed the IKV currents (Fig. 7D). The mean current density–voltage relationship (I–V) curve (Fig. 7E) of IKV derived from chronic intermittent hypoxia shifted downward (P < 0.01), and TIIA shifted upward the (I–V) curve (P < 0.05). With respect to sustained hypoxia experiments, sustained hypoxia obviously inhibited the IKV currents (Fig. 7H) as well, while TIIA significantly up-regulated the IKV currents (Fig. 7I), which were evidenced by the mean current density–voltage relationship (I–V) curve (Fig. 7J, P < 0.01, P < 0.05, respectively). 4. Discussion Nowadays, the pharmacological properties of TIIA have attracted great interests. Plenty of experimental studies and clinical trials have demonstrated the efficacy of TIIA on coronary artery disease, hypertension stroke, hyperlipidemia, and other cardiovascular diseases (Chan et al., 2004; Ji et al., 2000). It has been demonstrated that TIIA elicited vasodilatory effects on isolated coronary arteries and aorta through nitric oxide (NO)-mediated vasodilation and activation of K+ channels in the vascular smooth muscle cells (Kim et al., 2007; Wu et al., 2009). Moreover, the vasorelaxant effects of danshen and its fractions were also produced by inhibition of Ca 2+ influx, opening of BKca channels, and release of calcitnonin gene-related peptide from sensory nerves (Cao et al., 2003; Lam et al., 2005, 2006). Recently, the abilities of TIIA to modulate the pulmonary vascular function have also been proved. Wang et al. showed that TIIA prevented both hypoxia and monocrotaline induced pulmonary hypertension (PH) development (Wang et al., 2013). Our previous study also found that TIIA played an important role in the treatment of CH-PH in rats (Huang et al., 2009; Luo et al., 2013; Wang et al., 2010). However, the underlying mechanism of TIIA against PH is not well documented. Further investigations are urgently needed. In the present study, we confirmed the protective effects of TIIA on chronic intermittent hypoxia and sustained hypoxia-induced PH rats, as is characterized by the assessment of RVSP, RV hypertrophy, and the detection of pulmonary artery histological changes. To our knowledge, O2 -sensitive K+ channels, especially KV channels, expressed in PASMCs are widely regarded as one of core mechanism of CH-PH. Hypoxia inhibits KV channels in PASMCs, in turn causes PASMCs’ membrane depolarization, opens the voltagedependent calcium ion (Ca2+ ) channels, promotes Ca2+ influx, increases cytosolic free Ca2+ concentration ([Ca2+ ]cyt ), and then triggers hypoxic pulmonary vasoconstriction (HPV) (Yuan, 1995). When chronic exposes to hypoxia, the vast and growing number

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Fig. 3. TIIA improved chronic intermittent hypoxia-induced PH and pulmonary artery remodeling in rats. Rats were housed intermittently in a hypobaric hypoxia chamber(10% oxygen) 8 h per day followed by 16 h in room air (21% oxygen) every day and continuing 4 weeks. After hypoxia exposure, they were injected with or without TIIA (10 mg/kg) via intraperitoneal daily continuing 4 weeks. Normoxia exposure with or without TIIA (10 mg/kg) acted as control and also continued 4 weeks. The mean carotid arterial pressure (mCAP) (A) and the right ventricular systolic pressure (RVSP) (B) were examined by catheterization to assess the changes of hemodynamics. Then the hearts were isolated and cut into the right ventricular (RV), the left ventricular (LV) and the interventricular septum (S) three parts, which were finally weighed, respectively to calculate the ratio of RV/(LV + S)%(C). Data are means ± S. EM. n = 8 rats/group, * P < 0.05, ** P < 0.01 vs. normoxia group, # P < 0.05, ## .P < 0.01 vs. intermittent hypoxia group. (D) Histopathologic examination of the small pulmonary arteries was performed. (a) Normoxia and (b) Normoxia/TIIA group: there were normal structures in the small pulmonary arterioles; (c) Hypoxia group: vessel wall were thickened with medial smooth muscle cell proliferation and hypertrophy, and the vessel lumina were deflated. (d) Hypoxia/TIIA group: The vessel changes as described above were significantly alleviated compared with hypoxia group. The bar represents 50 ␮m. The ratio of medial wall thickness (WT%) (E) and the ratio of medial wall area (WA%) (F) of the pulmonary arteries were calculated. Data are means ± S.EM., n = 72 small pulmonary arteries/group; 3 small pulmonary arteries/slides; 3 slides/rat; 8 rats/group. * P < 0.05, ** P < 0.01 vs. normoxia group, # P < 0.05, ## P < 0.01 vs. intermittent hypoxia group.

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Fig. 4. TIIA improved chronic sustained hypoxia-induced PH and pulmonary artery remodeling in rats. Rats were housed sustainedly in a hypobaric hypoxia chamber (10% oxygen) at all times continuing 4 weeks. After sustained hypoxia exposure, they were injected with or without TIIA (10 mg/kg) via intraperitoneal daily continuing another 2 weeks. mCAP (A), RVSP (B) and the ratio of RV/(LV + S)%(C) were examined. Data are means ± S. EM. n = 8 rats/group, * P < 0.05, ** P < 0.01 vs. normoxia group, # P < 0.05, ## P < 0.01 vs. sustained hypoxia group. (D) Histopathologic examination of the small pulmonary arteries was performed. The bar represents 50 ␮m. The ratio of WT% (E) and WA% (F) were calculated. Data are means ± S.EM., n = 72 small pulmonary arteries/group; 3 small pulmonary arteries/slides; 3slides/rat; 8 rats/group. * P < 0.05, ** P < 0.01 vs. normoxia group, # P < 0.05, ## P < 0.01 vs. sustained hypoxia group.

of [Ca2+ ]cyt in PASMC, not only forms sustained HPV, but also stimulates PASMC proliferation, which ultimately contributes to the development of pulmonary vascular remodeling (PVR) (Mandegar et al., 2004). Moreover, sustained HPV or chronic exposure to hypoxia decreases the expression of O2 -sensitive KV channels (Wang et al., 2005), which attenuate the programed cell death and inhibit the activity of cytoplasmic caspases (Remillard and Yuan, 2004, 2005). Consequently, the balance between PASMC proliferation and apoptosis is disrupted, and pulmonary vascular

medial hypertrophy is promoted, which further aggravates PVR and elevates the pulmonary arteries pressure (Platoshyn et al., 2000; Stenmark et al., 2006). Accordingly, recovering O2 -sensitive KV channels may represent a therapeutic approach to CH-PH. Among these O2 -sensitive KV channels, KV 1.5 and KV 2.1 are of great importance, for they are primarily expressed in small pulmonary arterioles (Archer et al., 1996, 2004), and control PASMC membrane potential (Archer et al., 1998). More studies demonstrated that hypoxia caused reduction of KV 1.5 and KV 2.1 mRNA

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Fig. 5. TIIA stabilized chronic intermittent hypoxia-induced the mRNA expression and protein expression of KV 1.5 and KV 2.1 in the pulmonary arteries. The pulmonary arteries (3th divisions) were carefully isolated from rats exposed to normoxia or intermittent hypoxia exposure for 4 weeks with or without TIIA (10 mg/kg) treatment. Total RNA of was extracted and the KV 2.1 and KV 1.5 mRNA levels were analyzed by RT-PCR. The KV 2.1 (A and B) and KV 1.5(C and D) mRNA were normalized against ␤-actin. Total proteins were extracted, and then were subjected to immunoblotting using antibodies against KV 2.1, KV 1.5 and ␤-actin(E) and the intensities of KV 2.1 and KV 1.5 protein relative content were quantified (F and G). Data are means ± S. EM. n = 4, each band has PAs pooled from 4 rats, 16 rats/group, * P < 0.05, ** P < 0.01 vs. normoxia group, # P < 0.05, ## P < 0.01 vs. intermittent hypoxia group.

with consequent membrane depolarization and increased [Ca2+ ]cyt in chronically hypoxic rats (Hong et al., 2004; Sweeney and Yuan, 2000). Also, the amplitude of IKV and the mRNA/protein expression level of KV 1.5 and KV 2.1 both significantly decreased in PASMC from patients with pulmonary hypertension (Yuan et al., 1998a,b). These data provide strong evidence for the role of O2 -sensitive KV channels in mediating pulmonary vasoconstriction and vascular remodeling. Bases on theses previous data, we investigated whether KV channels were responsible for the protective efficacy of TIIA on intermittent hypoxia and sustained hypoxia-induced PH. In acute hypoxia experiments, we found acute hypoxia inhibited the IKV currents in PASMCs, and 25 ␮g/ml of TIIA reversed the downregulation of IKV currents. Treated with 4-AP to block the IKV currents in the acute hypoxia exposure, the up-regulation effect of 25 ␮g/ml of TIIA on the IKV currents was relieved. Besides, we found TIIA (25 ␮g/ml) had no effects on normal PASMCs in normoxic conditions in agreement with our previous study(Wang et al., 2010) that inhibition KV channels with 4-AP had no significantly impact on IPA rings vasodilation caused by TIIA in normoxic exposure, which suggested that TIIA up-regulated KV channels in hypoxia, but not normoxia.

In the chronic hypoxia experiments, we confirmed that both intermittent hypoxia and sustained hypoxia decreased the mRNA and protein expressions of KV 1.5 and KV 2.1 in the pulmonary arterioles, and dramatically cut down the IKV currents in PASMCs, which may be the crucial factors to cause the development of PVR and PH. While treated with TIIA markedly upgraded the mRNA and protein expressions of KV 1.5 and KV 2.1 and reversed the IKV currents, no matter which method of administration was used, contemporaneously with intermittent hypoxia for 4 weeks or after sustained hypoxia-induced PH was almost established for another 2 weeks. Similarly, TIIA didn’t affect the expressions of KV 1.5 and KV 2.1 and the IKV currents in the normoxia exposure either. These results suggested that the protective effect of TIIA on PVR and intermittent hypoxia and sustained hypoxia-induced PH was probably through its modulating the expressions of KV channels in the pulmonary arterioles and reversing the down-regulation of the IKV currents in PASMCs under chronic hypoxia condition. There is limitation in our study. We did not evaluate the effect of TIIA on the proliferation, apoptosis and the expression of certain transcriptional factors in lung. Investigation whether TIIA has the potential to inhibit proliferation, promote apoptosis and regulate the expression of transcriptional factors via upgrading the

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Fig. 6. TIIA stabilized chronic sustained hypoxia-induced the mRNA expression and protein expression of KV 1.5 and KV 2.1 in the pulmonary arteries. The pulmonary arteries (3th divisions) were carefully isolated from rats exposed to normoxia or sustained hypoxia exposure with or without TIIA treatment. Total RNA of was extracted and the KV 2.1 and KV 1.5 mRNA levels were analyzed by RT-PCR. The KV 2.1 (A and B) and KV 1.5(C and D) mRNA were normalized against ␤-actin. Total proteins were extracted, and then were subjected to immunoblotting using antibodies against KV 2.1, KV 1.5 and ␤-actin(E) and the intensities of KV 2.1 and KV 1.5 protein relative content were quantified (F and G). Data are means ± S. EM. n = 4, each band has PAs pooled from 4 rats, 16 rats/group, * P < 0.05, ** P < 0.01 vs. normoxia group, # P < 0.05, ## P < 0.01 vs. sustained hypoxia group.

down-regulation of KV channels under hypoxia condition would be more persuasive, which needs to be addressed in further studies. Although it requires further investigation, we confirmed that the efficacy of TIIA against intermittent hypoxia and sustained hypoxia-induced PH were correlated with modulating KV channel. Acute hypoxia dramatically down-regulated IKV currents, while

chronic intermittent hypoxia or sustained hypoxia caused severe PH, evidenced by increasing RVSP and RV hypertrophy, and pulmonary artery wall remodeling, accompanied with decreasing the expression of KV 2.1 and KV 1.5 and the down-regulation of IKV currents. TIIA significantly inhibited pulmonary arterioles wall remodeling, and then reduced RVSP and RV hypertrophy, probably

Fig. 7. TIIA recovered chronic intermittent hypoxia and sustained hypoxia-induced IKV current down-regulation in PASMCs. PASMCs dispersed from the pulmonary arteries (3th divisions) were prepared and whole-cell patch-clamping were used to analyze IKV currents. BKCa channels were inhibited by 5 mM TEA. Cells were voltage-clamped at −70 mV and currents were evoked in 20 mV steps from −70 mV to +70 mV using 400 ms pulses. TIIA does not affect the IKV currents under normoxia conditions (A and B). The IKV currents were suppressed by chronic intermittent hypoxia or sustained hypoxia(C and H). The IKV currents were recovered by TIIA treatment (10 mg/kg) (D and I). Current-voltage (I–V) curve was recorded in PASMC (E and J). Data are expressed as means ± S.E.M. n = 8 rats/group, 6–8 replicates per rat. * P < 0.05, ** P < 0.01 vs. normoxia group, # P < 0.05, ## P < 0.01 vs. intermittent hypoxia or sustained hypoxia group.

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