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The regulatory effect of hydrogen sulfide on hypoxic pulmonary hypertension in rats
BBRC Biochemical and Biophysical Research Communications 302 (2003) 810–816 www.elsevier.com/locate/ybbrc
The regulatory effect of hydrogen sulfide on hypoxic pulmonary hypertension in ratsq Zhang Chunyu,a Du Junbao,a,* Bu Dingfang,b Yan Hui,a Tang Xiuying,c and Tang Chaoshud a
Department of Pediatrics, Peking University First Hospital, Xi-An Men Street No. 1, Beijing 100034, PeopleÕs Republic of China b Central Laboratory, Peking University First Hospital, PeopleÕs Republic of China c Laboratory of Electron Microscopy, Peking University First Hospital, PeopleÕs Republic of China d Institute of Cardiovascular Diseases, Peking University First Hospital, PeopleÕs Republic of China Received 1 February 2003
Abstract Hypoxic pulmonary hypertension (HPH) is an important pathophysiological process. The mechanism of HPH is still not fully understood. Recent studies showed that hydrogen sulfide (H2 S) could relax vascular smooth muscles and inhibit the proliferation of cultured vascular smooth muscle cells. Our study showed that both the gene expression of cystathionine c-lyase (CSE), one of the H2 S generating enzymes, and the activity of CSE were suppressed in lung tissues during HPH. And the plasma level of H2 S was decreased during HPH. Exogenous supply of H2 S could increase the plasma level of H2 S, enhance CSE activity, and up-regulate CSE gene expression in lung tissue. At the same time, exogenous supply of H2 S could oppose the elevation of pulmonary arterial pressure and lessen the pulmonary vascular structure remodeling during HPH. The results showed that endogenous H2 S system was involved and exogenous H2 S could exert beneficial effect on the pathogenesis of HPH. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Hypoxia; Hydrogen sulfide; Pulmonary hypertension
Hypoxic pulmonary hypertension (HPH) is an important pathophysiological process which could occur in many cardiac and pulmonary diseases. Acute or chronic exposure to hypoxia could result in HPH which is characterized by high pressure in pulmonary artery and high pulmonary vascular resistance. The rise in pulmonary arterial pressure and vascular resistance is the result of a combination of hypoxic vasoconstriction and vascular structure remodeling. But the mechanisms of HPH are still not fully understood. q
This study was supported by The National Natural Science Foundation of China (39870844, 30070796, and 30271373), the Doctorate Training Program Foundation of the Ministry of Education of China (20020001063), Peking University 985 Cardiovascular Research Grant, and The Major Basic Research Program of China (G2000056905). * Corresponding author. Fax: +86-10-6613-4261. E-mail addresses: [email protected], [email protected] (J. Du).
It has been proved by a variety of experiments and clinical materials that endogenous gaseous transmitters including nitric oxide (NO) and carbon monoxide (CO) in the body play important roles in the pathogenesis of HPH. And NO inhalation has become an important option of treating and preventing HPH [1–4]. Recently, it was indicated that hydrogen sulfide (H2 S) which has been known as toxic gas for a long time could also be endogenously generated. H2 S is generated from cysteine in a reaction catalyzed by cystathionine b-synthesis (CBS) and cystathionine c-lyase (CSE) [5–7]. Recent studies also showed that H2 S had important physiological functions including relaxing rat aortic artery in vitro and inhibiting cultured vascular smooth muscle cell proliferation [8–10]. H2 S at physiological concentrations could also facilitate the induction of hippocampus long-term potentiation (LTP) [11–13]. But to date, the role of H2 S in the pathogenesis of HPH has not been reported.
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00256-0
C. Zhang et al. / Biochemical and Biophysical Research Communications 302 (2003) 810–816
The present study was to investigate the changes of endogenous H2 S during the development of HPH and its possible role in the pathogenesis of HPH. Materials and methods Hypoxic pulmonary hypertension rat model. The study was approved by the Animal Research Committee of Peking University. The rats were exposed to hypoxia according to the methods of Xue [14]. Nineteen male Wistar rats (180–220 g) were randomly divided into Control group (n ¼ 6), Hypoxia group (n ¼ 7), and Hy þ NaHS group (n ¼ 6). The rats were exposed to normobaric hypoxia (10% O2 ) in a transparent plastic hypoxia chamber (for rats in Hypoxia group and Hy þ NaHS group) for 3 weeks and 6 h everyday. Rats in Control group were housed in identical cages adjacent to the hypoxia chamber breathing room air. Hypoxia was generated by infusing N2 into the chamber. The degree of hypoxia was maintained by the balance between nitrogen infusing and the inward leak of air through holes in the chamber. For rats in Hy þ NaHS group, NaHS dissolved in physiological saline at a dosage of 14 lmol/kg body weight was injected intraperitoneally before hypoxia everyday. The same volume of physiological saline was injected for rats in the other two groups. Measurement of hemodynamic parameters and sample preparation. Three weeks after hypoxic exposure, the rats were anesthetized with urethane (1 g/kg body weight) intraperitoneally. A silicone catheter (outer diameter 0.9 mm) was introduced into the right jugular vein via a venotomy and passed across the tricuspid valve and right ventricle into pulmonary artery. The mean pulmonary artery pressure (mPAP) was simultaneously recorded. The mean aortic pressure (mAP) was also recorded by carotid artery catheterization. After the chest was opened, the right lower part of the lung tissue was rapidly removed to liquid nitrogen for quick freezing and then stored at )70 °C. Then the heart was excised and the atrias were removed. The free wall of right ventricle (RV) was separated from the heart. These tissues were blotted and weighed using an electronic scale. The wet weight ratio of RV against left ventricle plus septum, RV/(LV þ SP), was calculated as an indicator of right ventricular hypertrophy. The blood plasma was also prepared for the measurement of plasma H2 S concentration. Measurement of H2 S concentration in the plasma. In a test tube containing 0.5 ml of 1% zinc acetate and 2.5 ml of distilled water, 0.1 ml of plasma was added. Then 0.5 ml of 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 M HCl and 0.4 ml of 30 mM FeCl3 in 1.2 M HCl were also added to the test tube for 20 min of incubation at room temperature. The protein in the plasma was removed by adding 1 ml of 10% trichloroacetic acid to the solution and centrifuged. The optical absorbance of the resulting solution at 670 nm was measured with a spectrometer (Shimadzu UV 2100, Japan). The H2 S concentration in the solution was calculated against the calibration curve of the standard H2 S solution. Measurement of H2 S production. According to StipanukÕs methods [7], the lung tissues were homogenized in ice-cold 50 mM potassium phosphate buffer (PH 6.8). Reactions were performed in 25 ml Erlenmeyer flasks. The reaction mixture contained (mM): 10 L-cysteine, 2 pyridoxal 50 -phosphate, 100 potassium phosphate buffer (PH 7.4), and 10% (w/v) homogenates. The total volume of the reaction mixture was 1 ml. A small piece of filter paper was put into the central well of the flask and 0.5 ml of 1% zinc acetate was also added in the central well for trapping evolved H2 S in the mixture. The flasks were then flushed with N2 before being sealed with a double layer of parafilm. The catalytic reaction was initiated by transferring the flasks from an ice bath to a 37 °C shaking water bath. After 90 min at 37 °C, the reactions were stopped by injecting 0.5 ml of 50% trichloroacetic acid. Flasks were incubated in the shaking water bath for an additional hour at 37 °C to complete trapping of H2 S. The content of the central well was trans-
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ferred to test tubes and mixed with 3.5 ml of distilled water and 0.5 ml of 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 M HCl. To each tube, 0.4 ml of 30 mM FeCl3 in 1.2 M HCl was added immediately. After 20 min of incubation at room temperature, the optical absorbance of the resulting solution at 670 nm was measured with a spectrometer (Shimadzu UV 2100, Japan). The H2 S concentration in the solution was calculated against the calibration curve of the standard H2 S solution. For each sample, the measurement was done in duplicate. The H2 S production was expressed in a unit of nmol/mg wet tissue min. Measurement of CSE mRNA in lung tissue by quantitative reverse transcription-polymerase chain reaction. Total RNA was extracted from rat lung tissue using TRIzol reagent. The cDNA was synthesized using oligo ðdTÞ15 primer and M-MLV reverse transcriptase. The polymerase chain reaction (PCR) primers used to amplify the fragment of CSE cDNA were: CSE-S : 50 -TCCGG ATGGA GAAAC ACTTC CSE-A : 50 -GCTGC CTTTA AAGCT TGACC PCR using these two primers yielded a 400 bp fragment of wildtype rat CSE cDNA. The competitive internal standard for the measurement of CSE cDNA had the same sequences as the 400 bp fragment of wild-type rat CSE cDNA, except that a fragment of 39 bp at the downstream site of CSE-S primer was deleted [15,16]. Quantitative PCR was performed in a 0.2 ml of PCR tube containing 1 ll of rat lung cDNA, 1 ll of 3.9 fmol/liter competitive internal standard, 1 ll of 5 lM/each CSE-S and CSE-A mixture, 1 ll of 2.5 mM/each dNTP mixture, 2 mM MgCl2 , 2.5 ll of 10 PCR buffer, and 1.25 U Taq DNA polymerase, in a total volume of 25 ll. PCR products were separated in a 2% agarose gel and stained by ethidium bromide. The ratio of optical density of the two DNA bands was measured by a Gel Image Analysis System (AlphaImager, Alpha Innotech, CA, USA) under UV light. A standard curve of the ratio was drawn using the same condition as described above except that rat lung cDNA was changed to a series of dilutions of the plasmid containing the 400 bp wild-type CSE cDNA fragment. The relative amount of CSE cDNA in samples was then obtained from the standard curve [17]. To calibrate the amount of sample loaded in PCR mixture, b-actin cDNA was measured using the same method after the quantitative PCR. Three ll of PCR product was amplified again using the two rat b-actin primers (b-actin-S: 50 -ATC TG GCACC ACACC TTC, b-actin-A: 50 -AGCCA GGTCC AGACG CA). Relative amount of b-actin cDNA in loaded sample was then obtained from a pre-made standard curve of b-actin cDNA measurement. Standardized relative amount of CSE cDNA was used for further analysis. Morphological analysis of small pulmonary arteries. The left lower part of lung tissue was removed to 10% (wt/vol) paraformaldehyde for fixation. Then the lung tissues were dehydrated, embedded in paraffin, and sectioned at a thickness of 4 lm. The elastic fiber in lung tissues was stained according to the modified WeigertÕs method and counterstained with Van Gieson solution. Morphological analysis was performed using a video-linked microscope digitizing board system (Leica Q550CW, Germany). Only vessels showing clearly defined external and internal elastic lamina were used in analysis. According to BathÕs methods [18], the relative medial thickness (RMT) and relative medial areas (RMA) were calculated. A small part of lung tissue was cut from the upper part of left lung lobe and quickly immersed into 3% glutaraldehyde. And then it was cut into pieces of 1 mm 1 mm 1 mm in size and post–fixed in 1% phosphate-buffered osmium tetroxide for 6 h. Then it was rinsed again (overnight) and dehydrated in a graded series of ethanol. These specimens were infiltrated with propylene oxide and embedded with Epon812. These procedures were all done at room temperature. Finally, specimens were embedded in new batches of Epon812 and polymerized at 40°C (24 h) and 60°C (48 h). From selected blocks, series of transverse or longitudinal sections were made. Semi thin sections
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(1 lm) were stained with azur-II and methylene blue. Ultra thin sections (60–90 nm) were made with an ultra microtome and it was mounted on formvar-coated copper grids (75 meshes). Then it was stained with uranylacetate and lead citrate, and examined under a transmission electron microscope (JEM-100CX, JEOL, Japan) in detail. Chemicals and reagents. NaHS solution was freshly prepared by mixing the stock solution of sodium sulfide and hydrochloric acid. Phosphate 50 -pyridoxal was purchased from Sigma Corporation. TRIzol reagent was bought from Invitrogen, Oligo dðTÞ15 primer, dNTP, and M-MLV reverse transcriptase were bought from Promega Corporation. All the other chemicals were purchased from Beijing Chemical Reagents, China. Data analysis. The results were expressed as means SD. For comparison of the differences among the three groups, ANOVA followed by a post hoc analysis (Bonferroni test) was used by SPSS 10.0 statistic analysis software. A value of P < 0:05 was considered statistically significant.
Results and discussion Pulmonary artery hypertension refers to a group of diseases characterized by high pressure in the pulmonary artery and high pulmonary vascular resistance. Pulmonary artery hypertension is diagnosed when the systolic pulmonary artery pressure is higher than 30 mmHg (4.0 kPa), or the mean pulmonary artery pressure (mPAP) is higher than 20 mmHg (2.6 kPa). Chronic exposure to hypoxia results in hypoxic pulmonary artery hypertension. It was reported that after intermittent exposure to 10% of oxygen 6 h daily for 2–4 weeks, the rat could develop constant chronic pulmonary hypertension with right ventricular hypertrophy [14]. In our study, after 3 weeks of hypoxia, mPAP increased significantly compared with rats without hypoxia (23:7 2:2 vs. 16:3 3:7 mmHg, P < 0:01). We used RV/(LV þ SP), which stood for the wet weight ratio of right ventricular free wall vs. left ventricular plus septum. The ratio of RV/(LV þ SP), an indicator of right ventricular hypertrophy, also increased markedly in rats of Hypoxia group compared with that in Control group (0:41 0:03 vs. 0:29 0:02, P < 0:01). These results showed that HPH rat model was reproduced successfully, see Table 1.
Recent studies showed that gaseous transmitters, nitric oxide (NO), and carbon monoxide (CO), generated from vessels played important roles in the pathogenesis of HPH. And NO has now become an important target in the prevention and treatment of pulmonary and systemic hypertension [2,4]. But the mechanisms of HPH are still not fully understood. Hydrogen sulfide (H2 S) has been best known for decades as a toxic gas. Less recognized, however, is the fact that H2 S is also a biological gas endogenously generated from thiol-containing amino acids in a reaction catalyzed by cystathionine b-synthesis (CBS) and cystathionine c-lyase (CSE). It was reported that there were rich of CSE mRNA in the wall of pulmonary arteries, aorta, tail arteries, mesenteric arteries, and portal vein. But there were no CSE mRNA expressions in the vascular endothelium [11]. The serum level of H2 S was found to be 45:6 14:2 lmol/liter. The H2 S production in homogenates of portal vein and thoracic aorta was 19:6 2:8 and 33:7 2:2 nmol/min/g protein, respectively, in the presence of 10 mM L-cysteine and 2 mM pyridoxal 50 -phosphate [12]. In our study, we found that H2 S concentration in plasma of rats in Hypoxia group was significantly decreased compared with that in Control group (192:2 22:1 vs. 301:6 32:4 lmol/liter, P < 0:01). We also found that H2 S production in homogenates of lung tissues decreased significantly by 53.5% in rats of Hypoxia group compared with that in Control group (0:127 0:023 nmol/mg wet tissue.min vs. 0:278 0:099 nmol/mg wet tissue.min, P < 0:01). It was demonstrated that the activity of H2 S generating enzymes in lung tissues could be represented by H2 S production in homogenates of lung tissue in the presence of 10 mM Lcysteine and 2 mM pyridoxal 50 -phosphate [7]. And the H2 S generating enzymes existed in pulmonary artery were CSE instead of CBS. Therefore, it was suggested that the inhibition of CSE activity in lung tissues resulted in a decreased level of H2 S in plasma during hypoxia, see Table 2. We also detected the CSE gene expression in lung tissue by competitive RT-PCR. The relative amount of CSE mRNA in lung tissue of rats in Hypoxia group
Table 1 Comparison of hemodynamic parameters among the three groups of rats Group
N
mPAP (mmHg)
mAP (mmHg)
RV/LV + SP (mg/mg)
Control Hypoxia Hypoxia + NaHS
6 7 6
16:3 3:7 23:7 2:2a 16:3 2:8b
108:9 17:5 124:1 14:3 117:3 13:0
0:29 0:02 0:41 0:03a 0:31 0:02b
mPAP: mean pulmonary arterial pressure, mAP: mean aortic pressure, RV/(LV þ SP): the wet weight of right ventricle/(left ventricle þ septum). Compared with rats in Control group, the mPAP, and the ratio of RV/(LV þ SP) for rats in Hypoxia group increased significantly (P all < 0:01). Compared with rats in Hypoxia group, the mPAP and the ratio of RV/(LV þ SP) in rats of Hy þ NaHS group decreased significantly (P all < 0:01). There were no differences in the mean aortic pressure among the three groups of rats (P > 0:05). a vs. Control group, P < 0:01. b vs. Hypoxia group, P < 0:01.
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Table 2 Comparison of the plasma level of H2 S, H2 S production, and the amount of CSE mRNA in lung tissues among the three groups of rats Group
N
Plasma level of H2 S (lM)
H2 S production (nmol/mg wet tissue.min)
CSE mRNA (10 6 fmol)
Control Hypoxia Hy þ NaHS
6 7 6
301:6 32:4 192:2 22:1a 317:2 36:4b
0:278 0:099 0:127 0:023a 0:213 0:038b
2:17 0:22 1:02 0:15a 2:13 0:20b
The plasma level of H2 S, the H2 S production, and the amount of CSE mRNA in lung tissues all decreased significantly in Hypoxia group compared with that in Control group. The plasma level of H2 S, the H2 S production, and the amount of CSE mRNA in lung tissues all increased markedly in Hy þ NaHS group compared with that in Hypoxia group. a vs. Control group, P < 0:01. b vs. Hypoxia group, P < 0:01.
decreased by 53% compared with that in Control group (1:02 0:15 10 6 vs. 2:17 0:22 10 6 fmol, P < 0:01). It was indicated that CSE gene expression was down-regulated under hypoxia, see Table 2, Figs. 1 and 2. The down-regulation of CSE gene expression resulted in a suppression of H2 S production which might be responsible for the decreased H2 S level in plasma under hypoxia. These results suggested that the endogenous H2 S system might be involved in the pathogenesis of HPH. To further explore the role of H2 S during HPH, we attempted to supply exogenously H2 S to examine if it could reverse HPH. It was reported that H2 S existed in the body in two forms. In physiological saline, about one-third of the H2 S existed as the undissociated form (H2 S) and the remaining two-thirds as HS at equilibrium with H2 S [11]. Sodium hydrosulfide (NaHS) dissociates to Naþ and HS in solution and then HS associates with Hþ to produce H2 S. The use of NaHS as the donor of H2 S enabled us to define the concentration of H2 S in solution more accurately and reproducibly
Fig. 1. Comparison of relative amount of CSE mRNA in lung tissues among the three groups of rats. Legends for the figure: , control: Control group; , hypoxia: Hypoxia group; , hy þ NaHS: Hy þ NaHS group. *P < 0:01 vs. Control group; xP < 0:01 vs. Hypoxia group. The amount of CSE mRNA in lung tissue decreased significantly in rats of Hypoxia group compared with that in rats of Control group (P < 0:01); while the CSE mRNA amount increased markedly in rats of Hypoxia + NaHS group compared with that in rats of Hypoxia group (P < 0:01).
Fig. 2. Electrophoresis of CSE RT-PCR products in agarose gel after competitive PCR. Marker: DNA marker; Control: Control group; Hypoxia: Hypoxia group; Hy þ NaHS: Hy þ NaHS group. The two bands in agarose gel stained by ethidium bromide are the CSE wildtype fragment (400 bp) and CSE competitive internal standard (361 bp), respectively. The ratio of the optical density of the two bands (400/361 bp) represents the ratio of the amount of (CSE wild-type/ competitive internals) in samples. For the three groups, since the amount of competitive internals added is identical in each PCR, therefore, the ratio of the optical density of the two bands (400/361 bp) represents the relative amount of CSE wild-type in sample, which represents the CSE mRNA amounts. We can see that the relative amount of CSE mRNA is decreased in Hypoxia group compared with that in Control group; while it is increased in Hy þ NaHS group than that in Hypoxia group.
than bubbling H2 S gas into water. For these reasons, NaHS solution was used to provide exogenous H2 S. We selected the dosage of 14 lmol/kg body weight intraperitoneally to supply exogenous H2 S for rats in Hy þ NaHS group referring to ZhaoÕs study [9]. For rats in Hy þ NaHS group, both the mPAP and the ratio of RV/(LV þ SP) decreased markedly compared with rats in Hypoxia group (16:3 2:8 vs. 23:7 2:2 mmHg, and 0:31 0:02 vs. 0:41 0:03, respectively, P all < 0:01). The results showed that exogenously applied H2 S could exert protective effect on the development of HPH, see Table 1.
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In our subsequent study, we found that both the plasma levels of H2 S and H2 S production in lung tissues in rats of Hy þ NaHS group increased compared with those in Hypoxia group (317:2 36:4 vs. 192:2 22:1 lmol/liter, and 0:213 0:038 nmol/mg wet tissue.min vs. 0:127 0:023 nmol/mg wet tissue, respectively, P all < 0:01). The level of CSE gene expression which was represented by the amount of CSE mRNA was also enhanced significantly in rats of Hy þ NaHS group compared with those in Hypoxia group (2:13 0:20 10 6 vs. 1:02 0:15 10 6 fmol, P all < 0:01), see Table 2, Figs. 1 and 2. The above results showed that exogenously supplied NaHS could increase both the plasma level of H2 S and the activity of CSE. The CSE gene expression was also up-regulated by exogenously applied NaHS. And we assumed that the up-regulation of CSE gene expression and subsequent reinforcement of CSE activity were responsible for the increase of plasma level of H2 S. Table 3 Comparison of the relative medial thickness (RMT) and relative medial areas (RMA) of the pulmonary small arteries among the three groups of rats Group
N
RMT (%)
RMA (%)
Control Hypoxia Hy þ NaHS
6 7 6
6:80 1:37 9:56 1:37a 6:14 0:83b
11:77 1:11 16:34 2:57a 11:27 1:81b
Both the RMT and RMA of the pulmonary small arteries in Hypoxia group increased significantly compared with that in Control group. Both the RMT and RMA of the pulmonary small arteries in Hy þ NaHS group decreased significantly compared with that in Hypoxia group. a vs. Control group, P < 0:01. b vs. Hypoxia group, P < 0:01.
Since H2 S could relax the smooth muscle of ileum, portal vein, and thoracic aorta in vitro, and decrease the mean arterial pressure in vivo [8,9], the up-regulation of H2 S system was responsible for the alleviation of HPH in rats of Hy þ NaHS group after the administration of NaHS. The intracellular signal pathway of H2 S was not fully clear. Zhao et al. found that specific KATP channel blocker, glibenclamide, could inhibit H2 S-induced vasorelaxation. It was suggested that H2 S was one of the specific KATP channel openers. Additionally, we assumed that exogenously applied NaHS might exert positive feedback on the gene expression and the subsequent enzymatic activity of CSE. The up-regulation of both CSE gene expression and enzymatic activity could result in more endogenous H2 S production and thus exert anti-HPH effect more effectively in collaboration with exogenously applied H2 S. In this study, we found that with exogenous application of H2 S, the pulmonary vascular structure remodeling which was identified by the thickness of vascular wall was also lessened. The RMT and RMA of the pulmonary small arteries whose outer diameter ranged from 20 to 50 lm decreased markedly by 36% and 31%, respectively, in rats of Hy þ NaHS group compared with those in Hypoxia group, see Table 3 and Figs. 3A–C. The ultrastructure changes of the pulmonary small arteries also improved significantly for rats in Hy þ NaHS group compared with rats in Hypoxia group. The endothelial cells were flat, the size of smooth muscle cells was small and in fusiform shape, with less organelles and much more dense bodies in rats of Hy þ NaHS group compared with that in rats of Hypoxia group. See Figs. 4A–C.
Fig. 3. Elastin fiber staining of ratÕs small pulmonary arteries under optical microscope. (A) Control group: the wall of small artery is thinner than that in hypoxic group. (B) Hypoxia group: the wall of small artery is thicker than that in Control group. (C) Hy þ NaHS group: the wall of small artery is thinner than that in Hypoxia group.
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Fig. 4. Ultrastructure of small pulmonary artery stained with uranylacetate and lead citrate under transmission electron microscope. EC, endothelial cell; SMC, smooth muscle cell; EL, internal elastic lamina. (A) Control group: the endothelial cells were relatively flat, the internal elastic laminar was regular, and the smooth muscle cells were small in size with many dense bodies and fewer organelles in cytoplasm. (B) Hypoxia group: the endothelial cells were hyperplastic, the internal elastic laminar was irregular with breaks, and the smooth muscle cells were hypertrophic with numerous organelles in cytoplasm. (C) Hy þ NaHS group: the endothelial cells were flat and the internal elastic laminar was regular with less breaks. The smooth muscle cells were small in size with many dense bodies and fewer organelles in cytoplasm.
Our previous study showed that H2 S could suppress the proliferation of vascular smooth muscle cells and the suppressive effect may be due to the inhibition of MAPK activity [10]. Therefore, the lessening of vascular structure remodeling may be due to the inhibitory effect of H2 S on proliferation of vascular smooth muscle cells. But there are still many questions that need to be studied. For example, what will happen if exogenous H2 S was supplied to the rats without hypoxia exposure, and what will happen if specific CSE inhibitor, D ,L propargylglycerine, was used to block the endogenous H2 S generation. Given the fact that H2 S, NO, and CO can all be gaseous transmitters, are there any interactions between them? Wang [19] hypothesized that NO and CO together with H2 S formed an interactive network. Published data [12] have shown that the endogenous production of H2 S from rat aortic tissue was enhanced by NO donor treatment. NO donor also enhanced the expression level of CSE in cultured vascular SMCs. Hosoki et al. [11] found that the vasorelaxant effect of sodium nitroprusside (SNP), a NO donor, was enhanced by incubating rat aortic tissues with 30 lM NaHS. On the contrary, in another study, pretreating aortic tissues with 60 lM H2 S inhibited the vasorelaxant effect of SNP [20]. In conclusion, the CSE gene expression was downregulated, the CSE activity was suppressed, and the plasma level of H2 S also decreased during HPH. Therefore, the present study showed that the down-
regulation of endogenous H2 S system was involved in the pathogenesis of HPH in rat. We also demonstrated that exogenously applied H2 S could exert protective effect on the pathogenesis of HPH. Whether H2 S could become a new target of prevention and treatment of HPH needs further studies.
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The regulatory effect of hydrogen sulfide on hypoxic pulmonary hypertension in ratsq Zhang Chunyu,a Du Junbao,a,* Bu Dingfang,b Yan Hui,a Tang Xiuying,c and Tang Chaoshud a
Department of Pediatrics, Peking University First Hospital, Xi-An Men Street No. 1, Beijing 100034, PeopleÕs Republic of China b Central Laboratory, Peking University First Hospital, PeopleÕs Republic of China c Laboratory of Electron Microscopy, Peking University First Hospital, PeopleÕs Republic of China d Institute of Cardiovascular Diseases, Peking University First Hospital, PeopleÕs Republic of China Received 1 February 2003
Abstract Hypoxic pulmonary hypertension (HPH) is an important pathophysiological process. The mechanism of HPH is still not fully understood. Recent studies showed that hydrogen sulfide (H2 S) could relax vascular smooth muscles and inhibit the proliferation of cultured vascular smooth muscle cells. Our study showed that both the gene expression of cystathionine c-lyase (CSE), one of the H2 S generating enzymes, and the activity of CSE were suppressed in lung tissues during HPH. And the plasma level of H2 S was decreased during HPH. Exogenous supply of H2 S could increase the plasma level of H2 S, enhance CSE activity, and up-regulate CSE gene expression in lung tissue. At the same time, exogenous supply of H2 S could oppose the elevation of pulmonary arterial pressure and lessen the pulmonary vascular structure remodeling during HPH. The results showed that endogenous H2 S system was involved and exogenous H2 S could exert beneficial effect on the pathogenesis of HPH. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Hypoxia; Hydrogen sulfide; Pulmonary hypertension
Hypoxic pulmonary hypertension (HPH) is an important pathophysiological process which could occur in many cardiac and pulmonary diseases. Acute or chronic exposure to hypoxia could result in HPH which is characterized by high pressure in pulmonary artery and high pulmonary vascular resistance. The rise in pulmonary arterial pressure and vascular resistance is the result of a combination of hypoxic vasoconstriction and vascular structure remodeling. But the mechanisms of HPH are still not fully understood. q
This study was supported by The National Natural Science Foundation of China (39870844, 30070796, and 30271373), the Doctorate Training Program Foundation of the Ministry of Education of China (20020001063), Peking University 985 Cardiovascular Research Grant, and The Major Basic Research Program of China (G2000056905). * Corresponding author. Fax: +86-10-6613-4261. E-mail addresses: [email protected], [email protected] (J. Du).
It has been proved by a variety of experiments and clinical materials that endogenous gaseous transmitters including nitric oxide (NO) and carbon monoxide (CO) in the body play important roles in the pathogenesis of HPH. And NO inhalation has become an important option of treating and preventing HPH [1–4]. Recently, it was indicated that hydrogen sulfide (H2 S) which has been known as toxic gas for a long time could also be endogenously generated. H2 S is generated from cysteine in a reaction catalyzed by cystathionine b-synthesis (CBS) and cystathionine c-lyase (CSE) [5–7]. Recent studies also showed that H2 S had important physiological functions including relaxing rat aortic artery in vitro and inhibiting cultured vascular smooth muscle cell proliferation [8–10]. H2 S at physiological concentrations could also facilitate the induction of hippocampus long-term potentiation (LTP) [11–13]. But to date, the role of H2 S in the pathogenesis of HPH has not been reported.
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00256-0
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The present study was to investigate the changes of endogenous H2 S during the development of HPH and its possible role in the pathogenesis of HPH. Materials and methods Hypoxic pulmonary hypertension rat model. The study was approved by the Animal Research Committee of Peking University. The rats were exposed to hypoxia according to the methods of Xue [14]. Nineteen male Wistar rats (180–220 g) were randomly divided into Control group (n ¼ 6), Hypoxia group (n ¼ 7), and Hy þ NaHS group (n ¼ 6). The rats were exposed to normobaric hypoxia (10% O2 ) in a transparent plastic hypoxia chamber (for rats in Hypoxia group and Hy þ NaHS group) for 3 weeks and 6 h everyday. Rats in Control group were housed in identical cages adjacent to the hypoxia chamber breathing room air. Hypoxia was generated by infusing N2 into the chamber. The degree of hypoxia was maintained by the balance between nitrogen infusing and the inward leak of air through holes in the chamber. For rats in Hy þ NaHS group, NaHS dissolved in physiological saline at a dosage of 14 lmol/kg body weight was injected intraperitoneally before hypoxia everyday. The same volume of physiological saline was injected for rats in the other two groups. Measurement of hemodynamic parameters and sample preparation. Three weeks after hypoxic exposure, the rats were anesthetized with urethane (1 g/kg body weight) intraperitoneally. A silicone catheter (outer diameter 0.9 mm) was introduced into the right jugular vein via a venotomy and passed across the tricuspid valve and right ventricle into pulmonary artery. The mean pulmonary artery pressure (mPAP) was simultaneously recorded. The mean aortic pressure (mAP) was also recorded by carotid artery catheterization. After the chest was opened, the right lower part of the lung tissue was rapidly removed to liquid nitrogen for quick freezing and then stored at )70 °C. Then the heart was excised and the atrias were removed. The free wall of right ventricle (RV) was separated from the heart. These tissues were blotted and weighed using an electronic scale. The wet weight ratio of RV against left ventricle plus septum, RV/(LV þ SP), was calculated as an indicator of right ventricular hypertrophy. The blood plasma was also prepared for the measurement of plasma H2 S concentration. Measurement of H2 S concentration in the plasma. In a test tube containing 0.5 ml of 1% zinc acetate and 2.5 ml of distilled water, 0.1 ml of plasma was added. Then 0.5 ml of 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 M HCl and 0.4 ml of 30 mM FeCl3 in 1.2 M HCl were also added to the test tube for 20 min of incubation at room temperature. The protein in the plasma was removed by adding 1 ml of 10% trichloroacetic acid to the solution and centrifuged. The optical absorbance of the resulting solution at 670 nm was measured with a spectrometer (Shimadzu UV 2100, Japan). The H2 S concentration in the solution was calculated against the calibration curve of the standard H2 S solution. Measurement of H2 S production. According to StipanukÕs methods [7], the lung tissues were homogenized in ice-cold 50 mM potassium phosphate buffer (PH 6.8). Reactions were performed in 25 ml Erlenmeyer flasks. The reaction mixture contained (mM): 10 L-cysteine, 2 pyridoxal 50 -phosphate, 100 potassium phosphate buffer (PH 7.4), and 10% (w/v) homogenates. The total volume of the reaction mixture was 1 ml. A small piece of filter paper was put into the central well of the flask and 0.5 ml of 1% zinc acetate was also added in the central well for trapping evolved H2 S in the mixture. The flasks were then flushed with N2 before being sealed with a double layer of parafilm. The catalytic reaction was initiated by transferring the flasks from an ice bath to a 37 °C shaking water bath. After 90 min at 37 °C, the reactions were stopped by injecting 0.5 ml of 50% trichloroacetic acid. Flasks were incubated in the shaking water bath for an additional hour at 37 °C to complete trapping of H2 S. The content of the central well was trans-
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ferred to test tubes and mixed with 3.5 ml of distilled water and 0.5 ml of 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 M HCl. To each tube, 0.4 ml of 30 mM FeCl3 in 1.2 M HCl was added immediately. After 20 min of incubation at room temperature, the optical absorbance of the resulting solution at 670 nm was measured with a spectrometer (Shimadzu UV 2100, Japan). The H2 S concentration in the solution was calculated against the calibration curve of the standard H2 S solution. For each sample, the measurement was done in duplicate. The H2 S production was expressed in a unit of nmol/mg wet tissue min. Measurement of CSE mRNA in lung tissue by quantitative reverse transcription-polymerase chain reaction. Total RNA was extracted from rat lung tissue using TRIzol reagent. The cDNA was synthesized using oligo ðdTÞ15 primer and M-MLV reverse transcriptase. The polymerase chain reaction (PCR) primers used to amplify the fragment of CSE cDNA were: CSE-S : 50 -TCCGG ATGGA GAAAC ACTTC CSE-A : 50 -GCTGC CTTTA AAGCT TGACC PCR using these two primers yielded a 400 bp fragment of wildtype rat CSE cDNA. The competitive internal standard for the measurement of CSE cDNA had the same sequences as the 400 bp fragment of wild-type rat CSE cDNA, except that a fragment of 39 bp at the downstream site of CSE-S primer was deleted [15,16]. Quantitative PCR was performed in a 0.2 ml of PCR tube containing 1 ll of rat lung cDNA, 1 ll of 3.9 fmol/liter competitive internal standard, 1 ll of 5 lM/each CSE-S and CSE-A mixture, 1 ll of 2.5 mM/each dNTP mixture, 2 mM MgCl2 , 2.5 ll of 10 PCR buffer, and 1.25 U Taq DNA polymerase, in a total volume of 25 ll. PCR products were separated in a 2% agarose gel and stained by ethidium bromide. The ratio of optical density of the two DNA bands was measured by a Gel Image Analysis System (AlphaImager, Alpha Innotech, CA, USA) under UV light. A standard curve of the ratio was drawn using the same condition as described above except that rat lung cDNA was changed to a series of dilutions of the plasmid containing the 400 bp wild-type CSE cDNA fragment. The relative amount of CSE cDNA in samples was then obtained from the standard curve [17]. To calibrate the amount of sample loaded in PCR mixture, b-actin cDNA was measured using the same method after the quantitative PCR. Three ll of PCR product was amplified again using the two rat b-actin primers (b-actin-S: 50 -ATC TG GCACC ACACC TTC, b-actin-A: 50 -AGCCA GGTCC AGACG CA). Relative amount of b-actin cDNA in loaded sample was then obtained from a pre-made standard curve of b-actin cDNA measurement. Standardized relative amount of CSE cDNA was used for further analysis. Morphological analysis of small pulmonary arteries. The left lower part of lung tissue was removed to 10% (wt/vol) paraformaldehyde for fixation. Then the lung tissues were dehydrated, embedded in paraffin, and sectioned at a thickness of 4 lm. The elastic fiber in lung tissues was stained according to the modified WeigertÕs method and counterstained with Van Gieson solution. Morphological analysis was performed using a video-linked microscope digitizing board system (Leica Q550CW, Germany). Only vessels showing clearly defined external and internal elastic lamina were used in analysis. According to BathÕs methods [18], the relative medial thickness (RMT) and relative medial areas (RMA) were calculated. A small part of lung tissue was cut from the upper part of left lung lobe and quickly immersed into 3% glutaraldehyde. And then it was cut into pieces of 1 mm 1 mm 1 mm in size and post–fixed in 1% phosphate-buffered osmium tetroxide for 6 h. Then it was rinsed again (overnight) and dehydrated in a graded series of ethanol. These specimens were infiltrated with propylene oxide and embedded with Epon812. These procedures were all done at room temperature. Finally, specimens were embedded in new batches of Epon812 and polymerized at 40°C (24 h) and 60°C (48 h). From selected blocks, series of transverse or longitudinal sections were made. Semi thin sections
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(1 lm) were stained with azur-II and methylene blue. Ultra thin sections (60–90 nm) were made with an ultra microtome and it was mounted on formvar-coated copper grids (75 meshes). Then it was stained with uranylacetate and lead citrate, and examined under a transmission electron microscope (JEM-100CX, JEOL, Japan) in detail. Chemicals and reagents. NaHS solution was freshly prepared by mixing the stock solution of sodium sulfide and hydrochloric acid. Phosphate 50 -pyridoxal was purchased from Sigma Corporation. TRIzol reagent was bought from Invitrogen, Oligo dðTÞ15 primer, dNTP, and M-MLV reverse transcriptase were bought from Promega Corporation. All the other chemicals were purchased from Beijing Chemical Reagents, China. Data analysis. The results were expressed as means SD. For comparison of the differences among the three groups, ANOVA followed by a post hoc analysis (Bonferroni test) was used by SPSS 10.0 statistic analysis software. A value of P < 0:05 was considered statistically significant.
Results and discussion Pulmonary artery hypertension refers to a group of diseases characterized by high pressure in the pulmonary artery and high pulmonary vascular resistance. Pulmonary artery hypertension is diagnosed when the systolic pulmonary artery pressure is higher than 30 mmHg (4.0 kPa), or the mean pulmonary artery pressure (mPAP) is higher than 20 mmHg (2.6 kPa). Chronic exposure to hypoxia results in hypoxic pulmonary artery hypertension. It was reported that after intermittent exposure to 10% of oxygen 6 h daily for 2–4 weeks, the rat could develop constant chronic pulmonary hypertension with right ventricular hypertrophy [14]. In our study, after 3 weeks of hypoxia, mPAP increased significantly compared with rats without hypoxia (23:7 2:2 vs. 16:3 3:7 mmHg, P < 0:01). We used RV/(LV þ SP), which stood for the wet weight ratio of right ventricular free wall vs. left ventricular plus septum. The ratio of RV/(LV þ SP), an indicator of right ventricular hypertrophy, also increased markedly in rats of Hypoxia group compared with that in Control group (0:41 0:03 vs. 0:29 0:02, P < 0:01). These results showed that HPH rat model was reproduced successfully, see Table 1.
Recent studies showed that gaseous transmitters, nitric oxide (NO), and carbon monoxide (CO), generated from vessels played important roles in the pathogenesis of HPH. And NO has now become an important target in the prevention and treatment of pulmonary and systemic hypertension [2,4]. But the mechanisms of HPH are still not fully understood. Hydrogen sulfide (H2 S) has been best known for decades as a toxic gas. Less recognized, however, is the fact that H2 S is also a biological gas endogenously generated from thiol-containing amino acids in a reaction catalyzed by cystathionine b-synthesis (CBS) and cystathionine c-lyase (CSE). It was reported that there were rich of CSE mRNA in the wall of pulmonary arteries, aorta, tail arteries, mesenteric arteries, and portal vein. But there were no CSE mRNA expressions in the vascular endothelium [11]. The serum level of H2 S was found to be 45:6 14:2 lmol/liter. The H2 S production in homogenates of portal vein and thoracic aorta was 19:6 2:8 and 33:7 2:2 nmol/min/g protein, respectively, in the presence of 10 mM L-cysteine and 2 mM pyridoxal 50 -phosphate [12]. In our study, we found that H2 S concentration in plasma of rats in Hypoxia group was significantly decreased compared with that in Control group (192:2 22:1 vs. 301:6 32:4 lmol/liter, P < 0:01). We also found that H2 S production in homogenates of lung tissues decreased significantly by 53.5% in rats of Hypoxia group compared with that in Control group (0:127 0:023 nmol/mg wet tissue.min vs. 0:278 0:099 nmol/mg wet tissue.min, P < 0:01). It was demonstrated that the activity of H2 S generating enzymes in lung tissues could be represented by H2 S production in homogenates of lung tissue in the presence of 10 mM Lcysteine and 2 mM pyridoxal 50 -phosphate [7]. And the H2 S generating enzymes existed in pulmonary artery were CSE instead of CBS. Therefore, it was suggested that the inhibition of CSE activity in lung tissues resulted in a decreased level of H2 S in plasma during hypoxia, see Table 2. We also detected the CSE gene expression in lung tissue by competitive RT-PCR. The relative amount of CSE mRNA in lung tissue of rats in Hypoxia group
Table 1 Comparison of hemodynamic parameters among the three groups of rats Group
N
mPAP (mmHg)
mAP (mmHg)
RV/LV + SP (mg/mg)
Control Hypoxia Hypoxia + NaHS
6 7 6
16:3 3:7 23:7 2:2a 16:3 2:8b
108:9 17:5 124:1 14:3 117:3 13:0
0:29 0:02 0:41 0:03a 0:31 0:02b
mPAP: mean pulmonary arterial pressure, mAP: mean aortic pressure, RV/(LV þ SP): the wet weight of right ventricle/(left ventricle þ septum). Compared with rats in Control group, the mPAP, and the ratio of RV/(LV þ SP) for rats in Hypoxia group increased significantly (P all < 0:01). Compared with rats in Hypoxia group, the mPAP and the ratio of RV/(LV þ SP) in rats of Hy þ NaHS group decreased significantly (P all < 0:01). There were no differences in the mean aortic pressure among the three groups of rats (P > 0:05). a vs. Control group, P < 0:01. b vs. Hypoxia group, P < 0:01.
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Table 2 Comparison of the plasma level of H2 S, H2 S production, and the amount of CSE mRNA in lung tissues among the three groups of rats Group
N
Plasma level of H2 S (lM)
H2 S production (nmol/mg wet tissue.min)
CSE mRNA (10 6 fmol)
Control Hypoxia Hy þ NaHS
6 7 6
301:6 32:4 192:2 22:1a 317:2 36:4b
0:278 0:099 0:127 0:023a 0:213 0:038b
2:17 0:22 1:02 0:15a 2:13 0:20b
The plasma level of H2 S, the H2 S production, and the amount of CSE mRNA in lung tissues all decreased significantly in Hypoxia group compared with that in Control group. The plasma level of H2 S, the H2 S production, and the amount of CSE mRNA in lung tissues all increased markedly in Hy þ NaHS group compared with that in Hypoxia group. a vs. Control group, P < 0:01. b vs. Hypoxia group, P < 0:01.
decreased by 53% compared with that in Control group (1:02 0:15 10 6 vs. 2:17 0:22 10 6 fmol, P < 0:01). It was indicated that CSE gene expression was down-regulated under hypoxia, see Table 2, Figs. 1 and 2. The down-regulation of CSE gene expression resulted in a suppression of H2 S production which might be responsible for the decreased H2 S level in plasma under hypoxia. These results suggested that the endogenous H2 S system might be involved in the pathogenesis of HPH. To further explore the role of H2 S during HPH, we attempted to supply exogenously H2 S to examine if it could reverse HPH. It was reported that H2 S existed in the body in two forms. In physiological saline, about one-third of the H2 S existed as the undissociated form (H2 S) and the remaining two-thirds as HS at equilibrium with H2 S [11]. Sodium hydrosulfide (NaHS) dissociates to Naþ and HS in solution and then HS associates with Hþ to produce H2 S. The use of NaHS as the donor of H2 S enabled us to define the concentration of H2 S in solution more accurately and reproducibly
Fig. 1. Comparison of relative amount of CSE mRNA in lung tissues among the three groups of rats. Legends for the figure: , control: Control group; , hypoxia: Hypoxia group; , hy þ NaHS: Hy þ NaHS group. *P < 0:01 vs. Control group; xP < 0:01 vs. Hypoxia group. The amount of CSE mRNA in lung tissue decreased significantly in rats of Hypoxia group compared with that in rats of Control group (P < 0:01); while the CSE mRNA amount increased markedly in rats of Hypoxia + NaHS group compared with that in rats of Hypoxia group (P < 0:01).
Fig. 2. Electrophoresis of CSE RT-PCR products in agarose gel after competitive PCR. Marker: DNA marker; Control: Control group; Hypoxia: Hypoxia group; Hy þ NaHS: Hy þ NaHS group. The two bands in agarose gel stained by ethidium bromide are the CSE wildtype fragment (400 bp) and CSE competitive internal standard (361 bp), respectively. The ratio of the optical density of the two bands (400/361 bp) represents the ratio of the amount of (CSE wild-type/ competitive internals) in samples. For the three groups, since the amount of competitive internals added is identical in each PCR, therefore, the ratio of the optical density of the two bands (400/361 bp) represents the relative amount of CSE wild-type in sample, which represents the CSE mRNA amounts. We can see that the relative amount of CSE mRNA is decreased in Hypoxia group compared with that in Control group; while it is increased in Hy þ NaHS group than that in Hypoxia group.
than bubbling H2 S gas into water. For these reasons, NaHS solution was used to provide exogenous H2 S. We selected the dosage of 14 lmol/kg body weight intraperitoneally to supply exogenous H2 S for rats in Hy þ NaHS group referring to ZhaoÕs study [9]. For rats in Hy þ NaHS group, both the mPAP and the ratio of RV/(LV þ SP) decreased markedly compared with rats in Hypoxia group (16:3 2:8 vs. 23:7 2:2 mmHg, and 0:31 0:02 vs. 0:41 0:03, respectively, P all < 0:01). The results showed that exogenously applied H2 S could exert protective effect on the development of HPH, see Table 1.
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In our subsequent study, we found that both the plasma levels of H2 S and H2 S production in lung tissues in rats of Hy þ NaHS group increased compared with those in Hypoxia group (317:2 36:4 vs. 192:2 22:1 lmol/liter, and 0:213 0:038 nmol/mg wet tissue.min vs. 0:127 0:023 nmol/mg wet tissue, respectively, P all < 0:01). The level of CSE gene expression which was represented by the amount of CSE mRNA was also enhanced significantly in rats of Hy þ NaHS group compared with those in Hypoxia group (2:13 0:20 10 6 vs. 1:02 0:15 10 6 fmol, P all < 0:01), see Table 2, Figs. 1 and 2. The above results showed that exogenously supplied NaHS could increase both the plasma level of H2 S and the activity of CSE. The CSE gene expression was also up-regulated by exogenously applied NaHS. And we assumed that the up-regulation of CSE gene expression and subsequent reinforcement of CSE activity were responsible for the increase of plasma level of H2 S. Table 3 Comparison of the relative medial thickness (RMT) and relative medial areas (RMA) of the pulmonary small arteries among the three groups of rats Group
N
RMT (%)
RMA (%)
Control Hypoxia Hy þ NaHS
6 7 6
6:80 1:37 9:56 1:37a 6:14 0:83b
11:77 1:11 16:34 2:57a 11:27 1:81b
Both the RMT and RMA of the pulmonary small arteries in Hypoxia group increased significantly compared with that in Control group. Both the RMT and RMA of the pulmonary small arteries in Hy þ NaHS group decreased significantly compared with that in Hypoxia group. a vs. Control group, P < 0:01. b vs. Hypoxia group, P < 0:01.
Since H2 S could relax the smooth muscle of ileum, portal vein, and thoracic aorta in vitro, and decrease the mean arterial pressure in vivo [8,9], the up-regulation of H2 S system was responsible for the alleviation of HPH in rats of Hy þ NaHS group after the administration of NaHS. The intracellular signal pathway of H2 S was not fully clear. Zhao et al. found that specific KATP channel blocker, glibenclamide, could inhibit H2 S-induced vasorelaxation. It was suggested that H2 S was one of the specific KATP channel openers. Additionally, we assumed that exogenously applied NaHS might exert positive feedback on the gene expression and the subsequent enzymatic activity of CSE. The up-regulation of both CSE gene expression and enzymatic activity could result in more endogenous H2 S production and thus exert anti-HPH effect more effectively in collaboration with exogenously applied H2 S. In this study, we found that with exogenous application of H2 S, the pulmonary vascular structure remodeling which was identified by the thickness of vascular wall was also lessened. The RMT and RMA of the pulmonary small arteries whose outer diameter ranged from 20 to 50 lm decreased markedly by 36% and 31%, respectively, in rats of Hy þ NaHS group compared with those in Hypoxia group, see Table 3 and Figs. 3A–C. The ultrastructure changes of the pulmonary small arteries also improved significantly for rats in Hy þ NaHS group compared with rats in Hypoxia group. The endothelial cells were flat, the size of smooth muscle cells was small and in fusiform shape, with less organelles and much more dense bodies in rats of Hy þ NaHS group compared with that in rats of Hypoxia group. See Figs. 4A–C.
Fig. 3. Elastin fiber staining of ratÕs small pulmonary arteries under optical microscope. (A) Control group: the wall of small artery is thinner than that in hypoxic group. (B) Hypoxia group: the wall of small artery is thicker than that in Control group. (C) Hy þ NaHS group: the wall of small artery is thinner than that in Hypoxia group.
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Fig. 4. Ultrastructure of small pulmonary artery stained with uranylacetate and lead citrate under transmission electron microscope. EC, endothelial cell; SMC, smooth muscle cell; EL, internal elastic lamina. (A) Control group: the endothelial cells were relatively flat, the internal elastic laminar was regular, and the smooth muscle cells were small in size with many dense bodies and fewer organelles in cytoplasm. (B) Hypoxia group: the endothelial cells were hyperplastic, the internal elastic laminar was irregular with breaks, and the smooth muscle cells were hypertrophic with numerous organelles in cytoplasm. (C) Hy þ NaHS group: the endothelial cells were flat and the internal elastic laminar was regular with less breaks. The smooth muscle cells were small in size with many dense bodies and fewer organelles in cytoplasm.
Our previous study showed that H2 S could suppress the proliferation of vascular smooth muscle cells and the suppressive effect may be due to the inhibition of MAPK activity [10]. Therefore, the lessening of vascular structure remodeling may be due to the inhibitory effect of H2 S on proliferation of vascular smooth muscle cells. But there are still many questions that need to be studied. For example, what will happen if exogenous H2 S was supplied to the rats without hypoxia exposure, and what will happen if specific CSE inhibitor, D ,L propargylglycerine, was used to block the endogenous H2 S generation. Given the fact that H2 S, NO, and CO can all be gaseous transmitters, are there any interactions between them? Wang [19] hypothesized that NO and CO together with H2 S formed an interactive network. Published data [12] have shown that the endogenous production of H2 S from rat aortic tissue was enhanced by NO donor treatment. NO donor also enhanced the expression level of CSE in cultured vascular SMCs. Hosoki et al. [11] found that the vasorelaxant effect of sodium nitroprusside (SNP), a NO donor, was enhanced by incubating rat aortic tissues with 30 lM NaHS. On the contrary, in another study, pretreating aortic tissues with 60 lM H2 S inhibited the vasorelaxant effect of SNP [20]. In conclusion, the CSE gene expression was downregulated, the CSE activity was suppressed, and the plasma level of H2 S also decreased during HPH. Therefore, the present study showed that the down-
regulation of endogenous H2 S system was involved in the pathogenesis of HPH in rat. We also demonstrated that exogenously applied H2 S could exert protective effect on the pathogenesis of HPH. Whether H2 S could become a new target of prevention and treatment of HPH needs further studies.
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