Mechanical stretch regulates the expression of specific miRNA in extracellular vesicles released from lung epithelial cells.

The underlying mechanism of normal lung organogenesis is not well understood. An increasing number of studies are demonstrating that extracellular vesicles (EVs) play critical roles in organ development by delivering microRNAs (miRNA) to neighboring and distant cells. miRNAs are important for fetal lung growth; however, the role of miRNA-EVs (miRNAs packaged inside the EVs) during fetal lung development is unexplored. The aim of this study was to examine the expression of miRNA-EVs in MLE-12, a murine lung epithelial cell line subjected to mechanical stretch in vitro with the long-term goal to investigate their potential role in the fetal lung development. Both cyclic and continuous mechanical stretch regulate miRNA differentially in EVs released from MLE-12 and intracellularly, demonstrating that mechanical signals regulate the expression of miRNA-EVs in lung epithelial cells. These results provide a proof-of-concept for the potential role that miRNA-EVs could play in the development of fetal lung.

Mechanotransduction via TRPV4 regulates inflammation and differentiation in fetal mouse distal lung epithelial cells.

BACKGROUND: Mechanical ventilation plays a central role in the injury of premature lungs. However, the mechanisms by which mechanical signals trigger an inflammatory cascade to promote lung injury are not well-characterized. Transient receptor potential vanilloid 4 (TRPV4), a calcium-permeable mechanoreceptor channel has been shown to be a major determinant of ventilator-induced acute lung injury in adult models. However, the role of these channels as modulators of inflammation in immature lungs is unknown. In this study, we tested the hypothesis that TRPV4 channels are important mechanotransducers in fetal lung injury. METHODS: Expression of TRPV4 in the mouse fetal lung was investigated by immunohistochemistry, Western blot and qRT-PCR. Isolated fetal epithelial cells were exposed to mechanical stimulation using the Flexcell Strain Unit and inflammation and differentiation were analyzed by ELISA and SP-C mRNA, respectively. RESULTS: TRPV4 is developmentally regulated in the fetal mouse lung; it is expressed in the lung epithelium and increases with advanced gestation. In contrast, in isolated epithelial cells, TRPV4 expression is maximal at E17-E18 of gestation. Mechanical stretch increases TRPV4 in isolated fetal epithelial cells only during the canalicular stage of lung development. Using the TRPV4 agonist GSK1016790A, the antagonist HC-067047, and the cytokine IL-6 as a marker of inflammation, we observed that TRPV4 regulates release of IL-6 via p38 and ERK pathways. Interestingly, stretch-induced differentiation of fetal epithelial cells was also modulated by TRPV4. CONCLUSION: These studies demonstrate that TRPV4 may play an important role in the transduction of mechanical signals in the fetal lung epithelium by modulating not only inflammation but also the differentiation of fetal epithelial cells.

Efficient replication of Epstein-Barr virus-derived plasmids requires tethering by EBNA1 to host chromosomes.

The EBNA1 protein of Epstein-Barr virus enables plasmids carrying oriP both to duplicate and to segregate efficiently in proliferating cells. EBNA1 recruits the origin recognition complex (ORC) to establish a replication origin at one element of oriP, DS (dyad symmetry); at another element, FR (family of repeats), EBNA1 binds to an array of sites from which it tethers plasmids to host chromosomes for mitotic stability. We report experiments leading to the conclusion that tethering by EBNA1 to host chromosomes is also needed within interphase nuclei in order for plasmids to be replicated efficiently from oriP. The DNA-binding domain of EBNA1, which lacks chromosome-binding ability, was found to support weak, DS-specific replication in HEK293 cells after transient transfection, being 17% as active as wild-type EBNA1. The low efficiency of replication was not due to the failure of the DNA-binding domain to retain plasmids within nuclei, because plasmids were recovered in similar amounts and entirely from the nuclear fraction of these transiently transfected cells. A derivative of EBNA1 with its chromosome-tethering domains replaced by a 22-amino-acid nucleosome-binding domain was fully active in supporting oriP functions. The implication is that EBNA1's DNA-binding domain is able to recruit ORC to DS, but either this step or subsequent replication is only efficient if the plasmid is tethered to a host chromosome. Finally, with some cell lines, DS can hardly support even transient plasmid replication without FR. A loss of plasmids lacking FR from nuclei cannot account for this requirement, suggesting that the stronger tethering to chromosomes by FR is needed for plasmid replication within the nuclei of such cells.

Immunization with PfGBP130 generates antibodies that inhibit RBC invasion by P. falciparum parasites.

Background: Despite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions. Methods and results: To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies. Conclusion: We are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates.

Prolyl hydroxylase 2 dependent and Von-Hippel-Lindau independent degradation of Hypoxia-inducible factor 1 and 2 alpha by selenium in clear cell renal cell carcinoma leads to tumor growth inhibition.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) accounts for more than 80% of the cases of renal cell carcinoma. In ccRCC deactivation of Von-Hippel-Lindau (VHL) gene contributes to the constitutive expression of hypoxia inducible factors 1 and 2 alpha (HIF-α), transcriptional regulators of several genes involved in tumor angiogenesis, glycolysis and drug resistance. We have demonstrated inhibition of HIF-1α by Se-Methylselenocysteine (MSC) via stabilization of prolyl hydroxylases 2 and 3 (PHDs) and a significant therapeutic synergy when combined with chemotherapy. This study was initiated to investigate the expression of PHDs, HIF-α, and VEGF-A in selected solid cancers, the mechanism of HIF-α inhibition by MSC, and to document antitumor activity of MSC against human ccRCC xenografts. METHODS: Tissue microarrays of primary human cancer specimens (ccRCC, head & neck and colon) were utilized to determine the incidence of PHD2/3, HIF-α, and VEGF-A by immunohistochemical methods. To investigate the mechanism(s) of HIF-α inhibition by MSC, VHL mutated ccRCC cells RC2 (HIF-1α positive), 786-0 (HIF-2α positive) and VHL wild type head & neck cancer cells FaDu (HIF-1α) were utilized. PHD2 and VHL gene specific siRNA knockdown and inhibitors of PHD2 and proteasome were used to determine their role in the degradation of HIF-1α by MSC. RESULTS: We have demonstrated that ccRCC cells express low incidence of PHD2 (32%), undetectable PHD3, high incidence of HIF-α (92%), and low incidence of VEGF-A compared to head & neck and colon cancers. This laboratory was the first to identify MSC as a highly effective inhibitor of constitutively expressed HIF-α in ccRCC tumors. MSC did not inhibit HIF-1α protein synthesis, but facilitated its degradation. The use of gene knockdown and specific inhibitors confirmed that the inhibition of HIF-1α was PHD2 and proteasome dependent and VHL independent. The effects of MSC treatment on HIF-α were associated with significant antitumor activity against ccRCC xenograft. CONCLUSIONS: Our results show the role of PHD2/3 in stable expression of HIF-α in human ccRCC. Furthermore, HIF-1α degradation by MSC is achieved through PHD2 dependent and VHL independent pathway which is unique for HIF-α regulation. These data provide the basis for combining MSC with currently used agents for ccRCC.

Proteomic analysis of a murine model of lung hypoplasia induced by oligohydramnios.

Severe oligohydramnios (OH) due to prolonged loss of amniotic fluid can cause pulmonary hypoplasia. Animal model of pulmonary hypoplasia induced by amniotic fluid drainage is partly attributed to changes in mechanical compression of the lung. Although numerous studies on OH-model have demonstrated changes in several individual proteins, however, the underlying mechanisms for interrupting normal lung development in response to a decrease of amniotic fluid volume are not fully understood. In this study, we used a proteomic approach to explore differences in the expression of a wide range of proteins after induction of OH in a mouse model of pulmonary hypoplasia to find out the signaling/molecular pathways involved in fetal lung development. Liquid chromatography-massspectromery/mass spectrometry analysis found 474 proteins that were differentially expressed in OH-induced hypoplastic lungs in comparison to untouched (UnT) control. Among these proteins, we confirmed the downregulation of AKT1, SP-D, and CD200, and provided proof-of-concept for the first time about the potential role that these proteins could play in fetal lung development.

Oligohydramnios compromises lung cells size and interferes with epithelial-endothelial development.

BACKGROUND AND OBJECTIVE: Severe oligohydramnios can induce pulmonary hypoplasia. However, the mechanisms by which leaking of fluids cause lung hypoplasia are not well defined. The objective of this study was to characterize a mouse model of pulmonary hypoplasia induced by oligohydramnios. METHODS: Amniotic sacs were punctured on E14.5 of gestation. Untouched fetuses were used as control. Pregnancy was allowed to continue until E18.5 in which lung tissue was collected and evaluated for morphometry, proliferation, differentiation, apoptosis, and angiogenesis. RESULTS: Our results found that lung weight, lung to total body weight ratio, and lung water content were reduced in oligohydramnios when compared to controls. In contrast, oligohydramnios did not affect the DNA content. Morphometric studies confirmed that oligohydramnios fetuses had smaller air spaces than control. Interestingly, cells from oligohydramnios fetuses have smaller size and less regular shapes. Oligohydramnios decreased the differentiation of type I epithelial cells and compromised apoptosis and angiogenesis while proliferation was not affected. CONCLUSIONS: Although, the smaller size of the lung could be explained by a decreased of lung fluids, our data suggest that increased of external compression secondary to severe oligohydramnios can compromise cell size and interfere with epithelial and endothelial development. Type I epithelial cells could have an unrecognized key role in the differentiation of the distal lung mediated by mechanical signals. Pediatr Pulmonol. 2017;52:746-756. © 2017 Wiley Periodicals, Inc.

Mechanotransduction as an Adaptation to Gravity.

Gravity has played a critical role in the development of terrestrial life. A key event in evolution has been the development of mechanisms to sense and transduce gravitational force into biological signals. The objective of this manuscript is to review how living organisms on Earth use mechanotransduction as an adaptation to gravity. Certain cells have evolved specialized structures, such as otoliths in hair cells of the inner ear and statoliths in plants, to respond directly to the force of gravity. By conducting studies in the reduced gravity of spaceflight (microgravity) or simulating microgravity in the laboratory, we have gained insights into how gravity might have changed life on Earth. We review how microgravity affects prokaryotic and eukaryotic cells at the cellular and molecular levels. Genomic studies in yeast have identified changes in genes involved in budding, cell polarity, and cell separation regulated by Ras, PI3K, and TOR signaling pathways. Moreover, transcriptomic analysis of late pregnant rats have revealed that microgravity affects genes that regulate circadian clocks, activate mechanotransduction pathways, and induce changes in immune response, metabolism, and cells proliferation. Importantly, these studies identified genes that modify chromatin structure and methylation, suggesting that long-term adaptation to gravity may be mediated by epigenetic modifications. Given that gravity represents a modification in mechanical stresses encounter by the cells, the tensegrity model of cytoskeletal architecture provides an excellent paradigm to explain how changes in the balance of forces, which are transmitted across transmembrane receptors and cytoskeleton, can influence intracellular signaling pathways and gene expression.