Application of EPX as molecular target in screening of drugs for preventing and treating pulmonary arterial hypertension

Abstract

The invention relates to application of EPX as a molecular target to screening of drugs for preventing and treating pulmonary arterial hypertension, and belongs to the technical field of biological medicines. According to the invention, it is found for the first time that EPX regulates and controls an ACVRs-Smad downstream signal channel through specific binding with an ACVR2A / ACVR1B complex, resulting in abnormal proliferation of PASMCs, and participates in pulmonary vascular remodeling and the pathological process of PAH. According to the invention, EPX is used as a molecular target to screen small molecule compound components, the screened small molecule compound components, such as salvianolic acid B, neochlorogenic acid and hyperoside, can be competitively combined with EPX to block the combination with ACVR2A / ACVR1B complexes, so that an EPX-mediated ACVRs-Smad downstream signal channel is interfered, and finally, the abnormal proliferation of PASMCs is inhibited. The screened small molecule compound component can be used as a potential drug for preventing and treating PAH, so as to effectively improve PAH.

Description

Technical Field

[0001] The present invention relates to the application of EPX as a molecular target in screening drugs for preventing and treating pulmonary arterial hypertension, and belongs to the field of biomedical technology. Background Art

[0002] Pulmonary arterial hypertension (PAH) is mainly characterized by progressive elevation of pulmonary vascular resistance and right heart failure. The core pathological mechanism is pulmonary vascular remodeling, in which abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is a key driving factor. Although current therapeutic drugs can relieve symptoms through vasodilation, their effect on reversing vascular structural remodeling is limited. Research has found that abnormal activation of the activin receptor (ACVRs) signaling pathway plays an important role in the pathogenesis of PAH. In particular, overactivation of the ACVR2A / ACVR2B-Smad2 / 3 signaling axis can significantly promote the proliferation of PASMCs. The novel drug sotatercept, as an ACVR2A-Fc fusion protein, can specifically antagonize the binding of activin to the receptor and inhibit the proliferation of PASMCs by inhibiting the Smad2 / 3 signaling pathway, thereby effectively reversing vascular remodeling. The ACVRs-Smad2 / 3 pathway, as a key target for the treatment of PAH, provides a new direction for the development of therapies targeting the reversal of vascular remodeling.

[0003] Eosinophils, as the core effector cells of allergic reactions, are mainly distributed in the respiratory tract and lymphoid organs and play an important role in immune regulation and inflammatory responses. Recent studies have shown that the number of eosinophils in the peripheral blood and lung tissue of PAH patients is significantly positively correlated with the disease severity, and their secreted metabolites (such as 14-HDHA, 17-HDHA) and granule contents may participate in pulmonary vascular remodeling by regulating the proliferation of PASMCs. It is worth noting that although eosinophil peroxidase (EPX), as a characteristic granule protein of eosinophils, has been proven to play a role in respiratory diseases such as asthma and chronic rhinosinusitis, the mechanism of action of EPX in PAH is still unclear and requires further study. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides the application of EPX as a molecular target in screening drugs for the prevention and treatment of pulmonary arterial hypertension. The present invention has found through research that EPX plays an important role in the occurrence and development of PAH, and may specifically bind to the ACVR2A / ACVR1B receptors in the ACVRs-Smad signaling pathway, regulate the downstream signaling pathway, and participate in the pathological processes of pulmonary vascular remodeling and PAH. The present invention screens small molecule compound components as potential drugs for the prevention and treatment of pulmonary arterial hypertension through this mechanism, with the expectation of effectively improving PAH.

[0005] The technical solution of the present invention is as follows: The application of EPX as a molecular target in screening drugs for the prevention and treatment of pulmonary arterial hypertension.

[0006] Preferably according to the present invention, the EPX regulates the downstream ACVRs-Smad signaling pathway by specifically binding to the ACVR2A / ACVR1B complex, resulting in abnormal proliferation of PASMCs.

[0007] Preferably according to the present invention, the drug competitively binds to EPX, blocks its binding to the ACVR2A / ACVR1B complex, thereby interfering with the downstream ACVRs-Smad signaling pathway mediated by EPX, and finally inhibiting the abnormal proliferation of PASMCs.

[0008] More preferably, the EPX sites to which the drug competitively binds include one or more of ARG-84, ARG-324, ASP-352, ASN-353, ARG-340, ASN-494, and SER-491.

[0009] A drug for the prevention and treatment of pulmonary arterial hypertension, the active ingredient of which includes components that can competitively bind to EPX and block the binding of EPX to the ACVR2A / ACVR1B complex.

[0010] Preferably according to the present invention, the EPX sites to which the competitive binding occurs include one or more of ARG-84, ARG-324, ASP-352, ASN-353, ARG-340, ASN-494, and SER-491.

[0011] Preferably according to the present invention, the components include one or more of salvianolic acid B, neochlorogenic acid, and hyperin.

[0012] Preferably according to the present invention, the component is a combination of salvianolic acid B, neochlorogenic acid, and hyperin.

[0013] The application of one or more of salvianolic acid B, neochlorogenic acid, and hyperin in the preparation of drugs for the prevention and treatment of pulmonary arterial hypertension.

[0014] Preferably according to the present invention, salvianolic acid B, neochlorogenic acid and hyperoside can competitively bind to EPX, block its binding to the ACVR2A / ACVR1B complex, thereby interfering with the EPX-mediated ACVRs-Smad downstream signaling pathway, and finally inhibiting the abnormal proliferation of PASMCs.

[0015] Beneficial effects: The present invention firstly discovers that EPX plays an important role in the occurrence and development of PAH. EPX specifically binds to the ACVR2A / ACVR1B complex, regulates the ACVRs-Smad downstream signaling pathway, causes abnormal proliferation of PASMCs, and participates in the pathological processes of pulmonary vascular remodeling and PAH. The present invention screens small molecule compound components using EPX as a molecular target. The screened small molecule compound components, such as salvianolic acid B, neochlorogenic acid and hyperoside, can competitively bind to EPX, block its binding to the ACVR2A / ACVR1B complex, thereby interfering with the EPX-mediated ACVRs-Smad downstream signaling pathway, and finally inhibiting the abnormal proliferation of PASMCs. The screened small molecule compound components can be used as potential drugs for the prevention and treatment of PAH, with the expectation of effectively improving PAH. Description of the drawings

[0016] Figure 1 It is the H&E staining results of pulmonary distal arteriole vascular samples of the PAH group and the Non-PAH group; among them, A is the H&E staining picture, and B is the bar chart of the proportion of the media in the whole vascular thickness.

[0017] Figure 2 It is the Masson staining results of pulmonary distal arteriole vascular samples of the PAH group and the Non-PAH group; among them, A is the Masson staining picture, and B is the bar chart of the proportion of collagen fibers.

[0018] Figure 3 It is the α-SMA immunofluorescence staining results of pulmonary distal arteriole vascular samples of the PAH group and the Non-PAH group; among them, A is the α-SMA immunofluorescence staining picture, and B is the bar chart of the α-SMA expression level.

[0019] Figure 4 It is the immunohistochemical detection results of ACVR2A, ACVR1B, and ki67 in pulmonary distal arteriole vascular samples of the PAH group and the Non-PAH group; in the figure, A is the immunohistochemical picture, and B is the bar chart of the ACVR2A, ACVR1B, and ki67 expression levels.

[0020] Figure 5WB detection results of proteins in distal pulmonary arteriole vascular samples of PAH group and Non-PAH group; among them, A is the WB detection picture, and B is the bar chart of the protein expression levels of p-Smad2, p-Smad3, and Cleaved-caspase-3.

[0021] Figure 6 ELISA detection results of EPX expression levels in serum and lung tissue samples of PAH group and Non-PAH group; among them, A is the bar chart of EPX expression level in serum, and B is the bar chart of EPX expression level in lung tissue.

[0022] Figure 7 WB detection results of EPX expression levels in lung tissue samples of PAH group and Non-PAH group; among them, A is the WB detection picture, and B is the bar chart of EPX expression level.

[0023] Figure 8 Immunohistochemical detection results of EPX expression levels in lung tissue samples of PAH group and Non-PAH group; among them, A is the immunohistochemical detection picture, and B is the bar chart of EPX expression level.

[0024] Figure 9 For EPX in blood samples of PAH group and Non-PAH group + Flow cytometry detection results of eosinophils; among them, A is the flow cytometry detection picture, and B is EPX + Bar chart of the proportion of eosinophils.

[0025] Figure 10 Interaction conformation map of EPX and ACVR2A / ACVR1B complex predicted by AlphaFold 3.

[0026] Figure 11 Immunofluorescence confocal pictures of the binding of EPX to ACVR2A and EPX to ACVR1B.

[0027] Figure 12 Co-IP detection pictures of the binding of EPX to ACVR2A and ACVR1B in PASMCs.

[0028] Figure 13 Results of Scatchard plot evaluation of the binding ability of EPX to PASMCs; among them, A is the Scatchard plot curve under the condition of Control SiRNA, B is the Scatchard plot curve under the conditions of ACVR2A SiRNA and ACVR1B SiRNA, and C is the binding ability curve under different knockout conditions.

[0029] Figure 14Molecular docking simulation analysis diagrams of salvianolic acid B, neochlorogenic acid and hyperoside binding to EPX. Among them, A is the molecular docking diagram of salvianolic acid B and EPX, B is the molecular docking diagram of neochlorogenic acid and EPX, and C and D are the molecular docking diagrams of hyperoside and EPX at different angles.

[0030] Figure 15 Analysis diagrams of the binding sites at different parts of the EPX and ACVR2A / ACVR1B complex predicted by AlphaFold 3.

[0031] Figure 16 Bar graphs of the cell proliferation rates of the control group (Normoxia), hypoxia group (Hppoxia), and EPX hypoxia group (EPX).

[0032] Figure 17 Inhibitory curves of salvianolic acid B, neochlorogenic acid and hyperoside on the activity of PASMCs; among them, A is the inhibitory curve of salvianolic acid B, B is the inhibitory curve of neochlorogenic acid, and C is the inhibitory curve of hyperoside. Detailed implementation mode

[0033] The technical solutions of the present invention will be further described below in conjunction with the embodiments and the accompanying drawings, but the protection scope of the present invention is not limited thereto. The reagents and drugs involved in the embodiments are all conventional commercially available products without special instructions. The experimental steps involved in the embodiments are all conventional experimental operations in the art without special instructions.

[0034] Source of materials: All clinical samples of the present invention were taken from the Department of Cardiology, Guangdong Provincial People's Hospital. After obtaining the consent of the patients, 36 blood samples of PAH patients, 25 control blood samples of healthy people (Non-PAH), 8 lung tissue samples of PAH patients and 7 control lung tissue samples of Non-PAH were included.

[0035] Example 1: Analysis of the effect of the ACVRs-Smad signaling pathway on PAH in clinical samples 1. Take the lung tissue samples of the PAH group and the Non-PAH group, isolate the distal pulmonary arterioles therein, and perform H&E staining, Masson staining and α-SMA immunofluorescence staining respectively to conduct histological morphology research on the distal pulmonary arterioles of PAH patients. Among them, the α-SMA monoclonal antibody used in the immunofluorescence staining was purchased from Cell Signaling Technology (D4K9N, 1:300).

[0036] The H&E staining results of the distal pulmonary arteriole vascular samples are as Figure 1 shown. The number of vascular wall cells in the PAH group was significantly increased, indicating that the vascular wall of the distal pulmonary arterioles of PAH patients was significantly thickened.

[0037] The Masson staining results of the distal pulmonary arteriole vascular samples are as Figure 2 shown. The collagen fiber content in the vascular wall of the PAH group was significantly increased, suggesting that the collagen deposition in the distal pulmonary arterioles of PAH patients was significantly increased.

[0038] The α-SMA immunofluorescence staining results of the distal pulmonary arteriole vascular samples are as Figure 3 shown. The expression level of α-SMA in the vascular wall of the PAH group was significantly increased, suggesting that the proliferation of smooth muscle cells in the distal pulmonary arterioles of PAH patients was significantly increased.

[0039] The above results of histomorphological studies showed that compared with the Non-PAH group, the PAH group showed significant remodeling of the distal pulmonary arterioles, mainly manifested as thickening of the vascular wall, increased collagen deposition, and proliferation of smooth muscle cells (increased α-SMA expression).

[0040] 2. Take lung tissue samples from the PAH group and the Non-PAH group for immunohistochemical analysis to detect the expression of ACVR2A, ACVR1B, and ki67 in the distal pulmonary arterioles. Among them, the polyclonal antibody of ACVR2A used for immunohistochemical analysis was purchased from Thermo Fisher Scientific Invitrogen (3AB2BA67, 1:300), the monoclonal antibody of ACVR1B was purchased from ThermoFisher Scientific Invitrogen (3AB2DA07, 1:300), and the polyclonal antibody of ki67 was purchased from Abcam (ab15580, 1:100).

[0041] The immunohistochemical analysis results of the distal pulmonary arteriole vascular samples are as Figure 4 shown. Compared with the Non-PAH group, the expressions of ACVR2A and ACVR1B in the distal pulmonary arterioles of the PAH group were significantly up-regulated, and at the same time, the cell proliferation marker ki67 + was increased.

[0042] 3. Take the lung tissue samples of the PAH group and the Non-PAH group, isolate the distal pulmonary arterioles therein, extract proteins and perform WB detection to analyze the expression of related proteins (p-Smad2, p-Smad3) and Cleaved-caspase-3 in the ACVRs-Smad signaling pathway. Among them, the monoclonal antibody of p-Smad2 used in WB detection was purchased from Cell Signaling Technology (138D4, 1:1000), the monoclonal antibody of p-Smad3 was purchased from Cell Signaling Technology (C25A9, 1:1000), and the monoclonal antibody of Cleaved-caspase-3 was purchased from Cell Signaling Technology (D175, 1:1000).

[0043] The results of WB detection are as Figure 5 shown. In the PAH group, the protein expressions of p-Smad2 and p-Smad3 were up-regulated, and the protein expression of Cleaved-caspase-3 was down-regulated, further confirming that the phosphorylation level of Smad2 / 3 in the distal pulmonary arteriole tissue was significantly increased.

[0044] The above results indicate that the ACVRs-Smad signaling pathway plays an important role in the process of PAH-related pulmonary vascular remodeling. Activation of the ACVRs-Smad signaling pathway can promote the proliferation of PASMCs and inhibit apoptosis.

[0045] Example 2: Analysis of the effect of EPX on PAH in clinical samples 1. Take the blood samples of the PAH group and the Non-PAH group, separate the serum and perform ELISA detection. At the same time, take the lung tissue samples of the PAH group and the Non-PAH group, perform ELISA detection and immunohistochemical analysis respectively, and extract the proteins of the lung tissue for WB detection to analyze the EPX expression in PAH patients; among them, the monoclonal antibody of EPX used in immunohistochemical analysis was purchased from Proteintech (29755-1-AP, 1:300), and the monoclonal antibody of EPX used in WB detection was purchased from Proteintech (29755-1-AP, 1:1000).

[0046] The results of ELISA detection are as Figure 6 shown, the results of WB detection are as Figure 7 shown, and the results of immunohistochemical analysis are as Figure 8 shown. From the above detection results, it can be seen that compared with the Non-PAH group, the expression level of EPX in the serum and lung tissue of the PAH group was significantly increased.

[0047] 2. Isolate eosinophils from the blood samples of the PAH group and the Non-PAH group, and detect the expression of eosinophil EPX by flow cytometry.

[0048] The results of flow cytometry detection are as Figure 9 shown. In the blood eosinophils of the PAH group, the proportion of eosinophils expressing EPX increased significantly.

[0049] The above experimental results indicate that EPX is abnormally highly expressed in PAH patients, suggesting that it may participate in the pathological process of the disease by regulating eosinophil function, and preliminarily confirming that EPX promotes the occurrence and development of PAH.

[0050] Example 3: Specific binding of EPX to ACVR2A / ACVR1B The present invention has confirmed that both the ACVRs-Smad signaling pathway and EPX are related to the occurrence and development of PAH. To further verify whether there is an interaction between EPX and the ACVRs-Smad signaling pathway, AlphaFold 3 was used to predict the interaction relationship between EPX and ACVR2A / ACVR1B, which was significantly highly expressed in the ACVRs-Smad signaling pathway of PAH patients. The results of protein-protein interaction prediction analysis are as Figure 10 shown. EPX (blue) can form a stable complex structure with the tetramer of ACVR2A (pink) and ACVR1B (green).

[0051] Lung tissue samples of the PAH group and the Non-PAH group were taken for immunofluorescence experiments. The results of laser confocal microscopy imaging are as Figure 11 shown. EPX and ACVR2A / ACVR1B are co-expressed in the distal pulmonary arterioles, which is consistent with the results of protein-protein interaction prediction.

[0052] In addition, after co-incubating the EPX recombinant protein (purchased from Wuhan Yunclon Technology Co., Ltd., human source, RPJ138Hu01) with human pulmonary artery smooth muscle cells (PASMCs, purchased from ATCC) and then performing Co-IP experiments, the results of Co-IP detection are as Figure 12 shown, confirming that EPX can bind to the ACVR2A and ACVR1B complexes in PASMCs.

[0053] Furthermore, human pulmonary artery smooth muscle cells (PASMCs, ATCC) were transfected with Control siRNA, ACVR2A siRNA, ACVR1B siRNA, or co-transfected with ACVR2A siRNA and ACVR1B siRNA to knockdown the expression levels of ACVR2A or / and ACVR1B in PASMCs. After 48 hours, they were incubated with fluorescent / biotin-labeled EPX recombinant protein (RPJ138Hu01) at gradient concentrations (0 - 100 nM) at 4°C for 2 hours. After washing, the binding signals were detected; the dissociation constant (Kd) and the maximum binding capacity (Bmax) were calculated by Scatchard plot (Bound / Free vs Bound) to verify the change in the affinity between EPX and PASMCs in the knockdown group.

[0054] Among them, the nucleotide sequence of Control siRNA is as follows: 5′-UUCUCCGAACGUGUCACGU(dT)(dT)-3′, The nucleotide sequence of ACVR2A siRNA is as follows: Sense strand: 5′-GGAUCAAGCUCAUCAAGAA(dT)(dT)-3′, Antisense strand: 5′-UUCUUGAUGAGCUUGAUCC(dT)(dT)-3′, The nucleotide sequence of ACVR1B siRNA is as follows: Sense strand: 5′-CCACCAAGAUUCUACGAAA(dT)(dT)-3′, Antisense strand: 5′-UUUCGUAGAAUCUUGGUGG(dT)(dT)-3′.

[0055] The results of ligand-receptor binding experiment combined with Scatchard plot analysis are as Figure 13 shown. After knockdown of ACVR2A or ACVR1B, the affinity between EPX and PASMCs decreased significantly, especially after simultaneous knockdown of ACVR2A and ACVR1B, where the affinity between EPX and PASMCs was the lowest.

[0056] The above results indicate that EPX can specifically interact with the ACVR2A / ACVR1B complex at the PASMCs level, thereby affecting PASMCs proliferation.

[0057] Example 4: Screening of potential drugs for inhibiting EPX-induced PASMCs proliferation Autodock was used to simulate molecular docking to analyze the binding of different small molecule drugs to EPX. Finally, three small molecule compounds were screened out: Salvianolic acid B, Neochlorogenic acid, and Hyperoside. All of the above compounds were purchased from Shanghai TargetMol Co., Ltd. The simulated molecular docking of the three small molecule compounds with EPX is as shown in Figure 14 . Among them, Salvianolic acid B binds to the ARG-84, ARG-324, ASP-352, and ASN-353 sites of EPX; Neochlorogenic acid mainly acts on ASN-353 and ARG-340 of EPX; while Hyperoside binds to ASN-494, SER-491, ARG-324, and ARG-340 of EPX. The above results indicate that Salvianolic acid B, Neochlorogenic acid, and Hyperoside can all target and bind to the key sites of EPX. These interactions may inhibit the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by EPX by blocking the active conformation of EPX or interfering with its binding to downstream effector molecules.

[0058] In addition, AlphaFold 3 predicted that there are multiple binding sites between EPX and the ACVR2A / ACVR1B complex. The prediction results are as shown in Figure 15 . The binding sites include ARG-84, ARG-324, ASP-352, ASN-353, ARG-340, ASN-494, and SER-491. It is worth noting that the binding sites of the three small molecule compounds screened above with EPX completely overlap with the binding sites of the EPX and ACVR2A / ACVR1B complex, suggesting that these three compounds may competitively bind to EPX, block its binding to the ACVR2A / ACVR1B complex, and then interfere with the downstream signaling pathway mediated by EPX, ultimately inhibiting the abnormal proliferation of PASMCs. Moreover, due to the complete overlap of the binding sites of these three compounds with EPX and the binding sites of the EPX and ACVR2A / ACVR1B complex, their competitive inhibition effect is stronger.

[0059] Example 5: Verification of the effects of three small molecule compounds To further verify the effects of the three small molecule compounds screened in Example 4 on inhibiting the proliferation of PASMCs, the CCK-8 method was used to detect the effects of Salvianolic acid B, Neochlorogenic acid, and Hyperoside on the proliferation of PASMCs induced by EPX recombinant protein. The specific experimental steps are as follows: Human pulmonary artery smooth muscle cells (PASMCs, ATCC) were seeded in Smooth Muscle Cell Medium (purchased from ScienCell) containing 10% FBS and cultured at 37°C and 5% CO2 until 80% confluence. PASMCs were randomly divided into a control group (21% O2), a hypoxia group (1% O2), and an EPX hypoxia group (1% O2 + 1 μg / mL EPX). At the same time, EPX hypoxia combined with different concentrations of salvianolic acid B (0, 0.25, 0.5, 1, 2.5, 5, 10, 20, 50, 100, and 200 μM), chlorogenic acid (0, 0.5, 1, 2, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM), or hyperoside (0, 0.5, 1, 2, 3.125, 6.25, 12.5, 25, 50, and 100 μM) treatment groups were set up. After all treatment groups were cultured at 37°C, 5% CO2, and the corresponding oxygen content for 24 hours, the cell viability was detected by the CCK-8 method.

[0060] The results showed that hypoxia culture increased the proliferation rate of PASMCs to 148%, and it further increased to 189% after EPX stimulation (as Figure 16 ); while salvianolic acid B, chlorogenic acid, and hyperoside could all significantly inhibit the proliferation of PASMCs induced by EPX. The results were as shown in Figure 17 . Among them, hyperoside had the strongest inhibitory effect (EC50 = 6.11 μM), followed by salvianolic acid B (EC50 = 8.74 μM) and chlorogenic acid (EC50 = 11.93 μM). These results confirmed that these three compounds could effectively inhibit the abnormal proliferation of PASMCs mediated by EPX under hypoxic conditions.

Claims

1. Use of EPX as a molecular target in screening drugs for preventing and treating pulmonary arterial hypertension.

2. The application according to claim 1, wherein The said EPX regulates the downstream ACVRs-Smad signaling pathway by specifically binding to the ACVR2A / ACVR1B complex, resulting in abnormal proliferation of PASMCs.

3. The application according to claim 1, characterized in that The said drug competitively binds to EPX, blocks its binding to the ACVR2A / ACVR1B complex, thereby interfering with the downstream ACVRs-Smad signaling pathway mediated by EPX, and ultimately inhibits the abnormal proliferation of PASMCs.

4. The application according to claim 3, characterized in that, The EPX sites to which the said drug competitively binds include one or more of ARG-84, ARG-324, ASP-352, ASN-353, ARG-340, ASN-494, and SER-491.

5. A drug for preventing and treating pulmonary hypertension, characterized in that, The active ingredient includes a component that can competitively bind to EPX and block the binding of EPX to the ACVR2A / ACVR1B complex.

6. The drug according to claim 5, characterized in that, The EPX sites for the said competitive binding include one or more of ARG-84, ARG-324, ASP-352, ASN-353, ARG-340, ASN-494, and SER-491.

7. The drug according to claim 5, characterized in that, The said component includes one or more of salvianolic acid B, neochlorogenic acid, and hyperin.

8. The drug according to claim 5, characterized in that, The said component is a combination of salvianolic acid B, neochlorogenic acid, and hyperin.

9. Use of one or more of salvianolic acid B, neochlorogenic acid, and hyperin in preparing drugs for preventing and treating pulmonary arterial hypertension.

10. The application according to claim 9, characterized in that, The said salvianolic acid B, neochlorogenic acid, and hyperin can competitively bind to EPX, block its binding to the ACVR2A / ACVR1B complex, thereby interfering with the downstream ACVRs-Smad signaling pathway mediated by EPX, and ultimately inhibit the abnormal proliferation of PASMCs.

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