Int J Clin Exp Pathol 2014;7(12):8763-8769 www.ijcep.com /ISSN:1936-2625/IJCEP0002845
Original Article Transfer of human hepatocyte growth factor reduces inflammation and prevents pulmonary arterial remodeling in monocrotaline-induced pulmonary arterial hypertensive rats Jianying Chen1*, Hongzhe Zhang1*, Rujun Zhang1*, Zhenjun Liu1, Junxian Wang2, Mengyuan Xiao1, Mingchuan Ba1, Feng Yao1, Jinghu Liu1, Shi’an Huang1, Jixin Zhong1,3 Division of Cardiology, Department of Internal Medicine, Affiliated Hospital of Guangdong Medical College, Zhangjiang 524001, Guangdong, China; 2Department of Geriatrics, Affiliated Hospital of Guangdong Medical College, Zhangjiang 524001, Guangdong, China; 3Division of Cardiology, Department of Medicine, University of Maryland, Baltimore, Maryland 21201, USA. *Equal contributors. 1
Received September 28, 2014; Accepted November 26, 2014; Epub December 1, 2014; Published December 15, 2014 Abstract: Inflammation and endothelial dysfunction contribute to the pathogenesis and development of pulmonary arterial hypertension (PAH). This study was to investigate the therapeutic effect of human hepatocyte growth factor (HGF) gene transfer on monocrotaline (MCT) induced PAH rat models. PAH was induced by injecting MCT for 4 weeks. The rats were randomly assigned to phosphate buffered saline control group, MCT group, and HGF treatment group. After 2 weeks of induction, measures of mean pulmonary artery pressure (mPAP), weight ratio of the RV to the LV plus septum, percent wall thickness index (TI) and area index (AI) were significantly increased in MCT-group and HGF treatment-group compared with those in control group (P < 0.05). Those measurements in MCT-group were significantly higher than those in HGF treatment-group (P < 0.05). IL-6 significantly decreased in HGF treatmentgroup compared with MCT-group, but higher than that of control group (all P < 0.05). IL-10 in HGF treatment-group significantly increased compared with MCT-group, but lower than that of control group (all P < 0.05). Endothelial microparticles (EMP) started to decrease in the HGF treatment-group 3 days after treatment and was most significant after 1 and 2 weeks of treatment (all P < 0.05). Our results showed that transfer of human HGF may attenuate the inflammatory cell infiltrate, reduce the expression of inflammatory factors, and those effects are possibly due to the inhibition of EMP production which may decrease pulmonary vascular wall damage in PAH. Keywords: Human hepatocyte growth factor, pulmonary arterial hypertension, endothelial microparticles, monocrotaline
Introduction Pulmonary arterial hypertension (PAH) is a progressive unruly disease with a poor forecast. PAH is characterized by deregulated proliferation of vascular endothelial and intimal smooth muscle cells, with an increased pulmonary arterial pressure [1]. Moreover, it is noteworthy that a variety of autoimmune disorders and infectious diseases have been reported to be associated with PAH [2]. Despite recent advances in therapies including endothelin-receptor antagonists and phosphodiesterase type-5 inhibitors [3-5], the effects of these drugs are not satisfied, especially in long-term effect.
Recent studies found that HGF as a specific vascular endothelial growth factor has the functions of stimulating endothelial cell proliferation and migration, promoting new vessels formation, and inhibiting cell apoptosis and tissue reconstruction [6]. Many investigations have reported that HGF gene transfer attenuates medial hyperplasia and matrix accumulation in PAH of rats [7, 8]. However, it’s not clear whether HGF was able to attenuate the inflammatory response in PAH. Microparticles (MP) are shed membrane vesicles released during the apoptosis and activation of various cell types [9]. Moreover, circulat-
Hepatocyte growth factor and pulmonary arterial hypertension
Figure 1. Rats with MCT-induced PAH were treated with HGF or PBS for 2 weeks. A. Mean pulmonary artery pressure (MPAP), B. Right ventricular hypertrophy (RVH) were significantly lower in the HGF-treated groups compared with MCT-injected group (P < 0.05).
ing endothelial microparticles (EMPs) have been reported as a marker of endothelial injury and systemic vascular remodeling [10]. Vascular remodeling and endothelial dysfunction are involved in pulmonary hypertension, but treatment of HGF in regulating the release of EMPs and vascular remodeling is not reported. The purpose of this study was to investigate changes of pulmonary hemodynamics, inflammatory response, and prevention pulmonary arterial remodeling in monocrotaline (MCT)induced PAH after human HGF gene transfer. Materials and methods Reagents Monocrotaline (MCT) (Sigma company); IL-6, IL-10 ELISA reagent kit (Excell Bio company); HGF ELISA reagent kit (BG company); Standard particles 0.8 μm, 3.0 μm (Sigma company); PE-CD31 antibody, FITC-CD42b antibody (eBioscience company); HGF gene-recombined adenovirus (Ad-HGF), virus activity 6.3×109 IU/ml (Benyuan Zhengyang genentech limited company). Animals and treatment protocols All procedures were approved by Institutional Animal Care and Use Committee, Affiliated Hospital of Guangdong Medical College (AIACUC-10-7-1). Thirty male SD rats with the weight ranges from 250 g to 300 g were pur8764
chased from the Animal Center of Guangdong Province. The rats were randomly divided into the following 3 groups: control group (n = 10), MCT group (n = 10), and HGF treatment group (n = 10). Rats in control groups were raised in normal condition. Pulmonary hypertension was induced by injection of MCT at 50 mg/kg for 3 weeks. In the meantime, rats were intratracheally given 1.3×109 IU Ad-HGF (HGF treatment group) or 0.2 ml PBS (control group and MCT group). Pulmonary blood pressure measurements Two weeks after the treatment of Ad-HGF, pulmonary artery pressure was measured in anesthetic rats by intraperitoneal injection of 7% chloral hydrate. A ployethylene cannula filled with 2% heparin sodium was inserted into the right external jugular vein and forwarded to the right ventricular (RV). The data were recorded after stabilization of the tracing using a liquid pressure transducer, which was interfaced to a Med lab electrophysiolograph. Index of right ventricular hypertrophy measurements The extent of right ventricular hypertrophy was evaluated by the ratio of right ventricle to left ventricle plus septum weight (RV/LV+S). Histopathological analysis The lung was washed with 4°C 0.1% PBS and physiological saline repeatedly, fixed in 10% Int J Clin Exp Pathol 2014;7(12):8763-8769
Hepatocyte growth factor and pulmonary arterial hypertension
Figure 2. Representative images of lung HE staining. The medial layer of the pulmonary arterioles was progressively thickened after MCT injection. The medial wall area and thickness of the MCT group was significantly attenuated in HGFtreated group (P < 0.05).
formalin solutions overnight, and embedded in paraffin. For identification of vascularity and the thickness of pulmonary arteriolar, formalinfixed paraffin-embedded lungs were sectioned, deparaffinized, and stained for elastic lamina by hematoxylin and eosin (HE). We calculated the thickness index (TI) and area index (AI) of pulmonary arterioles (diameter < 500 μm) wall by computer image analysis system (light microscopy, ×200, selected 5 cross-sections for each section). 8765
Inflammatory index Lung tissue (1 g) was cut into pieces, homogenized at 4°C, and resuspended with 5 ml physiological saline. After centrifugation at 4°C to get rid of debris, the content of HGF in the lung tissue was determined by ELISA. Blood samples were obtained before, 7 days, and 14 days after MCT injection through posterior orbital venous. After centrifugation, sera Int J Clin Exp Pathol 2014;7(12):8763-8769
Hepatocyte growth factor and pulmonary arterial hypertension wall thickness of the pulmonary arterioles appeared to be less in the HGF treated group (Figure 2A).
Figure 3. Content of HGF in lung tissues. HGF level in the lung of HGF group was significantly higher than that of MCT-injected group (P < 0.05).
were collected for the detection of IL-6 and IL-10 (ELISA). Circulated endothelial microparticles (EMP) measurement Blood samples were centrifuged at 1000 rpm for 10 min at room temperature to remove hemocyte and then centrifuged at 3400 rpm for 10 min to remove blood platelet. The surface marker phenotype of these EMP was analyzed by flow cytometry using PE-CD31 and FITC-CD42b. Statistical analysis All quantitative data were expressed as mean ± standard deviation (SD). To compare values among these groups, analysis of variance (AVOVA) was applied. The statistical significance level was defined as P < 0.05. Analyses were performed using SPASS 17.0. Results Hemodynamic and RV weight analysis Two weeks after treatment, the mean pulmonary artery pressure (MPAP) and right ventricular hypertrophy (RVH) were significantly lower in the HGF-treatment PAH groups compared with MCT-induced PAH group (P < 0.05), but were significantly higher compared with the control group (Figure 1A and 1B). Pulmonary artery wall observation There was a significant medial wall thickening in the pulmonary arterioles of MCT-injected rats compared with that of control group 2 weeks after MCT injection. In contrast, medial
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The ratio of medial wall area index and thickness index in the MCT-injected group significantly increased compared with control group 2 weeks after MCT injection (Figure 2B, P < 0.05). In the HGF treatment group, the ratio of medial thickness index of the pulmonary artery was significantly lower than that of MCT group (Figure 2B, P < 0.05). Inflammatory index analysis The content of HGF in the lung tissue was significantly higher in HGF treatment group than that of MCT group (0.35 ± 0.02 vs. 0.12 ± 0.04, P < 0.05, Figure 3). IL-6 significantly decreased in HGF group compared with MCT group, but higher than that of control group (all P < 0.05, Table 1). In contrast, IL-10 significantly increased HGF group compared with MCT group, but lower than that of control group (all P < 0.05, Table 1). Circulated microparticles (EMP) There was no significant difference in initial EMP between the two groups (MCT-injected & HGF treatment groups, P < 0.05). However, EMP was significantly lower in HGF treatment group compared with MCT group after treatment (P < 0.05) (Table 1); The values decreased time-dependently until 2 weeks after HGF treatment (Table 1). Discussion In the rats with MCT-induced PAH, the elevation of pulmonary arterial pressure correlates with a thickening of the medial wall in small pulmonary arteries and arterioles due to the proliferation of vascular smooth muscle cells [11, 12]. Previous studies have reported that transaction of the lung with the HGF gene via the pulmonary artery reduces medial hyperplasia in lung arterioles and inhibits overgrowth of pulmonary arterial smooth muscle cells [7, 8]. Here we demonstrated human HGF transfer reduced pulmonary IL-6 expression and adventitial infiltration of IL-6-expressing inflammatory cells and prevented pulmonary arterial remodeling in MCT-induced PAH rat model. Administration of MCT in rats, can cause endothelial cell injury and elicit massive mononucle-
Int J Clin Exp Pathol 2014;7(12):8763-8769
Hepatocyte growth factor and pulmonary arterial hypertension Table 1. Expression of IL-6 and IL-10 were after HGF transfusion Day Control MCT-injected HGF-treated IL-6 0 40.5 ± 20.1 237.6 ± 53.2* 223.8 ± 37.8* 7 36.2 ± 26.8 327.7 ± 32.4* 151.0 ±30.8*,# 14 42.7 ± 28.4 398.8 ± 54.9* 101.6 ± 14.9*,# IL-10 0 102.4 ± 46.6 56.4 ± 7.4* 40.5 ± 22.4* 7 98.7 ± 25.4 41.9 ± 6.6* 62.2 ± 19.4* 14 99.7 ± 23.0 25.3 ± 7.5* 71.8 ± 23.4# EMP 0 35.7 ± 10.8 68.1 ± 3.2* 69.5 ± 2.8* 7 35.2 ±11.1 75.4 ± 3.3* 59.1 ± 2.8*,# 14 39.9 ± 10.2 78.8 ± 3.0* 55.9 ± 3.7*,# *P < 0.05 when compared control group; #P < 0.05 when compared MCT group.
ar infiltration into perivascular regions of arterioles and muscular arteries, leading to the development of severe PAH after MCT exposure. A growing number of evidence supported that inflammatory mediators including IL-6 and IL-10 play a significant role in PAH [13-18]. The combination of HGF and c-Met phosphorylates GSK3β, NF-κB p65 in cytoplasm to depress the expression of IL-6 and raise the expression of IL-10 [15, 16]. Our study showed that after two weeks of Ad-HGF transfection in PAH rats, the expression of IL-10 increased while IL-6 decreased, accompanied with ameliorated PAH symptoms. Normal pulmonary vascular endothelium release bioactive substances towards part vessels to regulate vascular tone and the growth of smooth muscle cells, maintaining the stability of the whole vessel. More and more studies demonstrated that endothelial dysfunction is an important aspect of pathogenesis of pulmonary hypertension. EMP was released into the microenvironment by endothelium cells that were activated or undergoing apoptosis [19]. These particles carried surface proteins and biological information of their parent cells and could regard as potential biomarkers for monitoring endothelial cell functional status [20]. These results demonstrated that monitoring the levels of circulating EMP could evaluate the relationship between pulmonary hypertension and severe endothelial injury. In addition, in hypoxia-induced pulmonary hypertensive rats, EMP induces endothelial cell dysfunction by reducing the NO generation and increasing oxidative stress [21]. In our study, EMP was significantly higher in MCT-induced pulmonary hyper8767
tension. However, Ad-HGF transfer downregulated the level of EMP. It’s possible that HGF could protect and repair pulmonary vascular endothelium by decreasing EMP levels and promote no generation, which may delay the development of pulmonary hypertension. However, further studies are required to explore the underlying mechanisms. The production of EMP also has a close relationship with inflammation. EMP may promote inflammatory reaction. A large amount of studies demonstrated that EMP has the ability to activate neutrophilic granulocyte, facilitating the interaction of lymphomonocyte and endothelial cells (EC), and mediating chemotactic effect on neutrophils [22]. These affects may be mediated through oxidized phospholipids. The interaction between EMP and monocyte also makes the latter to release mediators of inflammation, causing endothelial cells and monocytes paracrine and anticrime [23]. The level of EMP is positively correlated with IL-6 level, suggesting a connection between EMP and inflammation. In conclusion, our results showed that transfer human HGF may attenuate the inflammatory cell infiltration, reduce the expression of inflammation factors, and those effects are possibly due to the inhibition of EMP production which decreased pulmonary vascular wall damage in PAH. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (81370242 and 81101553), Guangdong Natural Science Foundation (S2013010015005), Science and Technology Planning Project of Guangdong (2009B030801378 and 2012B031800224), Science and Technology Innovation Fund of Guangdong Medical College (STIF201128), Financial Foundation of Zhanjiang [(2011) 79 and (2010) 174], and American Heart Association (13POST17210033). Disclosure of conflict of interest None. Address correspondence to: Dr. Jianying Chen or Dr. Shi’an Huang, Division of Cardiology, Affiliated Hospital of Guangdong Medical College, 57 South
Int J Clin Exp Pathol 2014;7(12):8763-8769
Hepatocyte growth factor and pulmonary arterial hypertension Renmin Ave, Xiashan District, Zhanjiang 524001, Guangdong Province, China. Tel: 86-759-2369047; E-mail: [email protected] (JYC); [email protected] (SAH)
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Barst RJ, McGoon M, Torbicki A, Sitbon O, Krowka MJ, Olschewski H and Gaine S. Diagnosis and differential assessment of pulmonary arterial hypertension. J Am Coll Cardiol 2004; 43: 40S-47S. [2] Dorfmuller P, Perros F, Balabanian K and Humbert M. Inflammation in pulmonary arterial hypertension. Eur Respir J 2003; 22: 358363. [3] Barst RJ, McGoon M, McLaughlin V, Tapson V, Rich S, Rubin L, Wasserman K, Oudiz R, Shapiro S, Robbins IM, Channick R, Badesch D, Rayburn BK, Flinchbaugh R, Sigman J, Arneson C and Jeffs R. Beraprost therapy for pulmonary arterial hypertension. J Am Coll Cardiol 2003; 41: 2119-2125. [4] Humbert M, Segal ES, Kiely DG, Carlsen J, Schwierin B and Hoeper MM. Results of European post-marketing surveillance of bosentan in pulmonary hypertension. Eur Respir J 2007; 30: 338-344. [5] Croom KF and Curran MP. Sildenafil: a review of its use in pulmonary arterial hypertension. Drugs 2008; 68: 383-397. [6] Tahara Y, Ido A, Yamamoto S, Miyata Y, Uto H, Hori T, Hayashi K and Tsubouchi H. Hepatocyte growth factor facilitates colonic mucosal repair in experimental ulcerative colitis in rats. J Pharmacol Exp Ther 2003; 307: 146-151. [7] Ono M, Sawa Y, Mizuno S, Fukushima N, Ichikawa H, Bessho K, Nakamura T and Matsuda H. Hepatocyte growth factor suppresses vascular medial hyperplasia and matrix accumulation in advanced pulmonary hypertension of rats. Circulation 2004; 110: 2896-2902. [8] Ono M, Sawa Y, Fukushima N, Suhara H, Nakamura T, Yokoyama C, Tanabe T and Matsuda H. Gene transfer of hepatocyte growth factor with prostacyclin synthase in severe pulmonary hypertension of rats. Eur J Cardiothorac Surg 2004; 26: 1092-1097. [9] Boulanger CM, Amabile N and Tedgui A. Circulating microparticles: a potential prognostic marker for atherosclerotic vascular disease. Hypertension 2006; 48: 180-186. [10] Amabile N, Guerin AP, Leroyer A, Mallat Z, Nguyen C, Boddaert J, London GM, Tedgui A and Boulanger CM. Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure. J Am Soc Nephrol 2005; 16: 3381-3388.
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[11] Lim KA, Kim KC, Cho MS, Lee BE, Kim HS and Hong YM. Gene expression of endothelin-1 and endothelin receptor a on monocrotalineinduced pulmonary hypertension in rats after bosentan treatment. Korean Circ J 2010; 40: 459-464. [12] Koo HS, Kim KC and Hong YM. Gene expressions of nitric oxide synthase and matrix metalloproteinase-2 in monocrotaline-induced pulmonary hypertension in rats after bosentan treatment. Korean Circ J 2011; 41: 83-90. [13] Price LC, Wort SJ, Perros F, Dorfmuller P, Huertas A, Montani D, Cohen-Kaminsky S and Humbert M. Inflammation in pulmonary arterial hypertension. Chest 2012; 141: 210-221. [14] Miyata M, Sakuma F, Yoshimura A, Ishikawa H, Nishimaki T and Kasukawa R. Pulmonary hypertension in rats. 2. Role of interleukin-6. Int Arch Allergy Immunol 1995; 108: 287-291. [15] Ito T, Okada T, Miyashita H, Nomoto T, NonakaSarukawa M, Uchibori R, Maeda Y, Urabe M, Mizukami H, Kume A, Takahashi M, Ikeda U, Shimada K and Ozawa K. Interleukin-10 expression mediated by an adeno-associated virus vector prevents monocrotaline-induced pulmonary arterial hypertension in rats. Circ Res 2007; 101: 734-741. [16] Coudriet GM, He J, Trucco M, Mars WM and Piganelli JD. Hepatocyte growth factor modulates interleukin-6 production in bone marrow derived macrophages: implications for inflammatory mediated diseases. PLoS One 2010; 5: e15384. [17] Hall S, Brogan P, Haworth SG and Klein N. Contribution of inflammation to the pathology of idiopathic pulmonary arterial hypertension in children. Thorax 2009; 64: 778-783. [18] Hassoun PM, Mouthon L, Barbera JA, Eddahibi S, Flores SC, Grimminger F, Jones PL, Maitland ML, Michelakis ED, Morrell NW, Newman JH, Rabinovitch M, Schermuly R, Stenmark KR, Voelkel NF, Yuan JX and Humbert M. Inflammation, growth factors, and pulmonary vascular remodeling. J Am Coll Cardiol 2009; 54: S10-19. [19] Amabile N, Heiss C, Real WM, Minasi P, McGlothlin D, Rame EJ, Grossman W, De Marco T and Yeghiazarians Y. Circulating endothelial microparticle levels predict hemodynamic severity of pulmonary hypertension. Am J Respir Crit Care Med 2008; 177: 1268-1275. [20] Tual-Chalot S, Guibert C, Muller B, Savineau JP, Andriantsitohaina R and Martinez MC. Circulating microparticles from pulmonary hypertensive rats induce endothelial dysfunction. Am J Respir Crit Care Med 2010; 182: 261-268. [21] Huber J, Vales A, Mitulovic G, Blumer M, Schmid R, Witztum JL, Binder BR and Leitinger
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Hepatocyte growth factor and pulmonary arterial hypertension N. Oxidized membrane vesicles and blebs from apoptotic cells contain biologically active oxidized phospholipids that induce monocyte-endothelial interactions. Arterioscler Thromb Vasc Biol 2002; 22: 101-107. [22] Boulanger CM, Scoazec A, Ebrahimian T, Henry P, Mathieu E, Tedgui A and Mallat Z. Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction. Circulation 2001; 104: 2649-2652.
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[23] Chirinos JA, Zambrano JP, Virani SS, Jimenez JJ, Jy W, Ahn E, Horstman LL, Castellanos A, Myerburg RJ and Ahn YS. Correlation between apoptotic endothelial microparticles and serum interleukin-6 and C-reactive protein in healthy men. Am J Cardiol 2005; 95: 12581260.
Int J Clin Exp Pathol 2014;7(12):8763-8769
Original Article Transfer of human hepatocyte growth factor reduces inflammation and prevents pulmonary arterial remodeling in monocrotaline-induced pulmonary arterial hypertensive rats Jianying Chen1*, Hongzhe Zhang1*, Rujun Zhang1*, Zhenjun Liu1, Junxian Wang2, Mengyuan Xiao1, Mingchuan Ba1, Feng Yao1, Jinghu Liu1, Shi’an Huang1, Jixin Zhong1,3 Division of Cardiology, Department of Internal Medicine, Affiliated Hospital of Guangdong Medical College, Zhangjiang 524001, Guangdong, China; 2Department of Geriatrics, Affiliated Hospital of Guangdong Medical College, Zhangjiang 524001, Guangdong, China; 3Division of Cardiology, Department of Medicine, University of Maryland, Baltimore, Maryland 21201, USA. *Equal contributors. 1
Received September 28, 2014; Accepted November 26, 2014; Epub December 1, 2014; Published December 15, 2014 Abstract: Inflammation and endothelial dysfunction contribute to the pathogenesis and development of pulmonary arterial hypertension (PAH). This study was to investigate the therapeutic effect of human hepatocyte growth factor (HGF) gene transfer on monocrotaline (MCT) induced PAH rat models. PAH was induced by injecting MCT for 4 weeks. The rats were randomly assigned to phosphate buffered saline control group, MCT group, and HGF treatment group. After 2 weeks of induction, measures of mean pulmonary artery pressure (mPAP), weight ratio of the RV to the LV plus septum, percent wall thickness index (TI) and area index (AI) were significantly increased in MCT-group and HGF treatment-group compared with those in control group (P < 0.05). Those measurements in MCT-group were significantly higher than those in HGF treatment-group (P < 0.05). IL-6 significantly decreased in HGF treatmentgroup compared with MCT-group, but higher than that of control group (all P < 0.05). IL-10 in HGF treatment-group significantly increased compared with MCT-group, but lower than that of control group (all P < 0.05). Endothelial microparticles (EMP) started to decrease in the HGF treatment-group 3 days after treatment and was most significant after 1 and 2 weeks of treatment (all P < 0.05). Our results showed that transfer of human HGF may attenuate the inflammatory cell infiltrate, reduce the expression of inflammatory factors, and those effects are possibly due to the inhibition of EMP production which may decrease pulmonary vascular wall damage in PAH. Keywords: Human hepatocyte growth factor, pulmonary arterial hypertension, endothelial microparticles, monocrotaline
Introduction Pulmonary arterial hypertension (PAH) is a progressive unruly disease with a poor forecast. PAH is characterized by deregulated proliferation of vascular endothelial and intimal smooth muscle cells, with an increased pulmonary arterial pressure [1]. Moreover, it is noteworthy that a variety of autoimmune disorders and infectious diseases have been reported to be associated with PAH [2]. Despite recent advances in therapies including endothelin-receptor antagonists and phosphodiesterase type-5 inhibitors [3-5], the effects of these drugs are not satisfied, especially in long-term effect.
Recent studies found that HGF as a specific vascular endothelial growth factor has the functions of stimulating endothelial cell proliferation and migration, promoting new vessels formation, and inhibiting cell apoptosis and tissue reconstruction [6]. Many investigations have reported that HGF gene transfer attenuates medial hyperplasia and matrix accumulation in PAH of rats [7, 8]. However, it’s not clear whether HGF was able to attenuate the inflammatory response in PAH. Microparticles (MP) are shed membrane vesicles released during the apoptosis and activation of various cell types [9]. Moreover, circulat-
Hepatocyte growth factor and pulmonary arterial hypertension
Figure 1. Rats with MCT-induced PAH were treated with HGF or PBS for 2 weeks. A. Mean pulmonary artery pressure (MPAP), B. Right ventricular hypertrophy (RVH) were significantly lower in the HGF-treated groups compared with MCT-injected group (P < 0.05).
ing endothelial microparticles (EMPs) have been reported as a marker of endothelial injury and systemic vascular remodeling [10]. Vascular remodeling and endothelial dysfunction are involved in pulmonary hypertension, but treatment of HGF in regulating the release of EMPs and vascular remodeling is not reported. The purpose of this study was to investigate changes of pulmonary hemodynamics, inflammatory response, and prevention pulmonary arterial remodeling in monocrotaline (MCT)induced PAH after human HGF gene transfer. Materials and methods Reagents Monocrotaline (MCT) (Sigma company); IL-6, IL-10 ELISA reagent kit (Excell Bio company); HGF ELISA reagent kit (BG company); Standard particles 0.8 μm, 3.0 μm (Sigma company); PE-CD31 antibody, FITC-CD42b antibody (eBioscience company); HGF gene-recombined adenovirus (Ad-HGF), virus activity 6.3×109 IU/ml (Benyuan Zhengyang genentech limited company). Animals and treatment protocols All procedures were approved by Institutional Animal Care and Use Committee, Affiliated Hospital of Guangdong Medical College (AIACUC-10-7-1). Thirty male SD rats with the weight ranges from 250 g to 300 g were pur8764
chased from the Animal Center of Guangdong Province. The rats were randomly divided into the following 3 groups: control group (n = 10), MCT group (n = 10), and HGF treatment group (n = 10). Rats in control groups were raised in normal condition. Pulmonary hypertension was induced by injection of MCT at 50 mg/kg for 3 weeks. In the meantime, rats were intratracheally given 1.3×109 IU Ad-HGF (HGF treatment group) or 0.2 ml PBS (control group and MCT group). Pulmonary blood pressure measurements Two weeks after the treatment of Ad-HGF, pulmonary artery pressure was measured in anesthetic rats by intraperitoneal injection of 7% chloral hydrate. A ployethylene cannula filled with 2% heparin sodium was inserted into the right external jugular vein and forwarded to the right ventricular (RV). The data were recorded after stabilization of the tracing using a liquid pressure transducer, which was interfaced to a Med lab electrophysiolograph. Index of right ventricular hypertrophy measurements The extent of right ventricular hypertrophy was evaluated by the ratio of right ventricle to left ventricle plus septum weight (RV/LV+S). Histopathological analysis The lung was washed with 4°C 0.1% PBS and physiological saline repeatedly, fixed in 10% Int J Clin Exp Pathol 2014;7(12):8763-8769
Hepatocyte growth factor and pulmonary arterial hypertension
Figure 2. Representative images of lung HE staining. The medial layer of the pulmonary arterioles was progressively thickened after MCT injection. The medial wall area and thickness of the MCT group was significantly attenuated in HGFtreated group (P < 0.05).
formalin solutions overnight, and embedded in paraffin. For identification of vascularity and the thickness of pulmonary arteriolar, formalinfixed paraffin-embedded lungs were sectioned, deparaffinized, and stained for elastic lamina by hematoxylin and eosin (HE). We calculated the thickness index (TI) and area index (AI) of pulmonary arterioles (diameter < 500 μm) wall by computer image analysis system (light microscopy, ×200, selected 5 cross-sections for each section). 8765
Inflammatory index Lung tissue (1 g) was cut into pieces, homogenized at 4°C, and resuspended with 5 ml physiological saline. After centrifugation at 4°C to get rid of debris, the content of HGF in the lung tissue was determined by ELISA. Blood samples were obtained before, 7 days, and 14 days after MCT injection through posterior orbital venous. After centrifugation, sera Int J Clin Exp Pathol 2014;7(12):8763-8769
Hepatocyte growth factor and pulmonary arterial hypertension wall thickness of the pulmonary arterioles appeared to be less in the HGF treated group (Figure 2A).
Figure 3. Content of HGF in lung tissues. HGF level in the lung of HGF group was significantly higher than that of MCT-injected group (P < 0.05).
were collected for the detection of IL-6 and IL-10 (ELISA). Circulated endothelial microparticles (EMP) measurement Blood samples were centrifuged at 1000 rpm for 10 min at room temperature to remove hemocyte and then centrifuged at 3400 rpm for 10 min to remove blood platelet. The surface marker phenotype of these EMP was analyzed by flow cytometry using PE-CD31 and FITC-CD42b. Statistical analysis All quantitative data were expressed as mean ± standard deviation (SD). To compare values among these groups, analysis of variance (AVOVA) was applied. The statistical significance level was defined as P < 0.05. Analyses were performed using SPASS 17.0. Results Hemodynamic and RV weight analysis Two weeks after treatment, the mean pulmonary artery pressure (MPAP) and right ventricular hypertrophy (RVH) were significantly lower in the HGF-treatment PAH groups compared with MCT-induced PAH group (P < 0.05), but were significantly higher compared with the control group (Figure 1A and 1B). Pulmonary artery wall observation There was a significant medial wall thickening in the pulmonary arterioles of MCT-injected rats compared with that of control group 2 weeks after MCT injection. In contrast, medial
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The ratio of medial wall area index and thickness index in the MCT-injected group significantly increased compared with control group 2 weeks after MCT injection (Figure 2B, P < 0.05). In the HGF treatment group, the ratio of medial thickness index of the pulmonary artery was significantly lower than that of MCT group (Figure 2B, P < 0.05). Inflammatory index analysis The content of HGF in the lung tissue was significantly higher in HGF treatment group than that of MCT group (0.35 ± 0.02 vs. 0.12 ± 0.04, P < 0.05, Figure 3). IL-6 significantly decreased in HGF group compared with MCT group, but higher than that of control group (all P < 0.05, Table 1). In contrast, IL-10 significantly increased HGF group compared with MCT group, but lower than that of control group (all P < 0.05, Table 1). Circulated microparticles (EMP) There was no significant difference in initial EMP between the two groups (MCT-injected & HGF treatment groups, P < 0.05). However, EMP was significantly lower in HGF treatment group compared with MCT group after treatment (P < 0.05) (Table 1); The values decreased time-dependently until 2 weeks after HGF treatment (Table 1). Discussion In the rats with MCT-induced PAH, the elevation of pulmonary arterial pressure correlates with a thickening of the medial wall in small pulmonary arteries and arterioles due to the proliferation of vascular smooth muscle cells [11, 12]. Previous studies have reported that transaction of the lung with the HGF gene via the pulmonary artery reduces medial hyperplasia in lung arterioles and inhibits overgrowth of pulmonary arterial smooth muscle cells [7, 8]. Here we demonstrated human HGF transfer reduced pulmonary IL-6 expression and adventitial infiltration of IL-6-expressing inflammatory cells and prevented pulmonary arterial remodeling in MCT-induced PAH rat model. Administration of MCT in rats, can cause endothelial cell injury and elicit massive mononucle-
Int J Clin Exp Pathol 2014;7(12):8763-8769
Hepatocyte growth factor and pulmonary arterial hypertension Table 1. Expression of IL-6 and IL-10 were after HGF transfusion Day Control MCT-injected HGF-treated IL-6 0 40.5 ± 20.1 237.6 ± 53.2* 223.8 ± 37.8* 7 36.2 ± 26.8 327.7 ± 32.4* 151.0 ±30.8*,# 14 42.7 ± 28.4 398.8 ± 54.9* 101.6 ± 14.9*,# IL-10 0 102.4 ± 46.6 56.4 ± 7.4* 40.5 ± 22.4* 7 98.7 ± 25.4 41.9 ± 6.6* 62.2 ± 19.4* 14 99.7 ± 23.0 25.3 ± 7.5* 71.8 ± 23.4# EMP 0 35.7 ± 10.8 68.1 ± 3.2* 69.5 ± 2.8* 7 35.2 ±11.1 75.4 ± 3.3* 59.1 ± 2.8*,# 14 39.9 ± 10.2 78.8 ± 3.0* 55.9 ± 3.7*,# *P < 0.05 when compared control group; #P < 0.05 when compared MCT group.
ar infiltration into perivascular regions of arterioles and muscular arteries, leading to the development of severe PAH after MCT exposure. A growing number of evidence supported that inflammatory mediators including IL-6 and IL-10 play a significant role in PAH [13-18]. The combination of HGF and c-Met phosphorylates GSK3β, NF-κB p65 in cytoplasm to depress the expression of IL-6 and raise the expression of IL-10 [15, 16]. Our study showed that after two weeks of Ad-HGF transfection in PAH rats, the expression of IL-10 increased while IL-6 decreased, accompanied with ameliorated PAH symptoms. Normal pulmonary vascular endothelium release bioactive substances towards part vessels to regulate vascular tone and the growth of smooth muscle cells, maintaining the stability of the whole vessel. More and more studies demonstrated that endothelial dysfunction is an important aspect of pathogenesis of pulmonary hypertension. EMP was released into the microenvironment by endothelium cells that were activated or undergoing apoptosis [19]. These particles carried surface proteins and biological information of their parent cells and could regard as potential biomarkers for monitoring endothelial cell functional status [20]. These results demonstrated that monitoring the levels of circulating EMP could evaluate the relationship between pulmonary hypertension and severe endothelial injury. In addition, in hypoxia-induced pulmonary hypertensive rats, EMP induces endothelial cell dysfunction by reducing the NO generation and increasing oxidative stress [21]. In our study, EMP was significantly higher in MCT-induced pulmonary hyper8767
tension. However, Ad-HGF transfer downregulated the level of EMP. It’s possible that HGF could protect and repair pulmonary vascular endothelium by decreasing EMP levels and promote no generation, which may delay the development of pulmonary hypertension. However, further studies are required to explore the underlying mechanisms. The production of EMP also has a close relationship with inflammation. EMP may promote inflammatory reaction. A large amount of studies demonstrated that EMP has the ability to activate neutrophilic granulocyte, facilitating the interaction of lymphomonocyte and endothelial cells (EC), and mediating chemotactic effect on neutrophils [22]. These affects may be mediated through oxidized phospholipids. The interaction between EMP and monocyte also makes the latter to release mediators of inflammation, causing endothelial cells and monocytes paracrine and anticrime [23]. The level of EMP is positively correlated with IL-6 level, suggesting a connection between EMP and inflammation. In conclusion, our results showed that transfer human HGF may attenuate the inflammatory cell infiltration, reduce the expression of inflammation factors, and those effects are possibly due to the inhibition of EMP production which decreased pulmonary vascular wall damage in PAH. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (81370242 and 81101553), Guangdong Natural Science Foundation (S2013010015005), Science and Technology Planning Project of Guangdong (2009B030801378 and 2012B031800224), Science and Technology Innovation Fund of Guangdong Medical College (STIF201128), Financial Foundation of Zhanjiang [(2011) 79 and (2010) 174], and American Heart Association (13POST17210033). Disclosure of conflict of interest None. Address correspondence to: Dr. Jianying Chen or Dr. Shi’an Huang, Division of Cardiology, Affiliated Hospital of Guangdong Medical College, 57 South
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Hepatocyte growth factor and pulmonary arterial hypertension Renmin Ave, Xiashan District, Zhanjiang 524001, Guangdong Province, China. Tel: 86-759-2369047; E-mail: [email protected] (JYC); [email protected] (SAH)
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