S Alex Mitsialis, PhD
Harvard Medical School, Newborn Medicine, BCH, Faculty Member
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Acute hypoxia in the lung causes arteriolar vasoconstriction whereas prolonged hypoxia promotes proliferation and migration of vascular smooth muscle cells (VSMC) and extracellular matrix deposition in the arterial wall, a process known... more
Acute hypoxia in the lung causes arteriolar vasoconstriction whereas prolonged hypoxia promotes proliferation and migration of vascular smooth muscle cells (VSMC) and extracellular matrix deposition in the arterial wall, a process known as vascular remodeling.1 These abnormalities are characteristic of pulmonary hypertension.2 Several clinical conditions characterized by lung inflammation have been linked to the development of chronic pulmonary hypertension.3 Interestingly, perivascular inflammatory cell infiltration as well as increased serum levels of pro-inflammatory cytokines, such as interleukin (IL)-lβ and IL-6, have been reported in clinical cases of primary pulmonary hypertension.4-5 However, little attention has been given up to now to the role of pulmonary inflammation in the pathogenesis of pulmonary hypertension induced by hypoxia.
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Supplemental Figure 1. Morphological and phenotypic characterization of bone marrow-derived macrophages (BMDM) from 6-8 week old FVB mice by flow cytometry.<br>Supplemental Figure 2. Detection of P50 nuclear staining (white arrows)... more
Supplemental Figure 1. Morphological and phenotypic characterization of bone marrow-derived macrophages (BMDM) from 6-8 week old FVB mice by flow cytometry.<br>Supplemental Figure 2. Detection of P50 nuclear staining (white arrows) in alveolar macrophages obtained from <i>Hmox1</i><sup>+/+</sup> and <i>Hmox1</i><sup>-/- </sup>mice at baseline normoxic conditions and expression of cytokines and markers of alveolar macrophage activation in normoxic <i>Hmox1</i><sup>+/+</sup> and <i>Hmox1</i><sup>-/- </sup>mice as determined by RT-qPCR.<b></b><i></i><sub></sub><sup></sup><br>
Avian tumor virus supercoiled DNA was isolated from infected quail tumor cells and molecularly cloned in pBR322. Four different recombinant clones denoted pATV-6, pATV-7, pATV-8, and pATV-9 were characterized in detail by restriction... more
Avian tumor virus supercoiled DNA was isolated from infected quail tumor cells and molecularly cloned in pBR322. Four different recombinant clones denoted pATV-6, pATV-7, pATV-8, and pATV-9 were characterized in detail by restriction endonuclease mapping and by DNA sequencing. The results of these studies indicate that (i) the two large terminal repeats (LTRs) present in PATV-6, are different sizes, (ii) pATV-8 and pATV-9 contain only one LTR, (iii) pATV-7 contains an inversion of 0.6 kilobase in the env gene and a deletion of the U3 region and the src gene, and (iv) the src gene is deleted in pATV-6 and pATV-9. Circle formation from linear molecules was also examined in several of the clones by DNA sequencing through the circle joint. pATV-6 is an example of one class of circular molecules and contains a partially repeated LTR similar to that reported by Ju and Skalka (Cell 22:379-386, 1980). A second class of circles was exemplified by pATV-8 and pATV-9, which contain a single cop...
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For the first time, we present evidence with restriction enzymes HpaII and MspI which indicates that the proviral DNA sequence of avian sarcoma virus is modified by methylation in a nonpermissive rat cell line but not in permissive... more
For the first time, we present evidence with restriction enzymes HpaII and MspI which indicates that the proviral DNA sequence of avian sarcoma virus is modified by methylation in a nonpermissive rat cell line but not in permissive chicken cells. Some of the endogenous viral sequences in the permissive cells were also methylated. No 5-methylcytosine could be detected in the unintegrated viral DNA.
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An increasing number of studies implicate heme oxygenase-1 (HO-1) in the regulation of inflammation. Although the mechanisms involved in this cytoprotection are largely unknown, HO-1 and its enzymatic products, carbon monoxide and... more
An increasing number of studies implicate heme oxygenase-1 (HO-1) in the regulation of inflammation. Although the mechanisms involved in this cytoprotection are largely unknown, HO-1 and its enzymatic products, carbon monoxide and bilirubin, downregulate the inflammatory response by either attenuating the expression of adhesion molecules and thus inhibiting leukocyte recruitment or by repressing the induction of cytokines and chemokines. In the present study we used genetically engineered mice that express high levels of a human cDNA HO-1 transgene in lung epithelium to assess the effect of HO-1 on lung inflammation. Two separate models of inflammation were studied: hypoxic exposure and lipopolysaccharide (LPS) challenge. We found that both mRNA and protein levels of specific cytokines and chemokines were significantly elevated in response to hypoxia in the lungs of wild-type mice after 2 and 5 days of exposure but significantly suppressed in the hypoxic lungs of transgenic mice, su...
Research Interests: Biology, Cytokines, Inflammation, Membrane Proteins, Lung Inflammation, and 15 moreMedicine, Lipopolysaccharide, Humans, Carbon Monoxide, Mice, Animals, Heme Oxygenase, Cytoprotection, Lung, Chemokine, Lipopolysaccharides, Gene Expression Regulation, Bronchoalveolar lavage, Inflammatory response, and Medical and Health Sciences
Research Interests: Physiology, Molecular Biology, Biology, Macrophages, Medicine, and 14 moreGene expression, Signal Transduction, Cell line, Mice, Animals, NF-kappa B, Medical Physiology, Mitogen Activated Protein Kinase, Chemokine, Oxygen, Chemokines, Biochemistry and cell biology, NFkB, and Gene Expression Regulation
The Drosophila chorion factor 1/ultraspiracle (CF1/USP) transcription factor, a homologue of the retinoid X receptor, is a developmentally important member of the family of nuclear (steroid) hormone receptors. Using newly developed... more
The Drosophila chorion factor 1/ultraspiracle (CF1/USP) transcription factor, a homologue of the retinoid X receptor, is a developmentally important member of the family of nuclear (steroid) hormone receptors. Using newly developed monoclonal antibodies and a full-length bacterially produced protein, we have studied in detail the in vitro DNA-binding properties of this factor and aspects of its distribution in vivo. During oogenesis, CF1/USP is present both in germline cells and in the somatic follicular epithelium. We have determined the optimal binding site of partially purified bacterially produced CF1/USP by an in vitro selection procedure and also have characterized its binding to the follicular-specific chorion s15 promoter. In vitro this bacterially produced factor is unusual in binding to a single element ("half-site"); simultaneous but noncoordinate binding to a second half-site is possible if these repeated elements are organized in direct orientation and spaced ...
Research Interests: Biology, Drosophila melanogaster, Cell Biology, Medicine, Multidisciplinary, and 15 moreNuclear Receptor, Female, Animals, Polymerase Chain Reaction, DNA binding, Monoclonal Antibody, Base Sequence, Ovary, Protein Binding, Epitopes, DNA binding proteins, Binding Site, In Vitro Selection, Nuclear hormone receptor, and Molecular Sequence Data
Retinoids have been shown to influence pattern formation during development and regeneration in numerous systems such as limbs, vertebrae, and neural tube although there is little information about the effects of retinoids on pattern... more
Retinoids have been shown to influence pattern formation during development and regeneration in numerous systems such as limbs, vertebrae, and neural tube although there is little information about the effects of retinoids on pattern formation in visceral organs. We investigated the effects of exogenous retinoic acid on the in vitro pattern of airway branching and on lung epithelial cell differentiation. Histology, [3H]thymidine autoradiographies and reverse transcriptase/polymerase chain reaction (RT/PCR) amplification were used to assess the effects of retinoids and the expression of lung epithelial markers of differentiation. We found that retinoic acid interferes, in a dose-dependent fashion, with the expression of epithelial genes that are found in distal segments of the fetal lung (surfactant-associated proteins SP-A, SP-B, and SP-C). At high concentrations, retinoic acid (RA) dramatically altered the developmental pattern of the lung, favoring growth of structures that resemble proximal airways and concomitantly suppressing distal epithelial buds. We hypothesize that this in vitro &quot;proximalizing&quot; effect on the developing lung may be related to alterations in the expression of pattern-related genes.
Research Interests: Biology, Medicine, Gene expression, In Situ Hybridization, In Vitro, and 13 moreAnimals, Cellular differentiation, Plant tissue Culture Techniques, American, Lung, Rats, Epithelial cells, Retinoic Acid, Tretinoin, Base Sequence, Retinoid, Molecular Sequence Data, and Cardiovascular medicine and haematology
Fredenburgh et al. – 1 Heme oxygenase (HO)-1, the inducible isoform of heme oxygenase, is a cytoprotective enzyme that plays a central role in the defense against oxidative and inflammatory insults in the lung. HO-1 catalyzes the... more
Fredenburgh et al. – 1 Heme oxygenase (HO)-1, the inducible isoform of heme oxygenase, is a cytoprotective enzyme that plays a central role in the defense against oxidative and inflammatory insults in the lung. HO-1 catalyzes the degradation of heme, a potent oxidant, into biliverdin, iron, and carbon monoxide (CO). These downstream products of heme catabolism have recently been found to mediate the anti-oxidant, anti-apoptotic, anti-proliferative, vasodilatory and antiinflammatory properties of HO-1. While absence of HO-1 is rare in humans, a number of HO-1 promoter polymorphisms have been identified which may influence HO-1 expression in vivo and lead to disease states. This review will summarize studies that implicate HO-1 and heme metabolites in the pathophysiology of pulmonary disease and discuss recent advances in the therapeutic applications of HO-1.The Role of HO-1 in Pulmonary Disease Fredenburgh et al. – 2 Heme oxygenase (HO)-1, the inducible isoform of heme oxygenase, pla...
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Exposure to hypoxia causes an inflammatory reaction in the mouse lung, and this response can be modulated by overexpressing the hypoxia-inducible stress-response enzyme, heme oxygenase-1 (HO-1). We hypothesized that the inflammasome... more
Exposure to hypoxia causes an inflammatory reaction in the mouse lung, and this response can be modulated by overexpressing the hypoxia-inducible stress-response enzyme, heme oxygenase-1 (HO-1). We hypothesized that the inflammasome activity may be a central pathway by which HO-1 controls pulmonary inflammation following alveolar hypoxia. Therefore, we investigated whether HO-1 controls inflammasome activation by altering its expression in macrophages primed with classic NOD-like receptor containing a pyrin domain 3 (NLRP3) inducers, and in murine lungs lacking HO-1 and exposed to acute hypoxia. We found that lack of HO-1 activated lipopolysaccharide (LPS) and ATP-treated bone marrow-derived macrophages, causing an increase in secreted levels of cleaved interleukin (IL)-1B, IL-18, and caspase-1, markers of increased inflammasome activity, whereas HO-1 overexpression suppressed IL-1B, NLRP3, and IL-18. The production of cleaved IL-1B and the activation of caspase-1 in LPS- and ATP-pr...
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Rationale: Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF), a chronic progressive lung disease characterized by interstitial fibrosis with decreasing lung volumes... more
Rationale: Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF), a chronic progressive lung disease characterized by interstitial fibrosis with decreasing lung volumes and hypoxemic respiratory failure. Recently, we reported that the therapeutic capacity of MSCs predominantly resides in the secretome, and that the chief therapeutic vector therein is represented by the exosomes. Objectives: To test the therapeutic effects of MSC-exosomes (MEx) in a bleomycin-induced pulmonary fibrosis model and investigate putative mechanisms of action. Methods: Exosomes were isolated from media conditioned by human bone marrow MSCs (MEx). Adult mice (C57BL/6 strain) were challenged with endotracheal instillation of bleomycin and treated with MEx concurrently or at day 7. Treated animals and appropriate control groups were assessed at day 7 and/or day 14. Results: Bleomycin-challenged mice presented with severe septal thickening and prominent fibrosis, and this was effectively prevented (day 0 treatment) or reversed (day 7 treatment) by a single dose of MEx. Furthermore, MEx therapy modulated whole lung macrophage phenotype, and shifted the proportion of lung ‘proinflammatory’ classical monocytes, ‘regulatory’ monocytes and alveolar macrophages to favor the monocyte/macrophage profiles of untreated-control mice, and, importantly a parallel immunomodulatory effect was demonstrated in the bone marrow. Notably, transplantation of MEx-preconditioned bone marrow-derived monocytes alleviated core features of pulmonary fibrosis. Conclusion: A bolus dose of MEx prevents and reverts core features of bleomycin-induced pulmonary fibrosis. The beneficial actions of MEx are mediated via the systemic modulation of monocyte phenotypes.
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Members of the E2F gene family are transcription factors that have been implicated in the control of genes essential for cell cycle progression. Regulation of E2F function is finely tuned by the retinoblastoma tumor suppressor gene... more
Members of the E2F gene family are transcription factors that have been implicated in the control of genes essential for cell cycle progression. Regulation of E2F function is finely tuned by the retinoblastoma tumor suppressor gene product and a small family of related "pocket proteins," with the participation of a number of cyclins and cyclin-dependent kinases. Perturbations of this regulatory network can lead to oncogenic transformation and, in certain systems, to the loss of the ability to maintain terminal differentiation. We describe here the cloning, structural characterization, and tissue expression pattern of a new member of the E2F family, E2F-5. We show that this protein is highly conserved between human and rat but exhibits considerable divergence from E2F-1, E2F-2, or E2F3. Together with the recently reported E2F-4, E2F-5 defines a new branch of the E2F family. The distribution of E2F-5 mRNA among adult rat tissues and the temporal pattern of its expression dur...
Research Interests: Biology, Cell Cycle, Cell Biology, Transcription Factors, Medicine, and 15 moreHumans, Sequence alignment, Animals, Lung, Transcription Factor, Rats, Age Factors, Amino Acid Sequence, Base Sequence, Cell Cycle Proteins, DNA binding proteins, Cellular Molecular Biology, Gene Expression Regulation, retinoblastoma protein (RB1)., and Molecular Sequence Data
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Supplemental Figure 1. Morphological and phenotypic characterization of bone marrow-derived macrophages (BMDM) from 6-8 week old FVB mice by flow cytometry.<br>Supplemental Figure 2. Detection of P50 nuclear staining (white arrows)... more
Supplemental Figure 1. Morphological and phenotypic characterization of bone marrow-derived macrophages (BMDM) from 6-8 week old FVB mice by flow cytometry.<br>Supplemental Figure 2. Detection of P50 nuclear staining (white arrows) in alveolar macrophages obtained from <i>Hmox1</i><sup>+/+</sup> and <i>Hmox1</i><sup>-/- </sup>mice at baseline normoxic conditions and expression of cytokines and markers of alveolar macrophage activation in normoxic <i>Hmox1</i><sup>+/+</sup> and <i>Hmox1</i><sup>-/- </sup>mice as determined by RT-qPCR.<b></b><i></i><sub></sub><sup></sup><br>
Additional file 4. TSC possess the capability of clone formation and differentiation of into alveolar epithelial cells in vitro. TSCs were cultured in a 100-mm dish at limited dilution of one cell every 60mm2 for 14 days. Representative... more
Additional file 4. TSC possess the capability of clone formation and differentiation of into alveolar epithelial cells in vitro. TSCs were cultured in a 100-mm dish at limited dilution of one cell every 60mm2 for 14 days. Representative phase contrast images of an entire clone (a), and a clone immunofluorescence stained for CD117 (green) and DAPI (blue, b). TSCs were seeded on the apical side of a clear12-transwell plate and exposed to differentiation medium of 2% FBS DMEM/F12 supplied with 0.005mg/mL insulin, 0.01mg/mL Tranferrin, 30nM Sodium selenite, 10nM Hydrocortisone, 10nM Beta-estradiol, 10nM HEPES, 2mM L-glutamine and 50ng/mL EGF for 6 days, following air-liquid interface for an additional 11-12 days. Representative images of these differentiated cells immuno-fluorescence stained for AQP5 (red, c and d), SPC (red, e and f). Scale bars represent 1000µm for a and b, 50µm for c~f. Bar graphs showing quantitation of qRT-PCR for AQP5 (g) and SPC (h) in fold change comparing diffe...
Additional file 3. TSCs attenuate BLM-induced lung fibrosis. The lungs were harvested at day 14 after BLM exposure. a~d) Representative image of Masson's trichrome blue staining for the lung. Scale bar represents 50µm. e) Quantitation... more
Additional file 3. TSCs attenuate BLM-induced lung fibrosis. The lungs were harvested at day 14 after BLM exposure. a~d) Representative image of Masson's trichrome blue staining for the lung. Scale bar represents 50µm. e) Quantitation of Masson's trichrome blue staining of the lung. Following Masson's trichrome staining, tissues were scanned using VS120 slides scanner, at the fully automated mode and 10x objective (39). Images were uploaded in ImgaeJ software. The collagen deposition was expressed as a percentage of collagen in total tissue area, and presented as mean±SEM. One-way analysis of variance was performed. *P<0.005 versus sham group. &P<0.05 versus ALI+PBS group, n = 3~4 mice for each group.
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Antenatal stressors such as chorioamnionitis (CA) increase the risk for bronchopulmonary dysplasia (BPD). Studies have shown that experimental BPD can be ameliorated by postnatal treatment with mesenchymal stromal cell-derived... more
Antenatal stressors such as chorioamnionitis (CA) increase the risk for bronchopulmonary dysplasia (BPD). Studies have shown that experimental BPD can be ameliorated by postnatal treatment with mesenchymal stromal cell-derived extracellular vesicles (MEx). However, the antenatal efficacy of MEx to prevent BPD is unknown. To determine whether antenatal MEx therapy attenuates intrauterine inflammation and preserves lung growth in a rat model of CA-induced BPD. At embryonic day ( E) 20, rat litters were treated with intra-amniotic injections of saline, endotoxin (ETX) to model chorioamnionitis, MEx, or ETX plus MEx followed by cesarean section delivery with placental harvest at E22. Placental and lung evaluations were conducted at day 0 and day 14, respectively. To assess the effects of ETX and MEx on lung growth in vitro, E15 lung explants were imaged for distal branching. Placental tissues from ETX-exposed pregnancies showed increased expression of inflammatory markers NLRP-3 and IL-...
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RATIONALE Mesenchymal stem/stromal cell (MSC)-small extracellular vesicle (MEx) treatment has shown promise in models of neonatal lung injury. The molecular mechanisms by which MEx afford beneficial effects remain incompletely understood.... more
RATIONALE Mesenchymal stem/stromal cell (MSC)-small extracellular vesicle (MEx) treatment has shown promise in models of neonatal lung injury. The molecular mechanisms by which MEx afford beneficial effects remain incompletely understood. OBJECTIVE To investigate the therapeutic mechanism of action through assessment of MEx biodistribution and impact on immune cell phenotypic heterogeneity. METHODS MEx were isolated from the conditioned medium of human umbilical cord Wharton's Jelly-derived MSCs. Newborn mice were exposed to hyperoxia (HYRX, 75% O2) from birth and returned to room air at postnatal day (PN) 14. Mice received either a bolus intravenous MEx dose at PN4 or bone marrow-derived myeloid cells (BMDMy) pretreated with MEx. Animals were harvested at PN4, 7, 14, or 28 to characterize MEx biodistribution or for assessment of pulmonary parameters. The therapeutic role of MEx-educated BMDMy was determined in vitro and in vivo. MEASUREMENTS AND RESULTS MEx therapy ameliorated core histological features of HYRX-induced neonatal lung injury. Biodistribution and mass cytometry studies demonstrated that MEx localize in the lung and interact with myeloid cells. MEx restored the apportion of alveolar macrophages in the HYRX injured lung and concomitantly suppressed inflammatory cytokine production. In vitro and ex vivo studies revealed that MEx promoted an immunosuppressive BMDMy phenotype. Functional assays demonstrated that the immunosuppressive actions of BMDMy are driven by phenotypically and epigenetically reprogrammed monocytes. Adoptive transfer of MEx-educated BMDMy, but not naïve BMDMy, restored alveolar architecture, blunted fibrosis and pulmonary vascular remodeling and improved exercise capacity. CONCLUSION MEx ameliorates hyperoxia-induced neonatal lung injury though epigenetic and phenotypic reprogramming of myeloid cells.
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ABSTRACT
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The regulatory elements present in the long terminal repeat (LTR) of avian sarcoma virus DNA were analyzed by recombinant DNA techniques coupled with DNA-mediated gene transfer in avian as well as mammalian cells. For this purpose, the... more
The regulatory elements present in the long terminal repeat (LTR) of avian sarcoma virus DNA were analyzed by recombinant DNA techniques coupled with DNA-mediated gene transfer in avian as well as mammalian cells. For this purpose, the neomycin resistance gene from transposon Tn5 was inserted downstream from the avian sarcoma virus LTR, and the recombinant plasmid DNA was introduced into cells by the calcium phosphate technique. Cells resistant to the drug G-418 were selected. Analysis of the RNA transcripts made in vivo in these transformants indicated that initiation and termination of the transcripts occurred in the LTR sequences. Deletions were then introduced into the LTR, and their effect on transcription was also studied. These results allowed us to identify a strong regulatory sequence between nucleotides -299 and -114 in the LTR of avian sarcoma virus.
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... sequences of 270 kb have been obtained thus far from one of these seg-ments by chromosomal walking (Eickbush and Kafa-tos 1982). ... of the segment of D. melanogaster chromosome III encompassing cho-rion genes s18-1, s15-1, and s19-1... more
... sequences of 270 kb have been obtained thus far from one of these seg-ments by chromosomal walking (Eickbush and Kafa-tos 1982). ... of the segment of D. melanogaster chromosome III encompassing cho-rion genes s18-1, s15-1, and s19-1 has been reported (Wong et al. ...
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Rationale: Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF), a chronic progressive lung disease characterized by interstitial fibrosis with decreasing lung volumes... more
Rationale: Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF), a chronic progressive lung disease characterized by interstitial fibrosis with decreasing lung volumes and hypoxemic respiratory failure. Recently, we reported that the therapeutic capacity of MSCs predominantly resides in the secretome, and that the chief therapeutic vector therein is represented by the exosomes. Objectives: To test the therapeutic effects of MSC-exosomes (MEx) in a bleomycin-induced pulmonary fibrosis model and investigate putative mechanisms of action. Methods: Exosomes were isolated from media conditioned by human bone marrow MSCs (MEx). Adult mice (C57BL/6 strain) were challenged with endotracheal instillation of bleomycin and treated with MEx concurrently or at day 7. Treated animals and appropriate control groups were assessed at day 7 and/or day 14. Results: Bleomycin-challenged mice presented with severe septal thickening a...
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Despite major advances in neonatal intensive care, infants born at extremely low birth weight still face an increased risk for chronic illness that may persist into adulthood. Pulmonary, retinal, and neurocognitive morbidities associated... more
Despite major advances in neonatal intensive care, infants born at extremely low birth weight still face an increased risk for chronic illness that may persist into adulthood. Pulmonary, retinal, and neurocognitive morbidities associated with preterm birth remain widespread despite interventions designed to minimize organ dysfunction. The design of therapeutic applications for preterm pathologies sharing common underlying triggers, such as fluctuations in oxygen supply or in the inflammatory state, requires alternative strategies that promote anti-inflammatory, pro-angiogenic, and trophic activities—ideally as a unitary treatment. Mesenchymal stem/stromal cell-derived extracellular vesicles (MEx) possess such inherent advantages, and they represent a most promising treatment candidate, as they have been shown to contribute to immunomodulation, homeostasis, and tissue regeneration. Current pre-clinical studies into the MEx mechanism of action are focusing on their restorative capabil...
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In preeclamptic pregnancies, a variety of intrauterine alterations lead to abnormal placentation, release of inflammatory/antiangiogenic factors, and subsequent fetal growth restriction with significant potential to cause a primary insult... more
In preeclamptic pregnancies, a variety of intrauterine alterations lead to abnormal placentation, release of inflammatory/antiangiogenic factors, and subsequent fetal growth restriction with significant potential to cause a primary insult to the developing fetal lung. Thus, modulation of the maternal intrauterine environment may be a key therapeutic avenue to prevent preeclampsia-associated developmental lung injury. A biologic therapy of interest are mesenchymal stromal cell-derived extracellular vesicles (MEx), which we have previously shown to ameliorate preeclamptic physiology through intrauterine immunomodulation. To evaluate the therapeutic potential of MEx to improve developmental lung injury in experimental preeclampsia. Using the heme oxygenase-1 null mouse (Hmox1-/-) model, preeclamptic pregnant dams were administered intravenous antenatal MEx treatment during each week of pregnancy followed by analysis of fetal and postnatal lung tissues, amniotic fluid protein profiles and lung explant/amniotic fluid co-cultures in comparison with control and untreated preeclamptic pregnancies. We first identified that a preeclamptic intrauterine environment had a significant adverse impact on fetal lung development including alterations in fetal lung developmental gene profiles in addition to postnatal alveolar and bronchial changes. Amniotic fluid proteomic analysis and fetal lung explant/amniotic fluid co-cultures further demonstrated that maternally administered MEx altered the expression of multiple inflammatory mediators in the preeclamptic intrauterine compartment resulting in normalization of fetal lung branching morphogenesis and developmental gene expression. Our evaluation of fetal and postnatal parameters overall suggests that antenatal MEx treatment may provide a highly valuable preventative therapeutic modality for amelioration of lung development in preeclamptic disease.
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... In contrast, the quantitative demands of choriogenesis are extremely high in Drosophila melanogaster: the genes are present in a ... In parallel, we will be attempting to identify and compare the trans-regulatory elements at the... more
... In contrast, the quantitative demands of choriogenesis are extremely high in Drosophila melanogaster: the genes are present in a ... In parallel, we will be attempting to identify and compare the trans-regulatory elements at the molecular level. ... tATg | AAA AAA caaa tgee | aca.. ...
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Background Acute lung injury (ALI) is a common lung disorder that affects millions of people every year. The infiltration of inflammatory cells into the lungs and death of the alveolar epithelial cells are key factors to trigger a... more
Background Acute lung injury (ALI) is a common lung disorder that affects millions of people every year. The infiltration of inflammatory cells into the lungs and death of the alveolar epithelial cells are key factors to trigger a pathological cascade. Trophoblast stem cells (TSCs) are immune privileged, and demonstrate the capability of self-renewal and multipotency with differentiation into three germ layers. We hypothesized that intratracheal transplantation of TSCs may alleviate ALI. Methods ALI was induced by intratracheal delivery of bleomycin (BLM) in mice. After exposure to BLM, pre-labeled TSCs or fibroblasts (FBs) were intratracheally administered into the lungs. Analyses of the lungs were performed for inflammatory infiltrates, cell apoptosis, and engraftment of TSCs. Pro-inflammatory cytokines/chemokines of lung tissue and in bronchoalveolar lavage fluid (BALF) were also assessed. Results The lungs displayed a reduction in cellularity, with decreased CD45+ cells, and les...
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Motile cilia and sperm flagella share an extremely conserved microtubule-based cytoskeleton, called the axoneme, which sustains beating and motility of both organelles. Ultra-structural and/or functional defects of this axoneme are... more
Motile cilia and sperm flagella share an extremely conserved microtubule-based cytoskeleton, called the axoneme, which sustains beating and motility of both organelles. Ultra-structural and/or functional defects of this axoneme are well-known to cause primary ciliary dyskinesia (PCD), a disorder characterized by recurrent respiratory tract infections, chronic otitis media, situs inversus, male infertility and in most severe cases, hydrocephalus. Only recently, mutations in genes encoding axonemal proteins with preferential expression in the testis were identified in isolated male infertility; in those cases, individuals displayed severe asthenozoospermia due to Multiple Morphological Abnormalities of the sperm Flagella (MMAF) but not PCD features. In this study, we performed genetic investigation of two siblings presenting MMAF without any respiratory PCD features, and we report the identification of the c.2018T > G (p.Leu673Pro) transversion in AK7, encoding an adenylate kinase,...
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The nucleotide sequences in the long terminal repeat of avian sarcoma virus that are recognized in vitro by HeLa cell RNA polymerase II have been identified. For this purpose, various 5' and 3' deletions were introduced into a... more
The nucleotide sequences in the long terminal repeat of avian sarcoma virus that are recognized in vitro by HeLa cell RNA polymerase II have been identified. For this purpose, various 5' and 3' deletions were introduced into a cloned long terminal repeat fragment. The effects of these deletions on transcription initiation in HeLa whole-cell extracts were then studied. Three specific transcripts have been identified. The major transcript is initiated at nucleotide +1 (relative to the cap site). Deletion of the upstream sequence between -299 and -55 has no effect on the level of transcription from this start site, whereas deletion of the sequence downstream of -14 drastically reduces the levels of transcription. In contrast, deletion of the sequence downstream from the TATA box has no effect on the initiation or efficiency of synthesis of the two minor RNA species, which are initiated at around nucleotides -260 and -105. The transcription of these RNA products, however, is abo...
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This review provides an updated perspective on rapidly proliferating efforts to harness extracellular vesicles (EVs) for therapeutic applications. We summarize current knowledge, emerging strategies, and open questions pertaining to... more
This review provides an updated perspective on rapidly proliferating efforts to harness extracellular vesicles (EVs) for therapeutic applications. We summarize current knowledge, emerging strategies, and open questions pertaining to clinical potential and translation. Potentially useful EVs comprise diverse products of various cell types and species. EV components may also be combined with liposomes and nanoparticles to facilitate manufacturing as well as product safety and evaluation. Potential therapeutic cargoes include RNA, proteins, and drugs. Strategic issues considered herein include choice of therapeutic agent, means of loading cargoes into EVs, promotion of EV stability, tissue targeting, and functional delivery of cargo to recipient cells. Some applications may harness natural EV properties, such as immune modulation, regeneration promotion, and pathogen suppression. These properties can be enhanced or customized to enable a wide range of therapeutic applications, includin...
Research Interests: Pharmacology, RNA, Nanoparticles, Nanotechnology, Medicine, and 15 moreBiological Sciences, DNA, Mesenchymal stem cells, Humans, Animals, Gene transfer techniques, Drug Design, Liposomes, Exosomes, Drug Carriers, Extracellular Vesicles, Microvesicles, Pharmaceutical preparations, Tissue distribution, and Medical and Health Sciences
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The combination of a vascular endothelial growth factor receptor antagonist, Sugen 5416 (SU5416), and chronic hypoxia is known to cause pronounced pulmonary hypertension (PH) with angioobliterative lesions in rats and leads to exaggerated... more
The combination of a vascular endothelial growth factor receptor antagonist, Sugen 5416 (SU5416), and chronic hypoxia is known to cause pronounced pulmonary hypertension (PH) with angioobliterative lesions in rats and leads to exaggerated PH in mice as well. We sought to determine whether weekly SU5416 injections during 3 weeks of hypoxia leads to long-term development of angioobliterative lesions and sustained or progressive PH in mice. Male C57BL/6J mice were injected with SU5416 (SuHx) or vehicle (VehHx) weekly during 3 weeks of exposure to 10% oxygen. Echocardiographic and invasive measures of hemodynamics and pulmonary vascular morphometry were performed after the 3-week hypoxic exposure and after 10 weeks of recovery in normoxia. SuHx led to higher right ventricular (RV) systolic pressure and RV hypertrophy than VehHx after 3 weeks of hypoxia. Ten weeks after hypoxic exposure, RV systolic pressure decreased but remained elevated in SuHx mice compared with VehHx or normoxic con...
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Pulmonary arterial hypertension (PAH) remains a serious disease, and although current treatments may prolong and improve quality of life, search for novel and effective therapies is warranted. Using genetically modified mouse lines, we... more
Pulmonary arterial hypertension (PAH) remains a serious disease, and although current treatments may prolong and improve quality of life, search for novel and effective therapies is warranted. Using genetically modified mouse lines, we tested the ability of bone marrow-derived stromal cells (mesenchymal stem cells [MSCs]) to treat chronic hypoxia-induced PAH. Recipient mice were exposed for 5 weeks to normobaric hypoxia (8%–10% O2), MSC preparations were delivered through jugular vein injection and their effect on PAH was assessed after two additional weeks in hypoxia. Donor MSCs derived from wild-type (WT) mice or heme oxygenase-1 (HO-1) null mice (Hmox1KO) conferred partial protection from PAH when transplanted into WT or Hmox1KO recipients, whereas treatment with MSCs isolated from transgenic mice harboring a human HO-1 transgene under the control of surfactant protein C promoter (SH01 line) reversed established disease in WT recipients. SH01-MSC treatment of Hmox1KO animals, whi...
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Fusions with the bacterial gene for chloramphenicol acetyltransferase followed by P-element-mediated germ-line transformation in Drosophila have permitted localization of the DNA sequence that confers a high degree of developmental... more
Fusions with the bacterial gene for chloramphenicol acetyltransferase followed by P-element-mediated germ-line transformation in Drosophila have permitted localization of the DNA sequence that confers a high degree of developmental specificity on a pair of silkmoth eggshell (chorion) genes. The short, 272-base-pair, 5'-flanking region shared by the divergently transcribed genes is sufficient for developmentally appropriate expression when placed upstream of the chloramphenicol acetyltransferase gene, in either orientation. A highly conserved motif within that region, TCACGT, is essential for chorion-specific expression.
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Urinary and renal rK1-kallikrein was studied in spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). It was demonstrated that the antiserum used for kallikrein radioimmunoassay (RIA) reacts with rK1- but not with... more
Urinary and renal rK1-kallikrein was studied in spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). It was demonstrated that the antiserum used for kallikrein radioimmunoassay (RIA) reacts with rK1- but not with rK7-protein. The specificity of the kininogenase assay was tested: rK7 had only 8% of the activity of rK1. Urinary kallikrein excretion by RIA was reduced by about two thirds in 5-week-old SHR compared WKY (11.5 versus 37.1µg/24h). On the contrary, the kidney content of rK1-kallikrein by RIA was increased by 40% in these rats (11.6 versus 8.4 ng/mg protein). The increase in kidney rK1 was confirmed by kininogenase assays. The same pattern of reduced urinary and increased renal rK1-kallikrein was observed in 8-week-old SHR rats. Kidney rK1-kallikrein mRNA tended to be lower (0.10 > p > 0.05) in SHR compared to WKY rats, suggesting that the increased kidney rK1 content is not due to increased rK1 synthesis. We hypothesize that the combination of ...
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Bone morphogenetic protein receptor 2 (BMPR2) mutations have been linked to familial pulmonary arterial hypertension (PAH), but the molecular pathways leading to this severe pathology remain poorly characterized. We report that hypoxia, a... more
Bone morphogenetic protein receptor 2 (BMPR2) mutations have been linked to familial pulmonary arterial hypertension (PAH), but the molecular pathways leading to this severe pathology remain poorly characterized. We report that hypoxia, a paramount stimulus for the development of pulmonary hypertension, suppresses the expression of inhibitor of differentiation 1 (Id1), a downstream target of the BMPR2 pathway, in human pulmonary artery smooth muscle cells (HPASMC). This attenuation of BMP signaling by hypoxia is conveyed through a repression of the transcriptional activity of the BMP responsive element (BRE) through mechanisms involving the transcriptional corepressor C-terminal–binding protein 1 (CtBP-1) and histone deacetylases (HDACs). Concordantly, overexpression of CtBP-1 suppressed BMP signaling, whereas small interfering RNA against CtBP-1 efficiently enhanced BMP stimulation of Id1 gene expression. Scavengers of reactive oxygen species had no effect on the hypoxic regulation...
Research Interests: Genetics, Biology, Cell Biology, Transcription Factors, Medicine, and 15 moreGene expression, Pulmonary Hypertension, Bone Morphogenetic Proteins, Signal Transduction, Humans, Anoxia, HIstone Deacetylase, Clinical Sciences, Circulation, NAD, DNA binding proteins, Pulmonary Artery, Signaling pathway, Pulmonary artery hypertension, and Cardiovascular medicine and haematology
Background— Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of proinflammatory mediators that are critical for the later development of hypoxic pulmonary... more
Background— Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of proinflammatory mediators that are critical for the later development of hypoxic pulmonary hypertension. Mesenchymal stromal cell transplantation inhibits lung inflammation, vascular remodeling, and right heart failure and reverses hypoxic pulmonary hypertension in experimental models of disease. In this study, we aimed to investigate the paracrine mechanisms by which mesenchymal stromal cells are protective in hypoxic pulmonary hypertension. Methods and Results— We fractionated mouse mesenchymal stromal cell–conditioned media to identify the biologically active component affecting in vivo hypoxic signaling and determined that exosomes, secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and the induction of proinflammatory and proproliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-ind...
Research Interests: Hypoxia, Innate immunity, Heart Failure, Humans, Anoxia, and 15 moreMice, Animals, Pneumonia, Exosomes, Clinical Sciences, microRNAs, Public health systems and services research, Circulation, Bronchopulmonary Dysplasia, Human Fibroblasts, Mesenchymal Stromal Cells, Extracellular Vesicles, Microvesicles, fibroblasts, and Cardiovascular medicine and haematology
ABSTRACT
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Research Interests: Pathology, Cytokines, Inflammation, Medicine, Mesenchymal stem cells, and 15 moreMice, Animals, Male, American, Lung, Bone Marrow Transplantation, Analysis of Variance, Lung Injury, Chronic lung disease, Murine Model, Hyperoxia, Bone Marrow Cells, Bone marrow stromal cells, Medical and Health Sciences, and Cell culture techniques
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Bronchopulmonary dysplasia (BPD) remains a major complication of prematurity resulting in significant morbidity and mortality. The pathology of BPD is multifactorial and leads to alveolar simplification and distal lung injury. Previous... more
Bronchopulmonary dysplasia (BPD) remains a major complication of prematurity resulting in significant morbidity and mortality. The pathology of BPD is multifactorial and leads to alveolar simplification and distal lung injury. Previous studies have shown a beneficial effect of systemic treatment with bone marrow-derived mesenchymal stromal cells (MSCs) and MSC-conditioned media (MSC-CM) leading to amelioration of the lung parenchymal and vascular injury in vivo in the hyperoxia murine model of BPD. It is possible that the beneficial response from the MSCs is at least in part due to activation of endogenous lung epithelial stem cells. Bronchioalveolar stem cells (BASCs) are an adult lung stem cell population capable of self-renewal and differentiation in culture, and BASCs proliferate in response to bronchiolar and alveolar lung injury in vivo. Systemic treatment of neonatal hyperoxia-exposed mice with MSCs or MSC-CM led to a significant increase in BASCs compared with untreated cont...
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Extracellular acidosis (EA) has profound effects on vascular homeostasis, including vascular bed-specific alterations in vascular tone. Regulation of gene expression by EA has been observed in a variety of cells including vascular... more
Extracellular acidosis (EA) has profound effects on vascular homeostasis, including vascular bed-specific alterations in vascular tone. Regulation of gene expression by EA has been observed in a variety of cells including vascular endothelial cells. Whether EA regulates gene expression in vascular smooth muscle cells (VSMCs) is not known. Heme oxygenase (HO)-1 is expressed in vascular cells, and its expression is regulated by cellular stressors such as heat, radiation, and hypoxia. Increased HO-1 expression in VSMCs leads to increased production of CO and its second messenger cGMP, which are important regulators of vascular tone and paracrine interactions in the vasculature. We examined whether EA regulates the expression of HO-1 in VSMCs. Exposure of VSMCs to acidic medium (pH 6.8) significantly increased HO-1 mRNA and protein compared with exposure to medium of physiological pH (pH 7.4). The acidic induction of HO-1 expression was time dependent and involved both transcriptional a...
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Additional file 5. Assessment of PKH67 dye leakage into surrounding cells in vitro. TSCs and MLE-12 cells were pre-labeled with PKH67 (green) and PKH26 (red), respectively. TSCs were mixed with MLE-12 cells at a ratio of 1:10 and... more
Additional file 5. Assessment of PKH67 dye leakage into surrounding cells in vitro. TSCs and MLE-12 cells were pre-labeled with PKH67 (green) and PKH26 (red), respectively. TSCs were mixed with MLE-12 cells at a ratio of 1:10 and co-cultured for 3 days. Cells were harvested and a cytospin performed to concentrate the cells. Representative image showing a) mixture unlabeled MLE-12 cells and TSCs staining for DAPI (blue); b) TSCs pre-labeled with PKH67; c) MLE-12 cells pre-labeled with PKH26; d~f) co-cultured pre-labeled TSCs and MLE-12 cells in FITC channel (d), in red channel (e) and merged image of green, red and blue (DAPI, f). White arrowheads point to the green TSCs and arrows point to the red MLE-12 cells. Scale bar represents 50µm.
© 2020 International Society for Cell and Gene Therapy STATEMENT: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular... more
© 2020 International Society for Cell and Gene Therapy STATEMENT: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.
Background—Lung inflammation precedes the development of hypoxia-induced pulmonary hypertension (HPH); however its role in the pathogenesis of HPH is poorly understood. We sought to characterize the hypoxic inflammatory response and... more
Background—Lung inflammation precedes the development of hypoxia-induced pulmonary hypertension (HPH); however its role in the pathogenesis of HPH is poorly understood. We sought to characterize the hypoxic inflammatory response and elucidate its role in the development of HPH. We also aimed to investigate the mechanisms by which heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, is protective in HPH. Methods and Results—We generated bitransgenic mice that overexpress human HO-1 under doxycycline (dox) control in an inducible, lung-specific manner. Hypoxic exposure of mice in the absence of dox resulted in early transient accumulation of monocytes/macrophages in the bronchoalveolar lavage. Alveolar macrophages acquired an alternatively activated phenotype (M2) in response to hypoxia, characterized by the expression of Found in Inflammatory Zone-1, Arginase-1 and Chitinase-3-like-3. A brief, two-day pulse of dox delayed but did not prevent the peak of hypoxic inflammation, and cou...
Research Interests: Carbon Dioxide, Medicine, Pulmonary Hypertension, Transgenic Mice, Cell Division, and 14 moreHumans, Anoxia, Mice, Animals, Pneumonia, Heme Oxygenase, Clinical Sciences, Monocytes, Public health systems and services research, Circulation, Interleukin, Pulmonary Artery, Macrophage activation, and Cardiovascular medicine and haematology
Treating premature infants with high oxygen is a routine intervention in the context of neonatal intensive care. Unfortunately, the increase in survival rates is associated with various detrimental sequalae of hyperoxia exposure, most... more
Treating premature infants with high oxygen is a routine intervention in the context of neonatal intensive care. Unfortunately, the increase in survival rates is associated with various detrimental sequalae of hyperoxia exposure, most notably bronchopulmonary dysplasia (BPD), a disease of disrupted lung development. The effects of high oxygen exposure on other developing organs of the infant, as well as the possible impact such disrupted development may have on later life remain poorly understood. Using a neonatal mouse model to investigate the effects of hyperoxia on the immature immune system we observed a dramatic involution of the thymic medulla, and this lesion was associated with disrupted FoxP3+ regulatory T cell generation and T cell autoreactivity. Significantly, administration of mesenchymal stromal cell-derived extracellular vesicles (MEx) restored thymic medullary architecture and physiological thymocyte profiles. Using single cell transcriptomics, we further demonstrate...
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Oral pioglitazone reverses pulmonary hypertensive vascular disease and prevents right heart failure via epigenetic, transcriptional, and metabolic mechanisms.
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Acute hypoxia in the lung causes arteriolar vasoconstriction whereas prolonged hypoxia promotes proliferation and migration of vascular smooth muscle cells (VSMC) and extracellular matrix deposition in the arterial wall, a process known... more
Acute hypoxia in the lung causes arteriolar vasoconstriction whereas prolonged hypoxia promotes proliferation and migration of vascular smooth muscle cells (VSMC) and extracellular matrix deposition in the arterial wall, a process known as vascular remodeling.1 These abnormalities are characteristic of pulmonary hypertension.2 Several clinical conditions characterized by lung inflammation have been linked to the development of chronic pulmonary hypertension.3 Interestingly, perivascular inflammatory cell infiltration as well as increased serum levels of pro-inflammatory cytokines, such as interleukin (IL)-lβ and IL-6, have been reported in clinical cases of primary pulmonary hypertension.4-5 However, little attention has been given up to now to the role of pulmonary inflammation in the pathogenesis of pulmonary hypertension induced by hypoxia.
Research Interests: Evolutionary Biology, Genetics, Molecular Evolution, Gene expression, DNA, and 13 moreDrosophila, Protein-DNA interaction, Animals, Biological evolution, Base Sequence, Sperm Dna Fragmentation, Molecular Interactions, Egg shell, Biochemistry and cell biology, Gene Expression Regulation, Bombyx mori, DNA fragmentation, and Molecular Sequence Data
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A sequence in the long terminal repeat (LTR) of avian tumor virus (ATV) DNA was shown to contain a promoter acute in Escherichia coli. For this analysis the bacterial promoters for the tetracycline (Tc) and neomycin (Nm) resistance genes... more
A sequence in the long terminal repeat (LTR) of avian tumor virus (ATV) DNA was shown to contain a promoter acute in Escherichia coli. For this analysis the bacterial promoters for the tetracycline (Tc) and neomycin (Nm) resistance genes were deleted from different plasmids and replaced with various fragments derived from the ATV DNA. Expression of the drug-resistant phenotype in the recombinant plasmids at levels comparable to or greater than those found with parental bacterial promotes was shown to be dependent on the presence of an intact sequence ranging from nucleotide +19 to -23 (relative to the cap site) in the ATV DNA. Comparison of the consensus bacterial promoter with the nucleotide sequence in this region revealed strong similarities.
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Chronic hypoxia causes pulmonary hypertension with smooth muscle cell proliferation and matrix deposition in the wall of the pulmonary arterioles. We demonstrate here that hypoxia also induces a pronounced inflammation in the lung before... more
Chronic hypoxia causes pulmonary hypertension with smooth muscle cell proliferation and matrix deposition in the wall of the pulmonary arterioles. We demonstrate here that hypoxia also induces a pronounced inflammation in the lung before the structural changes of the vessel wall. ...
Research Interests: Biology, Membrane Proteins, Medicine, Gene expression, Multidisciplinary, and 15 morePulmonary Hypertension, Transgenic Mice, Humans, Anoxia, Mice, Animals, Heme Oxygenase, Lung, Structural Change, Time Factors, Ct Image of Pulmonary Artery, Chronic Hypoxia, Pulmonary Artery, Smooth muscle cell, and Tumor necrosis factor
SUMMARY The extent of methylation of several avian oncogenic proviruses was determined by using the restriction endonucleases HpalI and MspI. The results indicated that the transformation-defective proviruses (RAV-O or B77-td), which are... more
SUMMARY The extent of methylation of several avian oncogenic proviruses was determined by using the restriction endonucleases HpalI and MspI. The results indicated that the transformation-defective proviruses (RAV-O or B77-td), which are exogenously ...
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Research Interests: Biology, Medicine, Humans, Mice, Female, and 15 moreAnimals, Male, American, Phenotype, Adenylate Kinase, Isoenzymes, Cilia, Amino Acid Sequence, Base Sequence, Molecular Sequence Data, DNA mutational analysis, Tissue distribution, frameshift mutation, Primary Ciliary Dyskinesia, and Cardiovascular medicine and haematology
Bronchopulmonary dysplasia (BPD) is characterized by simplified alveolarization and arrested vascular development of the lung with associated evidence of endothelial dysfunction, inflammation, increased oxidative damage, and iron... more
Bronchopulmonary dysplasia (BPD) is characterized by simplified alveolarization and arrested vascular development of the lung with associated evidence of endothelial dysfunction, inflammation, increased oxidative damage, and iron deposition. Heme oxygenase-1 (HO-1) has been reported to be protective in the pathogenesis of diseases of inflammatory and oxidative etiology. Because HO-1 is involved in the response to oxidative stress produced by hyperoxia and is critical for cellular heme and iron homeostasis, it could play a protective role in BPD. Therefore, we investigated the effect of HO-1 in hyperoxia-induced lung injury using a neonatal transgenic mouse model with constitutive lung-specific HO-1 overexpression. Hyperoxia triggered an increase in pulmonary inflammation, arterial remodeling, and right ventricular hypertrophy that was attenuated by HO-1 overexpression. In addition, hyperoxia led to pulmonary edema, hemosiderosis, and a decrease in blood vessel number, all of which w...
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Pulmonary arterial hypertension (PAH) is a progressive disease characterized by remodeling of the pulmonary arteries, increased pulmonary infiltrates, loss of vascular cross-sectional area, and elevated pulmonary vascular resistance.... more
Pulmonary arterial hypertension (PAH) is a progressive disease characterized by remodeling of the pulmonary arteries, increased pulmonary infiltrates, loss of vascular cross-sectional area, and elevated pulmonary vascular resistance. Despite recent advances in the management of PAH, there is a pressing need for the development of new tools to effectively treat and reduce the risk of further complications. Dysregulated immunity underlies the development of PAH, and macrophages orchestrate both the initiation and resolution of pulmonary inflammation, thus, manipulation of lung macrophage function represents an attractive target for emerging immunomodulatory therapies, including cell-based approaches. Indeed, mesenchymal stem cell (MSC)-based therapies have shown promise, effectively modulating the macrophage fulcrum to favor an anti-inflammatory, pro-resolving phenotype, which is associated with both histological and functional benefits in preclinical models of pulmonary hypertension ...
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Pulmonary hypertension (PH) is characterized by pulmonary arteriolar remodeling with excessive pulmonary vascular smooth muscle cell (VSMC) proliferation. This results in decreased responsiveness of pulmonary circulation to vasodilator... more
Pulmonary hypertension (PH) is characterized by pulmonary arteriolar remodeling with excessive pulmonary vascular smooth muscle cell (VSMC) proliferation. This results in decreased responsiveness of pulmonary circulation to vasodilator therapies. We have shown that extracellular acidosis inhibits VSMC proliferation and migration in vitro. Here we tested whether induction of nonhypercapnic acidosis in vivo ameliorates PH and the underlying pulmonary vascular remodeling and dysfunction. Adult male Sprague-Dawley rats were exposed to hypoxia (8.5% O2) for 2 wk, or injected subcutaneously with monocrotaline (MCT, 60 mg/kg) to develop PH. Acidosis was induced with NH4Cl (1.5%) in the drinking water 5 days prior to and during the 2 wk of hypoxic exposure (prevention protocol), or after MCT injection from day 21 to 28 (reversal protocol). Right ventricular systolic pressure (RVSP) and Fulton's index were measured, and pulmonary arteriolar remodeling was analyzed. Pulmonary and mesenter...
Research Interests: Physiology, Carbon Dioxide, Anoxia, Blood Pressure, Animals, and 15 moreMale, American, Acetylcholine, Medical Physiology, Hypertrophy, Lung, Ammonium Chloride, Phenylephrine, Ct Image of Pulmonary Artery, Heart Ventricles, nitroprusside, Antihypertensive agents, airway remodeling, monocrotaline, and In Vitro Techniques
Research Interests: Biomechanics, Hypoxia, Extracellular Matrix, Pulmonary Hypertension, Anoxia, and 15 moreCollagen, Mice, Blood Pressure, Chronic Disease, Animals, Clinical Sciences, Gels, Public health systems and services research, Circulation, Muscle contraction, Traction, Pulmonary Artery, Contractility, Cardiovascular medicine and haematology, and right ventricular hypertrophy
RATIONALE: Mesenchymal stem/stromal cell (MSC) therapies have shown promise in preclinical models of pathologies relevant to newborn medicine, such as bronchopulmonary dysplasia (BPD). We have reported that the therapeutic capacity of... more
RATIONALE:
Mesenchymal stem/stromal cell (MSC) therapies have shown promise in preclinical models of pathologies relevant to newborn medicine, such as bronchopulmonary dysplasia (BPD). We have reported that the therapeutic capacity of MSCs is comprised in their secretome, and demonstrated that the therapeutic vectors are exosomes produced by MSCs (MSC-exos).
OBJECTIVE:
To assess efficacy of MSC-exo treatment in a pre-clinical model of BPD and to investigate mechanisms underlying MSC-exo therapeutic action.
METHODS:
Exosomes were isolated from media conditioned by human MSC cultures. Newborn mice were exposed to hyperoxia (HYRX, 75% O2), treated with exosomes on postnatal day 4 (PN4) and returned to room air on PN7. Treated animals and appropriate controls were harvested on PN7, 14 or 42 for assessment of pulmonary parameters.
MEASUREMENTS AND MAIN RESULTS:
HYRX-exposed mice presented with pronounced alveolar simplification, fibrosis, and pulmonary vascular remodeling, which was effectively ameliorated by MSC-exo treatment. Pulmonary function tests and assessment of pulmonary hypertension showed functional improvements following MSC-exo treatment. Lung mRNA sequencing demonstrated that MSC-exo treatment induced pleiotropic effects on gene expression associated with HYRX-induced inflammation and immune responses. MSC-exos modulate the macrophage phenotype fulcrum, suppressing the proinflammatory M1 state and augmenting an anti-inflammatory M2-like state, both in vitro and in vivo.
CONCLUSION:
MSC-exo treatment blunts hyperoxia-associated inflammation and alters the hyperoxic lung transcriptome. This results in alleviation of HYRX-induced BPD, improvement of lung function, decrease in fibrosis and pulmonary vascular remodeling, and amelioration of pulmonary hypertension. The MSC exosome mechanism-of-action is associated with modulation of lung macrophage phenotype.
Mesenchymal stem/stromal cell (MSC) therapies have shown promise in preclinical models of pathologies relevant to newborn medicine, such as bronchopulmonary dysplasia (BPD). We have reported that the therapeutic capacity of MSCs is comprised in their secretome, and demonstrated that the therapeutic vectors are exosomes produced by MSCs (MSC-exos).
OBJECTIVE:
To assess efficacy of MSC-exo treatment in a pre-clinical model of BPD and to investigate mechanisms underlying MSC-exo therapeutic action.
METHODS:
Exosomes were isolated from media conditioned by human MSC cultures. Newborn mice were exposed to hyperoxia (HYRX, 75% O2), treated with exosomes on postnatal day 4 (PN4) and returned to room air on PN7. Treated animals and appropriate controls were harvested on PN7, 14 or 42 for assessment of pulmonary parameters.
MEASUREMENTS AND MAIN RESULTS:
HYRX-exposed mice presented with pronounced alveolar simplification, fibrosis, and pulmonary vascular remodeling, which was effectively ameliorated by MSC-exo treatment. Pulmonary function tests and assessment of pulmonary hypertension showed functional improvements following MSC-exo treatment. Lung mRNA sequencing demonstrated that MSC-exo treatment induced pleiotropic effects on gene expression associated with HYRX-induced inflammation and immune responses. MSC-exos modulate the macrophage phenotype fulcrum, suppressing the proinflammatory M1 state and augmenting an anti-inflammatory M2-like state, both in vitro and in vivo.
CONCLUSION:
MSC-exo treatment blunts hyperoxia-associated inflammation and alters the hyperoxic lung transcriptome. This results in alleviation of HYRX-induced BPD, improvement of lung function, decrease in fibrosis and pulmonary vascular remodeling, and amelioration of pulmonary hypertension. The MSC exosome mechanism-of-action is associated with modulation of lung macrophage phenotype.
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Research Interests: Humans, Mice, Female, Animals, Male, and 7 moreAmerican, Phenotype, Adenylate Kinase, Isoenzymes, Cilia, Amino Acid Sequence, and Base Sequence
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Background: Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of pro-inflammatory mediators that are critical for the later development of hypoxic pulmonary... more
Background: Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of pro-inflammatory mediators that are critical for the later development of hypoxic pulmonary hypertension (HPH). Mesenchymal stromal cell (MSC) transplantation inhibits lung inflammation, vascular remodeling and right heart failure, and reverses HPH in experimental models of disease. In this study, we aimed to investigate the paracrine mechanisms by which MSCs are protective in HPH.
Methods and Results: We fractionated mouse MSC-conditioned media to identify the biologically-active component affecting in vivo hypoxic signaling and determined that exosomes, secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and the induction of pro-inflammatory and pro-proliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-inducible mitogenic factor, in the murine model of HPH. Intravenous delivery of MSC exosomes (MEX) inhibited vascular remodeling and HPH, whereas MEX-depleted media or fibroblast-derived exosomes had no effect. MEX suppressed the hypoxic activation of signal transducer and activator of transcription 3 (STAT3) and the upregulation of the miR-17 superfamily of microRNA clusters, whereas it increased lung levels of miR-204, a key microRNA whose expression is decreased in human PH. MEX produced by human umbilical cord MSCs inhibited STAT3 signaling in isolated human pulmonary artery endothelial cells demonstrating a direct effect of MEX on hypoxic vascular cells.
Conclusions: This study indicates that MEX exert a pleiotropic protective effect on the lung and can prevent PH through suppression of hyperproliferative pathways, including STAT-3 mediated signaling induced by hypoxia.
Methods and Results: We fractionated mouse MSC-conditioned media to identify the biologically-active component affecting in vivo hypoxic signaling and determined that exosomes, secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and the induction of pro-inflammatory and pro-proliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-inducible mitogenic factor, in the murine model of HPH. Intravenous delivery of MSC exosomes (MEX) inhibited vascular remodeling and HPH, whereas MEX-depleted media or fibroblast-derived exosomes had no effect. MEX suppressed the hypoxic activation of signal transducer and activator of transcription 3 (STAT3) and the upregulation of the miR-17 superfamily of microRNA clusters, whereas it increased lung levels of miR-204, a key microRNA whose expression is decreased in human PH. MEX produced by human umbilical cord MSCs inhibited STAT3 signaling in isolated human pulmonary artery endothelial cells demonstrating a direct effect of MEX on hypoxic vascular cells.
Conclusions: This study indicates that MEX exert a pleiotropic protective effect on the lung and can prevent PH through suppression of hyperproliferative pathways, including STAT-3 mediated signaling induced by hypoxia.