Peptides 54 (2014) 101–107
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Adrenomedullin alleviates pulmonary artery collagen accumulation in rats with pulmonary hypertension induced by high blood flow Lulu Pang, Jianguang Qi ∗ , Yang Gao, Hongfang Jin, Junbao Du Department of Pediatrics, Peking University First Hospital, Beijing 100034, China
a r t i c l e
i n f o
Article history: Received 5 August 2013 Received in revised form 8 January 2014 Accepted 8 January 2014 Available online 27 January 2014 Keywords: Pulmonary hypertension High pulmonary blood flow Collagen Adrenomedullin
a b s t r a c t Collagen accumulation is one of the important pathologic changes in the development of pulmonary hypertension. Previous research showed that adrenomedullin (ADM) mitigates the development of pulmonary hypertension. The present study explored the role of ADM in the development of pulmonary artery collagen accumulation induced by high pulmonary blood flow, by investigating the effect of ADM [1.5 g/(kg h)] subcutaneously administered by mini-osmotic pump on pulmonary hemodynamics, pulmonary vascular structure and pulmonary artery collagen accumulation and synthesis in rats with high pulmonary blood flow induced by aortocaval shunting. The results showed that ADM significantly decreased mean pulmonary artery pressure (mPAP) and the ratio of right ventricular mass to left ventricular plus septal mass [RV/(LV + SP)], attenuated the muscularization of small pulmonary vessels and relative medial thickness (RMT) of pulmonary arteries in rats with high pulmonary blood flow. Meanwhile, ADM ameliorated pulmonary artery collagen deposition represented by a decrease in lung tissue hydroxyproline, collagens I and III content and pulmonary artery collagens I and III expression, reduced collagen synthesis represented by a decrease in lung tissue procollagens I and III mRNA expression. The results suggest that ADM plays a protective role in the development of pulmonary hypertension induced by high blood flow, by inhibiting pulmonary procollagen synthesis and alleviating pulmonary artery collagen accumulation. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Pulmonary hypertension induced by high pulmonary blood flow is one of the common complications of congenital heart disease (CHD) with systemic to pulmonary shunt [23]. Pulmonary vascular structural remodeling is the major pathological basis of pulmonary hypertension induced by high blood flow, characterized by medial hypertrophy, muscularization of small peripheral arteries, and increased deposition of extracellular matrix components. The status of the pulmonary vascular structure is often the important determinant of the clinical course, feasibility of surgical treatment, and long-term survival after cardiac operations [1]. Collagen accumulation plays an important role in the development
Abbreviations: ADM, adrenomedullin; TGF-1, transforming growth factor-1; mPAP, mean pulmonary artery pressure; RV/(LV + S), the ratio of right ventricle mass to left ventricle with septum mass; RMT, relative medial thickness; MA, muscularized artery; PMA, partially muscularized artery; NMV, non-muscularized vessel. ∗ Corresponding author. Tel.: +86 10 83573238; fax: +86 10 66530532. E-mail addresses: [email protected] (L. Pang), [email protected] (J. Qi). 0196-9781/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2014.01.013
of pulmonary hypertension and pulmonary vascular structural remodeling [18]. Although numerous studies were carried out and remarkable progress was made in understanding the pathophysiologic and molecular mechanisms responsible for pulmonary hypertension, the pulmonary vascular collagen remodeling induced by high pulmonary blood flow have not been fully elucidated. Adrenomedullin (ADM), originally isolated from human pheochromocytoma by Kitamura et al. [12], is a novel cardiovascular-active peptide associated with vasodilation, reducing blood pressure and inhibiting vascular smooth muscle cell migration and proliferation. ADM is distributed in a wide range of tissues and organs, including aorta, ventricles, lungs, and kidneys. It has been reported that there are abundant binding sites for ADM in the lungs [10]. Repeated inhalation of ADM, chronic infusion of ADM and intratrachieal gene transfer of ADM using polyplex nanomicelles attenuated monocrotaline induced pulmonary hypertension in rats [9,16,30]. Our previous study showed that chronic administration of ADM attenuated hypoxic pulmonary vascular structural remodeling and pulmonary hypertension [19]. In fact, intratracheal delivery of aerosolized ADM has beneficial effects on hemodynamics and exercise capacity in patients with idiopathic
102
L. Pang et al. / Peptides 54 (2014) 101–107
pulmonary hypertension [15]. These findings suggest that ADM plays an important role in the regulation of pulmonary hypertension and pulmonary vascular structural remodeling. Furthermore, Kach et al. observed that ADM significantly attenuated the expression of SMA, collagen-1, and fibronectin in pre-differentiated pulmonary myofibroblasts [11]. ADM was also reported to alleviate the collagen deposition of coronary arteries in Ang II-induced hypertensive rats [25], which suggested that ADM could intervene the excessive collagen deposition. So, we speculated that ADM might play an important role in the regulation of pulmonary hypertension induced by high pulmonary blood flow through alleviating pulmonary artery collagen accumulation. In the present study, we investigated the effects of ADM chronically administered by mini-osmotic pump on the pulmonary hemodynamics, pulmonary vascular structural changes and pulmonary vascular collagen metabolism in rats with aortocaval shunting, so as to explore the effects of ADM on the development of pulmonary hypertension and pulmonary vascular collagen remodeling induced by high pulmonary flow.
2. Materials and methods 2.1. Animal and agents Healthy male SD rats weighing 150–170 g were provided by China Academy of Military Medical Science (Clean grade, Certificate No. SCXK-2007-004). All animal experiments in this study were performed with the approval of Animal Care Committee of the Peking University First Hospital. Recombinant rat ADM was provided by Phoenix Pharmaceuticals, Inc. Rabbit–anti-rat antibody was provided by Beijing Hualisentai Bioscientific Co. Ltd. Goat serum and biotinylated anti-rabbit IgG were provided by ZSGB-BIO.
2.2. Animal model Twenty-four male SD rats were randomly divided into sham group (n = 8), shunt group (n = 8) and shunt with ADM group (n = 8). Abdominal aorta and inferior vena cava shunting was produced in rats of shunt group and shunt with ADM group according to the method that we previously described [20]. Rats were anesthetized using 10% chloral hydrate (3–4 mL/kg) intraperitoneally, and the abdominal aorta and inferior vena cava were exposed by opening the abdominal cavity via a midline incision. The aorta was clamped by using a bulldog vascular clamp caudal to the left renal artery. Then it was punctured at the union of the segment two thirds caudal to the renal artery and one third cephalic to the aortic bifurcation with an 18-gauge disposable needle. Then the needle was slowly withdrawn and a 9-0 silk thread was applied to stitch the puncture of the abdominal aorta. The clamp was removed and the patency of the shunt was verified visually by swelling of the vena cava and the mixing of arterial and venous blood. In the sham group, rats underwent the same experimental protocol as mentioned above except for the shunting procedure. All rats were housed under the same standard conditions and allowed free access to rat chow and water. After 8 weeks [13], ADM dissolved in 0.9% saline was administered into rats of shunt with ADM group subcutaneously implanted with mini-osmotic pump (model 2002, ALZA, Palo Alto, USA). The pumps, positioned in a pocket constructed in the subcutaneous tissue just below the back scapular region, were designed to release 1.5 g/(kg h) of the peptide for 14 days. For the sham group and shunt group, 0.9% saline was infused from the mini-osmotic pumps in a similar manner.
2.3. Measurement of hemodynamic parameters At the end of the 10-week experiment after the shunting operation (2-week after ADM administration), the rats were anesthetized with 10% chloral hydrate (3–4 ml/kg). To calculate the magnitude of the left-to-right shunt by oximetric data, blood specimens were drawn from the main pulmonary artery, aorta, and superior vena cava. Blood oxygen saturation was immediately determined using a gas analyzer (GASTAT-3, TMC, Tokyo, Japan). Pulmonary-to-systemic flow ratio (QP /QS ) was calculated according to the formula as follows [20]: Aorta saturation (%) − Superior vena cava saturation (%) Pulmonary venous saturation (%) − Pulmonary arterial saturation (%)
Mean pulmonary pressure (mPAP) of each rat was evaluated by using a right cardiac catheterization procedure. For the catheterization procedure, a polyethylene catheter was inserted into the external jugular vein and advanced under fluoroscopic guidance into right ventricle and pulmonary artery. The other end of the catheter was connected to a transducer (YZ-05-1, Beijing, China). The pressure tracings were simultaneously recorded on a physiologic recorder (BL-410, Chengdu Taimeng Software Co. Ltd, Chengdu, China) [4]. The thoracic cavity of each rat was opened. The heart was removed and prepared with the tissues including atrium, lipid, vessels removed. The right ventricle and the left ventricle with septum were isolated and individually weighted to calculate the ratio of right ventricle weight to left ventricle with septum weight [RV/(LV + SP)]. 2.4. Morphometric analysis of pulmonary arteries The same lung lobe of each rat was removed, fixed with formaldehyde, embedded in paraffin, and subsequently cut into sections. Sections for morphometric analysis were stained with aldehyde fuchsin dyeing methods. Small pulmonary vessels (15–50 m in external diameter) were divided into three types according to the degree of muscularization: muscularized artery (MA), with two distinct elastic lamina; partially muscularized artery (PMA), with one continuous external elastic lamina; and non-muscularized vessel (NMV), with only one single elastic lamina. The percentage of each type of small pulmonary vessels was determined. The relative medial thickness (RMT) of median (50–150 m in external diameter) and small (15–50 m in external diameter) muscularized pulmonary arteries with clearly defined internal elastic lamina and regular shape was determined by Image Processing And Analyzing System (CMIAS, Beijing, China) according to the method of Barth et al. [2]. 2.5. Hydroxyproline assay in the lung tissue Lung tissue was homogenized in a 10-fold volume (w/v) of buffer solution. The content of hydroxyproline in the lung tissue was determined by using a commercial hydroxyprolin eassay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The assays were performed according to the instructions. 2.6. Analysis of collagen I and collagen III contents in the lung tissue in addition to immunohistochemistry for collagen I and collagen III in pulmonary artery The contents of collagens I and III in the lung tissue were determined by using a commercial ELISA kit (Wuhan ElAnb Science Co. Ltd, Wuhan, China).
L. Pang et al. / Peptides 54 (2014) 101–107
103
Table 1 Primers and fluorescent probes. Gene Procollagen I
Procollagen III
-Actin
Sequence Forward Probe Reverse Forward Probe Reverse Forward Probe Reverse
5 -CTTGTTGCTGAGGGCAACAG-3 5 -FAM-ATTCACCTACACTGTCCTTGTCGATGGCTG-TMERA-3 5 -GCAGGCGAGATGGCTTATTC-3 5 -GAAAAAACCCTGCTCGGAATT-3 5 -FAM-AGAGACCTGAAATTCTGCCACCCTGAACTC-TMERA-3 5 -ATCCATCTTGCAGCCTTGGT-3 5 -ACCCGCGAGTACAACCTTCTT-3 5 -FAM-CCTCCGTCGCCGGTCCACAC-TMERA-3 5 -TATCGTCATCCATGGCGAACT-3
FAM, 5-carboxyfluorescern; TAMRA, 5-carboxytetramethylrhodamine.
The paraffin sections of lung tissue was dewaxed and hydrated, and then processed by 3% H2 O2 for 10 min at room temperature, followed by antigen repairing for 10 min (microwave heating method). The slides were washed twice with phosphate-buffered saline (PBS), then blocked with goat serum at 37 ◦ C for 20 min, and incubated overnight at 4 ◦ C with collagen I and collagen III antibodies (diluted at 1:300, 1:200, respectively). Then the slides were rinsed in PBS for three times, 3 min each. The biotinylated anti-rabbit IgG at 37 ◦ C was incubated for 60 min. After the slides were rinsed in PBS for three times, slides were stained with DAB to develop color. The yellow–brown granules under microscope were defined as the positive signals. For negative controls, sections were processed as above except that the primary incubation was performed with PBS instead of primary antibodies. For analysis of collagen I and collagen III expression, the mean optical destiny value per unit area of vessels was calculated by LeicaQ550cwImage Capturing and Analyzing System (Leica, German). Eight to twelve median and small pulmonary muscularized arteries of each rat were detected and the average values were calculated.
2.9. Statistical analysis All data were shown as mean ± S.D. ANOVA was performed to compare the differences among the three groups using SPSS 16.0 statistic analysis software. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. ADM improved the hemodynamics index QP /QS were significantly increased in rats of shunt and shunt + ADM group as compared with controls (p < 0.01, respectively). However, there was no significantly difference between shunt and shunt + ADM group (p > 0.05) (Table 2). The mPAP and RV/(LV + S) were significantly increased in rats of shunt group as compared with controls (p < 0.01, respectively). ADM, however, significantly decreased mPAP and RV/(LV + S) (p < 0.01, respectively) (Table 2). 3.2. ADM alleviated the pulmonary artery microstructure changes
2.7. Analysis of PINP and PIIINP in serum in addition to RT-PCR for procollagens I and III in lung tissue Blood samples were collected from external jugular arteries and centrifuged at 4 ◦ C to separate plasma. The content of N-terminal propeptides of types I and III (PINP and PIIINP), marker of collagen synthesis, was detected by using a commercial ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Total RNA in the lung was extracted by using trizol reagent (Invitrogen, CA, USA). Then the total RNA was reversed into cDNA by using moloney murine leukemia virus reverse transcription (MMLV-RT) and Oligo (dT) 15 or random primer. The rat collagens I and III and -actin mRNA levels were determined by using real-time PCR (Prism 7300 Sequence Detector, Applied Biosystems, Foster City, CA). PCR reaction volume was 25 L with contained 1 L of cDNA, 1 L of 2.5 mmol/L dNTP, 1 L of 5 pmol/L TaqMan probe, 1 L of 7.5 pmol/L sense and anti-sense primers, 2.5 L of 10 × PCR buffer containing 20 mmol/L MgCl2 , and 0.25 L TaqDNA polymerase. The mRNA levels were compared after they had been normalized relative to those of -actin. The amplification products were electrophoresed on 1.5% agarose gel and visualized by ethidium bromide staining. The primers and fluorescent probes used are shown in Table 1.
2.8. ELISA analysis of TGF-ˇ1 in the lung tissue The content of TGF-1 in the lung tissue was determined by using a commercial ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The assays were performed according to the instructions.
The percentage of MA of small pulmonary vessels was significantly increased in rats of shunt group as compared with controls (p < 0.01), but the percentage of NMV was significantly decreased (p < 0.01). ADM, however, significantly alleviated the muscularization of small pulmonary vessels (p < 0.01, respectively) (Table 3). RMT of median and small muscularized pulmonary arteries was both markedly increased in rats of shunt group compared with that of controls (p < 0.01, respectively). ADM, however, significantly reduced RMT of median pulmonary arteries (p < 0.05) and small pulmonary arteries (p < 0.01) (Table 3). 3.3. ADM alleviated the collagen accumulation in lung tissues The content of hydroxyproline in the lung homogenate was increased in rats of shunt group as compared with controls (p < 0.05). ADM, however, significantly decreased the content of hydroxyproline in the lung homogenate (p < 0.01) (Table 4).
Table 2 Hemodynamics parameters in rats. Groups
mPAP (mmHg)
RV/(LV + SP)
QP /QS
Sham Shunt Shunt + ADM
14.6 ± 2.4 25.5 ± 4.0** 16.4 ± 2.5##
0.22 ± 0.03 0.33 ± 0.06** 0.26 ± 0.03##
1.05 ± 0.12 2.11 ± 0.77** 2.27 + 0.60**
mPAP, mean pulmonary artery pressure; RV/(LV + S), the ratio of right ventricle mass to left ventricle with septum mass; QP /QS , pulmonary-to-systemic flow ratio. Values are expressed as mean ± S.D. ** p < 0.01 vs. sham group. ## p < 0.01 vs. shunt group.
104
L. Pang et al. / Peptides 54 (2014) 101–107
Fig. 1. Immunohistochemistry for collagen I expression in the pulmonary arteries of rats of sham (A), shunt (B) and shunt with ADM (C) groups. Collagen I expression by pulmonary arteries in rats of shunt group (B) was significantly strengthened as compared with that of sham group (A). ADM (C) decreased collagen I expression by pulmonary arteries in shunt rats. Magnification is 400×.
Fig. 2. Immunohistochemistry for collagen III expression in the pulmonary arteries of rats of sham (A), shunt (B) and shunt with ADM (C) groups. Collagen III expression by pulmonary arteries in rats of shunt group (B) was significantly strengthened as compared with that of sham group (A). ADM (C) decreased collagen III expression by pulmonary arteries in shunt rats. Magnification is 400×.
L. Pang et al. / Peptides 54 (2014) 101–107
105
Table 3 Pulmonary artery microstructure parameters in rats. Groups
Sham Shunt Shunt + ADM
Small pulmonary vessels (%)
Relative medial thickness (%)
MA
PMA
NMV
Median PA
Small PA
14.0 ± 1.7 23.1 ± 1.6** 17.6 ± 2.4** , ##
15.5 ± 2.8 26.1 ± 1.1** 21.3 ± 4.4** , ##
70.5 ± 4.1 50.9 ± 2.5** 61.1 ± 6.5** , ##
5.3 ± 1.1 8.5 ± 1.5** 7.3 ± 0.8** , #
7.6 ± 1.3 12.4 ± 2.1** 10.2 ± 2.1** , ##
MA, muscularized artery; PMA, partially muscularized artery; NMV, non-muscularized vessel; PA, pulmonary artery. Values are expressed as mean ± S.D. ** p < 0.01 vs. sham group. # p < 0.05 vs. shunt group. ## p < 0.01 vs. shunt group.
Table 4 Lung tissue concentration of hydroxyproline, collagen I and collagen III in rats. Groups Sham Shunt Shunt + ADM
Hydroxyproline (g/mg)
Collagen I (ng/g)
Collagen III (ng/g)
0.89 ± 0.07 1.09 ± 0.20* 0.84 ± 0.18##
15.66 ± 1.50 23.91 ± 4.03** 20.35 ± 2.60** , #
1.43 ± 0.31 2.28 ± 0.35** 1.95 ± 0.24** , #
Values are expressed as mean ± S.D. * p < 0.05 vs. sham group. ** p < 0.01 vs. sham group. # p < 0.05 vs. shunt group. ## p < 0.01 vs. shunt group.
Table 6 Plasma concentrations of PINP, PIIINP and lung tissue of TGF-1 in rats. Groups
PINP (ng/ml)
PIIINP (pg/ml)
TGF-1 (pg/mg)
Sham Shunt Shunt + ADM
14.0 ± 2.3 23.9 ± 6.0** 18.7 ± 5.3#
599 ± 179 923 ± 125** 738 ± 127#
67.8 ± 18.6 117.3 ± 32.7** 85.0 ± 25.1##
PINP, N-terminal propeptides of type I; PIIINP, N-terminal propeptides of type III. Values are expressed as mean ± S.D. ** p < 0.01 vs. sham group. # p < 0.05 vs. shunt group. ## p < 0.01 vs. shunt group.
3.5. ADM decreased the TGF-ˇ1 content in lung tissue The contents of collagen I and collagen III in the lung homogenate were significantly increased in rats of shunt group as compared with those of controls (p < 0.01, respectively). ADM, however, significantly decreased the contents of collagen I and collagen III in the lung homogenate (p < 0.05, respectively) (Table 4). Immunohistochemistry on lung sections from control animals showed expression of collegan I and collegan III protein in media and adventitia of pulmonary arteries and bronchia. The expressions of collagen I and collagen III in pulmonary arteries were significantly increased in rats of shunt group as compared with controls (p < 0.01, respectively). ADM, however, significantly decreased the expressions of collagens I and III in pulmonary arteries (p < 0.05, respectively) (Table 5, Figs. 1 and 2). 3.4. ADM alleviated the collagen synthesis in the lung tissue The concentrations of PINP and PIIINP in serum were significantly increased in rats of shunt group as compared with controls (p < 0.01, respectively). ADM, however, significantly decreased the concentrations of PINP and PIIINP in serum (p < 0.05, respectively) (Table 6). RT-PCR revealed significantly increased mRNA levels of procollagen I and procollagen III in the lung tissues of shunt rats compared with those of controls (p < 0.05, respectively). However, the mRNA levels of procollagen I and procollagen III in the lung tissues of shunt with ADM rats were significantly decreased as compared with those of shunt group (p < 0.05, respectively) (Fig. 3).
The content of TGF-1 in the lung homogenate was significantly increased in rats of shunt group compared with controls (p < 0.01). ADM, however, significantly decreased the content of TGF-1 (p < 0.01) (Table 6). 4. Discussion In the present study, we demonstrated that ADM administrated subcutaneously by mini-osmotic pump attenuated the development of pulmonary hypertension and pulmonary vascular structural remodeling induced by high blood flow, in association with the inhibition of collagen synthesis and accumulation in lung tissues of rats. Pulmonary hypertension is a progressive disease of the pulmonary vasculature that is associated with severe functional impairment and a poor prognosis. Pulmonary vascular structural remodeling, characterized by proliferation of endothelial cells and vascular smooth muscle cells, alterations in the extracellular matrix, thrombosis and fibrosis, is the important pathological change of pulmonary hypertension. In children, the most common etiology of pulmonary hypertension is high pulmonary blood flow secondary to congenital heart disease with systemic-topulmonary shunt [23]. Many vasoactive substances might be involved in the development of pulmonary hypertension induced by high pulmonary blood flow [7]. A variety of therapeutic agents, such as prostacyclin, endothelin-1, receptor antagonists,
Table 5 Immunohistochemical analysis of collagens I and III expression by pulmonary arteries in rats. Groups
Sham Shunt Shunt + ADM Values are expressed as mean ± S.D. * p < 0.05 vs. sham group. ** p < 0.01 vs. sham group. # p < 0.05 vs. shunt group.
Small pulmonary artery
Median pulmonary artery
Collagen I
Collagen III
Collagen I
Collagen III
0.357 ± 0.036 0.449 ± 0.049** 0.383 ± 0.056#
0.260 ± 0.018 0.312 ± 0.026** 0.285 ± 0.014* , #
0.378 ± 0.058 0.457 ± 0.039** 0.397 ± 0.050#
0.259 ± 0.011 0.310 ± 0.030** 0.280 ± 0.017* , #
106
L. Pang et al. / Peptides 54 (2014) 101–107
Fig. 3. Procollagen I (A) and procollagen III (B) mRNA expression in the lung tissue of rats. Procollagen I (A) and procollagen III (B) mRNA expression in the lung tissue of rats with shunt group was significantly increased as compared with that of sham group. ADM lowered the procollagen I (A) and procollagen III (B) mRNA expression in the lung tissue of shunting rats. * p < 0.05 vs. sham group. # p < 0.05 vs. shunt group.
and phosphodiesterase-type 5 inhibitors, are demonstrated leading to improvement of pulmonary hemodynamics, exercise capacity and survival in patients with pulmonary hypertension [14]. Despite therapeutic medication advances, for the majority of patients with pulmonary hypertension, disease progression is inevitable and long-term survival remains poor [27]. Thus, a novel therapeutic strategy is desirable for the treatment of pulmonary hypertension. ADM is a potent vasodilator peptide, belonging to calcitonin gene-related peptide superfamily [12]. Repeated inhalation of ADM, chronic infusion of ADM and intratrachieal gene transfer of ADM using polyplex nanomicelles attenuated monocrotalineinduced pulmonary hypertension in rats [9,16,30]. Our previous study showed that chronic administration of ADM attenuated hypoxic pulmonary vascular structural remodeling and pulmonary hypertension [19]. These findings suggested that ADM might play an important role in the regulation of pulmonary hypertension. In addition, Yoshibayash et al. found that plasma ADM level in pulmonary hypertension associated with congenital heart disease was significantly increased compared with those without pulmonary hypertension and normal control [29]. Zhao et al. found the mRNA and protein expressions of ADM in lung tissues of rats with pulmonary hypertension induced by high pulmonary blood flow were increased [6]. All above clues suggested that ADM might participate in the regulation of pulmonary hypertension induced by high blood flow. In the present study, we explored the effect of chronic ADM administration on pulmonary hypertension and pulmonary vascular remodeling induced by high pulmonary blood flow. The results showed that chronic ADM infusion administrated subcutaneously by mini-osmotic pump decreased mPAP and the ratio of RV/(LV + S), alleviated the muscularization of small pulmonary vessels, and decreased the RMT of pulmonary arteries. The results suggested that ADM ameliorated the development of pulmonary hypertension and pulmonary vascular remodeling induced by high pulmonary blood flow. The possible mechanisms by which ADM alleviates pulmonary hypertension and pulmonary vascular structural remodeling induced by high pulmonary blood flow have not been clarified. First of all, previous studies showed that ADM could dilate pulmonary artery through the nitric oxide-dependent mechanism [3,28]. ADM infusion could cause dose-related decreases in pulmonary vascular resistance under conditions of high pulmonary vascular tone, so as to reduce pulmonary artery pressure. Secondly, ADM could inhibit pulmonary artery smooth muscle cell DNA synthesis and
endothelin release [26], which suggested that ADM might function in the inhibition of pulmonary hypertension and pulmonary vascular remodeling through inhibiting pulmonary vascular smooth muscle cell proliferation. In addition, ADM could decrease AngIIinduced collagen deposition surrounding the coronary arteries, inhibiting myofibroblast differentiation and expressions of extracellular matrix-related genes in rats [25], which suggested ADM might alleviate pulmonary vascular structural remodeling as an antifibrotic factor. However, the effect of ADM on pulmonary vascular collagen accumulation has not been studied. Change of extracellular matrix is an important pathological alteration contributing a great deal to pulmonary vascular structural remodeling, and influences the structural and functional integrity of pulmonary vessels. Collagen I and collagen III are the most abundant components of the extracellular matrix of the vascular wall. Changes in the absolute or relative contents of collagen I and collagen III in pulmonary artery would result in changes in pulmonary vascular compliance and ultimately the development of pulmonary hypertension [18]. In the present study, the secretion of hydroxyproline, an inherent component of collagen, and the contents of collagen I and collagen III in the pulmonary arteries of rats with aortocaval shunting were increased as compared with controls. However, ADM decreased the contents of hydroxyproline, collagen I and collagen III in rats with high pulmonary blood flow, which suggested that ADM inhibited the anomalous deposition of collagen in the pulmonary arteries, so as to participating in the regulation of pulmonary hypertension and pulmonary vascular remodeling induced by high pulmonary blood flow. The imbalance between collagen synthesis and collagen degradation contributed to collagen accumulation [8]. It was reported that ADM inhibited the proliferation and differentiation of coronary adventitial fibroblasts, reduced the mRNA level of procollagen I, and alleviated the collagen deposition of coronary arteries in Ang IIinduced hypertensive rats [25], which suggested that ADM exerted a significantly inhibitory effect on the collagen synthesis in fibroblasts. It is unclear, however, whether ADM could alleviate collagen accumulation in pulmonary arteries by inhibiting collagen synthesis. Collagens I and III are secreted by interstitial fibroblasts as procollagens followed by splitting off of propeptides by endopeptidases and the release of their aminoterminal propeptides, PINP and PIIINP, and carboxyterminal propeptides, PICP and PIIICP, into the circulation [21]. So serum PINP and PIIINP are often used as the synthesis biomarkers of the two types of collagen. In our present study,
L. Pang et al. / Peptides 54 (2014) 101–107
mRNA levels of procollagen I and procollagen III in lung tissue, PINP and PIIINP in the serum were detected to be dramatically increased in rats of the shunt group. The mRNA levels of procollagen I and procollagen III in lung tissue, PINP and PIIINP in the serum were significantly decreased in rats of shunt with ADM group, which suggests that ADM inhibited the synthesis of collagen, so as to alleviate collagen accumulation in pulmonary arteries and pulmonary hypertension induced by high pulmonary blood flow. TGF-1 is a member of TGF- superfamily. TGF- is a major stimulus of extracellular matrix production in fibroproliferative diseases [5]. TGF-1 stimulation of fibrotic pathways occurs primarily through Smad 2/3 dependent pathways [22]. Knockdown of Smad2 and Smad3 inhibited proliferation and migration of adventitial fibroblasts and down-regulated the expression of and collagens I and III. Evidence showed that TGF-1/Smads pathway was activated in animals with MCT- and hypoxia-induced pulmonary hypertension and in patients with pulmonary arterial hypertension [24]. It is proved that ADM might serve as an antifibrotic factor to inhibit the synthesis of collagen through down-regulating the TGF-1 expression [17]. So, we hypothesized that TGF-1/Smads pathway activation might be involved in the regulation of ADM to collagen synthesis in the pulmonary hypertension and pulmonary vascular matrix remodeling. In this study, we detected the elevation of TGF-1 in the lung tissue of shunt rats, and the decrease of TGF-1 in the lung tissue after ADM administration. Therefore, we presumed that the effect of ADM on the regulation of collagen synthesis might involve decreasing TGF-1. However, further studies are needed to explain the mechanisms responsible for decrease of TGF-1 induced by ADM. As all we know, right cardiac catheter is the golden standard for the measurement of pulmonary artery pressure. Because the rats will be sacrificed after the measurements of the hemodynamics parameters, we can not measure hemodynamics parameters in the same rat twice, so the hemodynamics parameters data in rats at baseline and at the end of 8-week experiment were lack. This is the limitation of our study. But in the previous study of our research group, we observed that pulmonary hypertension and right ventricular hypertrophy were developed at the end of 8 weeks aortocaval shunting in rats [13], that is also our reason to administer ADM to rats at the end of 8 weeks aortocaval shunting, so as to explore the effects of ADM on pulmonary hypertension. In conclusion, this study suggests that ADM plays a protective role in the development of pulmonary hypertension and pulmonary vascular structural remodeling induced by high blood flow. ADM alleviates pulmonary artery collagen accumulation by inhibiting pulmonary procollagen synthesis. The effect of ADM on collagen metabolism might involve TGF-1, a key extracellular matrix regulator. Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant no. 30973226). References [1] Barst RJ, McGoon MD, Elliott CG, Foreman AJ, Miller DP, Ivy DD. Survival in childhood pulmonary arterial hypertension: insights from the registry to evaluate early and long-term pulmonary arterial hypertension disease management. Circulation 2012;125:113–22. [2] Barth PJ, Kimpel C, Roy S, Wagner C. An improved mathematical approach for the assessment of the medial thickness of pulmonary arteries. Pathol Res Pract 1993;189:567–76. [3] Bayram Z, Golbasi I, Ozdem SS. The role of nitric oxide and potassium channels in the effect of adrenomedullin in human internal thoracic arteries. Regul Pept 2010;161:92–6. [4] Bing W, Junbao D, Jianguang Q, Jian L, Chaoshu T. l-Arginine impacts pulmonary vascular structure in rats with an aortocaval shunt. J Surg Res 2002;108:20–31.
107
[5] Chen CP, Yang YC, Su TH, Chen CY, Aplin JD. Hypoxia and transforming growth factor-beta 1 act independently to increase extracellular matrix production by placental fibroblasts. J Clin Endocrinol Metab 2005;90:1083–90. [6] Cuifen Z, Lijuan W, Li G, Wei X, Zhiyu W, Fuhai L. Changes and distributions of peptides derived from proadrenomedullin in left-to-right shunt pulmonary hypertension of rats. Circ J 2008;72:476–81. [7] D’Alto M, Mahadevan VS. Pulmonary arterial hypertension associated with congenital heart disease. Eur Respir Rev 2012;21:328–37. [8] Estrada KD, Chesler NC. Collagen-related gene and protein expression changes in the lung in response to chronic hypoxia. Biomech Model Mechanobiol 2009;8:263–72. [9] Harada-Shiba M, Takamisawa I, Miyata K, Ishii T, Nishiyama N, Itaka K, et al. Intratracheal gene transfer of adrenomedullin using polyplex nanomicelles attenuates monocrotaline-induced pulmonary hypertension in rats. Mol Ther 2009;17:1180–6. [10] Ichiki Y, Kitamura K, Kangawa K, Kawamoto M, Matsuo H, Eto T. Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett 1994;338:6–10. [11] Kach J, Sandbo N, Sethakorn N, Williams J, Reed EB, La J, et al. Regulation of myofibroblast differentiation and bleomycin-induced pulmonary fibrosis by adrenomedullin. Am J Physiol Lung Cell Mol Physiol 2013;304: L757–64. [12] Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 1993;192:553–60. [13] Li XH, Du JB, Tang XY, Jin HF, Tang CS. Changes of pulmonary artery structural remodeling in pulmonary hypertension induced by high pulmonary flow in rats. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2005;27:446–51. [14] Malenfant S, Margaillan G, Loehr JE, Bonnet S, Provencher S. The emergence of new therapeutic targets in pulmonary arterial hypertension: from now to the near future. Expert Rev Respir Med 2013;7:43–55. [15] Nagaya N, Kyotani S, Uematsu M, Ueno K, Oya H, Nakanishi N, et al. Effects of adrenomedullin inhalation on hemodynamics and exercise capacity in patients with idiopathic pulmonary arterial hypertension. Circulation 2004;109: 351–6. [16] Nagaya N, Okumura H, Uematsu M, Shimizu W, Ono F, Shirai M, et al. Repeated inhalation of adrenomedullin ameliorates pulmonary hypertension and survival in monocrotaline rats. Am J Physiol Heart Circ Physiol 2003;285:H2125–31. [17] Niu P, Shindo T, Iwata H, Iimuro S, Takeda N, Zhang Y, et al. Protective effects of endogenous adrenomedullin on cardiac hypertrophy, fibrosis, and renal damage. Circulation 2004;109:1789–94. [18] Ooi CY, Wang Z, Tabima DM, Eickhoff JC, Chesler NC. The role of collagen in extralobar pulmonary artery stiffening in response to hypoxia-induced pulmonary hypertension. Am J Physiol Heart Circ Physiol 2010;299:H1823–31. [19] Qi JG, Ding YG, Tang CS, Du JB. Chronic administration of adrenomedullin attenuates hypoxic pulmonary vascular structural remodeling and inhibits proadrenomedullin N-terminal 20-peptide production in rats. Peptides 2007;28:910–9. [20] Qi J, Du J, Tang X, Li J, Wei B, Tang C. The upregulation of endothelial nitric oxide synthase and urotensin-II is associated with pulmonary hypertension and vascular diseases in rats produced by aortocaval shunting. Heart Vessels 2004;19:81–8. [21] Radovan J, Vaclav P, Petr W, Jan C, Michal A, Richard P, et al. Changes of collagen metabolism predict the left ventricular remodeling after myocardial infarction. Mol Cell Biochem 2006;293:71–8. [22] Ren M, Wang B, Zhang J, Liu P, Lv Y, Liu G, et al. Smad2 and Smad3 as mediators of the response of adventitial fibroblasts induced by transforming growth factor 1. Mol Med Rep 2011;4:561–7. [23] Rosenzweig EB, Barst RJ. Congenital heart disease and pulmonary hypertension: pharmacology and feasibility of late surgery. Prog Cardiovasc Dis 2012;55:128–33. [24] Thomas M, Docx C, Holmes AM, Beach S, Duggan N, England K, et al. Activin-like kinase 5 (ALK5) mediates abnormal proliferation of vascular smooth muscle cells from patients with familial pulmonary arterial hypertension and is involved in the progression of experimental pulmonary arterial hypertension induced by monocrotaline. Am J Pathol 2009;174:380–9. [25] Tsuruda T, Kato J, Hatakeyama K, Masuyama H, Cao YN, Imamura T, et al. Antifibrotic effect of adrenomedullin on coronary adventitia in angiotensin II-induced hypertensive rats. Cardiovasc Res 2005;65:921–9. [26] Upton PD, Wharton J, Coppock H, Davie N, Yang X, Yacoub MH, et al. Adrenomedullin expression and growth inhibitory effects in distinct pulmonary artery smooth muscle cell subpopulations. Am J Respir Cell Mol Biol 2001;24:170–8. [27] Vachiéry JL, Gaine S. Challenges in the diagnosis and treatment of pulmonary arterial hypertension. Eur Respir Rev 2012;21:313–20. [28] Yang BC, Lippton H, Gumusel B, Hyman A, Mehta JL. Adrenomedullin dilates rat pulmonary artery rings during hypoxia: role of nitric oxide and vasodilator prostaglandins. J Cardiovasc Pharmacol 1996;28:458–62. [29] Yoshibayashi M, Kamiya T, Kitamura K, Saito Y, Kangawa K, Nishikimi T, et al. Plasma levels of adrenomedullin in primary and secondary pulmonary hypertension in patients
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Adrenomedullin alleviates pulmonary artery collagen accumulation in rats with pulmonary hypertension induced by high blood flow Lulu Pang, Jianguang Qi ∗ , Yang Gao, Hongfang Jin, Junbao Du Department of Pediatrics, Peking University First Hospital, Beijing 100034, China
a r t i c l e
i n f o
Article history: Received 5 August 2013 Received in revised form 8 January 2014 Accepted 8 January 2014 Available online 27 January 2014 Keywords: Pulmonary hypertension High pulmonary blood flow Collagen Adrenomedullin
a b s t r a c t Collagen accumulation is one of the important pathologic changes in the development of pulmonary hypertension. Previous research showed that adrenomedullin (ADM) mitigates the development of pulmonary hypertension. The present study explored the role of ADM in the development of pulmonary artery collagen accumulation induced by high pulmonary blood flow, by investigating the effect of ADM [1.5 g/(kg h)] subcutaneously administered by mini-osmotic pump on pulmonary hemodynamics, pulmonary vascular structure and pulmonary artery collagen accumulation and synthesis in rats with high pulmonary blood flow induced by aortocaval shunting. The results showed that ADM significantly decreased mean pulmonary artery pressure (mPAP) and the ratio of right ventricular mass to left ventricular plus septal mass [RV/(LV + SP)], attenuated the muscularization of small pulmonary vessels and relative medial thickness (RMT) of pulmonary arteries in rats with high pulmonary blood flow. Meanwhile, ADM ameliorated pulmonary artery collagen deposition represented by a decrease in lung tissue hydroxyproline, collagens I and III content and pulmonary artery collagens I and III expression, reduced collagen synthesis represented by a decrease in lung tissue procollagens I and III mRNA expression. The results suggest that ADM plays a protective role in the development of pulmonary hypertension induced by high blood flow, by inhibiting pulmonary procollagen synthesis and alleviating pulmonary artery collagen accumulation. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Pulmonary hypertension induced by high pulmonary blood flow is one of the common complications of congenital heart disease (CHD) with systemic to pulmonary shunt [23]. Pulmonary vascular structural remodeling is the major pathological basis of pulmonary hypertension induced by high blood flow, characterized by medial hypertrophy, muscularization of small peripheral arteries, and increased deposition of extracellular matrix components. The status of the pulmonary vascular structure is often the important determinant of the clinical course, feasibility of surgical treatment, and long-term survival after cardiac operations [1]. Collagen accumulation plays an important role in the development
Abbreviations: ADM, adrenomedullin; TGF-1, transforming growth factor-1; mPAP, mean pulmonary artery pressure; RV/(LV + S), the ratio of right ventricle mass to left ventricle with septum mass; RMT, relative medial thickness; MA, muscularized artery; PMA, partially muscularized artery; NMV, non-muscularized vessel. ∗ Corresponding author. Tel.: +86 10 83573238; fax: +86 10 66530532. E-mail addresses: [email protected] (L. Pang), [email protected] (J. Qi). 0196-9781/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2014.01.013
of pulmonary hypertension and pulmonary vascular structural remodeling [18]. Although numerous studies were carried out and remarkable progress was made in understanding the pathophysiologic and molecular mechanisms responsible for pulmonary hypertension, the pulmonary vascular collagen remodeling induced by high pulmonary blood flow have not been fully elucidated. Adrenomedullin (ADM), originally isolated from human pheochromocytoma by Kitamura et al. [12], is a novel cardiovascular-active peptide associated with vasodilation, reducing blood pressure and inhibiting vascular smooth muscle cell migration and proliferation. ADM is distributed in a wide range of tissues and organs, including aorta, ventricles, lungs, and kidneys. It has been reported that there are abundant binding sites for ADM in the lungs [10]. Repeated inhalation of ADM, chronic infusion of ADM and intratrachieal gene transfer of ADM using polyplex nanomicelles attenuated monocrotaline induced pulmonary hypertension in rats [9,16,30]. Our previous study showed that chronic administration of ADM attenuated hypoxic pulmonary vascular structural remodeling and pulmonary hypertension [19]. In fact, intratracheal delivery of aerosolized ADM has beneficial effects on hemodynamics and exercise capacity in patients with idiopathic
102
L. Pang et al. / Peptides 54 (2014) 101–107
pulmonary hypertension [15]. These findings suggest that ADM plays an important role in the regulation of pulmonary hypertension and pulmonary vascular structural remodeling. Furthermore, Kach et al. observed that ADM significantly attenuated the expression of SMA, collagen-1, and fibronectin in pre-differentiated pulmonary myofibroblasts [11]. ADM was also reported to alleviate the collagen deposition of coronary arteries in Ang II-induced hypertensive rats [25], which suggested that ADM could intervene the excessive collagen deposition. So, we speculated that ADM might play an important role in the regulation of pulmonary hypertension induced by high pulmonary blood flow through alleviating pulmonary artery collagen accumulation. In the present study, we investigated the effects of ADM chronically administered by mini-osmotic pump on the pulmonary hemodynamics, pulmonary vascular structural changes and pulmonary vascular collagen metabolism in rats with aortocaval shunting, so as to explore the effects of ADM on the development of pulmonary hypertension and pulmonary vascular collagen remodeling induced by high pulmonary flow.
2. Materials and methods 2.1. Animal and agents Healthy male SD rats weighing 150–170 g were provided by China Academy of Military Medical Science (Clean grade, Certificate No. SCXK-2007-004). All animal experiments in this study were performed with the approval of Animal Care Committee of the Peking University First Hospital. Recombinant rat ADM was provided by Phoenix Pharmaceuticals, Inc. Rabbit–anti-rat antibody was provided by Beijing Hualisentai Bioscientific Co. Ltd. Goat serum and biotinylated anti-rabbit IgG were provided by ZSGB-BIO.
2.2. Animal model Twenty-four male SD rats were randomly divided into sham group (n = 8), shunt group (n = 8) and shunt with ADM group (n = 8). Abdominal aorta and inferior vena cava shunting was produced in rats of shunt group and shunt with ADM group according to the method that we previously described [20]. Rats were anesthetized using 10% chloral hydrate (3–4 mL/kg) intraperitoneally, and the abdominal aorta and inferior vena cava were exposed by opening the abdominal cavity via a midline incision. The aorta was clamped by using a bulldog vascular clamp caudal to the left renal artery. Then it was punctured at the union of the segment two thirds caudal to the renal artery and one third cephalic to the aortic bifurcation with an 18-gauge disposable needle. Then the needle was slowly withdrawn and a 9-0 silk thread was applied to stitch the puncture of the abdominal aorta. The clamp was removed and the patency of the shunt was verified visually by swelling of the vena cava and the mixing of arterial and venous blood. In the sham group, rats underwent the same experimental protocol as mentioned above except for the shunting procedure. All rats were housed under the same standard conditions and allowed free access to rat chow and water. After 8 weeks [13], ADM dissolved in 0.9% saline was administered into rats of shunt with ADM group subcutaneously implanted with mini-osmotic pump (model 2002, ALZA, Palo Alto, USA). The pumps, positioned in a pocket constructed in the subcutaneous tissue just below the back scapular region, were designed to release 1.5 g/(kg h) of the peptide for 14 days. For the sham group and shunt group, 0.9% saline was infused from the mini-osmotic pumps in a similar manner.
2.3. Measurement of hemodynamic parameters At the end of the 10-week experiment after the shunting operation (2-week after ADM administration), the rats were anesthetized with 10% chloral hydrate (3–4 ml/kg). To calculate the magnitude of the left-to-right shunt by oximetric data, blood specimens were drawn from the main pulmonary artery, aorta, and superior vena cava. Blood oxygen saturation was immediately determined using a gas analyzer (GASTAT-3, TMC, Tokyo, Japan). Pulmonary-to-systemic flow ratio (QP /QS ) was calculated according to the formula as follows [20]: Aorta saturation (%) − Superior vena cava saturation (%) Pulmonary venous saturation (%) − Pulmonary arterial saturation (%)
Mean pulmonary pressure (mPAP) of each rat was evaluated by using a right cardiac catheterization procedure. For the catheterization procedure, a polyethylene catheter was inserted into the external jugular vein and advanced under fluoroscopic guidance into right ventricle and pulmonary artery. The other end of the catheter was connected to a transducer (YZ-05-1, Beijing, China). The pressure tracings were simultaneously recorded on a physiologic recorder (BL-410, Chengdu Taimeng Software Co. Ltd, Chengdu, China) [4]. The thoracic cavity of each rat was opened. The heart was removed and prepared with the tissues including atrium, lipid, vessels removed. The right ventricle and the left ventricle with septum were isolated and individually weighted to calculate the ratio of right ventricle weight to left ventricle with septum weight [RV/(LV + SP)]. 2.4. Morphometric analysis of pulmonary arteries The same lung lobe of each rat was removed, fixed with formaldehyde, embedded in paraffin, and subsequently cut into sections. Sections for morphometric analysis were stained with aldehyde fuchsin dyeing methods. Small pulmonary vessels (15–50 m in external diameter) were divided into three types according to the degree of muscularization: muscularized artery (MA), with two distinct elastic lamina; partially muscularized artery (PMA), with one continuous external elastic lamina; and non-muscularized vessel (NMV), with only one single elastic lamina. The percentage of each type of small pulmonary vessels was determined. The relative medial thickness (RMT) of median (50–150 m in external diameter) and small (15–50 m in external diameter) muscularized pulmonary arteries with clearly defined internal elastic lamina and regular shape was determined by Image Processing And Analyzing System (CMIAS, Beijing, China) according to the method of Barth et al. [2]. 2.5. Hydroxyproline assay in the lung tissue Lung tissue was homogenized in a 10-fold volume (w/v) of buffer solution. The content of hydroxyproline in the lung tissue was determined by using a commercial hydroxyprolin eassay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The assays were performed according to the instructions. 2.6. Analysis of collagen I and collagen III contents in the lung tissue in addition to immunohistochemistry for collagen I and collagen III in pulmonary artery The contents of collagens I and III in the lung tissue were determined by using a commercial ELISA kit (Wuhan ElAnb Science Co. Ltd, Wuhan, China).
L. Pang et al. / Peptides 54 (2014) 101–107
103
Table 1 Primers and fluorescent probes. Gene Procollagen I
Procollagen III
-Actin
Sequence Forward Probe Reverse Forward Probe Reverse Forward Probe Reverse
5 -CTTGTTGCTGAGGGCAACAG-3 5 -FAM-ATTCACCTACACTGTCCTTGTCGATGGCTG-TMERA-3 5 -GCAGGCGAGATGGCTTATTC-3 5 -GAAAAAACCCTGCTCGGAATT-3 5 -FAM-AGAGACCTGAAATTCTGCCACCCTGAACTC-TMERA-3 5 -ATCCATCTTGCAGCCTTGGT-3 5 -ACCCGCGAGTACAACCTTCTT-3 5 -FAM-CCTCCGTCGCCGGTCCACAC-TMERA-3 5 -TATCGTCATCCATGGCGAACT-3
FAM, 5-carboxyfluorescern; TAMRA, 5-carboxytetramethylrhodamine.
The paraffin sections of lung tissue was dewaxed and hydrated, and then processed by 3% H2 O2 for 10 min at room temperature, followed by antigen repairing for 10 min (microwave heating method). The slides were washed twice with phosphate-buffered saline (PBS), then blocked with goat serum at 37 ◦ C for 20 min, and incubated overnight at 4 ◦ C with collagen I and collagen III antibodies (diluted at 1:300, 1:200, respectively). Then the slides were rinsed in PBS for three times, 3 min each. The biotinylated anti-rabbit IgG at 37 ◦ C was incubated for 60 min. After the slides were rinsed in PBS for three times, slides were stained with DAB to develop color. The yellow–brown granules under microscope were defined as the positive signals. For negative controls, sections were processed as above except that the primary incubation was performed with PBS instead of primary antibodies. For analysis of collagen I and collagen III expression, the mean optical destiny value per unit area of vessels was calculated by LeicaQ550cwImage Capturing and Analyzing System (Leica, German). Eight to twelve median and small pulmonary muscularized arteries of each rat were detected and the average values were calculated.
2.9. Statistical analysis All data were shown as mean ± S.D. ANOVA was performed to compare the differences among the three groups using SPSS 16.0 statistic analysis software. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. ADM improved the hemodynamics index QP /QS were significantly increased in rats of shunt and shunt + ADM group as compared with controls (p < 0.01, respectively). However, there was no significantly difference between shunt and shunt + ADM group (p > 0.05) (Table 2). The mPAP and RV/(LV + S) were significantly increased in rats of shunt group as compared with controls (p < 0.01, respectively). ADM, however, significantly decreased mPAP and RV/(LV + S) (p < 0.01, respectively) (Table 2). 3.2. ADM alleviated the pulmonary artery microstructure changes
2.7. Analysis of PINP and PIIINP in serum in addition to RT-PCR for procollagens I and III in lung tissue Blood samples were collected from external jugular arteries and centrifuged at 4 ◦ C to separate plasma. The content of N-terminal propeptides of types I and III (PINP and PIIINP), marker of collagen synthesis, was detected by using a commercial ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Total RNA in the lung was extracted by using trizol reagent (Invitrogen, CA, USA). Then the total RNA was reversed into cDNA by using moloney murine leukemia virus reverse transcription (MMLV-RT) and Oligo (dT) 15 or random primer. The rat collagens I and III and -actin mRNA levels were determined by using real-time PCR (Prism 7300 Sequence Detector, Applied Biosystems, Foster City, CA). PCR reaction volume was 25 L with contained 1 L of cDNA, 1 L of 2.5 mmol/L dNTP, 1 L of 5 pmol/L TaqMan probe, 1 L of 7.5 pmol/L sense and anti-sense primers, 2.5 L of 10 × PCR buffer containing 20 mmol/L MgCl2 , and 0.25 L TaqDNA polymerase. The mRNA levels were compared after they had been normalized relative to those of -actin. The amplification products were electrophoresed on 1.5% agarose gel and visualized by ethidium bromide staining. The primers and fluorescent probes used are shown in Table 1.
2.8. ELISA analysis of TGF-ˇ1 in the lung tissue The content of TGF-1 in the lung tissue was determined by using a commercial ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The assays were performed according to the instructions.
The percentage of MA of small pulmonary vessels was significantly increased in rats of shunt group as compared with controls (p < 0.01), but the percentage of NMV was significantly decreased (p < 0.01). ADM, however, significantly alleviated the muscularization of small pulmonary vessels (p < 0.01, respectively) (Table 3). RMT of median and small muscularized pulmonary arteries was both markedly increased in rats of shunt group compared with that of controls (p < 0.01, respectively). ADM, however, significantly reduced RMT of median pulmonary arteries (p < 0.05) and small pulmonary arteries (p < 0.01) (Table 3). 3.3. ADM alleviated the collagen accumulation in lung tissues The content of hydroxyproline in the lung homogenate was increased in rats of shunt group as compared with controls (p < 0.05). ADM, however, significantly decreased the content of hydroxyproline in the lung homogenate (p < 0.01) (Table 4).
Table 2 Hemodynamics parameters in rats. Groups
mPAP (mmHg)
RV/(LV + SP)
QP /QS
Sham Shunt Shunt + ADM
14.6 ± 2.4 25.5 ± 4.0** 16.4 ± 2.5##
0.22 ± 0.03 0.33 ± 0.06** 0.26 ± 0.03##
1.05 ± 0.12 2.11 ± 0.77** 2.27 + 0.60**
mPAP, mean pulmonary artery pressure; RV/(LV + S), the ratio of right ventricle mass to left ventricle with septum mass; QP /QS , pulmonary-to-systemic flow ratio. Values are expressed as mean ± S.D. ** p < 0.01 vs. sham group. ## p < 0.01 vs. shunt group.
104
L. Pang et al. / Peptides 54 (2014) 101–107
Fig. 1. Immunohistochemistry for collagen I expression in the pulmonary arteries of rats of sham (A), shunt (B) and shunt with ADM (C) groups. Collagen I expression by pulmonary arteries in rats of shunt group (B) was significantly strengthened as compared with that of sham group (A). ADM (C) decreased collagen I expression by pulmonary arteries in shunt rats. Magnification is 400×.
Fig. 2. Immunohistochemistry for collagen III expression in the pulmonary arteries of rats of sham (A), shunt (B) and shunt with ADM (C) groups. Collagen III expression by pulmonary arteries in rats of shunt group (B) was significantly strengthened as compared with that of sham group (A). ADM (C) decreased collagen III expression by pulmonary arteries in shunt rats. Magnification is 400×.
L. Pang et al. / Peptides 54 (2014) 101–107
105
Table 3 Pulmonary artery microstructure parameters in rats. Groups
Sham Shunt Shunt + ADM
Small pulmonary vessels (%)
Relative medial thickness (%)
MA
PMA
NMV
Median PA
Small PA
14.0 ± 1.7 23.1 ± 1.6** 17.6 ± 2.4** , ##
15.5 ± 2.8 26.1 ± 1.1** 21.3 ± 4.4** , ##
70.5 ± 4.1 50.9 ± 2.5** 61.1 ± 6.5** , ##
5.3 ± 1.1 8.5 ± 1.5** 7.3 ± 0.8** , #
7.6 ± 1.3 12.4 ± 2.1** 10.2 ± 2.1** , ##
MA, muscularized artery; PMA, partially muscularized artery; NMV, non-muscularized vessel; PA, pulmonary artery. Values are expressed as mean ± S.D. ** p < 0.01 vs. sham group. # p < 0.05 vs. shunt group. ## p < 0.01 vs. shunt group.
Table 4 Lung tissue concentration of hydroxyproline, collagen I and collagen III in rats. Groups Sham Shunt Shunt + ADM
Hydroxyproline (g/mg)
Collagen I (ng/g)
Collagen III (ng/g)
0.89 ± 0.07 1.09 ± 0.20* 0.84 ± 0.18##
15.66 ± 1.50 23.91 ± 4.03** 20.35 ± 2.60** , #
1.43 ± 0.31 2.28 ± 0.35** 1.95 ± 0.24** , #
Values are expressed as mean ± S.D. * p < 0.05 vs. sham group. ** p < 0.01 vs. sham group. # p < 0.05 vs. shunt group. ## p < 0.01 vs. shunt group.
Table 6 Plasma concentrations of PINP, PIIINP and lung tissue of TGF-1 in rats. Groups
PINP (ng/ml)
PIIINP (pg/ml)
TGF-1 (pg/mg)
Sham Shunt Shunt + ADM
14.0 ± 2.3 23.9 ± 6.0** 18.7 ± 5.3#
599 ± 179 923 ± 125** 738 ± 127#
67.8 ± 18.6 117.3 ± 32.7** 85.0 ± 25.1##
PINP, N-terminal propeptides of type I; PIIINP, N-terminal propeptides of type III. Values are expressed as mean ± S.D. ** p < 0.01 vs. sham group. # p < 0.05 vs. shunt group. ## p < 0.01 vs. shunt group.
3.5. ADM decreased the TGF-ˇ1 content in lung tissue The contents of collagen I and collagen III in the lung homogenate were significantly increased in rats of shunt group as compared with those of controls (p < 0.01, respectively). ADM, however, significantly decreased the contents of collagen I and collagen III in the lung homogenate (p < 0.05, respectively) (Table 4). Immunohistochemistry on lung sections from control animals showed expression of collegan I and collegan III protein in media and adventitia of pulmonary arteries and bronchia. The expressions of collagen I and collagen III in pulmonary arteries were significantly increased in rats of shunt group as compared with controls (p < 0.01, respectively). ADM, however, significantly decreased the expressions of collagens I and III in pulmonary arteries (p < 0.05, respectively) (Table 5, Figs. 1 and 2). 3.4. ADM alleviated the collagen synthesis in the lung tissue The concentrations of PINP and PIIINP in serum were significantly increased in rats of shunt group as compared with controls (p < 0.01, respectively). ADM, however, significantly decreased the concentrations of PINP and PIIINP in serum (p < 0.05, respectively) (Table 6). RT-PCR revealed significantly increased mRNA levels of procollagen I and procollagen III in the lung tissues of shunt rats compared with those of controls (p < 0.05, respectively). However, the mRNA levels of procollagen I and procollagen III in the lung tissues of shunt with ADM rats were significantly decreased as compared with those of shunt group (p < 0.05, respectively) (Fig. 3).
The content of TGF-1 in the lung homogenate was significantly increased in rats of shunt group compared with controls (p < 0.01). ADM, however, significantly decreased the content of TGF-1 (p < 0.01) (Table 6). 4. Discussion In the present study, we demonstrated that ADM administrated subcutaneously by mini-osmotic pump attenuated the development of pulmonary hypertension and pulmonary vascular structural remodeling induced by high blood flow, in association with the inhibition of collagen synthesis and accumulation in lung tissues of rats. Pulmonary hypertension is a progressive disease of the pulmonary vasculature that is associated with severe functional impairment and a poor prognosis. Pulmonary vascular structural remodeling, characterized by proliferation of endothelial cells and vascular smooth muscle cells, alterations in the extracellular matrix, thrombosis and fibrosis, is the important pathological change of pulmonary hypertension. In children, the most common etiology of pulmonary hypertension is high pulmonary blood flow secondary to congenital heart disease with systemic-topulmonary shunt [23]. Many vasoactive substances might be involved in the development of pulmonary hypertension induced by high pulmonary blood flow [7]. A variety of therapeutic agents, such as prostacyclin, endothelin-1, receptor antagonists,
Table 5 Immunohistochemical analysis of collagens I and III expression by pulmonary arteries in rats. Groups
Sham Shunt Shunt + ADM Values are expressed as mean ± S.D. * p < 0.05 vs. sham group. ** p < 0.01 vs. sham group. # p < 0.05 vs. shunt group.
Small pulmonary artery
Median pulmonary artery
Collagen I
Collagen III
Collagen I
Collagen III
0.357 ± 0.036 0.449 ± 0.049** 0.383 ± 0.056#
0.260 ± 0.018 0.312 ± 0.026** 0.285 ± 0.014* , #
0.378 ± 0.058 0.457 ± 0.039** 0.397 ± 0.050#
0.259 ± 0.011 0.310 ± 0.030** 0.280 ± 0.017* , #
106
L. Pang et al. / Peptides 54 (2014) 101–107
Fig. 3. Procollagen I (A) and procollagen III (B) mRNA expression in the lung tissue of rats. Procollagen I (A) and procollagen III (B) mRNA expression in the lung tissue of rats with shunt group was significantly increased as compared with that of sham group. ADM lowered the procollagen I (A) and procollagen III (B) mRNA expression in the lung tissue of shunting rats. * p < 0.05 vs. sham group. # p < 0.05 vs. shunt group.
and phosphodiesterase-type 5 inhibitors, are demonstrated leading to improvement of pulmonary hemodynamics, exercise capacity and survival in patients with pulmonary hypertension [14]. Despite therapeutic medication advances, for the majority of patients with pulmonary hypertension, disease progression is inevitable and long-term survival remains poor [27]. Thus, a novel therapeutic strategy is desirable for the treatment of pulmonary hypertension. ADM is a potent vasodilator peptide, belonging to calcitonin gene-related peptide superfamily [12]. Repeated inhalation of ADM, chronic infusion of ADM and intratrachieal gene transfer of ADM using polyplex nanomicelles attenuated monocrotalineinduced pulmonary hypertension in rats [9,16,30]. Our previous study showed that chronic administration of ADM attenuated hypoxic pulmonary vascular structural remodeling and pulmonary hypertension [19]. These findings suggested that ADM might play an important role in the regulation of pulmonary hypertension. In addition, Yoshibayash et al. found that plasma ADM level in pulmonary hypertension associated with congenital heart disease was significantly increased compared with those without pulmonary hypertension and normal control [29]. Zhao et al. found the mRNA and protein expressions of ADM in lung tissues of rats with pulmonary hypertension induced by high pulmonary blood flow were increased [6]. All above clues suggested that ADM might participate in the regulation of pulmonary hypertension induced by high blood flow. In the present study, we explored the effect of chronic ADM administration on pulmonary hypertension and pulmonary vascular remodeling induced by high pulmonary blood flow. The results showed that chronic ADM infusion administrated subcutaneously by mini-osmotic pump decreased mPAP and the ratio of RV/(LV + S), alleviated the muscularization of small pulmonary vessels, and decreased the RMT of pulmonary arteries. The results suggested that ADM ameliorated the development of pulmonary hypertension and pulmonary vascular remodeling induced by high pulmonary blood flow. The possible mechanisms by which ADM alleviates pulmonary hypertension and pulmonary vascular structural remodeling induced by high pulmonary blood flow have not been clarified. First of all, previous studies showed that ADM could dilate pulmonary artery through the nitric oxide-dependent mechanism [3,28]. ADM infusion could cause dose-related decreases in pulmonary vascular resistance under conditions of high pulmonary vascular tone, so as to reduce pulmonary artery pressure. Secondly, ADM could inhibit pulmonary artery smooth muscle cell DNA synthesis and
endothelin release [26], which suggested that ADM might function in the inhibition of pulmonary hypertension and pulmonary vascular remodeling through inhibiting pulmonary vascular smooth muscle cell proliferation. In addition, ADM could decrease AngIIinduced collagen deposition surrounding the coronary arteries, inhibiting myofibroblast differentiation and expressions of extracellular matrix-related genes in rats [25], which suggested ADM might alleviate pulmonary vascular structural remodeling as an antifibrotic factor. However, the effect of ADM on pulmonary vascular collagen accumulation has not been studied. Change of extracellular matrix is an important pathological alteration contributing a great deal to pulmonary vascular structural remodeling, and influences the structural and functional integrity of pulmonary vessels. Collagen I and collagen III are the most abundant components of the extracellular matrix of the vascular wall. Changes in the absolute or relative contents of collagen I and collagen III in pulmonary artery would result in changes in pulmonary vascular compliance and ultimately the development of pulmonary hypertension [18]. In the present study, the secretion of hydroxyproline, an inherent component of collagen, and the contents of collagen I and collagen III in the pulmonary arteries of rats with aortocaval shunting were increased as compared with controls. However, ADM decreased the contents of hydroxyproline, collagen I and collagen III in rats with high pulmonary blood flow, which suggested that ADM inhibited the anomalous deposition of collagen in the pulmonary arteries, so as to participating in the regulation of pulmonary hypertension and pulmonary vascular remodeling induced by high pulmonary blood flow. The imbalance between collagen synthesis and collagen degradation contributed to collagen accumulation [8]. It was reported that ADM inhibited the proliferation and differentiation of coronary adventitial fibroblasts, reduced the mRNA level of procollagen I, and alleviated the collagen deposition of coronary arteries in Ang IIinduced hypertensive rats [25], which suggested that ADM exerted a significantly inhibitory effect on the collagen synthesis in fibroblasts. It is unclear, however, whether ADM could alleviate collagen accumulation in pulmonary arteries by inhibiting collagen synthesis. Collagens I and III are secreted by interstitial fibroblasts as procollagens followed by splitting off of propeptides by endopeptidases and the release of their aminoterminal propeptides, PINP and PIIINP, and carboxyterminal propeptides, PICP and PIIICP, into the circulation [21]. So serum PINP and PIIINP are often used as the synthesis biomarkers of the two types of collagen. In our present study,
L. Pang et al. / Peptides 54 (2014) 101–107
mRNA levels of procollagen I and procollagen III in lung tissue, PINP and PIIINP in the serum were detected to be dramatically increased in rats of the shunt group. The mRNA levels of procollagen I and procollagen III in lung tissue, PINP and PIIINP in the serum were significantly decreased in rats of shunt with ADM group, which suggests that ADM inhibited the synthesis of collagen, so as to alleviate collagen accumulation in pulmonary arteries and pulmonary hypertension induced by high pulmonary blood flow. TGF-1 is a member of TGF- superfamily. TGF- is a major stimulus of extracellular matrix production in fibroproliferative diseases [5]. TGF-1 stimulation of fibrotic pathways occurs primarily through Smad 2/3 dependent pathways [22]. Knockdown of Smad2 and Smad3 inhibited proliferation and migration of adventitial fibroblasts and down-regulated the expression of and collagens I and III. Evidence showed that TGF-1/Smads pathway was activated in animals with MCT- and hypoxia-induced pulmonary hypertension and in patients with pulmonary arterial hypertension [24]. It is proved that ADM might serve as an antifibrotic factor to inhibit the synthesis of collagen through down-regulating the TGF-1 expression [17]. So, we hypothesized that TGF-1/Smads pathway activation might be involved in the regulation of ADM to collagen synthesis in the pulmonary hypertension and pulmonary vascular matrix remodeling. In this study, we detected the elevation of TGF-1 in the lung tissue of shunt rats, and the decrease of TGF-1 in the lung tissue after ADM administration. Therefore, we presumed that the effect of ADM on the regulation of collagen synthesis might involve decreasing TGF-1. However, further studies are needed to explain the mechanisms responsible for decrease of TGF-1 induced by ADM. As all we know, right cardiac catheter is the golden standard for the measurement of pulmonary artery pressure. Because the rats will be sacrificed after the measurements of the hemodynamics parameters, we can not measure hemodynamics parameters in the same rat twice, so the hemodynamics parameters data in rats at baseline and at the end of 8-week experiment were lack. This is the limitation of our study. But in the previous study of our research group, we observed that pulmonary hypertension and right ventricular hypertrophy were developed at the end of 8 weeks aortocaval shunting in rats [13], that is also our reason to administer ADM to rats at the end of 8 weeks aortocaval shunting, so as to explore the effects of ADM on pulmonary hypertension. In conclusion, this study suggests that ADM plays a protective role in the development of pulmonary hypertension and pulmonary vascular structural remodeling induced by high blood flow. ADM alleviates pulmonary artery collagen accumulation by inhibiting pulmonary procollagen synthesis. The effect of ADM on collagen metabolism might involve TGF-1, a key extracellular matrix regulator. Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant no. 30973226). References [1] Barst RJ, McGoon MD, Elliott CG, Foreman AJ, Miller DP, Ivy DD. Survival in childhood pulmonary arterial hypertension: insights from the registry to evaluate early and long-term pulmonary arterial hypertension disease management. Circulation 2012;125:113–22. [2] Barth PJ, Kimpel C, Roy S, Wagner C. An improved mathematical approach for the assessment of the medial thickness of pulmonary arteries. Pathol Res Pract 1993;189:567–76. [3] Bayram Z, Golbasi I, Ozdem SS. The role of nitric oxide and potassium channels in the effect of adrenomedullin in human internal thoracic arteries. Regul Pept 2010;161:92–6. [4] Bing W, Junbao D, Jianguang Q, Jian L, Chaoshu T. l-Arginine impacts pulmonary vascular structure in rats with an aortocaval shunt. J Surg Res 2002;108:20–31.
107
[5] Chen CP, Yang YC, Su TH, Chen CY, Aplin JD. Hypoxia and transforming growth factor-beta 1 act independently to increase extracellular matrix production by placental fibroblasts. J Clin Endocrinol Metab 2005;90:1083–90. [6] Cuifen Z, Lijuan W, Li G, Wei X, Zhiyu W, Fuhai L. Changes and distributions of peptides derived from proadrenomedullin in left-to-right shunt pulmonary hypertension of rats. Circ J 2008;72:476–81. [7] D’Alto M, Mahadevan VS. Pulmonary arterial hypertension associated with congenital heart disease. Eur Respir Rev 2012;21:328–37. [8] Estrada KD, Chesler NC. Collagen-related gene and protein expression changes in the lung in response to chronic hypoxia. Biomech Model Mechanobiol 2009;8:263–72. [9] Harada-Shiba M, Takamisawa I, Miyata K, Ishii T, Nishiyama N, Itaka K, et al. Intratracheal gene transfer of adrenomedullin using polyplex nanomicelles attenuates monocrotaline-induced pulmonary hypertension in rats. Mol Ther 2009;17:1180–6. [10] Ichiki Y, Kitamura K, Kangawa K, Kawamoto M, Matsuo H, Eto T. Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett 1994;338:6–10. [11] Kach J, Sandbo N, Sethakorn N, Williams J, Reed EB, La J, et al. Regulation of myofibroblast differentiation and bleomycin-induced pulmonary fibrosis by adrenomedullin. Am J Physiol Lung Cell Mol Physiol 2013;304: L757–64. [12] Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 1993;192:553–60. [13] Li XH, Du JB, Tang XY, Jin HF, Tang CS. Changes of pulmonary artery structural remodeling in pulmonary hypertension induced by high pulmonary flow in rats. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2005;27:446–51. [14] Malenfant S, Margaillan G, Loehr JE, Bonnet S, Provencher S. The emergence of new therapeutic targets in pulmonary arterial hypertension: from now to the near future. Expert Rev Respir Med 2013;7:43–55. [15] Nagaya N, Kyotani S, Uematsu M, Ueno K, Oya H, Nakanishi N, et al. Effects of adrenomedullin inhalation on hemodynamics and exercise capacity in patients with idiopathic pulmonary arterial hypertension. Circulation 2004;109: 351–6. [16] Nagaya N, Okumura H, Uematsu M, Shimizu W, Ono F, Shirai M, et al. Repeated inhalation of adrenomedullin ameliorates pulmonary hypertension and survival in monocrotaline rats. Am J Physiol Heart Circ Physiol 2003;285:H2125–31. [17] Niu P, Shindo T, Iwata H, Iimuro S, Takeda N, Zhang Y, et al. Protective effects of endogenous adrenomedullin on cardiac hypertrophy, fibrosis, and renal damage. Circulation 2004;109:1789–94. [18] Ooi CY, Wang Z, Tabima DM, Eickhoff JC, Chesler NC. The role of collagen in extralobar pulmonary artery stiffening in response to hypoxia-induced pulmonary hypertension. Am J Physiol Heart Circ Physiol 2010;299:H1823–31. [19] Qi JG, Ding YG, Tang CS, Du JB. Chronic administration of adrenomedullin attenuates hypoxic pulmonary vascular structural remodeling and inhibits proadrenomedullin N-terminal 20-peptide production in rats. Peptides 2007;28:910–9. [20] Qi J, Du J, Tang X, Li J, Wei B, Tang C. The upregulation of endothelial nitric oxide synthase and urotensin-II is associated with pulmonary hypertension and vascular diseases in rats produced by aortocaval shunting. Heart Vessels 2004;19:81–8. [21] Radovan J, Vaclav P, Petr W, Jan C, Michal A, Richard P, et al. Changes of collagen metabolism predict the left ventricular remodeling after myocardial infarction. Mol Cell Biochem 2006;293:71–8. [22] Ren M, Wang B, Zhang J, Liu P, Lv Y, Liu G, et al. Smad2 and Smad3 as mediators of the response of adventitial fibroblasts induced by transforming growth factor 1. Mol Med Rep 2011;4:561–7. [23] Rosenzweig EB, Barst RJ. Congenital heart disease and pulmonary hypertension: pharmacology and feasibility of late surgery. Prog Cardiovasc Dis 2012;55:128–33. [24] Thomas M, Docx C, Holmes AM, Beach S, Duggan N, England K, et al. Activin-like kinase 5 (ALK5) mediates abnormal proliferation of vascular smooth muscle cells from patients with familial pulmonary arterial hypertension and is involved in the progression of experimental pulmonary arterial hypertension induced by monocrotaline. Am J Pathol 2009;174:380–9. [25] Tsuruda T, Kato J, Hatakeyama K, Masuyama H, Cao YN, Imamura T, et al. Antifibrotic effect of adrenomedullin on coronary adventitia in angiotensin II-induced hypertensive rats. Cardiovasc Res 2005;65:921–9. [26] Upton PD, Wharton J, Coppock H, Davie N, Yang X, Yacoub MH, et al. Adrenomedullin expression and growth inhibitory effects in distinct pulmonary artery smooth muscle cell subpopulations. Am J Respir Cell Mol Biol 2001;24:170–8. [27] Vachiéry JL, Gaine S. Challenges in the diagnosis and treatment of pulmonary arterial hypertension. Eur Respir Rev 2012;21:313–20. [28] Yang BC, Lippton H, Gumusel B, Hyman A, Mehta JL. Adrenomedullin dilates rat pulmonary artery rings during hypoxia: role of nitric oxide and vasodilator prostaglandins. J Cardiovasc Pharmacol 1996;28:458–62. [29] Yoshibayashi M, Kamiya T, Kitamura K, Saito Y, Kangawa K, Nishikimi T, et al. Plasma levels of adrenomedullin in primary and secondary pulmonary hypertension in patients
