Monocrotaline-induced pulmonary hypertension correlates with upregulation of connective tissue growth factor expression in the lung

Abstract

Pulmonary hypertension (PH) is characterized by structural and functional changes in the lung including proliferation of vascular smooth muscle cells (VSMCs) and excessive collagen synthesis. Although connective tissue growth factor (CTGF) is known to promote cell proliferation, migration, adhesion, and extracellular matrix production in various tissues, studies on the role of CTGF in pulmonary hypertension have been limited. Here, we examined CTGF expression in the lung tissues of male Sprague Dawley rats treated with monocrotaline (MCT, 60 µgram/kg), a pneumotoxic agent known to induce PH in animals. Establishment of PH was verified by the significantly increased right ventricular systolic pressure and right ventricle/left ventricle weight ratio in the MCT-treated rats. Histological examination of the lung revealed profound muscular hypertrophy in the media of pulmonary artery and arterioles in MCT-treated group. Lung parenchyma, vein, and bronchiole did not appear to be affected. RT-PCR analysis of the lung tissue at 5 weeks indicated significantly increased expression of CTGF in the MCT-treated group. In situ hybridization studies also confirmed abundant CTGF mRNA expression in VSMCs of the arteries and arterioles, clustered pneumocytes, and infiltrated macrophages. Interestingly, CTGF mRNA was not detected in VSMCs of vein or bronchiole. In saline-injected control, basal expression of CTGF was seen in bronchial epithelial cells, alveolar lining cells, and endothelial cells. Taken together, our results suggest that CTGF upregulation in arterial VSMC of the lung might be important in the pathogenesis of pulmonary hypertension. Antagonizing the role of CTGF could thus be one of the potential approaches for the treatment of PH.

EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 37, No. 1, 27-35, February 2005

Monocrotaline-induced pulmonary hypertension correlates with upregulation of connective tissue growth factor expression in the lung

Young-Sam Lee1*, Jonghoe Byun1*, Jeong-A Kim 1, Jung-Sun Lee1, Koung Li Kim 1, Yeon-Lim Suh2, Jeong-M in Kim 1, Hyung-Suk Jang1, Jae-Young Lee1, In-Soon Shin1,W onhee Suh1, Eun-Seok Jeon1and Duk-Kyung Kim 1,3

1Department of Medicine Cardiac and Vascular Center

2Department of PathologySamsung Medical CenterSamsung Biomedical Research Institute Sungkyunkwan University School of Medicine50 Ilwon-dong, Kangnam-gu, Seoul 135-710, Korea

3Corresponding author: Tel, 82-2-3410-3413;

Fax, 82-2-3410-3417; E-mail, [email protected] *These authors contributed equally to this work.

Accepted 13 January 2005

Abbreviations: CTGF, connective tissue growth factor; ET-1, endothelin-1; MCT, monocrotaline; PH, pulmonary hypertension; TGF-1, transforming growth factor-1; VSMC, vascular smooth muscle cell

Abstract

Pulm onary hypertension (PH) is characterized by structural and functional changes in the lung including proliferation of vascular sm ooth m uscle cells (VSM Cs) and excessive collagen synthesis. Although connective tissue growth factor (CTG F) is know n to prom ote cell proliferation, m igration, adhesion, and extracellular matrix production in various tissues, studies on the role of CTG F in pulm onary hypertension have been lim ited. Here, w e exam ined CTG F expression in the lung tissues of m ale Sprague Daw ley rats treated w ith m onocrotaline (M C T, 60 g/kg), a pneum otoxic agent know n to induce PH in anim als. Establishm ent of PH w as verified by the significantly increased right ventricular systolic pressure and right ventricle/left ventricle weight ratio in the M CT-treated rats. Histological exam i-

nation of the lung revealed profound m uscular hypertrophy in the m edia of pulm onary artery and arterioles in M C T-treated group. Lung parenchym a, vein, and bronchiole did not appear to be affected. RT-PCR analysis of the lung tissue at 5 weeks indicated significantly increased expression of CTG F in the M CT-treated group. In situ hybridization studies also confirm ed abundant CTG F m RNA expression in VSM C s of the arteries and arterioles, clustered pneum ocytes, and infiltrated m acrophages. Interestingly, CTG F m R NA w as not detected in VSM Cs of vein or bronchiole. In saline-injected control, basal expression of CTGF was seen in bronchial epithelial cells, alveolar lining cells, and endothelial cells. Taken together, our results suggest that CTG F upregulation in arterial VSM C of the lung m ight be im portant in the pathogenesis of pulm onary hypertension. Antagonizing the role of CTG F could thus be one of the potential approaches for the treatm ent of PH.

Keywords: connective tissue growth factor; fibrosis; hypertrophy; monocrotaline; pulmonary hypertension

Introduction

Pulmonary hypertension (PH) is a rare lung disorder in which the blood pressure in the pulmonary artery rises far above normal levels and may become life threatening (D'Alonzo et al., 1991). It is characterized by structural and functional changes in pulmonary vasculature and proliferation of pulmonary artery smooth muscle cells as well as excess collagen synthesis in the lung (Botney et al., 1993). Although pathogenesis of PH involves a complex and multi-factorial process, current evidence suggests that endothelial dysfunction plays an integral role in the initiation and progress of PH (Veyssier-Belot and Cacoub, 1999; Budhiraja et al., 2004).

Monocrotaline (MCT), a pyrrolizidine alkaloid from Crotalaria spectabilis, is activated metabolically in the liver to monocrotaline pyrrole which is then transported to the lungs and becomes pneumotoxic (Wilson et al., 1992). It is thought to induce lung reactions such as interstitial edema, inflammation, hemorrhage and fibrosis (Kay and Heath, 1969). Since subcutaneous injection of MCT can cause PH, medial hypertrophy of the pulmonary arteries, and severe pres-

28 Exp. Mol. Med. Vol. 37(1), 27-35, 2005

sure overload-induced right ventricular hypertrophy, MCT has been widely used to establish an animal model of PH (Meyrick et al., 1980; Ghodsi and Will, 1981).

Connective tissue growth factor (CTGF) is a cysteine-rich, 38-kDa polypeptide that was originally isolated from human umbilical vein endothelial cells (Bradham et al., 1991) and subsequently found in many cells including fibroblasts, chondrocytes, and smooth muscle cells (Kothapalli et al., 1997; Nakanishi et al., 1997; Hishikawa et al., 1999b). CTGF is involved in many cellular processes underlying fibrosis such as cell proliferation, migration, adhesion, and the synthesis of extracellular matrix (ECM) (Brigstock, 1999). It induces expression of fibronectin and collagen type I, which are the molecules abnormally deposited in fibrotic lesions of major organs such as liver, kidney, lung, and skin (Igarashi et al., 1996; Lasky et al., 1998; Ito et al., 1998; Paradis et al., 1999). Accumulation of connective tissue in vessel wall is an important component of the alterations in PH and results from a complex interplay between synthesis and degradation of ECM (Vieillard-Baron et al., 2000). Although there have been many studies on CTGF with regard to the development of fibrotic pathology (Grotendorst, 1997), its role in pulmonary hypertension has remained unexplored.

In the present study, we investigated CTGF expression in a rat model of MCT-induced PH. We first identified the regions of the lung that are affected by MCT treatment and then examined the level and distribution of CTGF expression by RT-PCR and in situ hybridization, respectively. Elevated expression of CTGF was observed in the hypertrophic smooth muscle cells of arteries and arterioles and in the cells that had infiltrated into the alveolar space of the lung, but not in the vein and bronchus. Our results suggest potential relevance of increased level of CTGF to the development of pulmonary arterial hypertrophy and PH in response to MCT.

M aterials and M ethods

Rat model of PH

MCT (300 mg Crotaline; Sigma, St. Louis, MI) was

dissolved in 1.8 ml of 1 M HCl followed by addition of 3 to 4 ml of distilled water. This solution was adjusted to pH 7.4 using 1 M NaOH solution and filled up to 15 ml with distilled water (Hayashi et al., 1967). Male Sprague-Dawley rats (6-week-old) received a single subcutaneous injection of MCT solution (60 mg/kg) or saline solution. Rats were housed with a 12/12 - light/dark cycle and given water and standard rat chow ad libitum. At 2 or 5 weeks after injection, the rats were sacrificed and the organs harvested for the following analyses.

Measurement of hem odynamic parameters and assessm ent of right ventricular hypertrophy

Rats were anesthetized using an intramuscular injection of ketamine (70 mg/kg; Ketalar 50 mg/ml;Yuhan Co., Korea) and xylazine (10 mg/kg; Rompun 23.32 mg/ml; Bayer, Korea). After exposing the heart, a 21-gauge needle was inserted into the right ventricle (RV). Pulmonary arterial pressures were measured immediately after insertion of the needle using the compact configurable monitor (78354C, HP, Palo Alto, CA). After the pressure measurement, the heart was excised and weighed. The weight ratio of RV free wall to septum plus left ventricular (LV) free wall was also measured.

Histology

The lung was prepared for histopathologic examination as follows. For paraffin section, the lung was isolated and fixed in formalin for 24 h at room temperature, dehydrated in ethanol, cleared in xylene, and embedded in paraffin block. Sections were cut in 6 m thickness and subjected to Massons's tri-chrome staining for the presence of collagen, which is a typical indicator of fibrosis.

In situ hybridization

Non-radioactive RNA probes were generated by in vitro transcription from the plasmid containing the 5' portion of the CTGF cDNA using SP6 or T7 RNA polymerase (Promega, Madison, WI). The tissue

Table 1. Effects of MCT-treatment on hemodynamic and physiologic profiles of rat.

2 week 5 week

Saline MCT Saline MCT

Body weight (BW), g 267 5 254 8 331 11 174 30*

RV systolic pressure, mmHg 24 1 42 2* 32 2 78 26*

RV/BW, g/kg body wt 0.67 0.03 0.74 0.04 0.56 0.02 1.41 0.16*

LV/BW, g/kg body wt 2.48 0.04 2.33 0.05 2.21 0.04 2.53 0.18

RV/LV, g/g 0.27 0.01 0.32 0.02 0.26 0.01 0.57 0.11*

Abbreviations: RV, right ventricular weight; LV, left ventricular weight. Data are mean SEM. *: P 0.05 vs. saline group.

CTGF upregulation in pulmonary hypertension 29

sections were dewaxed in xylene and fixed for 10 min in PBS containing 4% paraformaldehyde. The sections were then treated with proteinase K (20 g/ml) for 15 min to permeabilize the tissue and postfixed for 10 min with 4% paraformaldehyde solution. The sections were acetylated for 10 min with 0.1 M triethanol amine and acetic anhydride, and pre-hybridized for 1 h at 68oC. Hybridization mixture (5Denhardt's solution containing 50% formamide,

2SSC, 5% Dextran sulfate, 150 g tRNA and 150 g/ ml denatured salmon sperm DNA) was then poured onto the section and incubated overnight at 68oC in a moisture chamber with a digoxigenin-labeled CTGF- specific riboprobe. The next day, sections were washed at 50oC with 4SSC solution for 30 min and then with 2SSC solution containing 50% formamide for 30 min. For immunohistochemical detection of digoxigenin, the

Figure 1. Photomicrograph of the lung sections stained with Masson's trich-rome. Two weeks after MCT injection, muscular hypertrophy in the media of pulmonary artery and arteriole is evident. However, in the lung parenchyma, pulmonary vein, and bronchiole, no remarkable differences are seen between the saline- and MCT-injected groups. Blue staining indicates collagen deposition. Magnification: 400.

30 Exp. Mol. Med. Vol. 37(1), 27-35, 2005

sections were initially incubated in buffer A (100 mM Tris-HCl, pH 7.5 containing 150 mM NaCl) and then blocked at room temperature for 1 h in normal sheep serum. The sections were then incubated at 4oC

overnight with alkaline phosphatase- conjugated sheep polyclonal anti-digoxigenin antibody (Roche, Mannheim, Germany) diluted to 1:1000 in buffer A containing 1% normal sheep serum. The following day, the sections were subjected to three cycles of 10 min washing with buffer A and buffer B (100 mM Tris-HCl, pH 9.5 containing 100 mM NaCl and 50 mM

MgCl2). Alkaline phosphatase detection was carried out in the presence of nitroblue tetrazolium, BCIP, and levamisol in buffer B at room temperature in the dark. The sections were then dehydrated and covered with glass coverslips.

RT-PCR

Total RNA was extracted from the lung using Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA). After pre-heating 1 g of RNA at 65oC for 10 min, first-strand

Figure 2. Photomicrograph of the lung sections stained with Masson's tri-chrome 5 weeks after MCT or saline injection. In addition to medial hyper-trophy of pulmonary artery and arteriole, there are thickening of the alveolar septa with fibrosis, and alveolar macrophage infiltration in the lung treated with MCT. Bronchiole and vein do not appear to be affected by MCT treatment. Magnification: 400.

CTGF upregulation in pulmonary hypertension 31

cDNA synthesis was carried out at 42oC for 1 h using a random 6-mer primer and AMV reverse transcriptase (Promega) followed by denaturation at 95oC

for 10 min. PCR amplification was performed using the CTGF-specific primers (5'-CCTGGTCCAGACCACAGAGT-3' and 5'-CCAAGCTTCATGCCATGTCT-3') and transforming growth factor (TGF)-1 primers (5'-GAACCAAGGAGACGGAATAC-3' and 5'-GACAGAAGTTGGCATGGTAG-3').

Statistics

The data are expressed as means SEM. Differences between experimental groups were evaluated for statistical significance using Student's t-test and one-way ANOVA (Newman-Keuls multiple comparison test). A P value less than 0.05 was considered statistically significant.

Results

Rat model of pulmonary hypertension

A single subcutaneous injection of MCT resulted in a significant increase in RV systolic pressure after 5 weeks (78 26 mm Hg) compared with the saline-injected control rats (32 2 mm Hg) (Table 1). This was paralleled by right ventricular hypertrophy with the RV/LV weight ratio increase at 5 week (MCT groups: 0.57 0.11; saline groups: 0.26 0.01, respectively). Moreover, the MCT-injected rats (174 30 g) had significantly lower body weight (BW) than the saline-injected controls (331 11 g) after 5 weeks. The same tendency was noted at 2 week. The RV systolic pressure of the MCT group was significantly increased (42 2 mm Hg) compared with that of the control (24 1 mm Hg). However, differences in RV/LV ratio and BW between the two groups were not statistically significant at 2 week. These hemodynamic parameters indicated that a rat model of pulmonary hypertension was successfully made.

Hypertrophy of muscular pulmonary artery and arteriole and interstitial fibrosis of the lung following MCT treatment

Histological examination of the lung at 2 weeks after MCT treatment revealed profound muscular hyper-trophy in the media of muscular pulmonary arteries and arterioles (Figure 1). In contrast, lung parenchyma, vein, and bronchiole did not appear to be affected. At 5 week, the MCT-treated lung displayed interstitial thickening and prominent medial hyper-trophy of muscular pulmonary artery and arterioles (Figure 2). Fibrosis was demonstrated in thickened alveolar septa by Masson's trichrome stain. Intra-alveolar hemorrhage and macrophages were also found. These observations are in accordance with the previous report that showed the collagen deposition in the MCT-treated animals (Mansoor et al., 1995).

CTGF upregulation in the lung of MCT-treated rat

CTGF has been implicated in the fibrosis of various tissues as described earlier. A possible effect of MCT treatment on the expression of CTGF mRNA was examined by RT-PCR analysis of the lung tissue. The result showed that higher level of CTGF mRNA was detected in the MCT-treated group at 2 and 5 week (Figure 3). In contrast, there was a little expression of CTGF mRNA in the saline-injected control group with negligible time-course changes. Interestingly, the level of TGF-1 was significantly increased at 4 week, which appeared to precede the upregulation of CTGF at 5 week (Figure 3). An earlier report of CTGF expression induced by TGF-1 (Hishikawa et al., 1999a; Chen et al., 2000) suggests that upregulation of TGF-1 by MCT treatment may be responsible for the induction of CTGF expression.

Next, by in situ hybridization analysis of tissue sections, CTGF expressing cells in the lung were identified. As shown in Figure 4, CTGF mRNA was basally expressed in the endothelial cells of pulmonary artery and arteriole. Basal expression of CTGF was also observed in bronchial epithelium and alveolar lining cells. However, following MCT treatment, abundant expression of CTGF was observed in hypertrophic VSMCs of pulmonary artery and arteriole and in the clustered pneumocytes and macrophages that had infiltrated into alveolar space. Positive staining was also observed in alveolar septum. Interestingly, CTGF mRNA was not detected in VSMCs of vein or bronchiole.

Figure 3. RT-PCR analysis of the lung tissue after saline or MCT treatment. Total RNA (1 g) was isolated from each sample, reverse-transcribed, and then aliquot of the cDNA amplified by PCR using CTGF- or TGF-1-specific primers. A second aliquot of the RT reaction was amplified using primers specific for -actin as an internal control. One of the representative results is shown and the number indicates fold-increase over the saline control (mean SEM, n = 6). *P 0.05 vs. 4 wk, P 0.05 vs. 2 wk

32 Exp. Mol. Med. Vol. 37(1), 27-35, 2005

Figure 4. In situ hybridization of the lung tissue with CTGF mRNA-specific riboprobe. Five weeks after MCT treatment, abundant expression of CTGF mRNA is seen in smooth muscle cells of the remodeling pulmonary artery and arteriole (marked by black arrowhead and black arrow, respectively) and in infiltrated macrophages (marked by white arrowhead) within alveolar space (AS). Positive staining at alveolar septum is also marked by wedge. Endothelial dysfunction in the MCT-treated pulmonary artery is seen in support of previous study (Jones et al., 1996). Basal expression of CTGF is noted in the endothelial cells (EC) of pulmonary artery and arteriole, bronchial epithelium (BE), and alveolar lining cells as shown in saline-injected controls. CTGF mRNA is not detected in vascular smooth muscle cells of the vein or bronchiole. Magnification: 400.

Discussion

In the present study, we demonstrated that CTGF was highly upregulated in the lung of rat treated with MCT. Although it is known that CTGF is one of the key molecules in the progression of tissue fibrosis, this study is the first to suggest direct correlation between CTGF and MCT-induced PH in vivo. In situ hybridization study of the lung section showed that CTGF expression was mainly localized in the hyper-trophic smooth muscle cells of pulmonary arteries and arterioles as well as in alveolar macrophages and

pneumocytes. In support of this result, obliteration of artery, arteriole, and significant fibrosis of alveolar septa were observed in MCT-treated rat lung. Recent report that adenovirus-mediated overexpression of CTGF caused increased deposition of collagen in vivo (Bonniaud et al., 2003) is consistent with our data and suggests that CTGF might play an important role in the development of PH.

Although a number of different mechanisms would be involved in CTGF induction, TGF-1 is one of the potential mediators of CTGF upregulation in PH. Consistent with our finding (Figure 3), TGF-1 ex-

CTGF upregulation in pulmonary hypertension 33

pression was shown to be elevated in the lung during development of PH (Arcot et al., 1993; Tanaka et al., 1996). In addition, TGF-1 can induce the synthesis of CTGF both transcriptionally and translationally (Igarashi et al., 1993; Grotendorst et al., 1996), thus promoting the production and accumulation of ECM components in certain pathological states including the fibrosis of the liver, kidney, and lung (Border and Noble, 1994). Another possible mediator of CTGF upregulation is endothelin (ET)-1, a 21-amino-acid peptide with potent vasoconstrictor activity and platelet-aggregating properties (Yanagisawa et al., 1988). ET-1 was shown to induce expression of matrix and matrix-associated genes including CTGF in various cell types (Chaqour et al., 2002; Shi-Wen et al., 2004). The importance of ET-1 is also suggested by the previous reports which showed that ET-1 is elevated in patients with PH (Giaid et al., 1993) as well as in animal models of PH (Mansoor et al., 1995; Frasch et al., 1999). Therefore, it is very likely that TGF-1, together with ET-1, plays an active role in the upregulation of CTGF in MCT-induced PH. MCT-induced PH is also associated with cardiac remodeling including RV hypertrophy and interstitial fibrosis as evidenced by our data and other studies (Ghodsi and Will, 1981; Honda et al., 1992). Previously, it was reported that expression of renninangiotensin system, TGF-1, and ET-1 was enhanced in RV hypertrophy of MCT-induced PH rats (Park et al., 2001). In a more recent publication, Ahmed et al. (2004) demonstrated that CTGF acts as a myocardial effector of Angiotensin II-induced myocardial remodeling in ischemic heart failure via AT1 receptors in cardiac fibroblasts. Since Angiotensin-II is also known to stimulate synthesis of TGF-1 and ET-1 (Ito et al., 1993; Weber, 1997), these evidences altogether suggest that CTGF might be a more downstream target contributing to cardiac remodeling after PH. However, further investigation is needed to elaborate on this point. There are two contradictory views regarding the function of CTGF in regulating the growth of VSMC. One report suggested that CTGF acts as an inhibitor of human aortic smooth muscle cell growth at least in part by inducing apoptosis (Hishikawa et al., 1999a; b). The other report, on the contrary, suggested that CTGF is a growth factor for VSMC and that it may play a similar role in promoting proliferation and migration of VSMC and production of extracellular matrix such as collagen type I and fibronectin (Fan et al., 2000). The results of present study support the latter hypothesis because CTGF was specifically expressed in VSMC of remodeling arteries and arterioles with medial hypertrophy. However, since CTGF may not be the sole factor contributing to hypertrophy of VSMC and lung fibrosis, more studies are required to resolve this controversy.

PH has been historically chronic and incurable with a poor survival rate. Recently, however, many approaches aimed at restoring the balance between various vasoactive mediators have shown beneficial effects (Budhiraja et al., 2004). Indeed, in patients

with PH, an altered production of various endothelial vasoactive mediators, such as nitric oxide, prostacycline, ET-1, serotonin, and thromboxane, has been increasingly recognized. As an extension of these approaches, a strategy that is based on antagonizing the role of CTGF would represent novel therapeutic modality for PH.

In conclusion, we present an evidence that CTGF expression is upregulated in the lung of MCT-induced pulmonary hypertensive rat and that it is localized in the remodeling pulmonary arteries and arterioles and in infiltrated inflammatory cells. Our results suggest that CTGF plays an important role in the development of lung pathogenesis caused by PH.

Acknowledgm ent

This work was supported by the National Research Laboratory Grant from the Korea Institute of Science and Technology Evaluation and Planning (M1-0412-00-0048) and the Korean Ministry of Health and Welfare Grant (01-PJ1-PG1-01CH06-0003) to DK Kim.

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