Antibody signatures in patients with histopathologically defined multiple sclerosis patterns.

Early active multiple sclerosis (MS) lesions can be classified histologically into three main immunopathological patterns of demyelination (patterns I-III), which suggest pathogenic heterogeneity and may predict therapy response. Patterns I and II show signs of immune-mediated demyelination, but only pattern II is associated with antibody/complement deposition. In pattern III lesions, which include Baló's concentric sclerosis, primary oligodendrocyte damage was proposed. Serum antibody reactivities could reflect disease pathogenesis and thus distinguish histopathologically defined MS patterns. We established a customized microarray with more than 700 peptides that represent human and viral antigens potentially relevant for inflammatory demyelinating CNS diseases, and tested sera from 66 patients (pattern I n = 12; II n = 29; III n = 25, including 8 with Baló's), healthy controls, patients with Sjögren's syndrome and stroke patients. Cell-based assays were performed for aquaporin 1 (AQP1) and AQP4 antibody detection. No single peptide showed differential binding among study cohorts. Because antibodies can react with different peptides from one protein, we also analyzed groups of peptides. Patients with pattern II showed significantly higher reactivities to Nogo-A peptides as compared to patterns I (p = 0.02) and III (p = 0.02). Pattern III patients showed higher reactivities to AQP1 (compared to pattern I p = 0.002, pattern II p = 0.001) and varicella zoster virus (VZV, compared to pattern II p = 0.05). In patients with Baló's, AQP1 reactivity was also significantly higher compared to patients without Baló's (p = 0.04), and the former revealed distinct antibody signatures. Histologically, Baló's patients showed loss of AQP1 and AQP4 in demyelinating lesions, but no antibodies binding conformational AQP1 or AQP4 were detected. In summary, higher reactivities to Nogo-A peptides in pattern II patients could be relevant for enhanced axonal repair and remyelination. Higher reactivities to AQP1 peptides in pattern III patients and its subgroup of Baló's patients possibly reflect astrocytic damage. Finally, latent VZV infection may cause peripheral immune activation.

Aquaporin 1 alleviates acute kidney injury via PI3K-mediated macrophage M2 polarization.

BACKGROUND: Lipopolysaccharide (LPS)-induced acute kidney injury (AKI) is associated with an abnormal immune response. Accumulating evidence has demonstrated that aquaporin 1 (AQP1) prevents kidney tissue injury in LPS-induced AKI by mediating immune response. However, the underlying mechanisms remain obscure. Macrophages as immune cells with multiple phenotypes are important mediators in tissue homeostasis and host defense. We propose that macrophage polarization is implicated in AQP1-mediated immune response. METHODS: Herein we established sepsis-induced AKI model rats through intraperitoneal injection of LPS into Wistar rats to reveal immune mechanism of damage. We also used LPS-induced mouse RAW264.7 cells to elucidate the molecular mechanism of macropage polarization. RESULTS: Histopathology showed that renal tubular epithelial cells in the model group were swollen, inflammatory exudation was obvious and the inflammatory factors, interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were increased. Western blotting showed PI3K was upregulated in the model group. Serum creatinine and urea nitrogen increased after LPS injection. Renal AQP1 mRNA is downregulated and serum AQP1 protein increased first and then decreased in LPS-induced AKI rats. M2 macrophage markers (Arg-1, CD206) were increased in repair stage. In addition, treatment of murine macrophages (RAW264.7) with AQP1 siRNA resulted in decreased PI3K activation and M2 polarization, but increased IL-6 and TNF-α. Moreover, inhibiting PI3K with wortmannin imitated the results of AQP1 silencing. CONCLUSIONS: Macrophage M2 polarization is likely the cellular mechanism underlying the anti-AKI property of AQP1, and PI3K activation is involved in the AQP1-induced M2 phenotype switch.

Neoepitope targets of tumour-infiltrating lymphocytes from patients with pancreatic cancer.

BACKGROUND: Pancreatic cancer exhibits a poor prognosis and often presents with metastasis at diagnosis. Immunotherapeutic approaches targeting private cancer mutations (neoantigens) are a clinically viable option to improve clinical outcomes. METHODS: 3/40 TIL lines (PanTT26, PanTT39, PanTT77) were more closely examined for neoantigen recognition. Whole-exome sequencing was performed to identify non-synonymous somatic mutations. Mutant peptides were synthesised and assessed for antigen-specific IFN-γ production and specific tumour killing in a standard Cr51 assay. TIL phenotype was tested by flow cytometry. Lymphocytes and HLA molecules in tumour tissue were visualised by immunohistochemistry. RESULTS: PanTT26 and PanTT39 TILs recognised and killed the autologous tumour cells. PanTT26 TIL recognised the KRASG12v mutation, while a PanTT39 CD4+ TIL clone recognised the neoepitope (GLLRYWRTERLF) from an aquaporin 1-like protein (gene: K7N7A8). Repeated stimulation of TILs with the autologous tumour cells line lead to focused recognition of several mutated targets, based on IFN-γ production. TILs and corresponding PBMCs from PanTT77 showed shared as well as mutually exclusively tumour epitope recognition (TIL-responsive or PBMC-responsive). CONCLUSION: This study provides methods to robustly screen T-cell targets for pancreatic cancer. Pancreatic cancer is immunogenic and immunotherapeutic approaches can be used to develop improved, targeted therapies.

Aquaporin-1 attenuates macrophage-mediated inflammatory responses by inhibiting p38 mitogen-activated protein kinase activation in lipopolysaccharide-induced acute kidney injury.

OBJECTIVE: This study was designed to investigate the role of AQP1 in the development of LPS-induced AKI and its potential regulatory mechanisms in the inflammatory responses of macrophages. METHODS: Male Wistar rats were injected intraperitoneally with LPS, and biochemical and histological renal damage was assessed. The levels of inflammatory mediators, macrophage markers and AQP1 in blood and kidney tissues were assessed by ELISA. RTPCR was used to assess changes in the relative levels of AQP1 mRNA induced by LPS. Western blot and immunofluorescence analyses were performed to assay the activation of the p38 MAPK and NF-κB pathways, respectively. The same detection methods were used in vitro to determine the regulatory mechanisms underlying AQP1 function. RESULTS: AQP1 mRNA levels were dramatically decreased in AKI rats following the increased expression of inflammatory factors. In vitro experiments demonstrated that silencing the AQP1 gene increased inflammatory mediator secretion, altered the classical activation of macrophages, greatly enhanced the phosphorylation of p38 and accelerated the translocation of NF-κB. Furthermore, these results were blocked by doramapimod, a p38 inhibitor. Therefore, these effects were mediated by the increased phosphorylation of p38 MAPK. CONCLUSION: Our results suggest that altered AQP1 expression may be associated with the development of inflammation in AKI. AQP1 plays a protective role in modulating acute renal injury and can attenuate macrophage-mediated inflammatory responses by downregulating p38 MAPK activity in LPS-induced RAW264.7 cells. The pharmacological targeting of AQP1-mediated p38 MAPK signalling may provide a novel treatment approach for AKI.

Comparative Analysis for the Presence of IgG Anti-Aquaporin-1 in Patients with NMO-Spectrum Disorders.

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected.

Antiangiogenic tumor therapy by DNA vaccine inducing aquaporin-1-specific CTL based on ubiquitin-proteasome system in mice.

Aquaporin-1 (AQP-1) is a water channel protein highly expressed in the vascular endothelial cells of proliferating tissues including malignant cancers. Given that in APC ubiquitinated peptides are effectively introduced into proteasomes from which CD8 epitopes are excised, we fused ubiquitin with AQP-1 (pUB-AQP-1) to produce a DNA vaccine. In C57BL/6J mice immunized with pUB-AQP-1, the growth of B16F10 melanoma was profoundly inhibited. The antitumor effect of the pUB-AQP-1 DNA vaccine was largely mediated by CD8 T cells, which secrete IFN-γ, perforin, and granzyme-B in the presence of APCs transfected with pUB-AQP-1. AQP-1-specific CD8 T cells possessed cytotoxic activity both in vivo and in vitro. After tumor challenge, the microvessel density decreased and the ratio of total blood vessel area to tumor area was significantly reduced as compared with control mice, resulting in a dramatic suppression of tumor growth. The immunization effect was completely abrogated in immunoproteasome-deficient mice. Strikingly this pUB-AQP-1 DNA vaccine was also effective against Colon 26 colon tumors (BALB/c) and MBT/2 bladder tumors (C3H/HeN). Thus, this ubiquitin-conjugated DNA immunization-targeting tumor vasculature is a valid and promising antitumor therapy. This vaccine works across the barriers of tumor species and MHC class I differences in host mice.

Aquaporin-1 antibody in neuromyelitis optical patients.

BACKGROUND/METHODS: To find out the prevalence of aquaporin-antibody (Aqp-Ab) and characterize Aqp-Ab associated clinical features in NMO, Aqp-1 and Aqp-4-Abs were examined using radioimmunoprecipitation and cell-based assays, respectively. RESULTS: Aqp-4 and Aqp-1-Abs were detected in 20/30 and 8/30 NMO patients, respectively. One patient was Aqp-1-Ab single-positive, 13 patients were Aqp-4-Ab single-positive, 7 patients were Aqp-4/Aqp-1-Ab double-positive and 9 patients were seronegative. All double-positive patients had optic neuritis during the first attack. Only 2/29 MS patients and none of the control idiopathic intracranial hypertension patients were Aqp-1-Ab positive. CONCLUSION: Aqp-1-Ab is usually detected in Aqp-4-Ab positive NMO patients and might be involved in optic neuritis pathogenesis.

A new AQP1 null allele identified in a Gypsy woman who developed an anti-CO3 during her first pregnancy.

BACKGROUND AND OBJECTIVES: The Colton blood group antigens are carried by the AQP1 water channel. AQP1(-/-) individuals, also known as Colton-null since they express no Colton antigens, do not suffer any apparent clinical consequence but may develop a clinically significant alloantibody (anti-CO3) induced by transfusion or pregnancy. Identification and transfusion support of Colton-null patients are highly challenging, not only due to the extreme rarity of this phenotype, the lack of appropriate reagents in most laboratories, as well as the possibility of confusing it with the recently described CO:-1,-2,3,-4 phenotype where AQP1 is present. This study investigated a new Colton-null case and evaluated three commercially available anti-AQP1s to identify Colton-null red blood cell samples. METHODS: The Colton-null phenotype was investigated by standard serological techniques, AQP1 sequencing, immunoblot and flow cytometry analyses. RESULTS: We identified and characterized the Colton-null phenotype in a Gypsy woman who developed an anti-CO3 during her first pregnancy. After developing a simple and robust method to sequence AQP1, we showed that she was apparently homozygous for a new AQP1 null allele, AQP1 601delG, whose product is not expressed in her red blood cells. We also established the Colton specificity of three commercially available anti-AQP1s in immunoblot and/or flow cytometry analyses. CONCLUSION: This Gypsy woman represents the sixth Colton-null case characterized at the serological, genetic and biochemical levels. The validation here of new reagents and methods should facilitate the identification of Colton-null individuals.

The conjugation of an AQP1-directed immunotoxin in the study of site-directed therapy within the CNS.

PURPOSE: The water channel, aquaporin (AQP)1, is highly specific to the choroid plexus (CP) epithelium within the brain. It is therefore a potential target through which therapeutic agents could be selectively directed to the CP. Here we describe the conjugation of a monoclonal antibody (mAb), raised against an extra-cellular domain of AQP1, to the A chain of ricin (RCA). This reagent should allow study of a highly specific chemical lesion of the CP. METHODS: A published method was used to couple the anti-AQP1 mAb to the A chain of ricin using an SPDP cross-linker. The conjugate was purified by Superdex S-200 chromatography. Column fractions were analysed by SDS-PAGE and Western blotting. Fractions containing conjugate were assayed for cytotoxicity on rat CP cells in culture, using a WST-1 viability assay to assess cell death. RESULTS: SDS-PAGE and Western blotting demonstrated separation of conjugated anti-AQP1 mAb from unconjugated ricin by the Superdex column. Cultured rat CP cells were killed with an IC(50) of 1.81 µg/ml when exposed to the anti-AQP1 mAb-RCA conjugation reaction products. Selectivity of this reagent was demonstrated by the higher IC(50) exhibited by non-AQP1-expressing primary fibroblast cultures and cultures exposed to the reaction products from the non-specific IgG-RCA conjugation attempt. CONCLUSION: We have been able to demonstrate selective cytotoxicity in vitro of an anti-AQP1 mAb-RCA conjugate. We hope that this work will generate further interest in the use of this site-specific conjugate to target the CP in conditions such as hydrocephalus, tumours that arise from here, and other AQP1-expressing tumours.

Regulation of DAF-16-mediated Innate Immunity in Caenorhabditis elegans.

Activation of the innate immune system results in a rapid microbicidal response against microorganisms, which needs to be fine-tuned because uncontrolled immune responses can lead to infection and cancer, as well as conditions such as Crohn disease, atherosclerosis, and Alzheimer disease. Here we report that excessive activity of the conserved FOXO transcription factor DAF-16 enhances susceptibility to bacterial infections in Caenorhabditis elegans. We found that increased temperature activates not only DAF-16 nuclear import but also a control mechanism involved in DAF-16 nuclear export. The nuclear export of DAF-16 requires heat shock transcription factor HSF-1 and Hsp70/HSP-1. Furthermore, we show that increased expression of the water channel Aquoporin-1 is responsible for the deleterious consequences of excessive DAF-16-mediated immune response. These studies reveal a stress-inducible mechanism involved in the regulation of DAF-16 and indicate that uncontrolled DAF-16 activity and water homeostasis are a cause of the deleterious effects of excessive immune responses.

A functional AQP1 allele producing a Co(a-b-) phenotype revises and extends the Colton blood group system.

BACKGROUND: The Colton blood group system currently comprises three antigens, Co(a) , Co(b) , and Co3. The latter is only absent in the extremely rare individuals of the Colton "null" phenotype, usually referred to as Co(a-b-), which lack the water channel AQP1 that carries the Colton antigens. The discovery of a Co(a-b-) individual with no AQP1 deficiency suggested another molecular basis for the Co(a-b-) phenotype. STUDY DESIGN AND METHODS: Red blood cells were analyzed by stopped-flow light scattering and Western blotting and typed by hemagglutination and flow cytometry. Genotyping by sequencing and polymerase chain reaction-restriction fragment length polymorphism was applied. An expression system for Colton antigens was developed in mammalian cells. RESULTS: Although Co(a-b-), the proband expressed fully functional AQP1 and had developed a novel Colton alloantibody. Sequencing of AQP1 revealed a homozygous nucleotide change (140A>G) encoding the single-amino-acid substitution Q47R. A second case was identified due to the presence of this novel Colton alloantibody. By generating an expression system for Colton antigens in K-562 cells, the Q47R substitution was shown to inhibit the expression of both Co(a) and Co(b) antigens. Other naturally occurring single-amino-acid substitutions, that is, A45T, P38L, and N192K, were also studied in this Colton antigen expression system. CONCLUSIONS: The Co(a-b-) phenotype can be generated by a functional AQP1 allele, that is, AQP1 140G encoding AQP1 (Q47R) and allowing the development of a novel Colton alloantibody. This study also shows that the Co(b) antigen can be produced by at least two different substitutions at Amino Acid Position 45, that is, A45V and A45T.

The immunoexpression of aquaporin 1, PAX2, PAX8, connexin 36, connexin 43 in human fetal kidney.

INTRODUCTION: The kidney develops from two mesodermal primordia. Aquaporin 1 (AQP1) is a membrane protein characteristic to epithelial and endothelial cell of the human body. The Pax family of genes encodes transcription factors with important role in intrauterine development. Connexins are transmembrane proteins found in gap junctions. We monitored the changes in the expression of AQP1, paired box gene 2 (PAX2), paired box gene 8 (PAX8), connexin 36 (Cx36) and connexin 43 (Cx43) proteins in fetal renal tissue. MATERIALS AND METHODS: We studied 34 post mortem fetuses of 9 to 24 weeks from the Laboratory of Pathology, Emergency County Hospital of Târgu Mures, Romania, using immunohistochemistry. RESULTS: AQP1 expression appeared in the apical and basolateral parts of cells, lining the proximal convoluted tubules and the descending limb of Henle's loop, then in the tubule pole of Bowman's capsule also. Nuclear expression of PAX2 was observed in structures developed both from the ureteric bud and the metanephric mesenchyme, and of PAX8 was observed in the proximal convoluted tubule's epithelium, Henle's loop, and collecting ducts. Cytoplasmic expression of Cx36 was localized to nephrons in different developmental stages, glomerular vessels and collecting ducts, and of Cx43 was localized to the endothelium of glomerular and peritubular vessels, as well as to the epithelium of the proximal tubules. DISCUSSIONS AND CONCLUSIONS: Nephrogenesis begins in the embryonic period, and continues into the fetal period as well. It is regulated by a wide array of markers. The current study supplements literature data regarding immunoexpression of these markers during renal development in the fetal period.

[Clinical and biochemical characteristics of atypical variants of multiple sclerosis]. / Kliniko-biokhimicheskie kharakteristiki atipichnykh variantov rasseiannogo skleroza.

AIM: To study the clinical and biochemical features of atypical variants of multiple sclerosis (MS) (tumefactive demyelination (TD), Balo's concentric sclerosis (BCS)) and acute disseminated encephalomyelitis (ADEM)). MATERIAL AND METHODS: Forty-two patients were studied, including 32 patients with atypical variants of MS (6 patients with BCS and 26 patients with TD) and 10 patients with ADEM. The control group included 20 healthy volunteers. Clinical characteristics and EDSS scores were evaluated. Antibodies to aquaporin 1 (AQP1-IgG), aquaporin 4 (AQP4-IgG), antibodies to myelin oligodendrocyte glycoprotein (MOG-IgG) and aquaporin 1 (AQP1) in serum and cerebrospinal fluid (CSF) were detected using ELISA. RESULTS AND CONCLUSION: BCS and TD occurred both in isolation and comorbid with MS (in 50% of cases with BCS, 50% of cases with TD). Atypical symptoms of MS were detected in 50% of cases of CFS, 15.4% of cases of PD. The levels of CSF cytosis and CSF protein were not significantly different between the groups. The levels of AQP1-IgG, AQP4-IgG, AQP1, MOG-IgG in serum with BCS, TD and ADEM were significantly higher than in the control group. No significant differences were found between atypical variants of MS. A correlation between a high level of MOG-IgG and the EDSS score in BCS was shown. MOG-IgG may have a pathogenetic significance in BCS. Further studies of AQP1-IgG, AQP4-IgG and MOG-IgG in patients with atypical variants of MS are needed.

Expression and immunodetection of aquaporin 1 (AQP1) in canine spermatozoa.

The permeability of water and cryoprotectants through the plasma membrane is very important for cryopreservation of mammalian cells. Aquaporin 1 (AQP1) is one of the water channel proteins localized on the membranes of various cells including reproductive organs, allowing water to flow rapidly across the plasma membranes in the direction of osmotic gradients. Although mRNA expression of AQP1 was reported in the mammalian testis by reverse transcription polymerase chain reaction (RT-PCR), protein and mRNA expressions of AQP1 have not been confirmed to date in the sperm of any species. The present study was conducted to determine whether AQP1 mRNA is expressed and AQP1 protein exists in canine spermatozoa. Results from RT-PCR showed that AQP1 mRNA was expressed in canine spermatozoa as well as the testis. The size was similar to the one from canine genomic DNA as a positive control. In sperm, AQP1 protein was also detected by canine AQP1 specific antibody. From these results, both AQP1 mRNA and protein are expressed in male gamete in the dog. Expression of AQP1 may be involved in the flux of water during the cryopreservation of spermatozoa.

Study of the placentae of patients with neuromyelitis optica spectrum disorder.

Previous studies have shown that circulating AQP4-IgG may lead to negative consequences during pregnancy in patients with neuromyelitis optica spectrum disorder (NMOSD). The objective of this study was to explore whether AQP4-IgG influences pregnancy by affecting AQP4 expression and inducing placental inflammation in patients with NMOSD. We prospectively collected clinical data from six pregnant AQP4-IgG-seropositive NMOSD patients and their infants, and investigated AQP4 expression and placental inflammatory infiltration by comparing hematoxylin and eosin and immunohistochemical (AQP1, AQP4, C5b-9, IgG, CD3, CD8, CD20, and CD68) staining results with three normal controls. Four patients were term pregnant and their infants were normal for development, serum AQP4-IgG was positive at the time of birth, and three infants were negative for AQP4-IgG after 3 months. Two patients underwent induced abortion; one because of NMOSD relapse and another because of fetal malformation. Histological investigation showed normal structure of the chorionic villi, and no significant difference in the intensity of the immunohistochemical staining for AQP1, AQP4, and inflammatory markers in placentae of patients and the controls. Our results showed that there was no significant decrease in placental AQP4 expression, and no obvious placental inflammation or signs of damage in term placentae of NMOSD patients seropositive for AQP4-IgG.

Immunogenic potential of Rhipicephalus (Boophilus) microplus aquaporin 1 against Rhipicephalus sanguineus in domestic dogs.

This study evaluated a recombinant aquaporin 1 protein of Rhipicephalus (Boophilus) microplus (RmAQP1) as antigen in a vaccine against R. sanguineus. Five dogs were immunized with RmAQP1 (10 µg) + adjuvant (Montanide) (G1), and five were inoculated with adjuvant only (G2), three times. Twenty-one days after the last immunization, animals of both groups were challenged with R. sanguineus larvae, nymphs and adults, and their biotic potential was compared. Blood samples were collected before each immunization and every 28 days after the last immunization for 10 weeks. Serum antibody titers (IgG) were assessed by ELISA. We observed that: engorgement period of adult females from G1 was 12% shorter than G2; larvae from G1 had 8.7% longer engorgement period than G2 and weighed 7.2% less; nymphs from G1 had 4.5% shorter engorgement period than G2 and weighed 3.6% less; although the antibody titers increased following the second immunization, they rapidly decreased after the third immunization. Results indicated low immunoprotection of RmAQP1 against adult R. sanguineus ticks, and possible efficacy on larvae and nymphs fed on immunized dogs. Further studies should be performed for a full evaluation of the immunoprotection of RmAQP1 against R. sanguineus infestations in dogs.

Control of infestations by Ixodes ricinus tick larvae in rabbits vaccinated with aquaporin recombinant antigens.

BACKGROUND: Tick-borne diseases greatly impact human and animal health worldwide, and vaccines are an environmentally friendly alternative to acaricides for their control. Recent results have suggested that aquaporin (AQP) water channels have a key function during tick feeding and development, and constitute good candidate antigens for the control of tick infestations. METHODS: Here we describe the effect of vaccination with the Ixodes ricinus AQP1 (IrAQP) and a tick AQP conserved region (CoAQP) on I. ricinus tick larval mortality, feeding and molting. RESULTS: We demonstrated that vaccination with IrAQP and CoAQP had an efficacy of 32% and 80%, respectively on the control of I. ricinus larvae by considering the cumulative effect on reducing tick survival and molting. CONCLUSIONS: The effect of the AQP vaccines on larval survival and molting is essential to reduce tick infestations, and extended previous results on the effect of R. microplus AQP1 on the control of cattle tick infestations. These results supports that AQP, and particularly CoAQP, might be a candidate protective antigen for the control of different tick species.

PBEF promotes the apoptosis of pulmonary microvascular endothelial cells and regulates the expression of inflammatory factors and AQP1 through the MAPK pathways.

Pre-B cell colony-enhancing factor (PBEF) has been shown to have a variety of biological functions. Studies have proven that PBEF plays a functional role in acute lung injury (ALI). Therefore, in this study, we aimed to confirm the importance of PBEF in ALI. The effects of PBEF overexpression on the apoptosis of human pulmonary microvascular endothelial cells (HPMECs) were analyzed by flow cytometry, and the results indicated that PBEF promoted the apoptosis of HPMECs, which aggravated the development of ALI. Comparative experiments involving increasing and decreasing PBEF expression demonstrated that PBEF promoted the expression of inflammatory factors, such as interleukin (IL)­1ß, IL­6 and IL­8 in the HPMECs , thus intensifying the inflammatory response. PBEF also inhibited the expression of aquaporin 1 (AQP1), which caused a dysfunction and imbalance in water transport. Moreover, we also found that tumor necrosis factor (TNF)­α promoted the expression of PBEF in the HPMECs. After blocking the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways, we found that PBEF regulated the expression of inflammatory factors and AQP1, mainly through the MAPK pathways. Taken together, these results demonstrate that the increase in intracellular PBEF expression promoted the apoptosis of HPMECs and the expression of inflammatory factors and thus enhanced the inflammatory response and inhibited the expression of AQP1, which resulted in abnormal water transport, diminishing the regulatory effects of AQP1 on water transport.