Publications by authors named "Ashish K Sharma"

77 Publications

MicroRNA-206 antagomiR‒enriched extracellular vesicles attenuate lung ischemia‒reperfusion injury through CXCL1 regulation in alveolar epithelial cells.

J Heart Lung Transplant 2020 12 28;39(12):1476-1490. Epub 2020 Sep 28.

Department of Surgery, University of Florida, Gainesville, Florida; Department of Medicine, University of Florida, Gainesville, Florida. Electronic address:

Background: Our hypothesis is that the immunomodulatory capacities of mesenchymal stem cell‒derived extracellular vesicles (EVs) can be enhanced by specific microRNAs (miRNAs) to effectively attenuate post-transplant lung ischemia‒reperfusion (IR) injury.

Methods: The expression of miR-206 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on Days 0 and 1 after lung transplantation. Lung IR injury was evaluated in C57BL/6 mice using a left lung hilar-ligation model with or without treatment with EVs or antagomiR-206‒enriched EVs. Murine lung tissue was used for miRNA microarray hybridization analysis, and cytokine expression, lung injury, and edema were evaluated. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by enriched EVs against lung IR injury. In vitro studies analyzed type II epithelial cell activation after coculturing with EVs.

Results: A significant upregulation of miR-206 was observed in the BAL fluid of patients on Day 1 after lung transplantation compared with Day 0 and in murine lungs after IR injury compared with sham. Treatment with antagomiR-206‒enriched EVs attenuated lung dysfunction, injury, and edema compared with treatment with EVs alone after murine lung IR injury. Enriched EVs reduced lung injury and neutrophil infiltration as well as improved allograft oxygenation after murine orthotopic lung transplantation. Enriched EVs significantly decreased proinflammatory cytokines, especially epithelial cell‒dependent CXCL1 expression, in the in vivo and in vitro IR injury models.

Conclusions: EVs can be used as biomimetic nanovehicles for protective immunomodulation by enriching them with antagomiR-206 to mitigate epithelial cell activation and neutrophil infiltration in the lungs after IR injury.
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http://dx.doi.org/10.1016/j.healun.2020.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704771PMC
December 2020

Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene.

Biotechnol Bioeng 2021 Jan 24;118(1):186-198. Epub 2020 Sep 24.

Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, Maryland, USA.

Cell lines used in bioproduction are routinely engineered to improve their production efficiency. Numerous strategies, such as random mutagenesis, RNA interference screens, and transcriptome analyses have been employed to identify effective engineering targets. A genome-wide small interfering RNA screen previously identified the CASP8AP2 gene as a potential engineering target for improved expression of recombinant protein in the HEK293 cell line. Here, we validate the CASP8AP2 gene as an engineering target in HEK293 cells by knocking it out using CRISPR/Cas9 genome editing and assessing the effect of its knockout on recombinant protein expression, cell growth, cell viability, and overall gene expression. HEK293 cells lacking CASP8AP2 showed a seven-fold increase in specific expression of recombinant luciferase and a 2.5-fold increase in specific expression of recombinant SEAP, without significantly affecting cell growth and viability. Transcriptome analysis revealed that the deregulation of the cell cycle, specifically the upregulation of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, contributed to the improvement in recombinant protein expression in CASP8AP2 deficient cells. The results validate the CASP8AP2 gene is a viable engineering target for improved recombinant protein expression in the HEK293 cell line.
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http://dx.doi.org/10.1002/bit.27561DOI Listing
January 2021

A novel knock out strategy to enhance recombinant protein expression in Escherichia coli.

Microb Cell Fact 2020 Jul 23;19(1):148. Epub 2020 Jul 23.

Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD, 20892, USA.

Background: The expression of recombinant proteins triggers a stress response which downregulates key metabolic pathway genes leading to a decline in cellular health and feedback inhibition of both growth and protein expression. Instead of individually upregulating these downregulated genes or improving transcription rates by better vector design, an innovative strategy would be to block this stress response thereby ensuring a sustained level of protein expression.

Results: We postulated that the genes which are commonly up-regulated post induction may play the role of signalling messengers in mounting the cellular stress response. We identified those genes which have no known downstream regulatees and created knock outs which were then tested for GFP expression. Many of these knock outs showed significantly higher expression levels which was also sustained for longer periods. The highest product yield (Y) was observed in a BW25113ΔcysJ knock out (Y 0.57) and BW25113ΔelaA (Y 0.49), whereas the Y of the control W3110 strain was 0.08 and BW25113 was 0.16. Double knock out combinations were then created from the ten best performing single knock outs leading to a further enhancement in expression levels. Out of 45 double knock outs created, BW25113ΔelaAΔyhbC (Y 0.7) and BW25113ΔcysJΔyhbC (Y 0.64) showed the highest increase in product yield compared to the single gene mutant strains. We confirmed the improved performance of these knock outs by testing and obtaining higher levels of recombinant asparaginase expression, a system better suited for analysing sustained expression since it gets exported to the extracellular medium.

Conclusion: Creating key knock outs to block the CSR and enhance expression is a radically different strategy that can be synergistically combined with traditional methods of improving protein yields thus helping in the design of superior host platforms for protein expression.
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http://dx.doi.org/10.1186/s12934-020-01407-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376861PMC
July 2020

Pharmacologic inhibition of transient receptor channel vanilloid 4 attenuates abdominal aortic aneurysm formation.

FASEB J 2020 07 7;34(7):9787-9801. Epub 2020 Jun 7.

Department of Surgery, University of Florida, Gainesville, FL, USA.

Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, leukocyte infiltration, and vascular remodeling. This study investigates the role of TRPV4 channels, which are transmembrane calcium channels that can regulate vascular tone, in modulating AAA formation. The elastase-treatment model of AAA in C57BL6 (WT) mice and Angiotensin II treatment model in ApoE mice were used to confirm our hypotheses. The administration of a specific TRPV4 antagonist, GSK2193874, in elastase-treated WT mice and in AngII-treated ApoE mice caused a significant attenuation of aortic diameter, decrease in pro-inflammatory cytokines (IL-1β, IL-6, IL-17, MCP-1, MIP-1α, MIP-2, RANTES, and TNF-α), inflammatory cell infiltration (CD3 + T cells, macrophages, and neutrophils), elastic fiber disruption, and an increase in smooth muscle cell α-actin expression compared to untreated mice. Similarly, elastase-treated TRPV4 mice had a significant decrease in AAA formation, aortic inflammation, and vascular remodeling compared to elastase-treated WT mice on Day 14. In vitro studies demonstrated that the inhibition of TRPV4 channels mitigates aortic smooth muscle cell-dependent inflammatory cytokine production as well as decreases neutrophil transmigration through aortic endothelial cells. Therefore, our results suggest that TRPV4 antagonism can attenuate aortic inflammation and remodeling via decreased smooth muscle cell activation and neutrophil transendothelial migration during AAA formation.
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http://dx.doi.org/10.1096/fj.202000251RDOI Listing
July 2020

Linking Phospho-Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) to Histone Ubiquitination and Acetylation Essential for Spermatid Development During Spermiogenesis.

Front Cell Dev Biol 2020 15;8:310. Epub 2020 May 15.

Section on Molecular Endocrinology, Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, United States.

GRTH/DDX25 is a testicular RNA helicase expressed in germ cells that plays a crucial role in completion of spermatogenesis. Previously, we demonstrated a missense mutation (RH) of GRTH gene in Japanese infertile patients (5.8%) with non-obstructive azoospermia. This mutation upon expression in COS-1 cells revealed absence of the 61 kDa phosphorylated GRTH in cytoplasm and the presence of the 56 kDa non-phosphorylated GRTH in the nucleus. GRTH knock-in (KI) mice carrying the human GRTH (RH) mutation, lack phosphorylated GRTH, and sperm due to failure of round spermatid elongation during spermiogenesis. To determine the impact of phosphorylated GRTH on molecular events/pathways participating in spermatid development during spermiogenesis, we analyzed transcriptome profiles obtained from RNA-Seq of germ cells from KI and WT mice. RNA-Seq analysis of 2624 differentially expressed genes revealed 1404 down-regulated and 1220 up-regulated genes in KI mice. Genes relevant to spermatogenesis, spermatid development and spermatid differentiation were significantly down-regulated. KEGG enrichment analysis showed genes related to ubiquitin-mediated proteolysis and protein processing in endoplasmic reticulum pathway genes were significantly down-regulated while the up-regulated genes were found to be involved in Focal adhesion and ECM-receptor interaction pathways. Real-Time PCR analysis confirmed considerable reduction in transcripts of ubiquitination related genes , , , , , and increased expression of , , , , , mRNA's in KI mice compared to WT. Also, marked reduction in protein expression of UBE2J1, RNF8, RNF138 (ubiquitination network), MOF (histone acetyltransferase), their modified Histone substrates (H2AUb, H2BUb) and H4Ac, H4K16Ac were observed in KI mice. GRTH-IP mRNA binding studies revealed that and mRNAs from WT mice associated with GRTH protein and the binding is greatly impaired in the KI mice. Immunohistochemistry analysis showed significantly reduced expression of RNF8, MOF, H4Ac and H4K16Ac in round spermatids of KI mice. Absence of phosphorylated GRTH impairs UBE2J1, RNF8 and MOF-dependent histone ubiquitination and acetylation essential for histone replacement, chromatin condensation and spermatid elongation during spermiogenesis.
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http://dx.doi.org/10.3389/fcell.2020.00310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242631PMC
May 2020

Single-Photon Emission Computed Tomography Imaging Using Formyl Peptide Receptor 1 Ligand Can Diagnose Aortic Aneurysms in a Mouse Model.

J Surg Res 2020 07 12;251:239-247. Epub 2020 Mar 12.

Department of Surgery, University of Florida, Gainesville, Florida. Electronic address:

Background: Our previous studies showed that neutrophil infiltration and activation plays an important role in the pathogenesis of abdominal aortic aneurysms (AAA). However, there is a lack of noninvasive, inflammatory cell-specific molecular imaging methods to provide early diagnosis of AAA formation. Formyl peptide receptor 1 (FPR1) is rapidly upregulated on neutrophils during inflammation. Therefore, it is hypothesized that the use of cinnamoyl-F-(D)L-F-(D)L-F-K (cFLFLF), a PEGylated peptide ligand that binds FPR1 on activated neutrophils, would permit accurate and noninvasive diagnosis of AAA via single-photon emission computed tomography (SPECT) imaging.

Materials And Methods: Male C57BL/6 (wild-type) mice were treated with topical elastase (0.4 U/mL type 1 porcine pancreatic elastase) or heat-inactivated elastase (control), and aortic diameter was measured by video micrometry. Comparative histology was performed on Day 14 to assess neutrophil infiltration in aortic tissue. We performed near-infrared fluorescence imaging using c-FLFLF-Cy7 probe on Days 7 and 14 postelastase treatment and measured fluorescence intensity ex vivo in excised aortic tissue. A separate group of animals were injected with Tc-c-FLFLF 2 h before SPECT imaging on Day 14 using a SPECT/computed tomography/positron emission tomography trimodal scanner. Coexpression of neutrophils with c-FLFLF was also performed on aortic tissue by immunostaining on Day 14.

Results: Aortic diameter was significantly increased in the elastase group compared with controls on Days 7 and 14. Simultaneously, a marked increase in neutrophil infiltration and elastin degradation as well as decrease in smooth muscle integrity were observed in aortic tissue after elastase treatment compared with controls. Moreover, a significant increase in fluorescence intensity of c-FLFLF-Cy7 imaging probe was also observed in elastase-treated mice on Day 7 (approximately twofold increase) and Day 14 (approximately 2.5-fold increase) compared with respective controls. SPECT imaging demonstrated a multifold increase in signal intensity for Tc-cFLFLF radiolabel probe in mice with AAA compared with controls on Day 14. Immunostaining of aortic tissue with c-FLFLF-Cy5 demonstrated a marked increase in coexpression with neutrophils in AAA compared with controls.

Conclusions: cFLFLF, a novel FPR1 ligand, enables quantifiable, noninvasive diagnosis and progression of AAAs. Clinical application of this inflammatory, cell-specific molecular probe using SPECT imaging may permit early diagnosis of AAA formation, enabling targeted therapeutic interventions and preventing impending aortic rupture.
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http://dx.doi.org/10.1016/j.jss.2020.01.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540921PMC
July 2020

Effect of restricted dissolved oxygen on expression of Clostridium difficile toxin A subunit from E. coli.

Sci Rep 2020 02 20;10(1):3059. Epub 2020 Feb 20.

Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.

The repeating unit of the C. difficile Toxin A (rARU, also known as CROPS [combined repetitive oligopeptides]) C-terminal region, was shown to elicit protective immunity against C. difficile and is under consideration as a possible vaccine against this pathogen. However, expression of recombinant rARU in E. coli using the standard vaccine production process was very low. Transcriptome and proteome analyses showed that at restricted dissolved oxygen (DO) the numbers of differentially expressed genes (DEGs) was 2.5-times lower than those expressed at unrestricted oxygen. Additionally, a 7.4-times smaller number of ribosome formation genes (needed for translation) were down-regulated as compared with unrestricted DO. Higher rARU expression at restricted DO was associated with up-regulation of 24 heat shock chaperones involved in protein folding and with the up-regulation of the global regulator RNA chaperone hfq. Cellular stress response leading to down-regulation of transcription, translation, and energy generating pathways at unrestricted DO were associated with lower rARU expression. Investigation of the C. difficile DNA sequence revealed the presence of cell wall binding profiles, which based on structural similarity prediction by BLASTp, can possibly interact with cellular proteins of E. coli such as the transcriptional repressor ulaR, and the ankyrins repeat proteins. At restricted DO, rARU mRNA was 5-fold higher and the protein expression 27-fold higher compared with unrestricted DO. The report shows a strategy for improved production of C. difficile vaccine candidate in E. coli by using restricted DO growth. This strategy could improve the expression of recombinant proteins from anaerobic origin or those with cell wall binding profiles.
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http://dx.doi.org/10.1038/s41598-020-59978-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033237PMC
February 2020

SPECT imaging of lung ischemia-reperfusion injury using [Tc]cFLFLF for molecular targeting of formyl peptide receptor 1.

Am J Physiol Lung Cell Mol Physiol 2020 02 4;318(2):L304-L313. Epub 2019 Dec 4.

Department of Surgery, University of Virginia School of Medicine, Charlottesville, Virginia.

Primary graft dysfunction after lung transplantation, a consequence of ischemia-reperfusion injury (IRI), is a major cause of morbidity and mortality. IRI involves acute inflammation and innate immune cell activation, leading to rapid infiltration of neutrophils. Formyl peptide receptor 1 (FPR1) expressed by phagocytic leukocytes plays an important role in neutrophil function. The cell surface expression of FPR1 is rapidly and robustly upregulated on neutrophils in response to inflammatory stimuli. Thus, we hypothesized that use of [Tc]cFLFLF, a selective FPR1 peptide ligand, would permit in vivo neutrophil labeling and noninvasive imaging of IRI using single-photon emission computed tomography (SPECT). A murine model of left lung IRI was utilized. Lung function, neutrophil infiltration, and SPECT imaging were assessed after 1 h of ischemia and 2, 12, or 24 h of reperfusion. [Tc]cFLFLF was injected 2 h before SPECT. Signal intensity by SPECT and total probe uptake by gamma counts were 3.9- and 2.3-fold higher, respectively, in left lungs after ischemia and 2 h of reperfusion versus sham. These values significantly decreased with longer reperfusion times, correlating with resolution of IRI as shown by improved lung function and decreased neutrophil infiltration. SPECT results were confirmed using Cy7-cFLFLF-based fluorescence imaging of lungs. Immunofluorescence microscopy confirmed cFLFLF binding primarily to activated neutrophils. These results demonstrate that [Tc]cFLFLF SPECT enables noninvasive detection of lung IRI and permits monitoring of resolution of injury over time. Clinical application of [Tc]cFLFLF SPECT may permit diagnosis of lung IRI for timely intervention to improve outcomes after transplantation.
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http://dx.doi.org/10.1152/ajplung.00220.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052676PMC
February 2020

Female Mice Exhibit Abdominal Aortic Aneurysm Protection in an Established Rupture Model.

J Surg Res 2020 03 4;247:387-396. Epub 2019 Nov 4.

Department of Surgery, College of Medicine, University of Florida, Gainesville, Florida. Electronic address:

Background: Male gender is a well-established risk factor for abdominal aortic aneurysm (AAA), whereas estrogen is hypothesized to play a protective role. Although rupture rates are higher in women, these reasons remain unknown. In the present study, we sought to determine if female mice are protected from AAA rupture.

Materials And Methods: Apolipoprotein E-deficient male and female mice (aged 7 wk; n = 25 per group) were infused with angiotensin II (AngII; 2000 ng/kg/min) plus β-aminopropionitrile (BAPN) in the drinking water for 28 d to test the effects of gender on AAA rupture. Separately, a second group of male apolipoprotein E-deficient mice underwent AngII infusion + BAPN while being fed high-fat phytoestrogen free or a high-fat phytoestrogen diet to assess effects of phytoestrogens on rupture. In a third group, female mice either underwent oophorectomy or sham operation 4 wk before infusion of AngII and BAPN to further test the effects of female hormones on AA rupture. Surviving mice abdominal aorta were collected for histology, cytokine array, and gelatin zymography on postoperative day 28.

Results: Female mice had decreased AAA rupture rates (16% versus 46%; P = 0.029). Female mice expressed fewer elastin breaks (P = 0.0079) and decreased smooth muscle cell degradation (P = 0.0057). Multiple cytokines were also decreased in the female group. Gelatin zymography demonstrated significantly decreased pro-matrix metalloproteinase 2 in female mice (P = 0.001). Male mice fed a high dose phytoestrogen diet failed to decrease AAA rupture. Female mice undergoing oophorectomy did not have accelerated aortic rupture.

Conclusions: These data are the first to attempt to tease out hormonal effects on AAA rupture and the possible role of gender in rupture.
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http://dx.doi.org/10.1016/j.jss.2019.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111562PMC
March 2020

Cyclophilin A contributes to aortopathy induced by postnatal loss of smooth muscle TGFBR1.

FASEB J 2019 10 16;33(10):11396-11410. Epub 2019 Jul 16.

Division of Vascular Surgery and Endovascular Therapy, University of Florida College of Medicine, Gainesville, Florida, USA.

Recent recognition that TGF-β signaling disruption is involved in the development of aortic aneurysms has led to renewed investigations into the role of TGF-β biology in the aortic wall. We previously found that the type I receptor of TGF-β (TGFBR2) receptor contributes to formation of ascending aortic aneurysms and dissections (AADs) induced by smooth muscle cell (SMC)-specific, postnatal deletion of (). Here, we aimed to decipher the mechanistic signaling pathway underlying the pathogenic effects of TGFBR2 in this context. Gene expression profiling demonstrated that triggers an acute inflammatory response in developing AADs, and SMCs express an inflammatory phenotype in culture. Comparative proteomics profiling and mass spectrometry revealed that SMCs respond to TGF-β1 stimulation robust up-regulation of cyclophilin A (CypA). This up-regulation is abrogated by inhibition of TGFBR2 kinase activity, small interfering RNA silencing of expression, or inhibition of SMAD3 activation. In mice, rapidly promotes CypA production in SMCs of developing AADs, whereas treatment with a CypA inhibitor attenuates aortic dilation by 56% ( = 0.003) and ameliorates aneurysmal degeneration ( = 0.016). These protective effects are associated with reduced aneurysm-promoting inflammation. Collectively, these results suggest a novel mechanism, wherein loss of type I receptor of TGF-β triggers promiscuous, proinflammatory TGFBR2 signaling in SMCs, thereby promoting AAD formation.-Zhou, G., Liao, M., Wang, F., Qi, X., Yang, P., Berceli, S. A., Sharma, A. K., Upchurch, G. R., Jr., Jiang, Z. Cyclophilin A contributes to aortopathy induced by postnatal loss of smooth muscle TGFBR1.
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http://dx.doi.org/10.1096/fj.201900601RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766662PMC
October 2019

Adenosine 2A Receptor Activation Attenuates Ischemia Reperfusion Injury During Extracorporeal Cardiopulmonary Resuscitation.

Ann Surg 2019 06;269(6):1176-1183

Department of Surgery, University of Virginia, Charlottesville, VA.

Objective: We tested the hypothesis that systemic administration of an A2AR agonist will reduce multiorgan IRI in a porcine model of ECPR.

Summary Background Data: Advances in ECPR have decreased mortality after cardiac arrest; however, subsequent IRI contributes to late multisystem organ failure. Attenuation of IRI has been reported with the use of an A2AR agonist.

Methods: Adult swine underwent 20 minutes of circulatory arrest, induced by ventricular fibrillation, followed by 6 hours of reperfusion with ECPR. Animals were randomized to vehicle control, low-dose A2AR agonist, or high-dose A2AR agonist. A perfusion specialist using a goal-directed resuscitation protocol managed all the animals during the reperfusion period. Hourly blood, urine, and tissue samples were collected. Biochemical and microarray analyses were performed to identify differential inflammatory markers and gene expression between groups.

Results: Both the treatment groups demonstrated significantly higher percent reduction from peak lactate after reperfusion compared with vehicle controls. Control animals required significantly more fluid, epinephrine, and higher final pump flow while having lower urine output than both the treatment groups. The treatment groups had lower urine NGAL, an early marker of kidney injury (P = 0.01), lower plasma aspartate aminotransferase, and reduced rate of troponin rise (P = 0.01). Pro-inflammatory cytokines were lower while anti-inflammatory cytokines were significantly higher in the treatment groups.

Conclusions: Using a novel and clinically relevant porcine model of circulatory arrest and ECPR, we demonstrated that a selective A2AR agonist significantly attenuated systemic IRI and warrants clinical investigation.
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http://dx.doi.org/10.1097/SLA.0000000000002685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6757347PMC
June 2019

Can Lung Transplant Surgeons Still Be Scientists? High Productivity Despite Competitive Funding.

Heart Surg Forum 2019 01 8;22(1):E001-E007. Epub 2019 Jan 8.

Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Virginia Health System, Charlottesville, Virginia, USA.

Background: Today's declining federal budget for scientific research is making it consistently more difficult to become federally funded. We hypothesized that even in this difficult era, surgeon-scientists have remained among the most productive and impactful researchers in lung transplantation.

Methods: Grants awarded by the NIH for the study of lung transplantation between 1985 and 2015 were identified by searching NIH RePORTER for 5 lung transplantation research areas. A grant impact metric was calculated for each grant by dividing the sum of impact factors for all associated manuscripts by the total funding for that grant. We used nonparametric univariate analysis to compare grant impact metrics by department.

Results: We identified 109 lung transplantation grants, totaling approximately $300 million, resulting in 2304 papers published in 421 different journals. Surgery has the third highest median grant impact metric (4.2 per $100,000). The department of surgery had a higher median grant impact metric compared to private companies (P <.0001). There was no statistical difference in the grant impact metric compared to all other medical specialties, individual departments with multiple grants, or all basic science departments (all P >.05).

Conclusions: Surgeon-scientists in the field of lung transplantation have received fewer grants and less total funding compared to other researchers but have maintained an equally high level of productivity and impact. The dual-threat academic surgeon-scientist is an important asset to the research community and should continue to be supported by the NIH.
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http://dx.doi.org/10.1532/hsf.2024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468987PMC
January 2019

Ex Vivo Assessment of Porcine Donation After Circulatory Death Lungs That Undergo Increasing Warm Ischemia Times.

Transplant Direct 2018 Dec 12;4(12):e405. Epub 2018 Nov 12.

Department of Surgery, University of Virginia School of Medicine, Charlottesville, VA.

Background: Increased utilization of donation after circulatory death (DCD) lungs may help alleviate the supply/demand mismatch between available donor organs and lung transplant candidates. Using an established porcine DCD model, we sought to determine the effect of increasing warm ischemia time (WIT) after circulatory arrest on lung function during ex vivo lung perfusion (EVLP).

Methods: Porcine donors (n = 15) underwent hypoxic cardiac arrest, followed by 60, 90, or 120 minutes of WIT before procurement and 4 hours of normothermic EVLP. Oxygenation, pulmonary artery pressure, airway pressure, and compliance were measured hourly. Lung injury scores were assessed histologically after 4 hours of EVLP.

Results: After EVLP, all 3 groups met all the criteria for transplantation, except for 90-minute WIT lungs, which had a mean pulmonary artery pressure increase greater than 15%. There were no significant differences between groups as assessed by final oxygenation capacity, as well as changes in pulmonary artery pressure, airway pressure, or lung compliance. Histologic lung injury scores as well as lung wet-to-dry weight ratios did not significantly differ between groups.

Conclusions: These results suggest that longer WIT alone (up to 120 minutes) does not predict worse lung function at the conclusion of EVLP. Expanding acceptable WIT after circulatory death may eventually allow for increased utilization of DCD lungs in procurement protocols.
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http://dx.doi.org/10.1097/TXD.0000000000000845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6283086PMC
December 2018

ZFP148 (Zinc-Finger Protein 148) Binds Cooperatively With NF-1 (Neurofibromin 1) to Inhibit Smooth Muscle Marker Gene Expression During Abdominal Aortic Aneurysm Formation.

Arterioscler Thromb Vasc Biol 2019 01;39(1):73-88

From the Department of Surgery, The Robert M. Berne Cardiovascular Research Center (G.K.O., G.A.).

Objective- The goal of this study was to determine the role of ZFP148 (zinc-finger protein 148) in aneurysm formation. Approach and Results- ZFP148 mRNA expression increased at day 3, 7, 14, 21, and 28 after during abdominal aortic aneurysm formation in C57BL/6 mice. Loss of ZFP148 conferred abdominal aortic aneurysm protection using ERTCre+ ZFP148 flx/flx mice. In a third set of experiments, smooth muscle-specific loss of ZFP148 alleles resulted in progressively greater protection using novel transgenic mice (MYH [myosin heavy chain 11] Cre+ flx/flx, flx/wt, and wt/wt). Elastin degradation, LGAL3, and neutrophil staining were significantly attenuated, while α-actin staining was increased in ZFP148 knockout mice. Results were verified in total cell ZFP148 and smooth muscle-specific knockout mice using an angiotensin II model. ZFP148 smooth muscle-specific conditional mice demonstrated increased proliferation and ZFP148 was shown to bind to the p21 promoter during abdominal aortic aneurysm formation. ZFP148 smooth muscle-specific conditional knockout mice also demonstrated decreased apoptosis as measured by decreased cleaved caspase-3 staining. ZFP148 bound smooth muscle marker genes via chromatin immunoprecipitation analysis mediated by NF-1 (neurofibromin 1) promote histone H3K4 deacetylation via histone deacetylase 5. Transient transfections and chromatin immunoprecipitation analyses demonstrated that NF-1 was required for ZFP148 protein binding to smooth muscle marker genes promoters during aneurysm formation. Elimination of NF-1 using shRNA approaches demonstrated that NF-1 is required for binding and elimination of NF-1 increased BRG1 recruitment, the ATPase subunit of the SWI/SWF complex, and increased histone acetylation. Conclusions- ZFP148 plays a critical role in multiple murine models of aneurysm formation. These results suggest that ZFP148 is important in the regulation of proliferation, smooth muscle gene downregulation, and apoptosis in aneurysm development.
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http://dx.doi.org/10.1161/ATVBAHA.118.311136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422047PMC
January 2019

Resolvin D1 decreases abdominal aortic aneurysm formation by inhibiting NETosis in a mouse model.

J Vasc Surg 2018 12;68(6S):93S-103S

Department of Surgery, University of Virginia, Charlottesville, Va; Department of Surgery, University of Florida, Gainesville, Fla. Electronic address:

Objective: Resolvins have been shown to attenuate inflammation, whereas NETosis, the process of neutrophils releasing neutrophil extracellular traps (NETs), produces increased inflammation. It is hypothesized that treatment of animals with resolvin D1 (RvD1) would reduce abdominal aortic aneurysm (AAA) formation by inhibiting NETosis.

Methods: Wild-type 8- to 12-week-old C57BL/6 male mice (n = 47) and apolipoprotein E-deficient (ApoE) mice (n = 20) were used in two models to demonstrate the effects of RvD1 on AAA growth. In the topical elastase AAA model, wild-type mice were divided into three groups: a deactivated elastase control group, in which sham surgery was performed using deactivated elastase and mice were intravenously injected with phosphate-buffered saline (PBS) once a day until harvest; an elastase group, in which active elastase was used to induce AAA and mice were injected with PBS daily until harvest; and an RvD1-treated group, in which AAA was induced and mice were injected with RvD1 daily until harvest. In the angiotensin II (Ang II)-induced AAA model, ApoE mice were fed a high-fat diet and implanted with osmotic infusion pumps containing Ang II (1000 ng/kg/min). The Ang II model was divided into two groups: an Ang II control group, in which Ang II was delivered and mice were injected with PBS daily until harvest; and an RvD1-treated group, in which Ang II was delivered and mice were injected with RvD1 daily until harvest. On postoperative day 3, day 14, or day 28, aortic and blood samples were collected for Western blot, histology, cytokine array, enzyme-linked immunosorbent assay, and gelatin zymography after aortic diameter measurement.

Results: The day 14 RvD1-treated group demonstrated 42% reduced AAA diameter compared with the elastase group (P < .001). On postoperative day 3, the RvD1-treated group showed decreased levels of NETosis markers citrullinated histone H3 (P = .04) and neutrophil elastase (P = .002) compared with the elastase group. Among important cytokines involved in AAA formation, interleukin (IL) 1β was downregulated (P = .02) whereas IL-10, a protective cytokine, was upregulated (P = .01) in the RvD1-treated group. Active matrix metalloproteinase 2 also decreased in the RvD1-treated group (P = .03). The RvD1-treated group in the Ang II AAA model, a second model, demonstrated reduced AAA diameter compared with the Ang II control group on day 28 (P < .046). The RvD1-treated group showed decreased levels of citrullinated histone H3 on day 3 (P = .002). Cytokines interferon γ, IL-1β, C-X-C motif chemokine ligand 10, monocyte chemotactic protein 1, and regulated on activation, normal T cell expressed and secreted (RANTES) were all decreased on day 28 (P < .05).

Conclusions: RvD1-mediated inhibition of NETosis may represent a future medical treatment for the attenuation of AAA growth.
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http://dx.doi.org/10.1016/j.jvs.2018.05.253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459013PMC
December 2018

Effect of amino acids on transcription and translation of key genes in E. coli K and B grown at a steady state in minimal medium.

N Biotechnol 2019 Mar 29;49:120-128. Epub 2018 Oct 29.

Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD, 20892, United States. Electronic address:

Growing E. coli to high densities is a common strategy for biologicals production. The process is implemented by using complex or minimal media with different feeding strategies. To understand the effect of amino acids, E. coli B and K were grown at a steady state of 0.35 h in glucose minimal medium with and without amino acids, while their metabolism, protein abundance and gene expression were compared. The results showed that amino acids promoted higher acetate excretion, higher fatty acid biosynthesis (K strain), repressed glucose uptake rate, and decreased expression of proteins associated with the TCA cycle, glyoxylate shunt and amino acid biosynthesis. In presence of amino acids, E. coli K upregulated fatty acid biosynthesis and repressed more genes and proteins involved in amino acid biosynthesis than E. coli B. These findings are correlated with higher yield on glucose (Y) and high specific biomass production rate (q) in K strain in the presence of amino acids. In contrast, pre-formed precursor molecules such as amino acids did not affect fatty acid biosynthesis in E. coli B or Y and q, which were higher than those of E. coli K, suggesting that constitutive synthesis of energetically demanding precursors and higher fatty acid β-oxidation activity is key for high biomass-performer E. coli B. Both strains turned off unnecessary pathways and directed their metabolism to proteome efficient overflow metabolism likely to generate energy and provide protein to functions supporting higher growth rate.
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http://dx.doi.org/10.1016/j.nbt.2018.10.004DOI Listing
March 2019

Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500.

Sci Rep 2018 10 31;8(1):16108. Epub 2018 Oct 31.

Biotechnology Core Laboratory, National Institute of Diabetes and Digestives and Kidney Diseases (NIDDK) NIH, Maryland, USA.

Protective antigen (PA) of Bacillus anthracis is being considered as a vaccine candidate against anthrax and its production has been explored in several heterologous host systems. Since the systems tested introduced adverse issues such as inclusion body formation and endotoxin contamination, the production from B. anthracis is considered as a preferred method. The present study examines the effect of PA expression on the metabolism of B. anthracis producing strain, BH500, by comparing it with a control strain carrying an empty plasmid. The strains were grown in a bioreactor and RNA-seq analysis of the producing and non-producing strain was conducted. Among the observed differences, the strain expressing rPA had increased transcription of sigL, the gene encoding RNA polymerase σ, sigB, the general stress transcription factor gene and its regulators rsbW and rsbV, as well as the global regulatory repressor ctsR. There were also decreased expression of intracellular heat stress related genes such as groL, groES, hslO, dnaJ, and dnaK and increased expression of extracellular chaperons csaA and prsA2. Also, major central metabolism genes belonging to TCA, glycolysis, PPP, and amino acids biosynthesis were up-regulated in the PA-producing strain during the lag phase and down-regulated in the log and late-log phases, which was associated with decreased specific growth rates. The information obtained from this study may guide genetic modification of B. anthracis to improve PA production.
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http://dx.doi.org/10.1038/s41598-018-34196-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6208434PMC
October 2018

Tamsulosin attenuates abdominal aortic aneurysm growth.

Surgery 2018 11 31;164(5):1087-1092. Epub 2018 Aug 31.

Department of Surgery, University of Florida, Gainesville. Electronic address:

Background: Tamsulosin, an α-adrenergic receptor inhibitor, is prescribed to treat benign prostatic hyperplasia in men >60 years of age, the same demographic most susceptible to abdominal aortic aneurysm. The goal of this study was to investigate the effect of tamsulosin on abdominal aortic aneurysm pathogenesis.

Methods: Abdominal aortic aneurysms were induced in WT C57BL/6 male mice (n = 9-18/group), using an established topical elastase abdominal aortic aneurysm model. Osmotic pumps were implanted in mice 5 days before operation to create the model, administering either low dose (0.125 µg/day tamsulosin), high dose (0.250µg/day tamsulosin), or vehicle treatments with and without topical application of elastase. Blood pressures were measured preoperatively and on postoperative days 0, 3, 7, and 14. On postoperative day 14, aortic diameter was measured before harvest. Sample aortas were prepared for histology and cytokine analysis.

Results: Measurements of systolic blood pressure did not differ between groups. Mice treated with the low dose of tamsulosin and with the high dose of tamsulosin showed decreased aortic diameter compared with vehicle-treated control (93% ± 24 versus 94% ± 30 versus 132% ± 24, respectively; P = .0003, P = .0003). Cytokine analysis demonstrated downregulation of pro-inflammatory cytokines in both treatment groups compared with the control (P < .05). Histology exhibited preservation of elastin in both low- and high-dose tamsulosin-treated groups (P = .0041 and P = .0018, respectively).

Conclusion: Tamsulosin attenuates abdominal aortic aneurysm formation with increased preservation of elastin and decreased production of pro-inflammatory cytokines. Further studies are necessary to elucidate the mechanism by which tamsulosin attenuates abdominal aortic aneurysm pathogenesis.
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http://dx.doi.org/10.1016/j.surg.2018.06.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459011PMC
November 2018

Changes in bone microarchitecture following kidney transplantation-Beyond bone mineral density.

Clin Transplant 2018 09 2;32(9):e13347. Epub 2018 Aug 2.

Department of Nephrology, The Royal Melbourne Hospital, Parkville, Victoria, Australia.

Bone disease in kidney transplant recipients (KTRs) is characterized by bone mineral density (BMD) loss but bone microarchitecture changes are poorly defined. In this prospective cohort study, we evaluated bone microarchitecture using non-invasive imaging modalities; high-resolution magnetic resonance imaging (MRI), peripheral quantitative computed tomography (pQCT), dual energy X-ray absorptiometry (DXA), and the trabecular bone score (TBS) following kidney transplantation. Eleven KTRs (48.3 ± 11.2 years) underwent MRI (tibia), pQCT (radius) and DXA at baseline and 12 months post-transplantation. Transiliac bone biopsies, performed at transplantation, showed 70% of patients with high/normal bone turnover. Compared with baseline, 12-month MRI showed deterioration in indices of trabecular network integrity-surface to curve ratio (S/C; -15%, P = 0.03) and erosion index (EI; +19%, P = 0.01). However, cortical area increased (+10.3%, P = 0.04), with a non-significant increase in cortical thickness (CtTh; +7.8%, P = 0.06). At 12 months, parathyroid hormone values (median 10.7 pmol/L) correlated with improved S/C (r = 0.75, P = 0.009) and EI (r = -0.71, P = 0.01) while osteocalcin correlated with CtTh (r = 0.72, P = 0.02) and area (r = 0.70, P = 0.02). TBS decreased from baseline (-5.1%, P = 0.01) with no significant changes in BMD or pQCT. These findings highlight a post-transplant deterioration in trabecular bone quality detected by MRI and TBS, independent of changes in BMD, underlining the potential utility of these modalities in evaluating bone microarchitecture in KTRs.
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http://dx.doi.org/10.1111/ctr.13347DOI Listing
September 2018

Magnetic resonance imaging based assessment of bone microstructure as a non-invasive alternative to histomorphometry in patients with chronic kidney disease.

Bone 2018 09 31;114:14-21. Epub 2018 May 31.

Departments of Radiology and Orthopaedic Surgery, University of Pennsylvania, PA, USA.

Background: Chronic kidney disease (CKD) adversely affects bone microarchitecture and increases fracture risk. Historically, bone biopsy has been the 'gold standard' for evaluating renal bone disease but is invasive and infrequently performed. High-resolution magnetic resonance imaging (MRI) quantifies bone microarchitecture noninvasively. In patients with CKD, it has not been compared with results derived from bone biopsy or with imaging using dual energy X-ray absorptiometry (DXA).

Methods: Fourteen patients with end-stage kidney disease (ESKD) underwent MRI at the distal tibia, bone mineral density (BMD) by dual energy X-ray absorptiometry (DXA; hip and spine) and transiliac bone biopsies with histomorphometry and microcomputed tomography (micro-CT). All patients had biomarkers of mineral metabolism. Associations were determined by Spearman's or Pearson's rank correlation coefficients.

Results: MRI indices of trabecular network integrity, surface to curve ratio (S/C) and erosion index (EI), correlated to histomorphometric trabecular bone volume (S/C r = 0.85, p = 0.0003; EI r = -0.82, p = 0.001), separation (S/C r = -0.58, p = 0.039; EI r = 0.79, p = 0.0012) and thickness (S/C, r = 0.65, p = 0.017). MRI EI and trabecular thickness (TbTh) also correlated to micro-CT trabecular separation (EI r = 0.63, p = 0.02; TbTh r = -0.60, p = 0.02). Significant correlations were observed between histomorphometric mineralization and turnover indices and various MRI parameters. MRI-derived trabecular parameters were also significantly related to femoral neck BMD.

Conclusions: This study highlights the heterogeneity of bone microarchitecture at differing skeletal sites. MRI demonstrates significant, relevant associations to important bone biopsy and DXA indices and warrants further investigation to assess its potential to non-invasively evaluate changes in bone structure and quality over time.
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http://dx.doi.org/10.1016/j.bone.2018.05.029DOI Listing
September 2018

Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.

FASEB J 2018 May 29:fj201701138RR. Epub 2018 May 29.

Department of Surgery, University of Virginia, Charlottesville, Virginia, USA.

The formation of an abdominal aortic aneurysm (AAA) is characterized by inflammation, macrophage infiltration, and vascular remodeling. In this study, we tested the hypothesis that mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) immunomodulate aortic inflammation, to mitigate AAA formation via modulation of microRNA-147. An elastase-treatment model of AAA was used in male C57BL/6 wild-type (WT) mice. Administration of EVs in elastase-treated WT mice caused a significant attenuation of aortic diameter and mitigated proinflammatory cytokines, inflammatory cell infiltration, an increase in smooth muscle cell α-actin expression, and a decrease in elastic fiber disruption, compared with untreated mice. A 10-fold up-regulation of microRNA (miR)-147, a key mediator of macrophage inflammatory responses, was observed in murine aortic tissue in elastase-treated mice compared with controls on d 14. EVs derived from MSCs transfected with miR-147 mimic, but not with miR-147 inhibitor, attenuated aortic diameter, inflammation, and leukocyte infiltration in elastase-treated mice. In vitro studies of human aortic tissue explants and murine-derived CD11b macrophages induced proinflammatory cytokines after elastase treatment, and the expression was attenuated by cocultures with EVs transfected with miR-147 mimic, but not with miR-147 inhibitor. Thus, our findings define a critical role of MSC-derived EVs in attenuation of aortic inflammation and macrophage activation via miR-147 during AAA formation.-Spinosa, M., Lu, G., Su, G., Bontha, S. V., Gehrau, R., Salmon, M. D., Smith, J. R., Weiss, M. L., Mas, V. R., Upchurch, G. R., Sharma, A. K. Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.
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http://dx.doi.org/10.1096/fj.201701138RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181641PMC
May 2018

Deterioration of Cortical Bone Microarchitecture: Critical Component of Renal Osteodystrophy Evaluation.

Am J Nephrol 2018 23;47(6):376-384. Epub 2018 May 23.

Osteoporosis and Bone Biology Division, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia.

Background: Cortical bone is a significant determinant of bone strength and its deterioration contributes to bone fragility. Thin cortices and increased cortical porosity have been noted in patients with chronic kidney disease (CKD), but the "Turnover Mineralization Volume" classification of renal osteodystrophy does not emphasize cortical bone as a key parameter. We aimed to assess trabecular and cortical bone microarchitecture by histomorphometry and micro-CT in patients with CKD G5 and 5D (dialysis).

Methods: Transiliac bone biopsies were performed in 14 patients undergoing kidney transplantation (n = 12) and parathyroidectomy (n = 2). Structural parameters were analysed by histomorphometry and micro-CT including trabecular bone volume, thickness (TbTh), number (TbN) and separation and cortical thickness (CtTh) and porosity (CtPo). Indices of bone remodelling and mineralisation were obtained and relationships to bone biomarkers examined. Associations were determined by Spearman's or Pearson's rank correlation coefficients.

Results: By micro-CT, trabecular parameters were within normal ranges in most patients, but all patients showed very low CtTh (127 ± 44 µm) and high CtPo (60.3 ± 22.5%). CtPo was inversely related to TbN (r = -0.56; p = 0.03) by micro-CT and to TbTh (r = -0.60; p = 0.024) by histomorphometry and correlated to parathyroid hormone values (r = 0.62; p = 0.021). By histomorphometry, bone turnover was high in 50%, low in 21% and normal in 29%, while 36% showed abnormal patterns of mineralization. Significant positive associations were observed between osteoblast surface, osteoclast surface, mineralization surface and bone turnover markers.

Conclusions: Deterioration of cortical -microarchitecture despite predominantly normal trabecular parameters reinforces the importance of comprehensive cortical evaluation in patients with CKD.
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http://dx.doi.org/10.1159/000489671DOI Listing
November 2019

Pannexin-1 channels on endothelial cells mediate vascular inflammation during lung ischemia-reperfusion injury.

Am J Physiol Lung Cell Mol Physiol 2018 08 10;315(2):L301-L312. Epub 2018 May 10.

Department of Surgery, University of Virginia School of Medicine , Charlottesville, Virginia.

Ischemia-reperfusion (I/R) injury (IRI), which involves inflammation, vascular permeability, and edema, remains a major challenge after lung transplantation. Pannexin-1 (Panx1) channels modulate cellular ATP release during inflammation. This study tests the hypothesis that endothelial Panx1 is a key mediator of vascular inflammation and edema after I/R and that IRI can be blocked by Panx1 antagonism. A murine hilar ligation model of IRI was used whereby left lungs underwent 1 h of ischemia and 2 h of reperfusion. Treatment of wild-type mice with Panx1 inhibitors (carbenoxolone or probenecid) significantly attenuated I/R-induced pulmonary dysfunction, edema, cytokine production, and neutrophil infiltration versus vehicle-treated mice. In addition, VE-Cad-Cre/Panx1 mice (tamoxifen-inducible deletion of Panx1 in vascular endothelium) treated with tamoxifen were significantly protected from IRI (reduced dysfunction, endothelial permeability, edema, proinflammatory cytokines, and neutrophil infiltration) versus vehicle-treated mice. Furthermore, extracellular ATP levels in bronchoalveolar lavage fluid is Panx1-mediated after I/R as it was markedly attenuated by Panx1 antagonism in wild-type mice and by endothelial-specific Panx1 deficiency. Panx1 gene expression in lungs after I/R was also significantly elevated compared with sham. In vitro experiments demonstrated that TNF-α and/or hypoxia-reoxygenation induced ATP release from lung microvascular endothelial cells, which was attenuated by Panx1 inhibitors. This study is the first, to our knowledge, to demonstrate that endothelial Panx1 plays a key role in mediating vascular permeability, inflammation, edema, leukocyte infiltration, and lung dysfunction after I/R. Pharmacological antagonism of Panx1 activity may be a novel therapeutic strategy to prevent IRI and primary graft dysfunction after lung transplantation.
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http://dx.doi.org/10.1152/ajplung.00004.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139659PMC
August 2018

Increasing circulating sphingosine-1-phosphate attenuates lung injury during ex vivo lung perfusion.

J Thorac Cardiovasc Surg 2018 08 11;156(2):910-917. Epub 2018 Mar 11.

Department of Surgery, University of Virginia School of Medicine, Charlottesville, Va. Electronic address:

Background: Sphingosine-1-phosphate regulates endothelial barrier integrity and promotes cell survival and proliferation. We hypothesized that upregulation of sphingosine-1-phosphate during ex vivo lung perfusion would attenuate acute lung injury and improve graft function.

Methods: C57BL/6 mice (n = 4-8/group) were euthanized, followed by 1 hour of warm ischemia and 1 hour of cold preservation in a model of donation after cardiac death. Subsequently, mice underwent 1 hour of ex vivo lung perfusion with 1 of 4 different perfusion solutions: Steen solution (Steen, control arm), Steen with added sphingosine-1-phosphate (Steen + sphingosine-1-phosphate), Steen plus a selective sphingosine kinase 2 inhibitor (Steen + sphingosine kinase inhibitor), or Steen plus both additives (Steen + sphingosine-1-phosphate + sphingosine kinase inhibitor). During ex vivo lung perfusion, lung compliance and pulmonary artery pressure were continuously measured. Pulmonary vascular permeability was assessed with injection of Evans Blue dye.

Results: The combination of 1 hour of warm ischemia, followed by 1 hour of cold ischemia created significant lung injury compared with lungs that were immediately harvested after circulatory death and put on ex vivo lung perfusion. Addition of sphingosine-1-phosphate or sphingosine kinase inhibitor alone did not significantly improve lung function during ex vivo lung perfusion compared with Steen without additives. However, group Steen + sphingosine-1-phosphate + sphingosine kinase inhibitor resulted in significantly increased compliance (110% ± 13.9% vs 57.7% ± 6.6%, P < .0001) and decreased pulmonary vascular permeability (33.1 ± 11.9 μg/g vs 75.8 ± 11.4 μg/g tissue, P = .04) compared with Steen alone.

Conclusions: Targeted drug therapy with a combination of sphingosine-1-phosphate + sphingosine kinase inhibitor during ex vivo lung perfusion improves lung function in a murine donation after cardiac death model. Elevation of circulating sphingosine-1-phosphate via specific pharmacologic modalities during ex vivo lung perfusion may provide endothelial protection in marginal donor lungs leading to successful lung rehabilitation for transplantation.
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http://dx.doi.org/10.1016/j.jtcvs.2018.02.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056006PMC
August 2018

Invited commentary.

J Vasc Surg 2018 03;67(3):909

Charlottesville, Va.

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http://dx.doi.org/10.1016/j.jvs.2016.12.064DOI Listing
March 2018

Expanding the donor lung pool: how many donations after circulatory death organs are we missing?

J Surg Res 2018 03 11;223:58-63. Epub 2017 Nov 11.

Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Virginia Health System, Charlottesville, Virginia. Electronic address:

Background: The number of patients with end-stage pulmonary disease awaiting lung transplantation is at an all-time high, while the supply of available organs remains stagnant. Utilizing donation after circulatory death (DCD) donors may help to address the supply-demand mismatch. The objective of this study is to determine the potential donor pool expansion with increased procurement of DCD organs from patients who die at hospitals.

Material And Methods: The charts of all patients who died at a single, rural, quaternary-care institution between August 2014 and June 2015 were reviewed for lung transplant candidacy. Inclusion criteria were age <65 y, absence of cancer and lung pathology, and cause of death other than respiratory or sepsis.

Results: A total of 857 patients died within a 1-year period and were stratified by age: pediatric <15 y (n = 32, 4%), young 15-64 y (n = 328, 38%), and old >65 y (n = 497, 58%). Those without cancer totaled 778 (90.8%) and 512 (59%) did not have lung pathology. This leaves 85 patients qualifying for DCD lung donation (pediatric n = 10, young n = 75, and old n = 0). Potential donors were significantly more likely to have clear chest X-rays (24.3% versus 10.0%, P < 0.0001) and higher mean PaO2/FiO2 (342.1 versus 197.9, P < 0.0001) compared with ineligible patients.

Conclusions: A significant number of DCD lungs are available every year from patients who die within hospitals. We estimate the use of suitable DCD lungs could potentially result in a significant increase in the number of lungs available for transplantation.
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http://dx.doi.org/10.1016/j.jss.2017.09.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475907PMC
March 2018

Mesenchymal stromal cell-derived extracellular vesicles attenuate lung ischemia-reperfusion injury and enhance reconditioning of donor lungs after circulatory death.

Respir Res 2017 12 21;18(1):212. Epub 2017 Dec 21.

Department of Surgery, University of Virginia, P.O. Box 801359, Charlottesville, VA, 22908, USA.

Background: Lung ischemia-reperfusion (IR) injury after transplantation as well as acute shortage of suitable donor lungs are two critical issues impacting lung transplant patients. This study investigates the anti-inflammatory and immunomodulatory role of human mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) to attenuate lung IR injury and improve of ex-vivo lung perfusion (EVLP)-mediated rehabilitation in donation after circulatory death (DCD) lungs.

Methods: C57BL/6 wild-type (WT) mice underwent sham surgery or lung IR using an in vivo hilar-ligation model with or without MSCs or EVs. In vitro studies used primary iNKT cells and macrophages (MH-S cells) were exposed to hypoxia/reoxygenation with/without co-cultures with MSCs or EVs. Also, separate groups of WT mice underwent euthanasia and 1 h of warm ischemia and stored at 4 °C for 1 h followed by 1 h of normothermic EVLP using Steen solution or Steen solution containing MSCs or EVs.

Results: Lungs from MSCs or EV-treated mice had significant attenuation of lung dysfunction and injury (decreased edema, neutrophil infiltration and myeloperoxidase levels) compared to IR alone. A significant decrease in proinflammatory cytokines (IL-17, TNF-α, CXCL1 and HMGB1) and upregulation of keratinocyte growth factor, prostaglandin E2 and IL-10 occurred in the BAL fluid from MSC or EV-treated mice after IR compared to IR alone. Furthermore, MSCs or EVs significantly downregulated iNKT cell-produced IL-17 and macrophage-produced HMGB1 and TNF-α after hypoxia/reoxygenation. Finally, EVLP of DCD lungs with Steen solution including MSCs or EVs provided significantly enhanced protection versus Steen solution alone. Co-cultures of MSCs or EVs with lung endothelial cells prevents neutrophil transendothelial migration after exposure to hypoxia/reoxygenation and TNF-α/HMGB1 cytomix.

Conclusions: These results suggest that MSC-derived EVs can attenuate lung inflammation and injury after IR as well as enhance EVLP-mediated reconditioning of donor lungs. The therapeutic benefits of EVs are in part mediated through anti-inflammatory promoting mechanisms via attenuation of immune cell activation as well as prevention of endothelial barrier integrity to prevent lung edema. Therefore, MSC-derived EVs offer a potential therapeutic strategy to treat post-transplant IR injury as well as rehabilitation of DCD lungs.
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http://dx.doi.org/10.1186/s12931-017-0704-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740880PMC
December 2017

In vivo lung perfusion rehabilitates sepsis-induced lung injury.

J Thorac Cardiovasc Surg 2018 01 14;155(1):440-448.e2. Epub 2017 Sep 14.

Department of Surgery, University of Virginia, Charlottesville, Va. Electronic address:

Background: Sepsis is the leading cause of lung injury in adults and can lead to acute respiratory distress syndrome (ARDS). Using a novel technique of isolated in vivo lung perfusion (IVLP), we hypothesized that normothermic IVLP will improve oxygenation and compliance in a porcine model of sepsis-induced lung injury.

Methods: Mature adult swine (n = 8) were administered lipopolysaccharide (LPS; 50 μg/kg over 2 hours) via the external jugular vein, followed by sternotomy and central extracorporeal membrane oxygenation (ECMO) cannulation (right atrium to ascending aorta). The left pulmonary artery (inflow) and left superior and inferior pulmonary veins (outflow) were dissected out and cannulated to deliver isolated perfusion to the left lung. After 4 hours of normothermic IVLP with Steen solution, the left lung then underwent 4 hours of reperfusion after IVLP decannulation. Airway pressures and lung-specific pulmonary vein blood gases from the right lung (LPS control) and left lung (LPS + IVLP) of the same animal were compared.

Results: All animals demonstrated a significant reduction in the ratio of partial pressure of oxygen in arterial blood (PaO)/fraction of inspired oxygen (FiO) (P/F ratio) and total lung compliance at 2 hours after the start of LPS infusion (mean, 469 ± 19.7 mm Hg vs 222.2 ± 21.4 mm Hg; P < .0001). After reperfusion, 6 animals (75%) exhibited improved lung function, allowing for ECMO decannulation. Lung-specific oxygenation was superior in the left lung after 4 hours of reperfusion (mean, 310.5 ± 54.7 mm Hg vs 201.1 ± 21.7 mm Hg; P = .01). Similarly, total lung compliance improved after IVLP of the left lung. The lung wet weight to dry weight ratio demonstrated reduced edema in rehabilitated left lungs (mean, 6.5 ± 0.3 vs 7.5 ± 0.4; P = .04).

Conclusions: IVLP successfully rehabilitated LPS-injured lungs compared to ECMO support alone in this preclinical porcine model.
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http://dx.doi.org/10.1016/j.jtcvs.2017.08.124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744259PMC
January 2018

Muscle-derived extracellular superoxide dismutase inhibits endothelial activation and protects against multiple organ dysfunction syndrome in mice.

Free Radic Biol Med 2017 12 2;113:212-223. Epub 2017 Oct 2.

Center for Skeletal Muscle Research at Robert Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA; Departments of Medicine, University of Virginia, Charlottesville, VA 22908, USA; Departments of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA; Departments of Molecular Physiology & Biological Physics, University of Virginia, Charlottesville, VA 22908, USA. Electronic address:

Multiple organ dysfunction syndrome (MODS) is a detrimental clinical complication in critically ill patients with high mortality. Emerging evidence suggests that oxidative stress and endothelial activation (induced expression of adhesion molecules) of vital organ vasculatures are key, early steps in the pathogenesis. We aimed to ascertain the role and mechanism(s) of enhanced extracellular superoxide dismutase (EcSOD) expression in skeletal muscle in protection against MODS induced by endotoxemia. We showed that EcSOD overexpressed in skeletal muscle-specific transgenic mice (TG) redistributes to other peripheral organs through the circulation and enriches at the endothelium of the vasculatures. TG mice are resistant to endotoxemia (induced by lipopolysaccharide [LPS] injection) in developing MODS with significantly reduced mortality and organ damages compared with the wild type littermates (WT). Heterogenic parabiosis between TG and WT mice conferred a significant protection to WT mice, whereas mice with R213G knock-in mutation, a human single nucleotide polymorphism leading to reduced binding EcSOD in peripheral organs, exacerbated the organ damages. Mechanistically, EcSOD inhibits vascular cell adhesion molecule 1 expression and inflammatory leukocyte adhesion to the vascular wall of vital organs, blocking an early step of the pathology in organ damage under endotoxemia. Therefore, enhanced expression of EcSOD in skeletal muscle profoundly protects against MODS by inhibiting endothelial activation and inflammatory cell adhesion, which could be a promising therapy for MODS.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.09.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740866PMC
December 2017