The protective role of raltegravir in experimental acute lung injury in vitro and in vivo.

Disruption of pulmonary endothelial permeability and associated barrier integrity increase the severity of acute respiratory distress syndrome (ARDS). This study investigated the potential ability of the human immunodeficiency virus-1 (HIV-1) integrase inhibitor raltegravir to protect against acute lung injury (ALI) and the underlying mechanisms. Accordingly, the impact of raltegravir treatment on an in vitro lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cell (HPMEC) model of ALI and an in vivo LPS-induced two-hit ALI rat model was examined. In the rat model system, raltegravir treatment alleviated ALI-associated histopathological changes, reduced microvascular permeability, decreased Evans blue dye extravasation, suppressed the expression of inflammatory proteins including HMGB1, TLR4, p-NF-κB, NLRP3, and MPO, and promoted the upregulation of protective proteins including claudin 18.1, VE-cadherin, and aquaporin 5 as measured via western blotting. Immunohistochemical staining further confirmed the ability of raltegravir treatment to reverse LPS-induced pulmonary changes in NLRP3, claudin 18.1, and aquaporin 5 expression. Furthermore, in vitro analyses of HPMECs reaffirmed the ability of raltegravir to attenuate LPS-induced declines in VE-cadherin and claudin 18.1 expression while simultaneously inhibiting NLRP3 activation and reducing the expression of HMGB1, TLR4, and NF-kB, thus decreasing overall vascular permeability. Overall, our findings suggested that raltegravir may represent a viable approach to treating experimental ALI that functions by maintaining pulmonary microvascular integrity.

AQP5 regulates the proliferation and differentiation of epidermal stem cells in skin aging.

The epidermis, the outermost layer of the skin, is the first barrier that comes into contact with the external environment. It plays an important role in resisting the invasion of harmful substances and microbial infections. The skin changes with age and external environmental factors. This study aimed to investigate epidermal stem cells during the process of aging. This study enrolled 9 volunteers with benign pigmented nevus for clinical dermatologic surgery. The phenotypes associated with skin aging changes such as skin wrinkles and elasticity of the unexposed/healthy parts near benign pigmented skin were measured, and epidermal stem cells from this region were isolated for transcriptome sequencing. The results showed that epidermal stem cells could be obtained by magnetic activated cell sorting (MACS) with high purity. Results of the transcriptome sequencing revealed that aquaporin (AQP)5 significantly decreased in the epidermal stem cells with age, and further functional experiments revealed that AQP5 could promote the proliferation and dedifferentiation of HaCaT, but did not influence cell apoptosis. In summary, AQP5 regulated the proliferation and differentiation of epidermal stem cells in skin aging, and it may play an important role in the balance of proliferation and differentiation. However, further studies are needed to determine the mechanism by which AQP5 regulates the proliferation and differentiation of epidermal skin cells in aging.

Aquaporin 1, 3, and 5 Patterns in Salivary Gland Mucoepidermoid Carcinoma: Expression in Surgical Specimens and an In Vitro Pilot Study.

Salivary gland aquaporins (AQPs) are essential for the control of saliva production and maintenance of glandular structure. However, little is known of their role in salivary gland neoplasia. Salivary gland tumors comprise a heterogeneous group of lesions, featuring variable histological characteristics and diverse clinical behaviors. Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. The aim of this study was to evaluate the expression of AQP1, AQP3, and AQP5 in 24 MEC samples by immunohistochemistry. AQP1 expression was observed in vascular endothelium throughout the tumor stroma. AQP3 was expressed in epidermoid and mucosal cells and AQP5 was expressed in mucosal cells of MEC. These proteins were expressed in the human MEC cell line UH-HMC-3A. Cellular ultrastructural aspects were analyzed by electron microscopy to certificate the tumor cell phenotype. In summary, our results show that, despite the fact that these molecules are important for salivary gland physiology, they may not play a distinct role in tumorigenesis in MEC. Additionally, the in vitro model may offer new possibilities to further investigate mechanisms of these molecules in tumor biology and their real significance in prognosis and possible target therapies.

AQP5 regulates the proliferation and differentiation of epidermal stem cells in skin aging

The epidermis, the outermost layer of the skin, is the first barrier that comes into contact with the external environment. It plays an important role in resisting the invasion of harmful substances and microbial infections. The skin changes with age and external environmental factors. This study aimed to investigate epidermal stem cells during the process of aging. This study enrolled 9 volunteers with benign pigmented nevus for clinical dermatologic surgery. The phenotypes associated with skin aging changes such as skin wrinkles and elasticity of the unexposed/healthy parts near benign pigmented skin were measured, and epidermal stem cells from this region were isolated for transcriptome sequencing. The results showed that epidermal stem cells could be obtained by magnetic activated cell sorting (MACS) with high purity. Results of the transcriptome sequencing revealed that aquaporin (AQP)5 significantly decreased in the epidermal stem cells with age, and further functional experiments revealed that AQP5 could promote the proliferation and dedifferentiation of HaCaT, but did not influence cell apoptosis. In summary, AQP5 regulated the proliferation and differentiation of epidermal stem cells in skin aging, and it may play an important role in the balance of proliferation and differentiation. However, further studies are needed to determine the mechanism by which AQP5 regulates the proliferation and differentiation of epidermal skin cells in aging.

Effect of low radiation dose on the expression and location of aquaporins in rat submandibular gland.

Head and neck cancers are common in several regions of the world and the treatment usually includes radiotherapy. This treatment can generate adverse effects to the salivary flow, with a relationship between the dose and the damage caused. Salivary gland cells are highly permeable to water and therefore, they express aquaporins (AQPs). This study analyzed changes in the expression and location of these proteins and identified morphological changes induced by low radiation in rat submandibular gland. Female rats were divided into control and irradiated groups. Immunohistochemistry analysis allowed confirming the presence of AQP1 in the blood vessel endothelium. Intense and steady labelling granules were also observed in the cytoplasm of submandibular gland ductal cells. In addition, there was AQP5 positive labelling in ductal cells delimiting the lumen of intercalated duct, in the cytoplasm and membrane of acinar cells. Finally, the decrease of AQP labelling in irradiated animal glands validated their radiosensitivity. Thus, the decrease in AQP1 protein levels in the endothelium and AQP5 in gland ductal cells of irradiated animals may have hindered the removal of water from the lumen of ductal cells, inducing a delay in water absorption and triggering a slight lumen increase.

Characterization of air-liquid interface culture of A549 alveolar epithelial cells

Alveolar epithelia play an essential role in maintaining the integrity and homeostasis of lungs, in which alveolar epithelial type II cells (AECII) are a cell type with stem cell potential for epithelial injury repair and regeneration. However, mechanisms behind the physiological and pathological roles of alveolar epithelia in human lungs remain largely unknown, partially owing to the difficulty of isolation and culture of primary human AECII cells. In the present study, we aimed to characterize alveolar epithelia generated from A549 lung adenocarcinoma cells that were cultured in an air-liquid interface (ALI) state. Morphological analysis demonstrated that A549 cells could reconstitute epithelial layers in ALI cultures as evaluated by histochemistry staining and electronic microscopy. Immunofluorescent staining further revealed an expression of alveolar epithelial type I cell (AECI) markers aquaporin-5 protein (AQP-5), and AECII cell marker surfactant protein C (SPC) in subpopulations of ALI cultured cells. Importantly, molecular analysis further revealed the expression of AQP-5, SPC, thyroid transcription factor-1, zonula occludens-1 and Mucin 5B in A549 ALI cultures as determined by both immunoblotting and quantitative RT-PCR assay. These results suggest that the ALI culture of A549 cells can partially mimic the property of alveolar epithelia, which may be a feasible and alternative model for investigating roles and mechanisms of alveolar epithelia in vitro.

Characterization of air-liquid interface culture of A549 alveolar epithelial cells.

Alveolar epithelia play an essential role in maintaining the integrity and homeostasis of lungs, in which alveolar epithelial type II cells (AECII) are a cell type with stem cell potential for epithelial injury repair and regeneration. However, mechanisms behind the physiological and pathological roles of alveolar epithelia in human lungs remain largely unknown, partially owing to the difficulty of isolation and culture of primary human AECII cells. In the present study, we aimed to characterize alveolar epithelia generated from A549 lung adenocarcinoma cells that were cultured in an air-liquid interface (ALI) state. Morphological analysis demonstrated that A549 cells could reconstitute epithelial layers in ALI cultures as evaluated by histochemistry staining and electronic microscopy. Immunofluorescent staining further revealed an expression of alveolar epithelial type I cell (AECI) markers aquaporin-5 protein (AQP-5), and AECII cell marker surfactant protein C (SPC) in subpopulations of ALI cultured cells. Importantly, molecular analysis further revealed the expression of AQP-5, SPC, thyroid transcription factor-1, zonula occludens-1 and Mucin 5B in A549 ALI cultures as determined by both immunoblotting and quantitative RT-PCR assay. These results suggest that the ALI culture of A549 cells can partially mimic the property of alveolar epithelia, which may be a feasible and alternative model for investigating roles and mechanisms of alveolar epithelia in vitro.

The Expression of Water and Ion Channels in Diffuse Alveolar Damage Is Not Dependent on DAD Etiology.

INTRODUCTION: Aquaporins and ion channels are membrane proteins that facilitate the rapid movement of water and solutes across biological membranes. Experimental and in vitro studies reported that the function of these channels and pulmonary edema resolution are impaired in acute lung injury (ALI). Although current evidence indicates that alveolar fluid clearance is impaired in patients with ALI/diffuse alveolar damage (DAD), few human studies have addressed the alterations in pulmonary channels in this clinical condition. Additionally, it is not known whether the primary cause of DAD is a relevant variable for the channel dysfunction. METHODS: Autopsied lungs of 43 patients with acute respiratory failure (ARF) due to DAD of three different etiologies, non-pulmonary sepsis, H1N1 viral infection and leptospirosis, were compared to 18 normal lungs. We quantified the expression of aquaporin (AQP) 1, AQP3, AQP5, epithelial Na+ channel (ENaC) and sodium potassium ATPase (Na-K-ATPase) in the alveolar septum using immunohistochemistry and image analysis. RESULTS: The DAD group presented with increased expression of AQP3, AQP5 and Na-K-ATPase and decreased expression of ENaC compared to controls. However, there was no difference in protein expression within the DAD groups of different etiologies. CONCLUSION: Water and ion channels are altered in patients with ARF due to DAD. The cause of DAD does not seem to influence the level of impairment of these channels.

Cyclic adenosine monophosphate-protein kinase A signal pathway may be involved in pulmonary aquaporin-5 expression in ischemia/reperfusion rats following deep hypothermia cardiac arrest.

We investigated the effects of cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) on the expression of aquaporin 5 (AQP5) in ischemia/reperfusion (I/R) rats following deep hypothermia cardiac arrest. Wistar rats were randomly divided into: a sham control group (subjected to a sham operation); an I/R group (subjected to occlusion of the bronchial arteries and the left inferior pulmonary artery); an H89 group (subjected to occlusion of the bronchial arteries and the left inferior pulmonary vein and artery, and treated with 5 mg/kg H89 for 2 days before the study); and a forskolin group (subjected to occlusion of the bronchial arteries and the left inferior pulmonary vein and artery, and treated with 5 mg/kg forskolin for 2 days before the study). Expression levels of AQP5 mRNA and protein were determined using reverse transcription-polymerase chain reaction and western blotting. Decreased expression of AQP5 was noted in the pulmonary tissues of the I/R group compared with the sham controls. Compared to that in the control group, there was a notable decrease in AQP5 expression in the I/R group. After treating with forskolin, AQP5 expression increased in the forskolin group compared with the I/R group. In the H89 group, AQP5 expression decreased compared with the I/R group. The decreased expression of AQP5 was possibly associated with acute pulmonary injury induced by I/R. The cAMP-PKA signal pathway may be involved in the expression of AQP5 in I/R rats after deep hypothermia cardiac arrest.

Comparative expression analysis of aquaporin-5 (AQP5) in keratoconic and healthy corneas.

PURPOSE: Keratoconus (KC) is a common progressive corneal disease characterized by excessive stromal thinning, central or paracentral conical protrusion, and disruptions in Bowman's layer. The etiology of KC is largely unknown, and a combination of genetic and environmental factors is believed to play a role in the origin of the disease. Recently, the absence of transcripts of the water channel, aquaporin-5 (AQP5), was demonstrated by reverse-transcription polymerase chain reaction (RT-PCR) in KC tissues and was proposed as a possible marker for KC. In this study, we sought to evaluate AQP5 mRNA and protein expression in KC and non-KC corneal tissues using a combination of techniques. METHODS: A total of 69 samples of corneal tissue were analyzed including 39 corneal buttons from patients with advanced KC, 16 samples of non-KC corneal epithelium belonging to patients who underwent surface refractive surgery, 12 sclerocorneal rims obtained from healthy donor subjects, and two healthy corneal buttons. Determination of AQP5 transcript and protein expression patterns was performed by means of real time RT-PCR, immunohistochemistry, immunocytochemistry, and flow cytometry methods. Cell culture was performed to identify AQP5 protein expression in KC epithelial cells. RESULTS: AQP5 mRNA was expressed with no significant differences between KC and non-KC tissues. Moreover, AQP5 protein expression analysis did not reveal differences in protein levels and/or cell location among KC and non-KC tissues. Interestingly, AQP5 expression continues for up to 21 days in the isolated KC corneal epithelial cells. CONCLUSIONS: Our results do not support a role for AQP5 in KC etiopathogeny or as a disease marker. Genetic background differences or a distinct pathogenetic KC cascade specific to the analyzed population could account for the dissimilarities observed in KC-related AQP5 expression.