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Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

This work was performed under the Project HPC-EUROPA3 (No. INFRAIA-2016-1-730897), with the support of the EC Research Innovation Action under the H2020 Programme; in particular, the authors gratefully acknowledge the support of M. S. P. Sansom and the computer resources and technical support provided by ARCHER. M.A.G. acknowledges the support of Ayuda Juan de la Cierva-Incorporación (Grant No. IJCI-2016-27497) from Ministerio de Ciencia, innovación y universidades (Spain). M. S. P. Sansom acknowledges the following funding Nos. BBSRC BB/N000145/1, EPSRC EP/R004722/1, and EP/V010948/1. C. Valeriani acknowledges the funding from MINECO No. PID2019-105343GB-I00., Water diffusion through membrane proteins is a key aspect of cellular function. Essential processes of cellular metabolism are driven by osmotic pressure, which depends on water channels. Membrane proteins such as aquaporins (AQPs) are responsible for enabling water permeation through the cell membrane. AQPs are highly selective, allowing only water and relatively small polar molecules to cross the membrane. Experimentally, estimation of water flux through membrane proteins is still a challenge, and hence, accurate simulations of water permeation are of particular importance. We present a numerical study of water diffusion through AQP1 comparing three water models: TIP3P, OPC, and TIP4P/2005. Bulk diffusion, diffusion permeability, and osmotic permeability are computed and compared among all models. The results show that there are significant differences between TIP3P (a particularly widespread model for simulations of biological systems) and the more recently developed TIP4P/2005 and OPC models. We demonstrate that OPC and TIP4P/2005 reproduce protein-water interactions and dynamics in very good agreement with experimental data. From this study, we find that the choice of the water model has a significant effect on the computed water dynamics as well as its molecular behavior within a biological nanopore., Unión Europea. Horizonte 2020, Ministerio de Ciencia e Innovación (MICINN), Ministerio de Economía y Competitividad (MINECO), BBSRC UK Research & Innovation (UKRI) Biotechnology and Biological Sciences Research Council (BBSRC), EPSRC UK Research & Innovation (UKRI) Engineering & Physical Sciences Research Council (EPSRC), Depto. de Estructura de la Materia, Física Térmica y Electrónica, Fac. de Ciencias Físicas, TRUE, pub

Tumour angiogenesis plays a key role in tumour growth and progression. The application of current anti-angiogenic drugs is accompanied by adverse effects and drug resistance. Therefore, finding safer effective treatments is needed. Ginsenoside Rg3 (Rg3) has two epimers, 20(S)-Rg3 (SRg3) and 20(R)-Rg3 (RRg3), with stereoselective activities. Using response surface methodology, we optimised a combination of these two epimers for the loop formation of human umbilical vein endothelial cell (HUVEC). The optimised combination (C3) was tested on HUVEC and two murine endothelial cell lines. C3 significantly inhibited the loop formation, migration, and proliferation of these cells, inducing apoptosis in HUVEC and cell cycle arrest in all of the cell lines tested. Using molecular docking and vascular endothelial growth factor (VEGF) bioassay, we showed that Rg3 has an allosteric modulatory effect on vascular endothelial growth factor receptor 2 (VEGFR2). C3 also decreased the VEGF expression in hypoxic conditions, decreased the expression of aquaporin 1 and affected AKT signaling. The proteins that were mostly affected after C3 treatment were those related to mammalian target of rapamycin (mTOR). Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) was one of the important targets of C3, which was affected in both hypoxic and normoxic conditions. In conclusion, these results show the potential of C3 as a novel anti-angiogenic drug

Aquaporin-1 (AQP1) is the main water channel responsible for water transport through many epithelia and endothelia. The latest evidence pointed toward an important role of this protein also in gas permeation, angiogenesis, cell proliferation and migration. In the present work we studied the expression of AQP1 by immunohistochemical staining of 92 lung biopsies from patients diagnosed with a pleuropulmonary tumor (71 lung and 21 pleural neoplasms). AQP1 expression was analyzed comparing the results among the different histological patterns and against 9 control cases (5 parenchyma and 4 healthy pleura). Clear staining of AQP1 was detected in 39 of the 92 tumors analyzed. In parenchyma, AQP1 was more frequently detected in primary lung denocarcinomas (55%,P<0.001); in contrast, small cell carcinomas were the least AQP1 expressive tumors studied (93% of negative staining, P<0.05). Carcinomas analyzed in pleura (mesotheliomas and metastatic adenocarcinomas) also revealed strong xpression of AQP1. High expression of this protein was detected in small capillaries in areas near or surrounding the tumor, and novel intense AQP1 immunostaining was detected over thicker alveolar walls in alveoli inside or next to the tumoral tissue regardless of the tumor type. An important role of AQP1 in tumor angiogenesis is sustained by the abundant expression of this protein in the endothelia of tumor capillaries. Further studies are necessary to elucidate the potential pathophysiological role of this protein in pleuropulmonary neoplasms.

Aquaporin-1 (AQP1) is the main water channel responsible for water transport through many epithelia and endothelia. The latest evidence pointed toward an important role of this protein also in gas permeation, angiogenesis, cell proliferation and migration. In the present work we studied the expression of AQP1 by immunohistochemical staining of 92 lung biopsies from patients diagnosed with a pleuropulmonary tumor (71 lung and 21 pleural neoplasms). AQP1 expression was analyzed comparing the results among the different histological patterns and against 9 control cases (5 parenchyma and 4 healthy pleura). Clear staining of AQP1 was detected in 39 of the 92 tumors analyzed. In parenchyma, AQP1 was more frequently detected in primary lung denocarcinomas (55%,P<0.001); in contrast, small cell carcinomas were the least AQP1 expressive tumors studied (93% of negative staining, P<0.05). Carcinomas analyzed in pleura (mesotheliomas and metastatic adenocarcinomas) also revealed strong xpression of AQP1. High expression of this protein was detected in small capillaries in areas near or surrounding the tumor, and novel intense AQP1 immunostaining was detected over thicker alveolar walls in alveoli inside or next to the tumoral tissue regardless of the tumor type. An important role of AQP1 in tumor angiogenesis is sustained by the abundant expression of this protein in the endothelia of tumor capillaries. Further studies are necessary to elucidate the potential pathophysiological role of this protein in pleuropulmonary neoplasms.

Aquaporin-1 (AQP1) is the main water channel responsible for water transport through many epithelia and endothelia. The latest evidence pointed toward an important role of this protein also in gas permeation, angiogenesis, cell proliferation and migration. In the present work we studied the expression of AQP1 by immunohistochemical staining of 92 lung biopsies from patients diagnosed with a pleuropulmonary tumor (71 lung and 21 pleural neoplasms). AQP1 expression was analyzed comparing the results among the different histological patterns and against 9 control cases (5 parenchyma and 4 healthy pleura). Clear staining of AQP1 was detected in 39 of the 92 tumors analyzed. In parenchyma, AQP1 was more frequently detected in primary lung denocarcinomas (55%,P<0.001); in contrast, small cell carcinomas were the least AQP1 expressive tumors studied (93% of negative staining, P<0.05). Carcinomas analyzed in pleura (mesotheliomas and metastatic adenocarcinomas) also revealed strong xpression of AQP1. High expression of this protein was detected in small capillaries in areas near or surrounding the tumor, and novel intense AQP1 immunostaining was detected over thicker alveolar walls in alveoli inside or next to the tumoral tissue regardless of the tumor type. An important role of AQP1 in tumor angiogenesis is sustained by the abundant expression of this protein in the endothelia of tumor capillaries. Further studies are necessary to elucidate the potential pathophysiological role of this protein in pleuropulmonary neoplasms.

Aquaporin proteins significantly increase water permeability across tissues and cell membranes. Ocular tissues, including the trabecular meshwork (TM) and retinal pigment epithelium (RPE), are especially reliant on aquaporin mediated water movement for ocular homeostasis. Even though bulk fluid movement is paracellular through the TM and transcellular through the RPE, both express aquaporin-1 (AQP1). The role and regulation of AQP1 as it relates to homeostasis in different ocular tissues is not well understood. I hypothesized that ocular tissues respond to external mechanical and molecular cues by altering AQP1 expression and function in order to regulate ocular fluid movement and maintain homeostasis.To test how AQP1 function is altered in response to external cues in order to maintain tissue-specific homeostasis, I addressed the following two aims. The first aim was directed at determining how mechanical strain, an external stimulus that routinely affects TM function, influences AQP1 expression and TM homeostasis. Primary cultures of human TM were subjected to static and cyclic stretch and then analyzed for changes in AQP1 expression by western blot and cell damage by activity of lactate dehydrogense (LDH) in conditioned media. The results show AQP1 expression and LDH release significantly increased with static stretch. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r² = 0.7780).The second aim was directed at characterizing signaling mechanisms responsible for regulating fluid transport in RPE, previously shown to be dependent upon AQP1. I treated primary cultures of human RPE with either atrial natriuretic peptide (ANP) or 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP) in the presence or absence of Anantin (ANP-receptor inhibitor) or H-8 (Protein Kinase G inhibitor). The results show that ANP and 8-Br-cGMP significantly increased apical to basal net fluid movement (p < 0.05, n = 3). Inhibition of these effect

Embryos of the marine killifish Fundulus heteroclitus are adapted to survive aerially. However, it is unknown if they are able to control development under dehydration conditions. Here, we show that air-exposed blastula embryos under saturated relative humidity were able to stimulate development, and hence the time of hatching was advanced with respect to embryos continuously immersed in seawater. Embryos exposed to air at later developmental stages did not hatch until water was added, while development was not arrested. Air-exposed embryos avoided dehydration probably because of their thickened egg envelope, although it suffered significant evaporative water loss. The potential role of aquaporins as part of the embryo response to dehydration was investigated by cloning the aquaporin-0 (FhAqp0), -1a (FhAqp1a), and -3 (FhAqp3) cDNAs. Functional expression in Xenopus laevis oocytes showed that FhaAqp1a was a water-selective channel, whereas FhAqp3 was permeable to water, glycerol, and urea. Expression of fhaqp0 and fhaqp1a was prominent during organogenesis, and their mRNA levels were similar between water- and air-incubated embryos. However, fhaqp3 transcripts were highly and transiently accumulated during gastrulation, and the protein product was localized in the basolateral membrane of the enveloping epithelial cell layer and in the membrane of ingressing and migrating blastomers. Interestingly, both fhaqp3 transcripts and FhAqp3 polypeptides were downregulated in air-exposed embryos. These data demonstrate that killifish embryos respond adaptively to environmental desiccation by accelerating development and that embryos are able to transduce dehydration conditions into molecular responses. The reduced synthesis of FhAqp3 may be one of these mechanisms to regulate water and/or solute transport in the embryo.

Aquaporin-1 (AQP1) is involved in the angiogenesis and structural modifications of microvessels and possibly also in the pathogenesis of idiopathic menhorrhagia, where a reduced AQP1 expression is seen in the endometrium. Mifepristone treatment induces reduced menstrual bleeding and amenorrhea and also has a direct effect on endometrial arterioles. Administered with gestagen-only contraceptive methods, antiprogestins improve the bleeding pattern. The objective of this study was to evaluate the AQP1 expression in endometrial blood vessels during normal cycle and after mifepristone treatment. Localization and expression of AQP1 was determined using immunohistochemistry and reverse transcriptase chain reaction (RT-PCR) in 43 biopsies from human endometrium taken during a normal cycle and after mifepristone treatment. AQP1 expression in human endometrial vessels is not cycle dependent and is stronger in capillaries and arteries than in veins. After mifepristone treatment the staining intensity was increased, but not the number of stained vessels. The presence of AQP1 was also confirmed using RT-PCR. The changes in AQP1 expression could contribute to the reduced bleeding seen following mifepristone treatment and could be an effect of either antagonizing progesterone or cortisol.

The treatment of most patients with head and neck cancer includes ionizing radiation (IR). Salivary glands in the IR field suffer significant and irreversible damage, leading to considerable morbidity. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat [C. Delporte, B.C. O'Connell, X. He, H.E. Lancaster, A.C. O'Connell, P. Agre, B.J. Baum, Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands. Proc. Natl. Acad. Sci. U S A. 94 (1997) 3268-3273] and miniature pig [Z. Shan, J. Li, C. Zheng, X. Liu, Z. Fan, C. Zhang, C.M. Goldsmith, R.B. Wellner, B.J Baum, S. Wang. Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands. Mol. Ther. 11 (2005) 444-451] salivary glands approximately 16 weeks following IR resulted in a dose-dependent increase in salivary flow to > or =80% control levels on day 3. A control Ad vector was without any significant effect on salivary flow. Additionally, after administration of Ad vectors to salivary glands, no significant lasting effects were observed in multiple measured clinical chemistry and hematology values. Taken together, the findings show that localized delivery of AdhAQP1 to IR-damaged salivary glands is useful in transiently increasing salivary secretion in both small and large animal models, without significant general adverse events. Based on these results, we are developing a clinical trial to test if the hAQP1 cDNA transfer strategy will be clinically effective in restoring salivary flow in patients with IR-induced parotid hypofunction., Journal Article, Review, info:eu-repo/semantics/published

The treatment of most patients with head and neck cancer includes ionizing radiation (IR). Salivary glands in the IR field suffer significant and irreversible damage, leading to considerable morbidity. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat [C. Delporte, B.C. O'Connell, X. He, H.E. Lancaster, A.C. O'Connell, P. Agre, B.J. Baum, Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands. Proc. Natl. Acad. Sci. U S A. 94 (1997) 3268-3273] and miniature pig [Z. Shan, J. Li, C. Zheng, X. Liu, Z. Fan, C. Zhang, C.M. Goldsmith, R.B. Wellner, B.J Baum, S. Wang. Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands. Mol. Ther. 11 (2005) 444-451] salivary glands approximately 16 weeks following IR resulted in a dose-dependent increase in salivary flow to > or =80% control levels on day 3. A control Ad vector was without any significant effect on salivary flow. Additionally, after administration of Ad vectors to salivary glands, no significant lasting effects were observed in multiple measured clinical chemistry and hematology values. Taken together, the findings show that localized delivery of AdhAQP1 to IR-damaged salivary glands is useful in transiently increasing salivary secretion in both small and large animal models, without significant general adverse events. Based on these results, we are developing a clinical trial to test if the hAQP1 cDNA transfer strategy will be clinically effective in restoring salivary flow in patients with IR-induced parotid hypofunction., Journal Article, Review, info:eu-repo/semantics/published

BACKGROUND: Animal models are important for understanding the physiology and pathophysiology of peritoneal transport during peritoneal dialysis (PD). Mechanistic investigations of rat and rabbit models of PD are mostly based on intervention studies using pharmacologic agents or blocking antibodies. These models may be limited by the time-course, lack of specificity, or side effects of such interventions. Genetically modified mice could provide an attractive alternative to the above models. In this study, we have characterized PD parameters and tested the effect of gender and dialysate volume and/or osmolality in the C57BL/6J mouse. METHODS: Mice were submitted to a 2-hour peritoneal equilibration test in order to obtain permeability parameters. The expression of the water channel aquaporin-1 (AQP1) and endothelial NO synthase (eNOS) was investigated at the protein (immunoblotting, immunostaining) and mRNA [real-time reverse-transcription-polymerase chain reaction (RT-PCR)] levels. The potential effect of gender on these parameters was also studied. RESULTS: Exposure of mice to 2 mL of 3.86% glucose dialysate yielded equilibration curves for urea and glucose, a sodium sieving, and a net ultrafiltration (UF) that were remarkably similar to those obtained in rats. The increase in dialysate volume (from 2 mL to 3 mL and 6 mL) resulted in a higher ultrafiltration and, for the highest volume, an increase in the diffusive mass transport coefficient (MTAC) for urea. The increase in dialysate glucose concentration (from 1.36% to 3.86% and 7%) resulted in increased sodium sieving and higher UF, whereas the MTAC for urea was unchanged. In comparison with males, females had a similar peritoneal transport rate for small solutes but a significantly lower sodium sieving, reflecting a lower AQP1 mRNA and protein expression in the peritoneum. CONCLUSION: These data demonstrate the structural and functional similarity between mouse and rat models of PD, and further emphasize the rele

New insights in the molecular mechanisms regulating peritoneal permeability

BACKGROUND: Ultrafiltration (UF) failure often complicates peritoneal dialysis (PD). At least two molecules might be involved in UF failure: aquaporin-1 (AQP1), a water channel thought to be the ultra small pore of the peritoneal membrane (PM), and nitric oxide (NO), which might regulate effective peritoneal surface area and microvascular permeability. METHODS: The contributions of AQP1 and NO in UF failure were evaluated by combining different experimental approaches. Specific antibodies were used to investigate the expression (immunoblotting) and localization (immunostaining) of AQP1 and NO synthase (NOS) isoforms in the peritoneum, in correlation with: (i) morphometric analyses; (ii) the l-citrulline assay, which specifically measures NOS enzymatic activities; and (iii) permeability parameters across the PM. RESULTS: AQP1 is located in the endothelium lining peritoneal capillaries, and its expression is remarkably stable in samples ranging from normal to highly inflamed peritoneum and even when transcellular water permeability is absent (loss of sodium sieving). A significant NOS activity, mediated by specific NOS isoforms, can be assayed in the peritoneum. The NOS activity significantly increases in conditions such as peritonitis and long-term PD, and this increase is mirrored by up-regulation of NOS isoforms, as well as angiogenesis and increased endothelial area. CONCLUSIONS: These data suggest that the NO-mediated increase in effective peritoneal surface area, followed by a dissipation of the osmotic gradient, is a major mechanism accounting for the loss of UF in PD. Other biological consequences of increased NO levels in the peritoneum might include initiation of angiogenesis or modification of functionally important proteins such as AQP1.

The mammalian peripheral lung contains at least three aquaporin (AQP) water channels: AQP1 in microvascular endothelia, AQP4 in airway epithelia, and AQP5 in alveolar epithelia. In this study, we determined the role of AQP4 in airspace-to-capillary water transport by comparing water permeability in wild-type mice and transgenic null mice lacking AQP1, AQP4, or AQP1/AQP4 together. An apparatus was constructed to measure lung weight continuously during pulmonary artery perfusion of isolated mouse lungs. Osmotically induced water flux (J(v)) between the airspace and capillary compartments was measured from the kinetics of lung weight change in saline-filled lungs in response to changes in perfusate osmolality. J(v) in wild-type mice varied linearly with osmotic gradient size (4.4 x 10(-5) cm(3) s(-1) mOsm(-1)) and was symmetric, independent of perfusate osmolyte size, weakly temperature dependent, and decreased 11-fold by AQP1 deletion. Transcapillary osmotic water permeability was greatly reduced by AQP1 deletion, as measured by the same method except that the airspace saline was replaced by an inert perfluorocarbon. Hydrostatically induced lung edema was characterized by lung weight changes in response to changes in pulmonary arterial inflow or pulmonary venous outflow pressure. At 5 cm H(2)O outflow pressure, the filtration coefficient was 4.7 cm(3) s(-1) mOsm(-1) and reduced 1.4-fold by AQP1 deletion. To study the role of AQP4 in lung water transport, AQP1/AQP4 double knockout mice were generated by crossbreeding of AQP1 and AQP4 null mice. J(v) were (cm(3) s(-1) mOsm(-1) x 10(-5), SEM, n = 7-12 mice): 3.8 +/- 0. 4 (wild type), 0.35 +/- 0.02 (AQP1 null), 3.7 +/- 0.4 (AQP4 null), and 0.25 +/- 0.01 (AQP1/AQP4 null). The significant reduction in P(f) in AQP1 vs. AQP1/AQP4 null mice was confirmed by an independent pleural surface fluorescence method showing a 1.6 +/- 0.2-fold (SEM, five mice) reduced P(f) in the AQP1/AQP4 double knockout mice vs. AQP1 null mice. These

The mammalian peripheral lung contains at least three aquaporin (AQP) water channels: AQP1 in microvascular endothelia, AQP4 in airway epithelia, and AQP5 in alveolar epithelia. In this study, we determined the role of AQP4 in airspace-to-capillary water transport by comparing water permeability in wild-type mice and transgenic null mice lacking AQP1, AQP4, or AQP1/AQP4 together. An apparatus was constructed to measure lung weight continuously during pulmonary artery perfusion of isolated mouse lungs. Osmotically induced water flux (J(v)) between the airspace and capillary compartments was measured from the kinetics of lung weight change in saline-filled lungs in response to changes in perfusate osmolality. J(v) in wild-type mice varied linearly with osmotic gradient size (4.4 x 10(-5) cm(3) s(-1) mOsm(-1)) and was symmetric, independent of perfusate osmolyte size, weakly temperature dependent, and decreased 11-fold by AQP1 deletion. Transcapillary osmotic water permeability was greatly reduced by AQP1 deletion, as measured by the same method except that the airspace saline was replaced by an inert perfluorocarbon. Hydrostatically induced lung edema was characterized by lung weight changes in response to changes in pulmonary arterial inflow or pulmonary venous outflow pressure. At 5 cm H(2)O outflow pressure, the filtration coefficient was 4.7 cm(3) s(-1) mOsm(-1) and reduced 1.4-fold by AQP1 deletion. To study the role of AQP4 in lung water transport, AQP1/AQP4 double knockout mice were generated by crossbreeding of AQP1 and AQP4 null mice. J(v) were (cm(3) s(-1) mOsm(-1) x 10(-5), SEM, n = 7-12 mice): 3.8 +/- 0. 4 (wild type), 0.35 +/- 0.02 (AQP1 null), 3.7 +/- 0.4 (AQP4 null), and 0.25 +/- 0.01 (AQP1/AQP4 null). The significant reduction in P(f) in AQP1 vs. AQP1/AQP4 null mice was confirmed by an independent pleural surface fluorescence method showing a 1.6 +/- 0.2-fold (SEM, five mice) reduced P(f) in the AQP1/AQP4 double knockout mice vs. AQP1 null mice. These

Aquaporin-1 (AQP1) has been claimed to be the molecular counterpart of the transcellular pathway for free-water movement across the peritoneum during peritoneal dialysis. We report the case of a 67-year-old man, on peritoneal dialysis for 11 years, in whom ultrafiltration failure due to an abolition of the transcellular water transfer (documented by a loss of sodium sieving) was associated with an apparently normal expression of AQP1. We suggest that an alteration of AQP1 structure, rather than of its expression, accounts for this observation.