The Increased Endogenous Sulfur Dioxide Acts as a Compensatory Mechanism for the Downregulated Endogenous Hydrogen Sulfide Pathway in the Endothelial Cell Inflammation

Endogenous hydrogen sulfide (H2S) and sulfur dioxide (SO2) are regarded as important regulators to control endothelial cell function and protect endothelial cell against various injuries. In our present study, we aimed to investigate the effect of endogenous H2S on the SO2 generation in the endothelial cells and explore its significance in the endothelial inflammation in vitro and in vivo. The human umbilical vein endothelial cell (HUVEC) line (EA.hy926), primary HUVECs, primary rat pulmonary artery endothelial cells (RPAECs), and purified aspartate aminotransferase (AAT) protein from pig heart were used for in vitro experiments. A rat model of monocrotaline (MCT)-induced pulmonary vascular inflammation was used for in vivo experiments. We found that endogenous H2S deficiency caused by cystathionine-γ-lyase (CSE) knockdown increased endogenous SO2 level in endothelial cells and enhanced the enzymatic activity of AAT, a major SO2 synthesis enzyme, without affecting the expressions of AAT1 and AAT2. While H2S donor could reverse the CSE knockdown-induced increase in the endogenous SO2 level and AAT activity. Moreover, H2S donor directly inhibited the activity of purified AAT protein, which was reversed by a thiol reductant DTT. Mechanistically, H2S donor sulfhydrated the purified AAT1/2 protein and rescued the decrease in the sulfhydration of AAT1/2 protein in the CSE knockdown endothelial cells. Furthermore, an AAT inhibitor l-aspartate-β-hydroxamate (HDX), which blocked the upregulation of endogenous SO2/AAT generation induced by CSE knockdown, aggravated CSE knockdown-activated nuclear factor-κB pathway in the endothelial cells and its downstream inflammatory factors including ICAM-1, TNF-α, and IL-6. In in vivo experiment, H2S donor restored the deficiency of endogenous H2S production induced by MCT, and reversed the upregulation of endogenous SO2/AAT pathway via sulfhydrating AAT1 and AAT2. In accordance with the results of the in vitro experiment, HDX exacerbated the pulmonary vascular inflammation induced by the broken endogenous H2S production in MCT-treated rat. In conclusion, for the first time, the present study showed that H2S inhibited endogenous SO2 generation by inactivating AAT via the sulfhydration of AAT1/2; and the increased endogenous SO2 generation might play a compensatory role when H2S/CSE pathway was downregulated, thereby exerting protective effects in endothelial inflammatory responses in vitro and in vivo.

Endogenous hydrogen sulfide (H2S) and sulfur dioxide (SO2) are regarded as important regulators to control endothelial cell function and protect endothelial cell against various injuries. In our present study, we aimed to investigate the effect of endogenous H2S on the SO2 generation in the endothelial cells and explore its significance in the endothelial inflammation in vitro and in vivo. The human umbilical vein endothelial cell (HUVEC) line (EA.hy926), primary HUVECs, primary rat pulmonary artery endothelial cells (RPAECs), and purified aspartate aminotransferase (AAT) protein from pig heart were used for in vitro experiments. A rat model of monocrotaline (MCT)-induced pulmonary vascular inflammation was used for in vivo experiments. We found that endogenous H2S deficiency caused by cystathionine-γ-lyase (CSE) knockdown increased endogenous SO2 level in endothelial cells and enhanced the enzymatic activity of AAT, a major SO2 synthesis enzyme, without affecting the expressions of AAT1 and AAT2. While H2S donor could reverse the CSE knockdown-induced increase in the endogenous SO2 level and AAT activity. Moreover, H2S donor directly inhibited the activity of purified AAT protein, which was reversed by a thiol reductant DTT. Mechanistically, H2S donor sulfhydrated the purified AAT1/2 protein and rescued the decrease in the sulfhydration of AAT1/2 protein in the CSE knockdown endothelial cells. Furthermore, an AAT inhibitor l-aspartateβ-hydroxamate (HDX), which blocked the upregulation of endogenous SO2/AAT generation induced by CSE knockdown, aggravated CSE knockdown-activated nuclear factor-κB pathway in the endothelial cells and its downstream inflammatory factors including ICAM-1, TNF-α, and IL-6. In in vivo experiment, H2S donor restored the deficiency of endogenous H2S production induced by MCT, and reversed the upregulation of endogenous SO2/AAT inTrODUcTiOn Hydrogen sulfide (H2S), a new member of gaseous signal mole cule family, has been found as a metabolic end product of sulfur containing amino acids and to be involved in various physiologic and pathophysiologic processes since the end of the last century (1,2). Cystathionineγlysase (CSE) is regarded as a predomi nant H2Sgenerating enzyme in the cardiovascular tissues, and H2S is generated with the substrates of cystathionine or cysteine, catalyzed by CSE (3,4). The regulatory effect of endogenous H2S on the endothelial cell function attracted great attention because of the importance of endothelial cells in the vascular injury and repair. H2S was reported to stimulate the proliferation and migra tion of endothelial cells, promote endothelial cell angiogenesis, inhibit the endothelial cell inflammation, protect mitochondrial function, and mediate endothelialdependent vasorelaxation, etc (5)(6)(7)(8). Plenty of research demonstrated that H2S protected the endothelial cells against various insults from hypoxia, high salt, highglucose, angiotensin II, and tumor necrosis factorα (TNFα), and so on (7,(9)(10)(11)(12). Impaired endogenous H2S produc tion, bioavailability, and its function were involved in the patho genesis of endothelium dysfunctionrelated diseases including hypertension, vascular complication of diabetes, atherosclerosis, restenosis, and aging, etc (13)(14)(15).
Recently, sulfur dioxide (SO2), a brother of H2S, attracted more and more concerns in the field (16,17). SO2 was found to be endogenously generated from the enzymatic reaction catalyzed by aspartate amino transferase (AAT) in the metabolic pathway of sulfurcontaining amino acids (18). Endogenous SO2/AAT path way was discovered to exist in the endothelium, vascular smooth muscles, fibroblasts, cardiac myocytes, adipocyte, and alveolar epithelial cells and play an important role in the cardiovascular homeostasis (19)(20)(21)(22)(23)(24). Our research group firstly put forward the hypothesis that endogenous SO2 might be the fourth gaseous sig nal molecule involved in the regulation of cardiovascular system (25). Endogenous SO2 was discovered to promote the nitric oxide production and enhance the nitric oxideinduced vasodialation (26). It could protect against acute lung injury induced by limb ischemic/reperfusion (I/R) or by lipopolysaccharide or by oleic acid in rats (27)(28)(29). Moreover, AAT1 overexpression could alleviate the lung inflammatory response caused by oleic acid in a mice model of acute lung injury (29).
Collectively, both H2S and SO2 are generated from the same metabolic pathway in the similar origin tissues and exert similar biological effect (24,(29)(30)(31)(32)(33). For instance, Xiao et al. discovered that H2S mitigated cardiomyocyte injury caused by hypoxicreoxygenation via decreasing autophagy (30), while Chen et al. demonstrated that SO2 also alleviated myocardial hypertrophy by inhibiting Ang IIactivated autophagy in mice (31). Furthermore, the two gasotransmitters sometimes share the same signaling pathway, and even the same target residue. The activation of PI3K/Akt pathway mediated the protective effect of H2S preconditioning on the cerebral I/R injury (32). Meanwhile, it was involved in SO2 preconditioninginduced protection against myocardial I/R injury (24). H2S can inactivate inflammatory response by inhibiting the phosphorylation and nuclear translocation of NFκB p65 via sulfhydrating NFκB p65 cysteine 38 (33), whereas SO2 suppresses inflammatory response by sulfenylating NFκB p65 at the same residue (29).
So, here comes the question that what is the significance of the coexistence of H2S and SO2 in the biologic tissues. Li and Luo et al. found that SO2 increased endogenous H2S production in the development of artherosclerosis and pulmonary hypertension, and the upregulation of endogenous H2S pathway might be one of protective mechanisms responsible for endogenous SO2 (23,34). However, the impact of endogenous H2S on the endogenous SO2 production and its significance have been unclear. In the present study, we attempted to construct an endogenous H2S defiency endothelial cell inflammation model by transfecting lentiviruscontaining CSE shRNA using human umbilical vein endothelial cell (HUVEC) line (EA.hy926), investigate the effect of endogenous H2S on the endotheliumderived SO2 generation and explore its significance in the development of inflammatory response induced by the H2S/CSE deficiency. In addition, we also used the primary HUVECs, rat pulmonary artery endothelial cells (RPAECs) and rats with pulmonary vascular inflammation in the study to verify the effect of H2S on the endogenous SO2 production and its implication.

MaTerials anD MeThODs cell culture
The HUVEC line (EA.hy926) was purchased from China Infrastructure of Cell Line Resources Center, China. The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% strepto mycin, and 1% penicillin (Gibco, USA). Primary HUVECs were kindly provided by professor Jing Zhou, Peking University Health Science Center, Beijing, China, and RPAECs (PriCells, Wuhan, China) were cultured in the specialized endothelial cell medium (PriCells, Wuhan, China) supplemented with 10% FBS and 100 pathway via sulfhydrating AAT1 and AAT2. In accordance with the results of the in vitro experiment, HDX exacerbated the pulmonary vascular inflammation induced by the broken endogenous H2S production in MCT-treated rat. In conclusion, for the first time, the present study showed that H2S inhibited endogenous SO2 generation by inactivating AAT via the sulfhydration of AAT1/2; and the increased endogenous SO2 generation might play a compensatory role when H2S/CSE pathway was downregulated, thereby exerting protective effects in endothelial inflammatory responses in vitro and in vivo.
Keywords: endothelial cells, inflammation, sulfhydration, h2s, sO2 IU/mL penicillinstreptomycin. The endothelial cells were main tained in a humidified atmosphere of 5% CO2 at 37°C. CSE knockdown endothelial cells were obtained by infecting lentivirus containing CSE shRNA plus green fluorescent protein (GFP) cDNA (Cyagen, China). For the purpose of determining the appropriate concentration of lentivirus used for the treat ment, the cells were seeded in 6well plate, grown to 60-70% confluence, and transfected with different viral titers of lentivirus (1 × 10 4 to 2 × 10 5 TU/mL). After 12 h of the infection, freshly completed culture medium was replaced. After another 72 h, the green fluorescence of GFP was observed in the successfully trans fected cells under fluorescence microscope. Moreover, the protein expression of CSE in the cells was detected by western blot and the H2S level in cell supernatant was detected by H2Sselective sensor. The screening results demonstrated that the appropriate con centration of lentivirus containing CSE shRNA plus GFP cDNA was 1 × 10 5 TU/mL ( Figure S1 in Supplementary Material). The endothelial cells were seeded in T25 flasks and infected with len tiviral CSE shRNA (1 × 10 5 TU/mL) at 60-70% confluency. G418 antibiotics (200 µg/mL) was used for EA.hy926 cell screening for 2 week and G418 antibiotics (300 µg/mL) was used for primary HUVECs and primary RPAECs screening for 1 week. At the same time, vehicle lentivirus was used to infect the endothelial cells as the control according to the same protocol.
To explore the effect of endogenous H2S deficiency on the SO2/ AAT pathway in the endothelial cell and its mechanism, cells were randomly divided into vehicle group, CSE shRNA group, and CSE shRNA plus H2S group. Cells in the CSE shRNA + H2S group were pretreated with 200 µM of H2S donor sodium hydro sulfide hydrate (NaHS) for 24 h. Cells in the vehicle group and CSE shRNA group were incubated with equal volume of ddH2O. NaHS was freshly dissolved in ddH2O.
To investigate the significance of the increased endogenous SO2 generation in the endothelial cell inflammation caused by CSE knockdown, cells were divided into vehicle group, CSE shRNA group, and CSE shRNA + Laspartateβhydroxamate (HDX) group. Cells in the CSE shRNA + HDX group were pre treated with 200 µM HDX for 24 h. Cells in the control group and CSE shRNA infected group were incubated with equal volume of ddH2O. HDX is an inhibitor of AAT and freshly prepared. Eighteen male Wistar rats provided by the Animal Research Committee of the First Hospital, Peking University, weighing 160 ± 20 g, were randomly divided into three groups (n = 6 each group): control group, monocrotaline (MCT) group, and MCT + H2S group. On the first day, the rats of MCT and MCT + H2S groups were administered with MCT (60 mg/kg) by intraperitoneal injection, while the rats of control group were injected with the same dose of saline (5,35). The rats of MCT + H2S group were injected daily with the H2S donor, NaHS (56 µmol/kg), for 21 days, while the rats of the control and MCT groups were given the same dose of saline.

animal Preparation and grouping
Another 21 male Wistar rats were divided into three groups (n = 7 each group): control group, MCT group, and MCT + HDX group. The rats in the MCT and MCT + HDX groups were administered with MCT (60 mg/kg) by intraperitoneal injection on day 1. The rats in the MCT + HDX group were given HDX orally at 25 mg/kg on days 0, 7, and 14 (36). The rats in the control group received the same dose of saline.
rat Pulmonary artery Pressure Measured by right heart catheterization Rats were anesthetized via intraperitoneal injection of 0.5% sodium pentobarbital (0.1 mL/100 g) after 21 days of MCT challenge. The pulmonary artery pressure was measured via right heart catheterization as previously described (22). Briefly, the right external jugular vein was exposed and a catheter was guided through the superior vena cava, right atrium, and right ventricle into the pulmonary artery. The extracorporal end of the catheter was connected to a pressure sensor (BL410, Chengdu TME Technology, China) to record the continuous changes of pulmonary artery pressure, including systolic pulmonary artery pressure, diastolic pulmonary artery pressure, and mean pulmo nary artery pressure.

Morphological change of Pulmonary arteries
The rat lung tissue was immersed in the 10% (wt/vol) paraform aldehyde for fixation and then embedded in paraffin. The lung tissue was sectioned at a thickness of 4 µm. The elastic fiber in the pulmonary artery was stained using the modified Weigert's elastic fiber staining kit according to the manufacturer's protocol (Leagene, Beijing, China). The internal and external elastic lamina were shown as darkpurple color under microscope.

Measurement of h 2 s level by an h 2 s-selective sensor
The H2S level in endothelial cell supernatant and rat lung tissues was measured using the free radical analyzer TBR4100 with an H2Sselective sensor (ISOH2S100, WPI, China) as previously described (38,39). The rat lung homogenate was prepared by grind ing with cold PBS buffer (pH 7.2, 0.01 M). Firstly, an H2Sselective sensor was polarized with PBS buffer (pH 7.2, 0.05 M) until a stable baseline current was reached, and then the calibration curve of pA-H2S concentration began to be plotted as follows. The sen sor tip was immersed by 10 mm into 20 mL of PBS buffer solution containing Na2S at different concentrations (0.5, 1, 4, 8, 16, and 32 µM) sequentially. Then the calibration curve was constructed by plotting the signal output (pA) against the concentration (μM) of H2S. Secondly, the sensor tip was immersed into each sample by 10 mm to detect the H2S content in the sample according the calibration curve of pA-H2S concentration. All experiments were performed independently for at least three times.

Measurement of sO 2 level by high-Performance liquid chromatography (hPlc) analysis
SO2 content in the supernatant of EA.hy926 cells, primary HUVECs and RPAECs supernatants, and rat lung tissues was examined by HPLC (Agilent 1100 series, Agilent Technologies, Palo Alto, CA, USA) as previously described (29). The rat lung homogenate was prepared by grinding with cold PBS buffer (pH 7.2, 0.01 M). In brief, the sample was mixed with 0.212 mM sodium borohydride in 0.05 M Tris-HCl (pH 8.5) and incubated at room temperature for 30 min; this mixture was subsequently combined with 70 mM monobromobimane in acetonitrile. Then, perchloric acid was added, followed by vortex mixing. After that, the mixtures were centrifuged at 12,400× g for 10 min, and the supernatant was neutralized by 2.0 M Tris and subsequently cen trifuged again at 12,400× g for 10 min. Eventually, the neutralized supernatant was transferred and injected into an HPLC column. Sulfitebimane adduct was detected by the excitation at 392 nm and the emission at 479 nm. All experiments were performed independently for at least three times.

In Situ Detection of h 2 s by Fluorescent Probe
The H2S generation in the endothelial cells was in situ detected by H2S fluorescent probe kindly provided by professor Xinjing Tang, Peking University Health Science Center, Beijing, China, as described previously (5). The cells were cultured using Lab Tek chambered coverglass (Thermo, USA), rinsed with PBS for twice before incubation with the H2S fluorescent probe, then subsequently incubated with H2S fluorescent probes (100 µM) for 30 min, and fixed with icecold 4% paraformaldehyde for 20 min. Immunofluorescent images were obtained using a confocal laserscanning microscope (TCS SP5, Leica, Wetzlar, Germany). Green fluorescent indicates endogenous H2S in the cells and the fluorescent signal intensity was measured using Image J software (NIH, Bethesda, MD, USA). All experiments were performed independently for at least three times.

In Situ Detection of endogenous sO 2 by Fluorescent Probe
The SO2 generation in endothelial cells was detected in situ by SO2 fluorescent probe kindly provided by Professor Kun Li, College of Chemistry of Sichuan University, Sichuan, China. The specificity and sensitivity of this probe were previously verified (40,41). The cells were cultured using LabTek chambered coverglass (Thermo, USA) and subsequently incubated with SO2 fluorescent probes (20 µM) for 30 min and then rinsed twice with PBS prior to fixation with icecold 4% paraformaldehyde for 20 min. Then, cells were rinsed twice with PBS, each for 5 min before testing. Immunofluorescent images were obtained using a confocal laser scanning microscope (TCS SP5, Leica Microsystems, Wetzlar, Germany). Blue fluorescent indicates endogenous SO2 in the cells. The fluorescent signal intensity was measured using Image J software (NIH, Bethesda, MD, USA). All experiments were performed independently for at least three times.

aaT activity Detected by colorimetry assay
The activity of AAT in endothelial cells, purified AAT protein from pig heart, and rat lung tissues was tested by colorimetry assay (JianCheng, Nanjing, China) according to the manufac turer's instructions as described previously (18). AAT catalyzes the transfer of amino group and keto group in αketoglutaric acid and aspartic acid to form glutamic acid and oxaloacetic acid. Oxaloacetic acid is then decarboxylated by itself to form pyruvic acid, and the latter was reacted with 2,4 dinitrophenylhydrazine to produce the 2,4dinitrophenylhydrazone which shows a red-brown color in alkaline solution and can be detected by colorimetric method. Pyruvic acid solution (2 mM) was used as the standard to plot the standard curve. Endothelial cells were homogenized in PBS with an icewater bath and centrifuged at 5,000× g for 10 min at 4°C to get the supernatant. Equivalent AAT purified proteins (0.375 µg, Sigma, USA) were incubated at different concentrations of H2S (100, 200, and 500 µM) or double distilled water for 2 hr in 37°C water bath. In the 200 µM H2S plus DTT treatment, purified AAT protein was pretreated with NaHS (200 µM) for 1 h and then incubated with 1 mM DTT for a further 1 h in the continuous presence of NaHS. The rat lung homogenate was prepared by grinding with cold PBS buffer (pH 7.2, 0.01 M). AAT activity was expressed as Carmen's unit, which was calculated according to the standard curve after colorimetric determination. One unit of Carmen's is defined as follows: NADH is oxidized to NAD + by pyruvic acid generated from 1 mL of the sample within 1 min at 25°C in the total reaction capacity of 3 mL, which causes the absorbance deceased by 0.001 at 340 nm wavelength using a light path length of 1 cm. All experiments were performed independently for at least three times.

s-sulfhydrtation Detected by Biotin switch analysis
Ssulfhydration of AAT1 and AAT2 in EA.hy926 cell line, primary HUVECs, RPAECs, and rat lung tissues was detected by biotin switch assay as described previously (33,42). Endothelial cells or rat lung tissues were homogenized in nondenaturing lysis buffer with protease inhibitors and centri fuged at 13,000× g for 20 min at 4°C. Supernatant reserved for sulfhydration analysis was incubated with blocking buffer (lysis buffer supplemented with 2.5% SDS and 20 mM Smethyl meth anethiosulfonate) at 50°C for 30 min with continuous vortexing. The sample was added with acetone for removing Smethyl methanethiosulfonate at −20°C for 2 h. After acetone removal by centrifuge, the protein was resuspended in lysis buffer and incubated with EZlink iodoacetylPEG2 biotin (10 mg/mL) at 4°C for 12 h. Biotinylated proteins were precipitated by UltraLink™ Immobilized Neutravidin™ for 4 h on a roller sys tem (100 rpm) at 4°C and then washed three times with PBS. The sulfhydrated proteins were boiled with loading buffer without βmercaptoethanol and centrifuged at 5,000× g for 10 min to get supernatant, and then subjected to western blot using 8% SDS PAGE as described previously. All experiments were performed independently for at least three times.

s-sulfhydrtation Detected by Biotin Thiol assay
Ssulfhydrtation of AAT1 and AAT2 in EA.hy926 cell line and purified AAT protein from pig heart was detected by the biotin thiol assay as described before (43). The schematic protocol is shown in Figure S2 in Supplementary Material. Cells were homogenized in nondenaturing lysis buffer with protease inhibi tors and centrifuged at 13,000× g for 20 min at 4°C. The protein concentrations were determined by the BCA assay. Equal amount (1 mg) of total protein was incubated with 100 µM maleimide PEG2biotin (Thermo, USA) for 0.5 h on a roller system (100 rpm) at room temperature. Subsequently, the mixture was added with acetone at −20°C for 20 min. After washing for three times with 70% precooled acetone, the sample was centrifuged at 12,000× g for 5 min. The precipitate was resuspended in the buffer (0.1% SDS, 150 mM NaCl, 1 mM EDTA and 0.5% Triton X100, 50 mM Tris-HCl, pH 7.5) mixed with streptavidin-agarose resin (Thermo, USA) and kept rotating overnight at 4°C. The beads were washed three times with PBS containing 0.5% Triton X100 and centrifuged at 5,000× g for 5 min, and then the precipitate was mixed with 50 µL of loading buffer containing or not contain ing DTT (20 mM) with gentle shaking for 1.0 h on a roller system (100 rpm) at room temperature. After centrifugation at 5,000× g for 10 min, supernatant subjected to western blot using 8% SDS PAGE as described previously. Equal amount (3 µg) of purified AAT protein (Sigma, USA) was incubated with NaHS (200 µM) for 2 h. The sulfhydrated AAT was separated and measured using the abovementioned protocol. All experiments were performed independently for at least three times.

expression of iκBα in Primary rPaecs Detected by immunofluorescence
Immunofluorescent imaging was obtained using a confocal laser scanning microscope (TCS SP5, Leica, Germany). Briefly, RPAECs were rinsed with PBS before the fixation with 4% paraformaldehyde. The RPAECs were then incubated with the antiIκBα antibody (1:50, CST, USA) at 4°C overnight. RPAECs were subsequently incubated with the antimouseFITC conjugated secondary antibody (Thermo, USA) at 37°C for 1 h. After washing, the slides were observed under confocal microscope (5). All experiments were performed independently for at least three times. inflammatory cytokine levels Detected by enzyme-linked immunosorbent assay (elisa) Inflammatory cytokines including TNFα, IL6, and ICAM1 in the cell supernatant and rat lung tissue homogenates were meas ured using ELISA kits (eBioscience, CA, USA). Recombinant TNFα, IL6, and ICAM1 were used as standard substances. Samples and standard substances were incubated separately with an equal volume of diluent in an microplate coated with specific primary antibody at room temperature for 2 h using a shaker. Subsequently, the supernatant was removed, and the wells were rinsed with washing solution and dried. Horseradish peroxidase conjugated primary antibody was then added to the wells and incubated for 1 h. After rinsing with washing solution, 100 µL of substrate solution was added to each well to develop the chromo genic reaction for 15 min. Then, 50 µL of stop solution was added to each well to stop the reaction. A standard curve was made by absorbance at 450 nm as the vertical axis and standard sub stance concentration as the horizontal axis. The concentrations of inflammatory cytokines in the samples were then calculated (5). The protein concentration of rat lung tissue homogenate was determined with Bradford kit and used for adjusting the content of cytokines in the rat lung tissue. All experiments were performed independently for at least three times.

statistical analysis
Data are expressed as mean ± SEM. Comparisons among groups were analyzed by oneway ANOVA using SPSS 17.0 (SPSS Inc., USA). Means between groups with equal variance were analyzed by leastsignificance difference (LSD). When equal variance not assumed, means between groups were analyzed using Tamhane. P < 0.05 was considered statistically significant.

resUlTs endogenous sO 2 Production Was increased in cse Knockdown endothelial cells
For the purpose of revealing the effect of endogenous CSE/H2S pathway on endogenous SO2 production, EA.hy926 cell line was treated with CSE shRNA followed by H2S donor supplement. Compared with vehicle group, H2S level in cell supernatant was decreased in endothelial cells of CSE shRNA group, while H2S donor reimbursed the H2S deficiency caused by CSE knockdown (Figure 1A). In in situ fluorescent probe experiment, the data showed that CSE knockdown decreased the endogenous H2S level but promoted endogenous SO2 production (Figures 1B,C). Moreover, H2S donor NaHS inhibited the increase in endogenous SO2 level in the CSE knockdown EA.hy926 cells (Figures 1B,C). To further confirm the above result, we selected primary HUVECs and RPAECs in addition to EA.hy926 cells (Figures 1D-I).
Interestingly, the results in both primary endothelial cells were   (Figures 1E,F,H,I). The results suggested that endogenous H2S suppressed the SO2 production in endothelial cells.

The Protein expression of aaT1 and aaT2 in endothelial cells Was not affected by cse Knockdown
In order to elucidate the target on which endogenous CSE/ H2S inhibited SO2 production, we first detected the protein expression of AAT1 and AAT2, the two key endogenous SO2 producing enzymes in EA.hy926 cells. Compared with vehicle group, the expression of CSE in the endothelial cells of CSE shRNA group was markedly decreased (Figure 2A). However, there was no difference in the expressions of AAT1 and AAT2 in the endothelial cells between vehicle group and CSE shRNA group (Figures 2B,C). Next, the same protocol of the experi ments was done on both kinds of primary cells. Compared with the vehicle group, the expression of CSE was both downregulated in primary HUVECs and primary RPAECs (Figures 2D,G), while the expression of AAT1 and AAT2 was not affected by CSE knockdown (Figures 2E,F,H,I). The results proved that AAT1 and AAT2 protein expressions were not involved in the inhibitory effect of endogenous H2S/CSE on the endogenous SO2 production.

The endogenous h 2 s/cse inhibited the aaT activity
Aspartate aminotransferase activity is another important element involved in the regulation of endogenous SO2 pro duction. Therefore, we further detected the activity of AAT in the HUVECs and purified AAT protein. The result showed that activity of AAT was significantly increased in the CSE knockdown EA.hy926 cells. While compared with the vehicle cells, the exogenous supplementation of NaHS (200 µM) reversed the increase in the AAT activity caused by CSE knockdown (Figure 3A). The similar results were observed in both primary endothelial cells as shown in Figures 3B,C. Furthermore, NaHS (100-500 µM) directly inhibited the AAT activity in a concentrationdependent manner in purified AAT protein (Figure 3D), which further supported the direct inhibi tory effect of H2S on the AAT activity.

h 2 s s-sulfhydrated aaT to inhibit aaT activity
In in vitro experiment, DTT, a thiol reductant, could reverse the impact of H2S on the AAT activity ( Figure 3D), suggest ing that the thiol group at the cysteine of AAT protein might be involved in the mechanisms by which H2S suppressed AAT activity. Considering that Ssulfhydration, a special posttransla tion modification on the thiol group at the cysteine, was reported to participate in the wide biological effects of H2S, we detected the Ssulfhydration of AAT in the EA.hy926 cell, using modi fied biotin switch assay. The data showed that compared with vehicle group, Ssulfhydration of AAT1 and AAT2 was sharply reduced in EA.hy926 cell of CSE shRNA group, while the sup plementation of NaHS significantly reversed the decrease in the Ssulfhydration of AAT in the EA.hy926 cell caused by CSE knockdown (Figure 4A). Furthermore, we also used another method for detecting Ssulfhydration, known as biotin thiol assay. The results in Figure 4B are in accordance with those shown in Figure 4A, suggesting that H2S can sulfhydrate AAT1 and AAT2. Next, we detected Ssulfhydration of AAT by H2S in both pri mary HUVECs and primary RPAECs using biotin switch assay. Compared with vehicle group, Ssulfhydration of AAT1 and AAT2 was decreased significantly in both primary endothelial cells of CSE shRNA group, while the supplementation of NaHS significantly reversed the decrease in the Ssulfhydration of AAT (Figures 4C,D). Moreover, NaHSinduced Ssulfhydration of AAT1 and AAT2 in the purified protein from pig heart, which was blocked by the treatment with a thiol reductant DTT ( Figure 4E).
Collectively, the above data suggested that H2S might inhibit the activity of AAT via the sulfhydration of AAT.

Upregulation of endogenous sO 2 Production exerted compensatory effects to inhibit inflammation caused by Downregulated h 2 s/cse Pathway In Vitro
In order to explore the biological significance of elevated endog enous SO2 levels induced by downregulation of endogenous H2S/ CSE pathway, CSE knockdown EA.hy926 cells were treated with HDX, an AAT inhibitor. The results showed that compared with vehicle group, the SO2 level in the EA.hy926 cell supernatant was sharply increased, and the further treatment by HDX reversed the increased SO2 caused by CSE knockdown (Figure 5A). Meanwhile, the phosphorylation of NFκB p65 (pp65/p65) and the expression of ICAM1 which denoted the inflammatory response in the EA.hy926 cell were also upregulated by CSE knockdown. However, the treatment of HDX aggravated the increase in the phosphorylation of NFκB p65 and the expression of ICAM1, which resulted from the deficiency of endogenous H2S/CSE pathway (Figures 5B,C).
Furthermore, the same protocol of the experiment was done on primary HUVECs. The ratio of phosphorylated IκBα/IκBα (pIκBα/IκBα), IκBα protein level, the ratio of pp65/p65, and the expression of ICAM1 were also detected by western blot. The inflammatory cytokines IL6 and TNFα in primary HUVEC supernatant were detected by ELISA. Compared with the vehicle group, the SO2 level in primary HUVECs was markedly increased by CSE knockdown, and HDX blocked the increase in SO2 con tent in cell supernatant ( Figure 6A). The ratio of pIκBα/IκBα, the ratio of pp65/p65, and the expression of ICAM1 were all upregulated but IκBα protein level was reduced by CSE knock down (Figures 6B-D). Meanwhile, the inflammatory cytokines, IL6 and TNFα, in primary HUVEC supernatant were elevated by CSE knockdown (Figures 6E,F). However, the treatment of HDX promoted the increase in IκBα and NFκB p65 phospho rylation and the level of inflammatory cytokines, and aggravated the decrease in IκBα protein level, which resulted from the defi ciency of endogenous H2S/CSE pathway in the primary HUVECs (Figures 6B-F).
The results observed in the following primary RPAECs were in accordance with those in both EA.hy926 cells and primary HUVECs (Figure 7). HDX inhibited the increased SO2 content in the supernatant of primary RPAECs but aggravated the decrease in IκBα protein level and the increase in the phosphorylation of p65 and the levels of ICAM1, IL6, and TNFα in cell superna tants induced by CSE knockdown.
Collectively, the above data implied that the upregulated endogenous SO2 production might exert compensatory effects to inhibit the inflammation caused by H2S/CSE deficiency.   In order to further elucidate the significance of upregulated endogenous SO2 production induced by the deficiency of endog enous H2S/CSE pathway in the development of vascular inflam mation, we constructed a rat model of pulmonary hypertension in which the endogenous H2S production was suppressed by MCT stimulation. The data showed that compared with the con trol group, the systolic, diastolic, and mean pulmonary arterial pressures in the rats of MCT group were increased, respectively (Figures 8A-C). Moreover, the thickened media of small pulmo nary artery and the increased inflammatory cytokines IL6 and TNFα in the lung tissue in MCTtreated rat were demonstrated (Figures 8D-F). Simultaneously, the H2S content in the lung tissue of rats in the MCT group was lower than that of the control group ( Figure 8G). The supplement of H2S donor NaHS rescued the pulmonary hypertension, pulmonary vascular remodeling and pulmonary vascular inflammation in the rats of MCT group (Figures 8A-F).
In the above rat model, the effect of H2S on the endogenous SO2/AAT pathway was examined. The results demonstrated that compared with the control group, SO2 level, and AAT activity in the lung tissues of rats in the MCT group were increased signifi cantly (Figures 8H,I). Moreover, the sulfhydration of AAT1 and AAT2 in the lung tissues of MCT rats was decreased compared with the control group (Figures 8J,K). Compared with MCT group, SO2 level and AAT activity in the lung tissues of the rats in the MCT + H2S group were reduced, while sulfhydrated AAT1 and AAT2 were increased (Figures 8H-K), suggesting that the supplement of H2S donor NaHS restored the H2S level in the lung tissue of MCT rats, and subsequently blocked the upregulation of endogenous SO2/AAT pathway. Furthermore, the significance of upregulated SO2/AAT path way in the pulmonary vascular inflammation associated with the downregulation of endogenous H2S production was explored in the MCT rats treated with an AAT inhibitor HDX. The data showed that compared with the MCT group, AAT activity and SO2 level were suppressed significantly in the lung tissue of rats in the MCT + HDX group (Figures 9A,B). While HDX aggravated the increase in the phosphoralyation of NFκB p65, ICAM1 protein expression, the level of IL6, and TNFα in the lung tissue of MCTtreated rats (Figures 9C-F). In addition, the thickened media of small pulmonary artery in MCTtreated rats was exacerbated by HDX (Figure 9G), suggesting that the upregulation of endogenous SO2 pathway might be an important compensatory response when the endogenous H2S pathway col lapsed in the development of pulmonary vascular inflammation and pulmonary vascular remodeling.

DiscUssiOn
The impaired H2S/CSE pathway was one of important pathogen esis of many cardiovascular diseases due to the lack of protective effect of endogenous H2S on the heart and vessel. The facts that CSE knockout mice exhibited a series of marked cardiovascular pathological phenotypes further supported the significance of endogenous H2S/CSE in the cardiovascular regulation and diseases. For example, Yuan et al. found that vascular endothelial growth factor (VEGF)induced vascular solute hyperpermeabil ity was blunted in the CSE gene deficient mice, suggesting that endotheliumderived H2S protected the endothelial solute barrier function (44). Mani et al. discovered that CSE gene depletion pro moted aortic intimal proliferation and accelerated atherosclerotic development in the ApoE knockout mice fed with atherogenic diet (45). CSE knockout mice were also found to exhibit a delayed  wound healing and a markedly reduced microvessel formation in response to VEGF (46). In the present study, we observed the variation of endogenous SO2/AAT pathway, another protector in the cardiovascular system, in a CSE knockdown endothelial cell model and further explored its pathological significance in the endothelial cell inflammation. Firstly, we observed the change of endogenous SO2 generation in CSE knockdown EA.hy926 cells using SO2 fluorescent probe. The results showed that endogenous SO2 level in the CSE knockdown endothelial cells was markedly higher than that in the vehicle endothelial cells, while H2S level in the culture super natant and endothelial cells of CSE shRNA group was decreased compared with vehicle group. Moreover, H2S donor NaHS raised the H2S level in the supernatant and endothelial cells of CSE shRNA group, and blocked the increase in the SO2 level caused by CSE knockdown. In accordance with the results obtained from the HUVEC line, the levels of SO2 in the primary HUVECs and RPAECs were also increased by the impaired H2S/CSE pathway, while the restoration of H2S content in the primary endothelial cells abolished the increase in the endogenous SO2 generation. The abovementioned data confirmed that the endogenous H2S inhibited endotheliumderived SO2 production.
Aspartate aminotransferase is regarded as the key enzyme generating endogenous SO2 in the mammal animals. There are two kinds of AAT isoenzymes: AAT1 locates in the cytoplasm and AAT2 in the mitochondria (18,47). Considering that the expression and activity of AAT are the major elements to control the endogenous SO2 production (48), we measured the expres sion and activity of AAT in the CSE knockdown EA.hy926 cells to explore the mechanism by which endotheliumderived H2S repressed endogenous SO2 generation. The western blot results showed that there was no difference in the expression of AAT1 and AAT2 in the EA.hy926 cells between vehicle group and CSE shRNA group, suggesting endogenous H2S did not affect the expression of AAT protein. We further investigated the role of endogenous H2S in the control of AAT activity. Interestingly, the enzymatic activity of AAT in the CSE shRNA endothelial cells was higher than that in the vehicle endothelial cells, while H2S donor supplement alleviated the enhancement of AAT activity induced by CSE knockdown. The discrete regulation of the AAT protein expression and activity by H2S in the EA.hy926 cells was completely reproduced in both primary endothelial cells. Moreover, in in vitro experiment, H2S donor was found to directly inhibit activity of purified AAT protein in a concentration dependent manner, which further supported the speculation that endogenous H2S suppressed endotheliumderived SO2 genera tion via inhibiting AAT activity. In fact, the detached regulation of the AAT expression and activity was reported, although the expression and activity of AAT were identically controlled in general. For example, Barouki et al. found that the regulation of AAT1 mRNA in the Fao rat hepatoma cell line by dexamethasone correlated with the variation of the AAT1 activity, suggesting that dexamethasone acted at the transcriptional level (49). However, cortisol acetate treatment did not alter AAT1 activity but reduced AAT1 mRNA in rat muscles (50). Therefore, we supposed that the discrete effects of H2S on the AAT1/2 protein expression, and activity might result from the fact that H2S regulated SO2/AAT at a posttranslational level.
Secondly, we tested how H2S inhibited AAT activity. It is well known that endogenous H2S regulates various cellular processes  via Ssulfhydration of target proteins, a posttranslational modi fication at the thiol group in the cysteine residue in the proteins such as Keap1, P66Shc, and NFκB (1,33,42,(51)(52)(53), while the thiol group in the cysteine residue is also the molecular target of redox regulation. Coincidentally, AAT expression and activity were controlled in an oxygenrelated manner in a rodent model of acute ischemic stroke (54). In the present study, we found that DTT, a thiol reductant, could reverse the H2Sinduced decrease in the AAT activity in the in vitro experiment, suggesting the thiol group might be involved in the regulation of the AAT activity by H2S. Therefore, we detected the sulfhydration of AAT1 and AAT2 in the CSE knockdown EA.hy926 cells using the modified biotin switch assay (33). The data showed that CSE knockdown reduced the sulfhydration of AAT1 and AAT2 in the endothelial cells, while H2S donor enhanced the sulfhydra tion of AAT1 and AAT2 in the EA.hy926 cells of CSE shRNA group. To confirm the fact that endogenous H2S sulfhydrated AAT protein, we used biotin thiol assay (43), another method for detecting sulfhydration, to investigate the modification of AAT by H2S. The change of sulfhydration of AAT detected by biotin thiol assay was similar to the results using modified biotin switch assay. Moreover, in the in vitro experiments we discovered that NaHS induced a marked sulfhydration of AAT1 and AAT2, which was blocked by DTT treatment. In the experi ment on the primary HUVECs and RPAECs, the decrease in the sulfhydration of AAT1 and AAT2 caused by CSE knockdown was also rescued by NaHS. Those data suggested that sulfhydra tion of AAT might mediate the inhibitory effect of endogenous H2S on the AAT activity.
On the basis of H2S/CSE deficiencyinduced inflammation endothelial cell model, we further investigated the pathological significance of CSE knockdownenhanced endogenous SO2 production. We used HDX, an AAT inhibitor, to block the increased endogenous SO2 production in the HUVEC line, primary HUVECs and RPAECs, and observed the changes of NFκB pathway, a pivot regulator of cellular inflammation, and its downstream target genes including inflammatory cytokine ICAM1, IL6, and TNFα. NFκB, consisting of p65 and p50 subunits, locates in the cytosol complexed with the inhibitory protein IκBα in an inactivated state. Inflammatory stimuli such as hypoxia can activate the phosphorylation of IκBα, leading to the IκBα degradation and dissociation from NFκB. The released NFκB is subsequently phosphorylated, translocates into the nucleus and increases the transcription of inflammatory cytokines (33). ICAM1 is typically expressed on the surface of endothelial cells and of other inflammatory cells and mediates the binding of leukocytes to endothelial cell by coupling its ligand integrin. ICAM1 is regarded as a classical marker of endothelial inflammation (55). Therefore, we detected the phosphorylated IκBα, total IκBα, phosphorylated NFκB p65, and ICAM1 pro tein in the endothelial cells and ICAM1, IL6, and TNFα levels in the supernatant to reflect the endothelial cell inflammation. As we expected, HDX aggravated the increase in the expression of ICAM1 and the phosphorylation of NFκB p65 in the CSE knockdown EA.hy926 cells. Moreover, HDX was found to pro mote phosphorylation of IκBα, decrease IκBα protein level, and raise the phosphorylated NFκB p65 in both primary endothelial cells. The effects of HDX on the inflammatory cytokines in the supernatant of primary endothelial cells were in line with the regulation on the NFκB pathway. Therefore, we supposed that endogenous SO2/AAT pathway was upregulated as a compensa tory mechanism for the downregulated endogenous H2S pathway in the endothelial cell inflammation.
Finally, we further explored the importance of upregulated SO2/ AAT pathway following the broken H2S/CSE pathway in the in vivo experiments. As previously reported, endogenous H2S production in the rat lung tissues was downregulated by MCT treatment in a rat model of pulmonary vascular inflammation (5). Conversely, SO2 content and AAT activity in the lung tissue of MCTtreated rats were enhanced, while the AAT1 and AAT2 sulfhydraton was reduced. More interestingly, the restoration of H2S level reversed the upregulation of endogenous SO2/AAT pathway, demonstrated by the facts that NaHS increased the sulfhydrated AAT1 and AAT2, inactivated the AAT activity and reduced SO2 level in the lung tissue of rats in the MCT groups. Furthermore, as designed in the endothelial cell experiments, we used HDX and found that it inhibited the upregulation of endogenous SO2/AAT pathway. As we expected, the pulmonary vascular inflammation reflected by the phosphorylation of NFκB p65 and the elevated inflammatory cytokines including ICAM1, IL6, and TNFα was exacerbated when the deficient H2Sinduced SO2/AAT pathway was blocked by HDX. Moreover, in our previous studies, HDX was also found to exacerbate the MCTinduced pulmonary vascular inflammation, demonstrated by the fact that HDX enhanced NFκB p65 and ICAM1 expression in the pulmonary artery endothelial cells in an immunohistochemical study (56). As a result, HDX aggravated the thickened media of pulmonary artery in the MCTtreated rats in accordance with the findings previously reported (56,57). The abovementioned results were in accordance with the data obtained from in vitro endothelial cell experiment. Therefore, we supposed that endogenous SO2/AAT pathway was upregulated as a compensatory mechanism for the downregulated endogenous H2S pathway in the endothelial cell inflammation.
In brief, we firstly demonstrated that endogenous H2S inhib ited endothelial cellderived SO2 generation through suppressing AAT activity via sulfhydration in vitro and in vivo. When injury factors impaired H2S/CSE pathway, the endogenous SO2 produc tion was subsequently induced as a reserved protector to protect the endothelial cell functions such as antiinflammatory effects. Our findings deepen the understanding of regulatory mecha nism responsible for cardiovascular homeostasis, providing a new insight for the exploration of interaction among bioactive small molecules. More molecular and cellular biological studies, however, need to be done for disclosing the precise target and mechanisms by which endogenous H2S functions.

eThics sTaTeMenT
This study was carried out in accordance with the Animal Management Rule of the Ministry of Health of the People's Republic of China. The protocol was approved by the Animal Research Ethics Committee of Peking University First Hospital.
aUThOr cOnTriBUTiOns DZ, XW, XT and CL carried out the experimental work. DZ wrote the paper. HJ and YH designed and supervised the experiments. HJ, KL, XY and XT revised the primary manu script. JZ, WK, JD and CT were responsible for the quality control and analysis. DZ, LZ, GY and YT participated in the data analysis. All authors approved the final version of the manuscript.   The images showed that more than 90% of cells showed green fluorescence at the lentivirus concentration of 1 × 10 5 and 2 × 10 5 TU/mL compared with control group, suggesting that the lentivirus was successfully transfected. (B) The expression of CSE in the EA.hy926 cells detected by western blot. Compared with the control group, the expression of CSE was decreased by 60.2% and 65.1% at the lentivirus concentration of 1 × 10 5 and 2 × 10 5 TU/mL. (c) The H2S level in the supernatant of EA.hy926 cells was detected by H2S-selective sensor. Compared with control group, the H2S level in EA.hy926 cell supernatant was significantly decreased by 63.6% and 75%, respectively, which was similar to the change of CSE expression. * P < 0.05. Data are expressed as means ± SEM, and all experiments were performed independently for at least three times.
FigUre s2 | The schematic protocol of biotin thiol assay to detect the S-sulfhydration. The sample protein was incubated with maleimide-PEG2-biotin to alkylate both cysteine residue and sulfhydrated cysteine residue at the first step. At the subsequent step, the high-capacity affinity streptavidin-agarose resin was used to pull down the proteins which were alkylated by maleimide-PEG2biotin at the first step. At the last step, the proteins were reacted with buffer with or without DTT, a thiol reductant, for 30 min followed by centrifugation. DTT was used for cleaving the mixed disulfide bond and releasing the sulfhydrated protein. Therefore, sulfhydrated protein separated from the mixture containing DTT was detected by western blot, while supernatant separated from the mixture without DTT was used as negative control.