Effect of Simvastatin on 5-HT and 5-HTT in a Rat Model of Pulmonary Artery Hypertension Open Access

Pulmonary artery hypertension (PAH) is the clinical term used to describe a rare condition associated with progressive elevation of pulmonary arterial pressure (larger or equal 25 mm Hg at rest or 30 mm Hg in exercise). In the last 30 years, research in PAH has dramatically increased survival time and quality of life for patients with PAH [1]. However, there remains no cure despite major advances in understanding of the pathogenesis of PAH with respect to genetics [2], inflammation [3], and cellular and molecular biology [4]. PAH is associated with pulmonary vascular remodeling [5], which occurs more commonly secondary to collagen vascular disease, congenital systemic to pulmonary shunt, chronic obstructive pulmonary disease (COPD), interstitial fibrosis and high left-sided filling pressures [6,7]. The earliest pathological alteration of PAH is muscularization of the terminal portion of pulmonary arterialole. The chronic hypoxia rat is a commonly studied model of pulmonary hypertension [8,9]. However, to our knowledge, cigarette smoke-induced PAH has not been reported.

Smoking derived substances and chemicals are harmful, and can cause airway and vasculature inflammatory responses, which ultimately result in endothelial cell damage, pulmonary vascular remodeling, and pulmonary hypertension following COPD and emphysema. Therefore, the current study was designed to investigate PAH following cigarette smoke exposure in rats.

Long-term smoking exposure result in infiltration of inflammatory cells into the lung tissues including vasculature adventitia, which initiates vascular injury and remodeling through stimulating release of blood vessel activating factors including 5-hydroxytryptamine (5-HT), endothelin (ET-1), and many other factors [10]. 5-HT is mainly secreted by gastrointestinal chromaffin cells, serotinergic neurons, pulmonary neuroendocrine cells and activated platelets. Nerve endothelial corpuscles that are located in the airway could also secrete 5-HT [11]. Increasing evidence has shown that 5-HT is an important factor in the development of PAH, which can singularly or synergistically (with other factors) induce the contraction of pulmonary arteries, proliferation of pulmonary arterial smooth muscle cells (PASMCs), and local micro thrombus formation. In this regard, 5-HT induced mitotic responses of PASMCs, which is mediated by mitogen activated protein kinase (MAPK)-dependent pathways [12], and links the signal transduction between the surface receptor and the expression of target genes to modulate cell proliferation, differentiation, and apoptosis. Studies also suggest that 5-HT induced mitogenesis in PASMCs requires cellular internalization of 5-HT through the 5-HT transporter (5-HTT) rather than binding to a membrane receptor [13,14].

Studies have also indicated that hyperplasia of pulmonary artery smooth muscle cells in primary or secondary PAH is attributed to the overexpression of the serotonin transporter (5-HTT) [15]. The 5-HTT is a membrane protein whose main function is transporting 5-HT into the cells. Recent studies found that 5-HTT in PASMCs exerted multiple properties, including transporting 5-HT to induce proliferation of PASMCs [16]. Studies have shown that 5-HTT expression in PASMCs of primary PAH patients was markedly increased, and considered 5-HT play an important role in the development of PAH [17]. Following entry of 5-HT into the cells through 5-HTT, the MAPK pathway is activated to modulate the phosphorylation of GTPase activating proteins (GAP), enhance the activity of Ras, and induce DNA synthesis [18].

Simvastatin is an HMG-CoA reductase inhibitor that blocks production of cholesterol. In addition to their hypolipidemic effects, statins have variety of biological effects including amelioration of endothelial cell function; stimulation of endothelial nitric oxide synthase (eNOS) expression in vascular endothelial cells; anti-inflammation; and inhibition of smooth muscle cell proliferation [19]. Both in vivo and in vitro studies have demonstrated the anti-inflammatory and immuno-regulatory properties of statins [20,21,22]. Previous studies have reported that simvastatin reverses pulmonary vascular remodeling in PAH animal models, although the mechanisms remain unclear. A non-randomized and controlled clinical study reported that simvastatin ameliorated symptoms in PAH patients [23]. Other investigators have observed that statins dampened the rise in PAH by inhibiting the hyperplasia of rat PASMCs [24], and that statins had therapeutic benefit and improved prognosis of patients with pulmonary hypertension [23]. It has also been reported that statin administration could change serotonergic pathway signaling in the central nervous system and it linked to the expression and activity of the 5-HTT [25].

In the current study, therefore, cigarette smoke-induced rat PAH model was established and effect of simvastatin on smoking-induced rat PAH was investigated. Specifically, level of plasma 5-HT and lung tissue 5-HTT expression were determined in the PAH model, and their correlationwith alteration of parameters of PAH including mean pulmonary artery pressure (mPAP), right ventricular hypertrophy index (RVHI), small artery wall thickness (WT%), and small artery wall area (WA%) were determined.

Healthy male SD rats, 6 weeks old, body weight 100±20 g, were maintained in the animal facilities, Hebei Medical University. Animals were accommodated for one week before the experiment. All procedures were in accordance with the institutional and national guidelines for the care and use of laboratory animals. The Study Protocol was approved by The Institutional Animal Care and Use Committee, Hebei Medical University. Animals were randomized into the following three groups (10 rats in each group): Control group, Smoking group and Smoking + simvastatin group. Rats in the groups of smoking only or smoking + simvastatin were exposed to cigarette smoke in a smoking device manufactured by Shijiazhuang Jinyang Science and Technology Inc. (model: JY-01, Shijiazhuang, Hebei, China). Briefly, 20 cigarettes were burnt continuously and blown into a box together with oxygen, and animals were whole-body exposed to the cigarette smoke in the box. Animals were exposed to the cigarette smoke for one hour each time, twice a day, 5 days a week for 16 weeks. Animals of smoking + simvastatin group were intragastrically administered simvastatin (MSD, Hangzhou, China) at a dose of 5mg/kg/day, each day prior to the smoke exposure. The control rats were not treated with any reagent.

Pulmonary arterial pressure (PAP) measurement. Under anesthesia, the right external carotid artery was exposed and a heparinized catheter was inserted into the right external carotid artery and advanced till pulmonary artery to obtain pulmonary artery pressure (PAP), which was recorded by a multichannel recorder through a pressotransducer. Mean of PAP (mPAP) was calculated.

The right ventricular hypertrophy index (RVHI). This was done by detaching and weighing the right ventricular free wall (RV) followed by calculating the right ventricular hypertrophy index (RVHI) = [RV / (LV + S)]. Briefly, right ventricular wall was free from its atrial, septal and valve ring attachments, and pericardial fat was carefully removed. The right ventricular wall (RV) was then flattened out (endocardial surface down) and blotted on a filter paper for mass determination by weight. The left ventricle, including septum (S) was similarly weighed after removal of atrial and valvular attachments and pericardial fat (LV+S). A right ventricular hypertrophy index (RVHI) was then calculated as shown above.

Percentage of wall thickness (WT %) and wall area (WA %). Select one HE-stained slide from each rat, and pick 15 small pulmonary arteries, which was less than 50 µm in external diameter; 15 medium pulmonary arteries (between 50-150 µm in external diameter); and 10 integrated arteries, measure the total area (TA) and inner-side area (IA) of the arteries, wall thickness (WT), and external diameter (ED). WT% and WA% were then calculated as following:

WT %=(2×WT/ED)×100%,

WA %=(TA-IA)/TA×100%.

Bronchoalveolar lavage was performed using a three-in/three-out pattern of intra-tracheal instillation and removal with a total volume of 3 mL of saline in order to enrich total cells and optimize protein recovery. BALF specimens were centrifuged at 1000 rpm x 10 min. Pelleted cells were re-suspended with PBS, and differentially counted by Wright-Giemsa staining. Total of 200 leukocytes was counted in each BALF sample, and percentage of macrophage, neutrophil, and lymphocyte was calculated.

Total leukocytes number in BALF = leukocytes number (/mL) x volume of BALF (mL).

Plasma 5-hydroxytryptamine (5-HT) level was quantified by ELISA using a commercially available kit (Walnut, CA, USA), following the manufacturer's instruction.

Expression of serotonin transporter (5-HTT) in rat lung was assessed by immunohistochemistry. Briefly, paraffin embedded lung tissue slice was de-paraffinized and hydrated. Antigen was retrieved using sodium citrate and with heat-induced retrieval. Anti-5-HTT antibody at 1:200 dilution (ZHGB-BIO, Beijing, China) was applied overnight at 4°C. After HRP-conjugated 2^nd antibody was applied, expression of 5-HTT was visualized using a DAB detection kit (CWBIO (Beijing, China).

Total RNA was extracted from rat lung tissues with Trizol reagent (SBS, Shanghai, China) following the manufacturer's instruction. After RNA quantification, 1µg total RNA was used for reverse transcription using high capacity reverse transcription kit (Applied Biosystem by Life Technology, Grand Island, NY, USA). Quantitative real time PCR was performed using primers/probe and reaction reagents, 7500 Gene Detection System (Applied Biosystem by Life Technology, Grand Island, NY, USA). ß-actin was used as internal control. Data expressed as fold change versus Control, using 2^(-∆∆Ct)method.

All data were analyzed by SPSS 13.0 statistical software, and presented as mean±SD. One-way ANOVA was used for comparison of multiple groups, and Dunnett'sT-test was used for comparison of paired groups. Correlation analysis was conducted between the indices by logistic regression analysis, and it was considered statistically significant when p < 0.05.

There was no differencein baseline body weight among the three groups (p>0.05). After 16 weeks of smoke exposure, however, body weightwas significantly reduced in the smoke-exposed ratscompared to the control rats (449 ± 15 vs 489 ± 17 g, p < 0.01). Simvastatin significantly prevented smoke-induced weight loss (470 ± 11 g, p < 0.01compared to the smoking group, Table 1).

Establishment of rat pulmonary hypertension model was demonstrated by the following three parameters.

Pulmonary arterial pressure (PAP). Compared to the control group (17.37 ± 0.87 mmHg), mPAP was significantly increased in the smoking group (29.97 ± 1.11 mmHg, p<0.01), and it was significantly blocked by simvastatin (20.69 ± 1.19 mmHg, p<0.01, Fig. 1A).

Right ventricular hypertrophy index (RVHI). Consistent with the change of mPAP, cigarette smoking significantly increased RVHI (0.223 ± 0.011 of smoking rat vs 0.199 ± 0.009 of control, p < 0.01), and simvastatin could significantly reduce smoking-induced increase of RVHI (0.208 ± 0.007, p < 0.01, Fig. 1B).

Wall thickness (WT %) and wall area (WA %). Compared to the control group, WT% and WA% were significantly increased in the smoking group (37.4 ± 8.5 and 48.8 ± 7.5, respectively) compared to control group (20.3 ± 5.0 and 24.8 ± 4.6, respectively, p < 0.01). Simvastatin could significantly protected vascular wall from cigarette smoke-induced increase of thickness and area (30.3 ± 8.9 and 40.8 ± 9.8, respectively, p < 0.05 as compared to the smoking group, Fig. 1C).

Fluid recovered from bronchoalveolar lavage was 78 ± 8 %, 77 ± 7 %, and 77 ± 9 % of injected volume in control group, smoking group and smoking plus simvastatin group, respectively, and there was no significant difference between the 3 groups (p > 0.05). As expected, number of total white blood cells was significantly increased in the BALF of smoking group (24.89 ± 1.47 x 10⁶ cells/L vs 20.82 ± 1.23 x 10⁶ cells/L of control, p < 0.01), and it was significantly reduced in the rats treated with simvastatin (22.56 ± 1.76 x 10⁶ cells/L, p < 0.01, Fig. 2). Furthermore, differential count of neutrophils, macrophages and lymphocytes revealed that cigarette smoke exposure resulted in significantly increased accumulation of neutrophils, macrophages and lymphocytes in the bronchial alveolar lavage (p < 0.01), and that simvastatin significantly reduced smoke-induced inflammatory cell accumulation in the BALF (p < 0.05 or 0.01, Fig. 2).

Since inflammatory cells may contribute to the pathogenesis of cigarette smoke-induced pulmonary hypertension, the correlationship between BALF inflammatory cell number and parameters of PAH was analyzed. As shown in Fig. 3, the number of leukocytes was significantly correlated with mPAP (r = 0.739, p = 0.000), RVHI (r = 0.524, p = 0.003), WT% (r = 0.514, p = 0.004), and WA% (r = 0.581, p = 0.001).

Since 5-HT has been reported to play important role in the pathogenesis of PAH [26], gene expression of 5-HTT in the lung was assessed by real time RT-PCR, and protein of 5-HTT was detected by immunohistochemistry of the lung tissue. As shown in Fig. 4, 5-HTT was undetectable in the lung tissue of control group (Fig. 4A). In contrast, 5-HTT was strongly positive in the smoke-exposed rat pulmonary artery endothelium cells and alveolar macrophages (Fig. 4B), and simvastatin treatment resulted in significant decrease 5-HTT expression in the pulmonary arterial endothelium cells and smooth muscle cells (Fig. 4C).

Next, gene expression of 5-HTT in the rat lung tissue was assessed by real time RT-PCR. As shown in Fig. 5A, expression of 5-HTT mRNA in the smoking group was significantly increased (7.43 ± 3.54 fold change versus control group, p < 0.01, Fig. 5A), and simvastatin could significantly blocked cigarette smoke-induced up-regulation of 5-HTT mRNA (2.81 ± 0.82 fold change versus control group, p < 0.01, Fig. 5A).

Consistent with alteration of 5-HTT mRNA and protein in the lung tissues, circulating level of 5-HT protein was significantly increased in the smoke-exposed rat (8.04 ± 1.32ng/mL) compared to control rats (4.04 ± 0.84 ng/mL, p<0.01), and it was significantly reduced by simvastatin (6.34 ± 1.17 ng/mL, p < 0.05 compared to the smoking group, Fig. 5B).

As was shown in Fig. 6, protein level of 5-HTT in the lung tissue had no correlation with RVHI (r = -0.323, p = 0.082), but it was significantly correlated with mPAP (r = 0.842, p < 0.001), WT% (r = 0.486, p = 0.006), and WA% (r = 0.5923, p = 0.001).

Furthermore, protein level of 5-HT showed positive correlation with mPAP (r = 0.816, p < 0.001), RVHI (r = 0.467, p = 0.009), WT% (r = 0.644, p < 0.001), and WA% (r = 0.744, p < 0.001, Fig. 7).

Numerous animal models of pulmonary artery hypertension (PAH) are currently available. The most commonly used animal models of PAH are the chronic hypoxic model and the monocrotaline injury model [9]. In the current study, however, cigarette smoke exposure was used to prepare PAH model in rats. Cigarette smoke exposure resulted in not only COPD/emphysema, which has been previously reported by us and other investigators [27,28,29,30], but also pulmonary arteriolar wall remodeling and PAH as demonstrated in the current study. Specifically, here, we report that cigarette smoke lead to increase of mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVHI), arterial wall thickness (WT%) and wall area (WA%). Simvastatin could significantly protect rats from cigarette smoke-induced PAH as evidenced by partially reducing mPAP, RVHI, WT% and WA%. Cigarette smoke exposure resulted in significant increase of inflammatory cells in the rat BALF, as well as significant up-regulation of 5-HTT in the lung tissue and 5-HT in the blood circulation. Furthermore, total number of BALF leukocytes as well as level of 5-HTT or 5-HT were significantly correlated with PAH parameters including mPAP, RVHI, WT% and WA%, suggesting inflammatory cells and 5-HTT/5-HT may contribute to the development of PAH in the rats exposed to cigarette smoke. Interestingly, simvastatin could significantly block cigarette smoke-induced infiltration of inflammatory cells into lung tissues as well as up-regulation of 5HTT- and 5-HT, indicating simvastatin may have preventive or therapeutic effect on the development of PAH.

PAH refers to a mean pulmonary artery pressure at rest of greater than or equal to 25 mm Hg or greater than 30 mm Hg with exercise [31]. To study pathogenesis of PAH, variety types of animal models of PAH have been used. Among them, hypoxia-induced PAH model is the most commonly used model, which is characterized by varying degrees of mild to moderate remodeling of small pulmonary arterioles, adventitial thickening and infiltration by inflammatory cells into the adventitia [32]. While hypoxia-induced PAH animal is useful because it is predictable and reproducible, but the reproducibility is observed only within selected animal strains and the response is also significantly affected by age, as younger individuals with rapidly maturing lungs are more susceptible [8,33]. In the current study, therefore, we explored a new PAH model using cigarette smoke exposure. Similar to the hypoxia-induced PAH model, cigarette smoke exposure in the current study could also lead to infiltration of inflammatory cells into alveolar space and thickening of the vascular walls in small pulmonary arterioles. Nevertheless, cigarette smoke exposure could result in not only PAH, but also chronic bronchitis and emphysema [27]. Difference between the hypoxia-induced PAH and cigarette smoke-induced PAH, however, remains to be compared.

It is well known that cigarette smoke exposure could result in COPD and emphysema in animals [28,29,30]. Consistent with these reports, we have also previously reported that cigarette smoke-exposed rats developed COPD/emphysema characterized by enlarged alveoli (increased mean linear intersection) [27]. In addition to injury of alveoli, the current study further reports that cigarette smoke exposure could also lead to PAH characterized by inflammatory infiltration into adventitia of small arterioles, vascular endothelial dysfunction, and vasoconstriction. Specifically, cigarette smoke resulted in significant increase in mPAP, RVHI, WA%, and WT%, which was dramatically blocked by simvastatin. Consistent with previous reports [23,24], the current study demonstrated that simvastatin could significantly inhibited cigarette smoke-induced inflammation of small pulmonary arteries. Furthermore, cigarette smoke-induced animal model of PAH has never been previously reported, and thus, finding of the inhibitory effect of simvastatin on cigarette smoke-induced PAH is novel.

Inflammatory cell numbers and differentials in BALF is an important index that can reflect airway inflammation [34]. In the current study, the total number of inflammatory cells and percentage of neutrophils in BALF were decreased by simvastatin, indicating simvastatin inhibit inflammatory cell accumulation, activation and secretion in the airways and lung tissues. This is consistent with the findings reported by Lee et al. [35].

Although in general, no one animal model of PAH recapitulates all aspects of the severe pathology of human disease [8], these experimental models support the idea that PAH is not so much a specific disease entity as it is a syndrome that may result from a number of disparate pathological states. While pathogenesis of PAH remains to be defined, our results showed that changes in RVHI, WA, WT, mPAP were positively correlated with total cell number and differential cell count in BALF. These results suggested that pulmonary inflammatory infiltration and inflammatory destruction play important role in pulmonary vascular remodeling. Studies have reported that lymphocytes widely reside in small airways and lung parenchyma, including the alveolar wall and artery, and inflammatory cell infiltration is positively correlated to the thickness of pulmonary arterial thickening [36,37], suggesting that consistent development of small airway inflammation is important not only in COPD, but also inPAH.

The “Serotonin hypothesis of PAH” was developed in the 1960's after an outbreak of PAH was observed in patients taking aminorex, a diet pill that increases 5-HT availability by inducing platelet release of 5-HT. Normally, 5-HT is stored within the platelets and thus, plasma levels of free 5-HT are very low. 5-HT is uptaken by the cells specifically through the 5-HT transporter (5-HTT). Circulating 5-HT is produced mainly in the enterochromaffin cells of the intestine. Under pathological conditions such as hypoxia, 5-HT is also locally released from pulmonary neuroendocrine cells and epithelial cells [26]. As one of the most important vasoconstriction and mitotic factors, 5-HT induces contraction and proliferation of PASMCs, and by which mechanism, 5-HT may contribute to the development of PAH. In the current study, we found that cigarette smokes significantly increased expression of 5-HT and 5-HTT in the smoking group, and that expression of 5-HT and 5-HTT were positively correlated with WA%, WT% and mPAP, indicating 5-HT and 5-HTT maybe associated with pulmonary vascular remodeling and development of PAH. In addition, simvastatin significantly reduced 5-HT and 5-HTT expression, and partially blocked cigarette smoke-induced arterial thickening and lumen contraction. These results suggested that statins could protect pulmonary arterioles from inflammatory injury and remodeling through reducing 5-HT circulation and 5-HTT expression in the lung. Consistently, Laudi et al. reported that atorvastatin significantly inhibited monocrotaline-stimulated 5-HTT and PAH in rat models [38]. However, the mechanism of inhibition of 5-HTT by statin in response to either cigarette smoke exposure or monocrotaline remains to be further investigated although it may be associated with inhibition of Ras homolog gene family member A (RhoA) / Rho-associated, coiled-coil containing protein kinase (ROCK) pathway by statins [39].

Taken together, the current study demonstrated that cigarette smoke exposure resulted in inflammatory cell infiltration into the lung tissue as well as pulmonary arteriole remodeling, which ultimately lead to development of PAH. While mechanism of cigarette smoke-induced PAH remains to be further defined, 5-HT and its transporter (5-HTT) may play an important role in the development of PAH in response to cigarette smoke exposure in the rats. Simvastatin could not only dramatically inhibit inflammatory cells infiltration into the lung and up-regulation of 5-HT/5-HTT in response to cigarette smoke exposure, but also partially blocked pulmonary arteriole wall thickening and wall area enlargement. While the molecular mechanism of inhibitory effect of statin on cigarette smoke-induced 5-HTT up regulation and PAH remains to be further investigated in the future study, findings of current study may provide potential therapeutic target of PAH, specifically, statin regulation on 5-HTT may become a therapeutic strategy in the PAH prevention and treatment.

All the authors declare that they have no conflict of interest.

X. Jiang and L. Yuan contributed equally to the work.