FGF2 is Secreted in Extracellular Vesicles from Lung Cells.

The 18-kD isoform of basic fibroblast growth factor (bFGF/FGF2) lacks a conventional signal peptide sequence and is exported by a novel membrane-associated transport pathway. Extracellular vesicles (EVs) are increasingly recognized as mediators of intercellular communication in the lung, and our prior work demonstrates that EVs carry cargo that contribute to hyperoxic lung injury and are biomarkers for bronchopulmonary dysplasia. We used primary human bronchial epithelial (HBE), pulmonary artery endothelial (HPAE) and fibroblast (HNF) cells to determine if FGF2 was secreted in EVs. EVs were isolated by ultracentrifugation from HBE, HPAE, and HNF exposed to either normoxia or hyperoxia, followed by nanoparticle tracking analysis and electron microscopy. Hyperoxia exposure increased total EV number. All three cell types released FGF2-18kDa both directly into the extracellular environment (secretome), as well as in EVs. HBE released more FGF2-18kDa in EVs during hyperoxia, and these were internalized and localized to both nuclei and cytoplasm of recipient cells. By co-immunoprecipitation, we identified potential binding partners of FGF2-18kDa in the nuclei, including histone 1.2 (H1.2) binding protein, that may mediate downstream effects that do not involve FGF2 binding to cell surface receptors. FGF2-18kDa interaction with H1.2 binding protein may indicate a mechanism by which FGF2 secreted in EVs modulates cellular processes. FGF2 was also found to increase angiogenesis by Matrigel assay. Further studies are necessary to determine the biological relevance of the FGF2 in EVs as modulators of lung injury and disease.

Association Between Urinary Biomarkers and CKD in Extremely Low Gestational Age Neonates.

RATIONALE & OBJECTIVE: Children born before 28 weeks' gestation are at increased risk of chronic kidney disease (CKD). Urine biomarkers may shed light on mechanistic pathways and improve the ability to forecast CKD. We evaluated whether urinary biomarkers in neonates of low gestational age (GA) are associated with a reduced estimated glomerular filtration rate (eGFR) over time. STUDY DESIGN: A cohort study of neonates with an exploratory case-control study of a subset of the cohort. SETTING & PARTICIPANTS: 327 neonates born at 24-27 weeks' gestation with 2-year eGFR data from the PENUT (Preterm Erythropoietin Neuroprotection Trial) and the REPaIReD (Recombinant Erythropoietin for Prevention of Infant Renal Disease) study. EXPOSURES: 11 urinary biomarkers measured at 27, 30, and 34 weeks' postmenstrual age for the primary cohort study and 10 additional biomarkers for the exploratory case-control study. OUTCOMES: eGFR<90mL/min/1.73m2 at 2 years corrected for GA. ANALYTICAL APPROACH: Linear mixed models to assess differences in biomarker values between neonates in whom CKD did and did not develop, accounting for multiple comparisons using Bonferroni-Holm correction in the cohort study only. Cohort analyses were adjusted for sex, GA, and body mass index. Cases were matched to controls on these variables in the case-control study. RESULTS: After adjusting for weeks of GA, urinary levels of α-glutathione-S-transferase (log difference, 0.27; 95% CI, 0.12-0.43), albumin (log difference, 0.13; 95% CI, 0.02-0.25), and cystatin C (log difference, 0.19; 95% CI, 0.04-0.34) were higher in those in whom CKD developed than in those in whom it did not. Urinary albumin and cystatin C levels did not remain significantly different after Bonferroni-Holm correction. In the exploratory case-control analysis, there were no differences in any biomarkers between cases and controls. LIMITATIONS: Early deaths and a high number of subjects without eGFR at 2 years corrected for GA. CONCLUSIONS: Measurement of urinary biomarkers may assist in monitoring neonates who are at risk for CKD. Additional studies are needed to confirm these findings. FUNDING: Grants from government (National Institutes of Health). TRIAL REGISTRATION: Registered at ClinicalTrials.gov with study number NCT01378273. PLAIN-LANGUAGE SUMMARY: Approximately 15 million neonates worldwide are born prematurely, and 2 million are born before 28 weeks' gestation. Many of these children go on to experience chronic kidney disease. Urine biomarkers may allow for early recognition of those at risk for the development of kidney disease. In this study of more than 300 children born before 28 weeks' gestational age, we found higher mean urinary levels of α-glutathione-S-transferase at 27, 30, and 34 weeks in children whose estimated glomerular filtration rate was<90mL/min/1.73m2 at 2 years compared with children whose estimated glomerular filtration rate was>90mL/min/1.73m2 at 2 years. Measurement of urinary biomarkers may assist in monitoring neonates who are at risk for chronic kidney disease. Additional studies are needed to confirm our findings.

Microbial-induced Redox Imbalance in the Neonatal Lung Is Ameliorated by Live Biotherapeutics.

Bronchopulmonary dysplasia (BPD) is a common lung disease of premature infants. Hyperoxia exposure and microbial dysbiosis are contributors to BPD development. However, the mechanisms linking pulmonary microbial dysbiosis to worsening lung injury are unknown. Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that regulates oxidative stress responses and modulates hyperoxia-induced lung injury. We hypothesized that airway dysbiosis would attenuate Nrf2-dependent antioxidant function, resulting in a more severe phenotype of BPD. Here, we show that preterm infants with a Gammaproteobacteria-predominant dysbiosis have increased endotoxin in tracheal aspirates, and mice monocolonized with the representative Gammaproteobacteria Escherichia coli show increased tissue damage compared with germ-free (GF) control mice. Furthermore, we show Nrf2-deficient mice have worse lung structure and function after exposure to hyperoxia when the airway microbiome is augmented with E. coli. To confirm the disease-initiating potential of airway dysbiosis, we developed a novel humanized mouse model by colonizing GF mice with tracheal aspirates from human infants with or without severe BPD, producing gnotobiotic mice with BPD-associated and non-BPD-associated lung microbiomes. After hyperoxia exposure, BPD-associated mice demonstrated a more severe BPD phenotype and increased expression of Nrf2-regulated genes, compared with GF and non-BPD-associated mice. Furthermore, augmenting Nrf2-mediated antioxidant activity by supporting colonization with Lactobacillus species improved dysbiotic-augmented lung injury. Our results demonstrate that a lack of protective pulmonary microbiome signature attenuates an Nrf2-mediated antioxidant response, which is augmented by a respiratory probiotic blend. We anticipate antioxidant pathways will be major targets of future microbiome-based therapeutics for respiratory disease.

Premature infants born <28 weeks with acute kidney injury have increased bronchopulmonary dysplasia rates.

BACKGROUND: Despite a growing understanding of bronchopulmonary dysplasia (BPD) and advances in management, BPD rates remain stable. There is mounting evidence that BPD may be due to a systemic insult, such as acute kidney injury (AKI). Our hypothesis was that severe AKI would be associated with BPD. METHODS: We conducted a secondary analysis of premature infants [24-27 weeks gestation] in the Recombinant Erythropoietin for Protection of Infant Renal Disease cohort (N = 885). We evaluated the composite outcome of Grade 2/3 BPD or death using generalized estimating equations. In an exploratory analysis, urinary biomarkers of angiogenesis (ANG1, ANG2, EPO, PIGF, TIE2, FGF, and VEGFA/D) were analyzed. RESULTS: 594 (67.1%) of infants had the primary composite outcome of Grade 2/3 BPD or death. Infants with AKI (aOR: 1.69, 95% CI: 1.16-2.46) and severe AKI (aOR: 2.05, 95% CI: 1.19-3.54). had increased risk of the composite outcome after multivariable adjustment Among 106 infants with urinary biomarkers assessed, three biomarkers (VEGFA, VEGFD, and TIE2) had AUC > 0.60 to predict BPD. CONCLUSIONS: Infants with AKI had a higher likelihood of developing BPD/death, with the strongest relationship seen in those with more severe AKI. Three urinary biomarkers of angiogenesis may have potential to predict BPD development. IMPACT: AKI is associated with lung disease in extremely premature infants, and urinary biomarkers may predict this relationship. Infants with AKI and severe AKI have higher odds of BPD or death. Three urinary angiogenesis biomarkers are altered in infants that develop BPD. These findings have the potential to drive future work to better understand the mechanistic pathways of BPD, setting the framework for future interventions to decrease BPD rates. A better understanding of the mechanisms of BPD development and the role of AKI would have clinical care, cost, and quality of life implications given the long-term effects of BPD.

Urine acute kidney injury biomarkers in extremely low gestational age neonates: a nested case control study of 21 candidate urine biomarkers.

BACKGROUND: Acute kidney injury (AKI) is common and is associated with poor clinical outcomes in premature neonates. Urine biomarkers hold the promise to improve our understanding and care of patients with kidney disease. Because kidney maturation and gender can impact urine biomarker values in extremely low gestational age neonates (ELGANs), careful control of gestational age (GA) and time is critical to any urine biomarker studies in neonates. METHODS: To improve our understanding of the potential use of urine biomarkers to detect AKI during the first postnatal weeks, we performed a nested case-control study to evaluate 21 candidate urine AKI biomarkers. Cases include 20 ELGANs with severe AKI. Each case was matched with 2 controls for the same GA week (rounded down to the nearest week), gender, and birth weight (BW) (± 50 g). RESULTS: Urine cystatin C, creatinine, ghrelin, fibroblast growth factor-23 (FGF23), tissue metalloproteinase 2 (TIMP2) and vascular endothelial growth factor A (VEGFa) concentrations were higher in ELGANs with early severe AKI compared to matched control subjects without AKI. Urine epidermal growth factor (EGF) and uromodulin (UMOD) concentrations are lower in cases than controls. Interleukin (IL)-15 was lower on day 1, but higher on day 8 in cases than controls; while VEGFa was lower on day 1, but higher on day 5 in cases than controls. CONCLUSION: Urine biomarkers hold the promise to improve our ability to reliably detect kidney injury. Interventional studies are needed to determine the biomarkers' ability to predict outcomes, enhance AKI phenotypes, and improve timely interventions which can prevent the sequalae of AKI in ELGANs. A higher resolution version of the Graphical abstract is available as Supplementary information.

Gestational age, sex, and time affect urine biomarker concentrations in extremely low gestational age neonates.

BACKGROUND: Our understanding of the normative concentrations of urine biomarkers in premature neonates is limited. METHODS: We evaluated urine from 750 extremely low gestational age (GA) neonates without severe acute kidney injury (AKI) to determine how GA affects ten different urine biomarkers at birth and over the first 30 postnatal days. Then, we investigated if the urine biomarkers changed over time at 27, 30, and 34 weeks postmenstrual age (PMA). Next, we evaluated the impact of sex on urine biomarker concentrations at birth and over time. Finally, we evaluated if urine biomarkers were impacted by treatment with erythropoietin (Epo). RESULTS: We found that all ten biomarker concentrations differ at birth by GA and that some urine biomarker concentrations increase, while others decrease over time. At 27 weeks PMA, 7/10 urine biomarkers differed by GA. By 30 weeks PMA, 5/10 differed, and by 34 weeks PMA, only osteopontin differed by GA. About half of the biomarker concentrations differed by sex, and 4/10 showed different rates of change over time between males vs. females. We found no differences in urine biomarkers by treatment group. CONCLUSIONS: The temporal patterns, GA, and sex differences need to be considered in urine AKI biomarker analyses. IMPACT: Urine biomarker concentrations differ by GA at birth. Some urine biomarkers increase, while others decrease, over the first 30 postnatal days. Most urine biomarkers differ by GA at 27 weeks PMA, but are similar by 34 weeks PMA. Some urine biomarkers vary by sex in premature neonates. Urine biomarkers did not differ between neonates randomized to placebo vs. Epo.

Physical pain and musculoskeletal discomfort in vascular surgeons.

OBJECTIVE: Work-related pain and disability have been reported in the literature among surgeons. This national survey was designed to identify the prevalence and severity of these symptoms in vascular surgeons. METHODS: A survey was emailed to the 2910 members of the Society for Vascular Surgery. Physical pain was evaluated based on body part, and type of vascular procedure performed using the Borg 0 to 10 pain scale. Wellness questions were also queried. RESULTS: A total of 775 of Society for Vascular Surgery members responded, with a 26.6% response rate. Retirees were excluded from the study (n = 39). Among those actively working (n = 736), surgeons have been practicing surgery, on average, for 17.2 ± 11.6 years, with a mean age of 51.4 ± 10.9 years, and 83.6% are male. After a full day of open surgery, the majority of the responding vascular surgeons are in a moderately strong amount of pain (mean score, 4.4 ± 2.3). After a full day of endovascular procedures, most vascular surgeons are in a moderately strong amount of pain (mean score, 3.9 ± 2.4). Pain after open surgery is greatest in the neck, and after endovascular surgery pain is highest in the lower back. Surgeons performing endovenous procedures demonstrated the lowest pain scores (2.0 ± 2.0). In total, 36.9% (242/655, 81 missing responses) have sought medical care for work-related pain, with 8.3% (61/736) taking time away from the operating room. Of those, 26.2% (193/736) report pain severe enough that it interferes with sleep. Seventy-two (10%) required surgery or other significant medical procedures. Of the 39 retirees, 26% ended their careers owing to physical disabilities from work-related pain. Out of the entire cohort, 52.7% (334/633,103 missing responses) feel that physical discomfort will affect the longevity of their careers. Additionally, we found that high work-related physical discomfort is significantly associated with burnout (burnout vs no burnout; P < .0001). CONCLUSIONS: Our study shows that the majority of practicing vascular surgeons responding to the survey are in pain after a day of operating. Addressing work-related pain serves to improve the lives and careers of vascular surgeons and enhance surgical longevity.

Vascular surgeon wellness and burnout: A report from the Society for Vascular Surgery Wellness Task Force.

INTRODUCTION: Physician burnout has been linked to medical errors, decreased patient satisfaction, and decreased career longevity. In light of the increasing prevalence of cardiovascular disease, vascular surgeon burnout presents a legitimate public health concern owing to the impact on the adequacy of the vascular surgery workforce. The aims of this study were to define the prevalence of burnout among practicing vascular surgeons and identify factors that contribute to burnout to facilitate future Society for Vascular Surgery (SVS) initiatives to mitigate this crisis. METHODS: In 2018, active SVS members were surveyed electronically and confidentially using the Maslach Burnout Inventory. The survey was tailored to explore specialty-specific issues, and to capture demographic and practice-related characteristics. Emotional exhaustion (EE) and depersonalization (DP) were analyzed as dimensions of burnout. Consistent with convention, surgeons with a high score on the DP and/or EE subscales of the Maslach Burnout Inventory were considered to have at least one manifestation of professional burnout. Risk factors associated with symptoms of burnout were identified using bivariate analyses (χ2, Kruskal-Wallis). Multivariate logistic regression models were developed to identify independent risk factors for burnout. RESULTS: Of 2905 active SVS members, 960 responded to the survey (34% participation rate). After excluding retired surgeons and incomplete submissions, responses from 872 practicing vascular surgeons were analyzed. The mean age was 49.7 ± 11.0 years; the majority of respondents (81%) were male. Primary practice settings were academic (40%), community practice (41%), veteran's hospital (3.3%), active military practice (1.5%), or other. Years in practice averaged 15.7 ± 11.7. Overall, 41% of respondents had at least one symptoms of burnout (ie, high EE and/or high DP), 37% endorsed symptoms of depression in the past month, and 8% indicated they had considered suicide in the last 12 months. In unadjusted analysis, factors significantly associated with burnout (P < .05) included clinical work hours, on-call frequency, electronic medical record and documentation requirements, work-home conflict, and work-related physical pain. On multivariate analysis, age, work-related physical pain and work-home conflict were independent predictors for burnout. CONCLUSIONS: Symptoms of burnout and depression are common among vascular surgeons. Advancing age, work-related physical pain, and work-home conflict are independent predictors for burnout among vascular surgeons. Efforts to promote vascular surgeon well-being must address specialty-specific challenges, including the high prevalence of work-home conflict and occupational factors that contribute to work-related pain.

MicroRNA 219-5p inhibits alveolarization by reducing platelet derived growth factor receptor-alpha.

BACKGROUND: MicroRNA (miR) are small conserved RNA that regulate gene expression post-transcription. Previous genome-wide analysis studies in preterm infants indicate that pathways of miR 219-5p are important in infants with Bronchopulmonary Dysplasia (BPD). METHODS: Here we report a prospective cohort study of extremely preterm neonates wherein infants diagnosed with severe BPD expressed increased airway miR-219-5p and decreased platelet derived growth factor receptor alpha (PDGFR-α), a target of mir-219-5p and a key regulator of alveolarization, compared to post-conception age-matched term infants. RESULTS: miR-219-5p was highly expressed in the pulmonary epithelial lining in lungs of infants with BPD by in situ hybridization of human infant lungs. In both in vitro and in vivo (mouse) models of BPD, miR-219-5p was increased on exposure to hyperoxia compared with the normoxia control, with a complementary decrease of PDGFR-α. To further confirm the target relationship between miR-219 and PDGFR-α, pulmonary epithelial cells (MLE12) and lung primary fibroblasts were treated with a mimic of miR-219-5p and a locked nucleic acid (LNA) based inhibitor of miR-219-5p. In comparison with the control group, the level of miR-219 increased significantly after miR-219 mimic treatment, while the level of PDGFR-α declined markedly. LNA exposure increased PDGFR-α. Moreover, in BPD mouse model, over-expression of miR-219-5p inhibited alveolar development, indicated by larger alveolar spaces accompanied by reduced septation. CONCLUSIONS: Taken together, our results demonstrate that increased miR-219-5p contributes to the pathogenesis of BPD by targeting and reducing PDGFR-α. The use of specific miRNA antagonists may be a therapeutic strategy for preventing the development of BPD.

MicroRNA-145 Antagonism Reverses TGF-ß Inhibition of F508del CFTR Correction in Airway Epithelia.

RATIONALE: MicroRNAs (miRNAs) destabilize mRNA transcripts and inhibit protein translation. miR-145 is of particular interest in cystic fibrosis (CF) as it has a direct binding site in the 3'-untranslated region of CFTR (cystic fibrosis transmembrane conductance regulator) and is upregulated by the CF genetic modifier TGF (transforming growth factor)-ß. OBJECTIVES: To demonstrate that miR-145 mediates TGF-ß inhibition of CFTR synthesis and function in airway epithelia. METHODS: Primary human CF (F508del homozygous) and non-CF airway epithelial cells were grown to terminal differentiation at the air-liquid interface on permeable supports. TGF-ß (5 ng/ml), a miR-145 mimic (20 nM), and a miR-145 antagonist (20 nM) were used to manipulate CFTR function. In CF cells, lumacaftor (3 µM) and ivacaftor (10 µM) corrected mutant F508del CFTR. Quantification of CFTR mRNA, protein, and function was done by standard techniques. MEASUREMENTS AND MAIN RESULTS: miR-145 is increased fourfold in CF BAL fluid compared with non-CF (P < 0.01) and increased 10-fold in CF primary airway epithelial cells (P < 0.01). Exogenous TGF-ß doubles miR-145 expression (P < 0.05), halves wild-type CFTR mRNA and protein levels (P < 0.01), and nullifies lumacaftor/ivacaftor F508del CFTR correction. miR-145 overexpression similarly decreases wild-type CFTR protein synthesis (P < 0.01) and function (P < 0.05), and eliminates F508del corrector benefit. miR-145 antagonism blocks TGF-ß suppression of CFTR and enhances lumacaftor correction of F508del CFTR. CONCLUSIONS: miR-145 mediates TGF-ß inhibition of CFTR synthesis and function in airway epithelia. Specific antagonists to miR-145 interrupt TGF-ß signaling to restore F508del CFTR modulation. miR-145 antagonism may offer a novel therapeutic opportunity to enhance therapeutic benefit of F508del CFTR correction in CF epithelia.

Iloprost attenuates hyperoxia-mediated impairment of lung development in newborn mice.

Cyclooxygenase-2 (COX-2/PTGS2) mediates hyperoxia-induced impairment of lung development in newborn animals and is increased in the lungs of human infants with bronchopulmonary dysplasia (BPD). COX-2 catalyzes the production of cytoprotective prostaglandins, such as prostacyclin (PGI2), as well as proinflammatory mediators, such as thromboxane A2. Our objective was to determine whether iloprost, a synthetic analog of PGI2, would attenuate hyperoxia effects in the newborn mouse lung. To test this hypothesis, newborn C57BL/6 mice along with their dams were exposed to normoxia (21% O2) or hyperoxia (85% O2) from 4 to 14 days of age in combination with daily intraperitoneal injections of either iloprost 200 µg·kg-1·day-1, nimesulide (selective COX-2 antagonist) 100 mg·kg-1·day-1, or vehicle. Alveolar development was estimated by radial alveolar counts and mean linear intercepts. Lung function was determined on a flexiVent, and multiple cytokines and myeloperoxidase (MPO) were quantitated in lung homogenates. Lung vascular and microvascular morphometry was performed, and right ventricle/left ventricle ratios were determined. We determined that iloprost (but not nimesulide) administration attenuated hyperoxia-induced inhibition of alveolar development and microvascular density in newborn mice. Iloprost and nimesulide both attenuated hyperoxia-induced, increased lung resistance but did not improve lung compliance that was reduced by hyperoxia. Iloprost and nimesulide reduced hyperoxia-induced increases in MPO and some cytokines (IL-1ß and TNF-α) but not others (IL-6 and KC/Gro). There were no changes in pulmonary arterial wall thickness or right ventricle/left ventricle ratios. We conclude that iloprost improves lung development and reduces lung inflammation in a newborn mouse model of BPD.

Exposure of neonatal mice to bromine impairs their alveolar development and lung function.

The halogen bromine (Br2) is used extensively in industry and stored and transported in large quantities. Its accidental or malicious release into the atmosphere has resulted in significant casualties. The pathophysiology of Br2-induced lung injury has been studied in adult animals, but the consequences of Br2 exposure to the developing lung are completely unknown. We exposed neonatal mouse littermates on postnatal day 3 (P3) to either Br2 at 400 ppm for 30 min (400/30), to Br2 at 600 ppm for 30 min (600/30), or to room air, then returned them to their dams and observed until P14. Mice exposed to Br2 had decreased survival (S) and had decreased weight (W) at P14 in the 400/30 group (S = 63.5%, W = 6.67 ± 0.08) and in the 600/30 group (S = 36.1%, W = 5.13 ± 0.67) as compared with air breathing mice (S = 100%, W = 7.96 ± 0.30). Alveolar development was impaired, as evidenced by increased mean linear intercept at P14. At P14, Br2 exposed mice also exhibited a decrease of arterial partial pressure of oxygen, decreased quasi-static lung compliance, as well as increased alpha smooth muscle actin mRNA and protein and increased mRNA for IL-1ß, IL-6, CXCL1, and TNFα. Global gene expression, evaluated by RNA sequencing and Ingenuity Pathway Analysis, revealed persistent abnormalities in gene expression profiles at P14 involving pathways of "formation of lung" and "pulmonary development." The data indicate that Br2 inhalation injury early in life results in severe lung developmental consequences, wherein persistent inflammation and global altered developmental gene expression are likely mechanistic contributors.

IGFBP6 controls the expansion of chemoresistant glioblastoma through paracrine IGF2/IGF-1R signaling.

BACKGROUND: Glioblastomas (GBMs), the most common and most lethal of the primary brain tumors, are characterized by marked intra-tumor heterogeneity. Several studies have suggested that within these tumors a restricted population of chemoresistant glioma cells is responsible for recurrence. However, the gene expression patterns underlying chemoresistance are largely unknown. Numerous efforts have been made to block IGF-1R signaling pathway in GBM. However, those therapies have been repeatedly unsuccessful. This failure may not only be due to the complexity of IGF receptor signaling, but also due to complex cell-cell interactions in the tumor mass. We hypothesized that differential expression of proteins in the insulin-like growth factor (IGF) system underlie cell-specific differences in the resistance to temozolomide (TMZ) within GBM tumors. METHODS: Expression of IGF-1R was analyzed in cell lines, patient-derived xenograft cell lines and human biopsies by cell surface proteomics, flow cytometry, immunofluorescence and quantitative real time polymerase chain reaction (qRT-PCR). Using gain-of-function and loss-of-function strategies, we dissected the molecular mechanism responsible for IGF-binding protein 6 (IGFBP6) tumor suppressor functions both in in vitro and in vivo. Site direct mutagenesis was used to study IGFBP6-IGF2 interactions. RESULTS: We determined that in human glioma tissue, glioma cell lines, and patient-derived xenograft cell lines, treatment with TMZ enhances the expression of IGF1 receptor (IGF-1R) and IGF2 and decreases the expression of IGFBP6, which sequesters IGF2. Using chemoresistant and chemosensitive wild-type and transgenic glioma cells, we further found that a paracrine mechanism driven by IGFBP6 secreted from TMZ-sensitive cells abrogates the proliferation of IGF-1R-expressing TMZ-resistant cells in vitro and in vivo. In mice bearing intracranial human glioma xenografts, overexpression of IGFBP6 in TMZ-resistant cells increased survival. Finally, elevated expression of IGF-1R and IGF2 in gliomas associated with poor patient survival and tumor expression levels of IGFBP6 directly correlated with overall survival time in patients with GBM. CONCLUSIONS: Our findings support the view that proliferation of chemoresistant tumor cells is controlled within the tumor mass by IGFBP6-producing tumor cells; however, TMZ treatment eliminates this population and enriches the TMZ-resistant cell populationleading to accelerated growth of the entire tumor mass.

Prophylactic Groin Wound Vacuum-assisted Therapy in Vascular Surgery Patients at Enhanced Risk for Postoperative Wound Infection.

BACKGROUND: Vascular groin wounds have higher than expected surgical site infection (SSI) rates and some patients are at enhanced risk. The Wiseman et al. paper suggests an objective scoring system that identifies patients at enhanced risk of postdischarge SSI. We hypothesize that prophylactic groin wound vacuum-assisted closure (VAC) therapy in enhanced risk patients will decrease SSI and readmission and the Wiseman model provides potential evidence that enhanced risk patients can be objectively identified. METHODS: A single institution, retrospective analysis was conducted from January 2013 to September 2016 utilizing procedure codes to identify patients with wound VACs placed in the operating room. Two distinct groups were identified. The first was a wound complication patient group with 15 limbs (13 patients) with a groin wound VAC placed within 45 days postoperatively for groin wound complications. Eleven of these limbs had the VAC placed at readmission. The second group was a prophylactic patient group that included 8 limbs (7 patients) who received a VAC prophylactically placed in the enhanced risk wounds. These wounds were determined to be enhanced risk based on clinical criteria judged by the operating surgeon such as a large overhanging panniculus and/or one of several ongoing medical issues. We calculated a Wiseman score for all patients, determined total cost of the readmissions, and determined 30-day postsurgical SSI incidence for the prophylactic VAC group. RESULTS: Per the Wiseman scores, 9 limbs with postoperative complications were high risk and 3 limbs were moderate/high risk. Eleven limbs had a VAC placed at readmission with an average readmission cost of $8876.77. For the prophylactic group, 8 limbs were high risk with no observed postdischarge SSI in the first 30 days from surgery. The Wiseman scores showed close correlation between the retrospective high and moderate/high risk groups versus the prophylactic VAC group (31.5 ± 7.3 vs. 32 ± 5.5, P = 0.87). CONCLUSIONS: The Wiseman scores showed objective validation in the prognosis of anticipating groin wound breakdown. Our initial results suggest that prophylactic groin wound VAC placement for enhanced risk vascular surgery patients may proactively decrease wound morbidity, decrease readmission secondary to groin wound complications, and provide some cost benefit.

Biomarkers associated with bronchopulmonary dysplasia/mortality in premature infants.

BACKGROUND: Bronchopulmonary dysplasia (BPD) portends lifelong organ impairment and death. Our ability to predict BPD in first days of life is limited, but could be enhanced using novel biomarkers. METHODS: Using an available clinical and urine biomarker database obtained from a prospective 113 infant cohort (birth weight ≤1,200 g and/or gestational age ≤31 wk), we evaluated the independent association of 14 urine biomarkers with BPD/mortality. RESULTS: Two of the 14 urine biomarkers were independently associated with BPD/mortality after controlling for gestational age (GA), small for gestational age (SGA), and intubation status. The best performing protein was clusterin, a ubiquitously expressed protein and potential sensor of oxidative stress associated with lung function in asthma patients. When modeling for BPD/mortality, the independent odds ratio for maximum adjusted urine clusterin was 9.2 (95% CI: 3.3-32.8, P < 0.0001). In this model, clinical variables (GA, intubation status, and SGA) explained 38.3% of variance; clusterin explained an additional 9.2%, while albumin explained an additional 3.4%. The area under the curve incorporating clinical factors and biomarkers was 0.941. CONCLUSION: Urine clusterin and albumin may improve our ability to predict BPD/mortality. Future studies are needed to validate these findings and determine their clinical usefulness.

Regulation of alveolar septation by microRNA-489.

MicroRNAs (miRs) are small conserved RNA that regulate gene expression. Bioinformatic analysis of miRNA profiles during mouse lung development indicated a role for multiple miRNA, including miRNA-489. miR-489 increased on completion of alveolar septation [postnatal day 42 (P42)], associated with decreases in its conserved target genes insulin-like growth factor-1 (Igf1) and tenascin C (Tnc). We hypothesized that dysregulation of miR-489 and its target genes Igf1 and Tnc contribute to hyperoxia-induced abnormal lung development. C57BL/6 mice were exposed to normoxia (21%) or hyperoxia (85% O2) from P4 to P14, in combination with intranasal locked nucleic acid against miR-489 to inhibit miR-489, cytomegalovirus promoter (pCMV)-miR-489 to overexpress miR-489, or empty vector. Hyperoxia reduced miR-489 and increased Igf1 and Tnc. Locked nucleic acid against miR-489 improved lung development during hyperoxia and did not alter it during normoxia, whereas miR-489 overexpression inhibited lung development during normoxia. The 3' untranslated region in vitro reporter studies confirmed Igf1 and Tnc as targets of miR-489. While miR-489 was of epithelial origin and present in exosomes, its targets Igf1 and Tnc were produced by fibroblasts. Infants with bronchopulmonary dysplasia (BPD) had reduced lung miR-489 and increased Igf1 and Tnc compared with normal preterm or term infants. These results suggest increased miR-489 is an inhibitor of alveolar septation. During hyperoxia or BPD, reduced miR-489 and increased Igf1 and Tnc may be inadequate attempts at compensation. Further inhibition of miR-489 may permit alveolar septation to proceed. The use of specific miRNA antagonists or agonists may be a therapeutic strategy for inhibited alveolarization, such as in BPD.

Alterations in gene expression and DNA methylation during murine and human lung alveolar septation.

DNA methylation, a major epigenetic mechanism, may regulate coordinated expression of multiple genes at specific time points during alveolar septation in lung development. The objective of this study was to identify genes regulated by methylation during normal septation in mice and during disordered septation in bronchopulmonary dysplasia. In mice, newborn lungs (preseptation) and adult lungs (postseptation) were evaluated by microarray analysis of gene expression and immunoprecipitation of methylated DNA followed by sequencing (MeDIP-Seq). In humans, microarray gene expression data were integrated with genome-wide DNA methylation data from bronchopulmonary dysplasia versus preterm and term lung. Genes with reciprocal changes in expression and methylation, suggesting regulation by DNA methylation, were identified. In mice, 95 genes with inverse correlation between expression and methylation during normal septation were identified. In addition to genes known to be important in lung development (Wnt signaling, Angpt2, Sox9, etc.) and its extracellular matrix (Tnc, Eln, etc.), genes involved with immune and antioxidant defense (Stat4, Sod3, Prdx6, etc.) were also observed. In humans, 23 genes were differentially methylated with reciprocal changes in expression in bronchopulmonary dysplasia compared with preterm or term lung. Genes of interest included those involved with detoxifying enzymes (Gstm3) and transforming growth factor-ß signaling (bone morphogenetic protein 7 [Bmp7]). In terms of overlap, 20 genes and three pathways methylated during mouse lung development also demonstrated changes in methylation between preterm and term human lung. Changes in methylation correspond to altered expression of a number of genes associated with lung development, suggesting that DNA methylation of these genes may regulate normal and abnormal alveolar septation.

Genetic polymorphisms of heme-oxygenase 1 (HO-1) may impact on acute kidney injury, bronchopulmonary dysplasia, and mortality in premature infants.

BACKGROUND: Heme oxygenase 1 (HO1) catalyzes heme degradation, and offers protection for several organs, including the kidney. Genetic polymorphisms of HO-1 are associated with poor clinical outcomes in several populations. POPULATION: We prospectively enrolled 117 premature infants (birth weight ≤1,200 g or postgestational age ≤31 wk) and evaluated two DNA genetic variants proximal to the promoter region of HO-1 (GT(n) repeats, and -413T>A SNP). We evaluated how these polymorphisms affect two clinical outcomes: (i) Acute Kidney Injury (AKI)-rise in serum creatinine (SCr) ≥ 0.3 mg/dl or ≥ 150-200% from lowest previous value, (ii) the composite of mortality and bronchopulmonary dysplasia (BPD) defined as receipt of oxygen at 36 wk postmenstrual age. RESULTS: AKI occurred in 34/117 (29%) of neonates; 12/117 (10%) died; 29/105 (28%) survivors had BPD. Neonates with TT genotype at 413T>A before the HO-1 promoter had higher rates of AKI (P < 0.05). There was no difference in number of GT(n) repeats and clinical outcomes. CONCLUSION: We did not find an association between the GT(n) tandem repeat of HO-1 and AKI nor BPD/mortality. However, infants with TT genotype of the 413T>A genetic alteration had lower incidence of AKI. Further studies using larger cohorts are needed to better understand these relationships.

VEGF mRNA and protein concentrations in the developing human eye.

BACKGROUND: Vascular endothelial growth factor (VEGF), a well-characterized regulator of angiogenesis, has been mechanistically implicated in retinal neovascularization and in the pathogenesis of retinopathy of prematurity. However, the ontogeny of VEGF expression in the human fetal retina is not well known. Because retinal vasculature grows with gestational maturation, we hypothesized that VEGF expression also increases in the midgestation human fetal eye as a function of gestational age. METHODS: To identify changes in VEGF gene expression during normal human development, we measured VEGF mRNA by quantitative PCR and measured VEGF protein by enzyme-linked immunosorbent assay and western blots in 10-24 wk gestation fetal vitreous, retina, and serum. RESULTS: VEGF mRNA expression in the retina increased with gestational age. VEGF isoform A, particularly its VEGF121 splice variant, contributed to this positive correlation. Consistent with these findings, we detected increasing VEGF121 protein concentrations in vitreous humor from fetuses of 10-24 wk gestation, while VEGF concentrations decreased in fetal serum. CONCLUSION: VEGF121 mRNA and protein concentrations increase with increasing gestational age in the developing human retina. We speculate that VEGF plays an important role in normal retinal vascular development, and that preterm delivery affects production of this vascular growth factor.