Browsing by Author "Haneline, Laura S."

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    Are Newborn Outcomes Different for Term Babies Who Were Exposed to Antenatal Corticosteroids?
    (American Journal of Obstetrics and Gynecology, 2021-05-03) McKinzie, Alexandra; Yang, Ziyi; Teal, Evgenia; Daggy, Joanne K.; Tepper, Robert S.; Quinney, Sarah K.; Rhoads, Eli; Haneline, Laura S.; Haas, David M.; Obstetrics and Gynecology, School of Medicine
    Background Antenatal corticosteroids improve newborn outcomes for preterm infants. However, predicting which women presenting for threatened preterm labor will have preterm infants is inaccurate and many women receive antenatal corticosteroids but then go on to deliver at term. Objective The purpose of this study was to compare the short-term outcomes of infants born at term to women who received betamethasone (BMZ) for threatened preterm labor to infants who were not exposed to BMZ in utero. Study Design We performed a retrospective cohort study of infants born at or after 37 weeks’ gestational age (GA) to mothers diagnosed with threatened preterm labor during pregnancy. The primary neonatal outcomes of interest included transient tachypnea of the newborn (TTN), neonatal intensive care unit (NICU) admission, and small for gestational age (SGA), and were evaluated for their association with BMZ exposure while adjusting for covariates using multiple logistic regression. Results Of 5330 women, 1459 (27.5%) women received BMZ at a mean GA of 32.2±3.3 weeks. The mean age of women was 27±5.9 years-old and the mean GA at delivery was 38.9±1.1 weeks. Women receiving BMZ had higher rates of maternal comorbidities (P<0.001 for diabetes, asthma, and hypertensive disorder) and were more likely to self-identify as white (P=0.022). BMZ-exposed neonates had increased rates of TTN, NICU admission, SGA, hyperbilirubinemia, and hypoglycemia (all P-values <0.05). Controlling for maternal characteristics and GA at delivery, BMZ exposure was not significantly associated with diagnosis of TTN (aOR 1.10, 95% CI 0.80-1.51), though it was associated with more NICU admissions (aOR 1.49, 95% CI 1.19-1.86) and higher odds of the baby being small for gestational age (SGA, aOR 1.78, 95%CI 1.48 to 2.14). Conclusions Compared to women evaluated for preterm labor that did not receive BMZ, women receiving BMZ had infants with higher rates of NICU admission and SGA. While the benefits of BMZ to infants born preterm are clear, there may be negative impacts for infants delivered at term.
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    Biomarkers for Diagnosis and Prognosis of Sinusoidal Obstruction Syndrome after Hematopoietic Cell Transplantation.
    (Elsevier, 2015-10) Akil, Ayman; Zhang, Qing; Mumaw, Christen L.; Raiker, Nisha; Yu, Jeffrey; de Mendizabal, Nieves Velez; Haneline, Laura S.; Robertson, Kent A.; Skiles, Jodi; Diaz-Ricart, Maribel; Carreras, Enric; Renbarger, Jamie; Hanash, Samir; Bies, Robert R.; Paczesny, Sophie; Department of Pediatrics, IU School of Medicine
    Reliable, non-invasive methods for diagnosing and prognosing sinusoidal obstruction syndrome (SOS) early after hematopoietic cell transplantation (HCT) are needed. We used a quantitative mass spectrometry-based proteomics approach to identify candidate biomarkers of SOS by comparing plasma pooled from 20 patients with and 20 patients without SOS. Of 494 proteins quantified, we selected six proteins [L-Ficolin, vascular-cell-adhesion-molecule-1 (VCAM1), tissue-inhibitor of metalloproteinase-1, von Willebrand factor, intercellular-adhesion-molecule-1, and CD97] based on a differential heavy/light isotope ratio of at least 2 fold, information from the literature, and immunoassay availability. Next, we evaluated the diagnostic potential of these six proteins and five selected from the literature [suppression of tumorigenicity-2 (ST2), angiopoietin-2 (ANG2), hyaluronic acid (HA), thrombomodulin, and plasminogen activator inhibitor-1] in samples from 80 patients. The results demonstrate that together ST2, ANG2,
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    Dishevelled-associated activator of morphogenesis 1 (Daam1) is required for heart morphogenesis
    (2011-01) Li, Deqiang; Hallett, Mark A.; Zhu, Wuqiang; Rubart, Michael; Liu, Ying; Yang, Zhenyun; Chen, Hanying; Haneline, Laura S.; Chan, Rebecca J.; Schwartz, Robert J.; Field, Loren J.; Atkinson, Simon J.; Shou, Weinian
    Dishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, plays an important role in regulating the actin cytoskeleton via mediation of linear actin assembly. Previous functional studies of Daam1 in lower species suggest its essential role in Drosophila trachea formation and Xenopus gastrulation. However, its in vivo physiological function in mammalian systems is largely unknown. We have generated Daam1-deficient mice via gene-trap technology and found that Daam1 is highly expressed in developing murine organs, including the heart. Daam1-deficient mice exhibit embryonic and neonatal lethality and suffer multiple cardiac defects, including ventricular noncompaction, double outlet right ventricles and ventricular septal defects. In vivo genetic rescue experiments further confirm that the lethality of Daam1-deficient mice results from the inherent cardiac abnormalities. In-depth analyses have revealed that Daam1 is important for regulating filamentous actin assembly and organization, and consequently for cytoskeletal function in cardiomyocytes, which contributes to proper heart morphogenesis. Daam1 is also found to be important for proper cytoskeletal architecture and functionalities in embryonic fibroblasts. Biochemical analyses indicate that Daam1 does not regulate cytoskeletal organization through RhoA, Rac1 or Cdc42. Our study highlights a crucial role for Daam1 in regulating the actin cytoskeleton and tissue morphogenesis.
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    Elevated transgelin reduces function of endothelial colony forming cells from gestational diabetic pregnancies
    (Office of the Vice Chancellor for Research, 2016-04-08) Varberg, Kaela M.; Garretson, Rashell O.; Blue, Emily K.; Haneline, Laura S.
    Fetal exposure to maternal diabetes predisposes children to future complications including hypertension and cardiovascular disease. A key mechanism by which these complications are thought to occur and persist is through the functional impairment of vascular progenitor cells, including endothelial colony forming cells (ECFCs). Previously, we showed that ECFCs exposed to gestational diabetes exhibit functional deficits, such as impaired vessel formation, but also differential gene expression compared to uncomplicated controls. One gene that was confirmed to be significantly upregulated in ECFCS from diabetic pregnancies was transgelin, an actin-binding smooth muscle protein. However, the functional consequences of increased transgelin in ECFCs are unknown. Therefore, to determine if transgelin is sufficient and required to induce dysfunction of ECFCs from diabetic pregnancies, transgelin protein levels were manipulated using genetic methods. Specifically, lentiviral overexpression and siRNA knockdown techniques were used in ECFCs from control and diabetic pregnancies respectively. Network formation assays and trans-well migration assays were performed to assess whether alteration of transgelin levels impact ECFC vasculogenesis and migration. Decreasing transgelin expression in diabetes-exposed ECFCs increased network formation (n=15, p<0.05) and cell migration (n=12, p<0.05). Conversely, overexpression of transgelin in ECFCs from uncomplicated pregnancies decreased network formation (n=12, p<0.05). Additional studies are underway to further elucidate intracellular signaling altered as a result of increased transgelin expression in diabetes-exposed ECFCs. Delineating the mechanisms underlying ECFC functional deficits will aid in the understanding of how and why chronic vascular complications persist in children born to mothers with diabetes.
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    Endothelial Colony-Forming Cell Function Is Reduced During HIV Infection
    (Oxford Academic, 2019-04-01) Gupta, Samir K.; Liu, Ziyue; Sims, Emily C.; Repass, Matthew J.; Haneline, Laura S.; Yoder, Mervin C.; Medicine, School of Medicine
    Background: Human immunodeficiency virus (HIV) may be related to cardiovascular disease through monocyte activation-associated endothelial dysfunction. Methods: Blood samples from 15 HIV-negative participants (the uninfected group), 8 HIV-positive participants who were not receiving antiretroviral therapy (ART) (the infected, untreated group), and 15 HIV-positive participants who were receiving ART (the infected, treated group) underwent flow cytometry of endothelial colony-forming cells (ECFCs) and monocyte proportions. IncuCyte live cell imaging of 8 capillary proliferative capacity parameters were obtained from cord blood ECFCs treated with participant plasma. Results: The ECFC percentage determined by flow cytometry was not different between the study groups; however, values of the majority of capillary proliferative capacity parameters (ie, cell area, network length, network branch points, number of networks, and average tube width uniformity) were significantly lower in infected, untreated participants as compared to values for uninfected participants or infected, treated participants (P < .00625 for all comparisons). CD14+CD16+ intermediate monocytes and soluble CD163 were significantly and negatively correlated with several plasma-treated, cord blood ECFC proliferative capacity parameters in the combined HIV-positive groups but not in the uninfected group. Conclusions: Cord blood ECFC proliferative capacity was significantly impaired by plasma from infected, untreated patients, compared with plasma from uninfected participants and from infected, treated participants. Several ECFC functional parameters were adversely associated with monocyte activation in the HIV-positive groups, thereby suggesting a mechanism by which HIV-related inflammation may impair vascular reparative potential and consequently increase the risk of cardiovascular disease during HIV infection.
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    Epigenetic regulation in neonatal ECFCs following intrauterine exposure to gestational diabetes
    (Office of the Vice Chancellor for Research, 2015-04-17) Blue, Emily K.; Sheehan, BreAnn M.; Nuss, Zia V.; Gohn, Cassandra R.; Varberg, Kaela M.; McClintick, Jeanette N.; Haneline, Laura S.
    Gestational diabetes (GDM) complicates up to 10% of pregnancies. In addition to acute risks, the children of diabetic mothers have an increased risk of obesity, diabetes, and hypertension, starting in childhood. While the causes of this increased risk are unknown, previous studies in our lab have identified functional deficits in endothelial colony forming cells (ECFCs) isolated from the cord blood of GDM pregnancies. This study focused on identifying genes that have altered epigenetic modifications that result in abnormal mRNA and protein expression in ECFCs from the cord blood GDM pregnancies. The objective of this study was to identify mRNA expression and DNA methylation alterations in ECFCs that may help identify the causes of ECFC dysfunction following intrauterine exposure to GDM. ECFCs were obtained from control and GDM pregnancies. DNA, RNA, and protein samples were isolated in parallel from ECFCs. RNA microarray analysis using the Affymetrix Human 1.0 Gene Array was used to identify gene expression alterations in GDM ECFCs compared to control ECFCs. Genome-wide DNA methylation was assessed using an Infinium 450K Methylation Array for DNA samples at >450,000 CpG sites. Correlation analysis was performed to identify possible sites that have altered CpG methylation and RNA expression. RNA expression results were validated using qRT-PCR and western blotting. Bisulfite sequencing of genomic DNA from the ECFCs was performed to identify additional sites with altered methylation for regions not included in the DNA methylation array. Of the 28,000 genetic loci tested, 596 mRNAs were altered between control and GDM ECFCs (p<0.01). More stringent criteria identified 38 genes for further investigation by limiting analysis to genes that exhibited increased or decreased expression by at least 50%, with a p<0.01. PLAC8 was identified as being increased 5-fold by microarray analysis, a result which was confirmed in two cohorts by qRT-PCR and western blotting. Analysis of the methylation array and bisulfite sequencing results revealed 3 regions surrounding the transcriptional start site of PLAC8 gene whose CpG methylation negatively correlate with RNA expression in samples from control and GDM ECFCs. In contrast, a CpG island is fully unmethylated in both control and GDM ECFCs. The discovery of CpG sites whose methylation correlates with PLAC8 mRNA expression in ECFCs is consistent with the hypothesis that intrauterine exposure to GDM results in epigenetic changes. Analysis of methylation at this site could be used as a biomarker for children of mothers with GDM who may be at risk for disease later in life. Using bisulfite pyrosequencing, we are currently developing assays to quickly determine if methylation of the PLAC8 putative promoter region is altered in cord blood mononuclear cells obtained from GDM or healthy control pregnancies. We are also investigating the role of methylation in regulating PLAC8 RNA expression, determining if there is altered histone modifications and transcription factor binding in these regions, and examining other genes that may comprise a molecular signature of ECFC dysfunction.
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    Epigenetic Regulation of Placenta-Specific 8 Contributes to Altered Function of Endothelial Colony-Forming Cells Exposed to Intrauterine Gestational Diabetes Mellitus
    (American Diabetes Association, 2015-07) Blue, Emily K.; Sheehan, BreAnn M.; Nuss, Zia V.; Boyle, Frances A.; Hocutt, Caleb M.; Gohn, Cassandra R.; Varberg, Kaela M.; McClintick, Jeanette N.; Haneline, Laura S.; Department of Pediatrics, IU School of Medicine
    Intrauterine exposure to gestational diabetes mellitus (GDM) is linked to development of hypertension, obesity, and type 2 diabetes in children. Our previous studies determined that endothelial colony-forming cells (ECFCs) from neonates exposed to GDM exhibit impaired function. The current goals were to identify aberrantly expressed genes that contribute to impaired function of GDM-exposed ECFCs and to evaluate for evidence of altered epigenetic regulation of gene expression. Genome-wide mRNA expression analysis was conducted on ECFCs from control and GDM pregnancies. Candidate genes were validated by quantitative RT-PCR and Western blotting. Bisulfite sequencing evaluated DNA methylation of placenta-specific 8 (PLAC8). Proliferation and senescence assays of ECFCs transfected with siRNA to knockdown PLAC8 were performed to determine functional impact. Thirty-eight genes were differentially expressed between control and GDM-exposed ECFCs. PLAC8 was highly expressed in GDM-exposed ECFCs, and PLAC8 expression correlated with maternal hyperglycemia. Methylation status of 17 CpG sites in PLAC8 negatively correlated with mRNA expression. Knockdown of PLAC8 in GDM-exposed ECFCs improved proliferation and senescence defects. This study provides strong evidence in neonatal endothelial progenitor cells that GDM exposure in utero leads to altered gene expression and DNA methylation, suggesting the possibility of altered epigenetic regulation.
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    The Exploration of an Effective Medical Countermeasure Enhancing Survival and Hematopoietic Recovery and Preventing Immune Insufficiency in Lethally-Irradiated Mice
    (2020-08) Wu, Tong; Orschell, Christie M.; Basile, David P.; Unthank, Joseph L.; Haneline, Laura S.; Pelus, Louis M.; MacVittie, Thomas J.
    There is an urgent demand for effective medical countermeasures (MCM) in the event of high-dose radiation exposure ranging from nuclear plant disasters to potential nuclear warfare. Victims of lethal-dose radiation exposure face multi-organ injuries including the hematopoietic acute radiation syndrome (H-ARS) and the delayed effects of acute radiation exposure (DEARE) years after irradiation. Defective lymphocyte reconstitution and its subsequent immune insufficiency are some of the most serious consequences of H-ARS and DEARE. In order to investigate potential MCMs to protect or mitigate these radiation injuries, the prolonged tissue-specific immunosuppression at all levels of lymphocyte development in established murine H-ARS and DEARE models was defined, along with unique sex-related and age-related changes present in some tissues but not others. The “double hits” of irradiation and age-related stress on lymphopoiesis led to significant myeloid skew and long-term immune involution. Different kinds and different combinations of hematopoietic growth factors, some in combination with angiotensin converting enzyme inhibitor, were administered to lethally irradiated mice. These radiomitigators were found to significantly increase survival and enhance hematopoiesis in H-ARS, but they did little to alleviate the severity of DEARE including immune insufficiency. 16,16 dimethyl-prostaglandin E2 (dmPGE2), a long-acting formulation of PGE2 with similar biological effects as PGE2, was found to enhance survival and hematopoiesis in lethal-irradiated mice when used as radiomitigator or radioprotectant. The optimum time window for administration of radioprotectant and radiomitigator dmPGE2 was defined, which is -3hr to -15min prior to irradiation and +6hr to +30hr post irradiation. Significant survival efficacy of radioprotectant dmPGE2 was also demonstrated in pediatric and geriatric mice. Using specific PGE2 receptor (EP) agonists, the EP4 receptor was defined as the PGE2 receptor potentially responsible for dmPGE2 radioprotection. Radioprotectant dmPGE2 was also found to prevent radiation-induced thymic involution and to ameliorate the long-term immune suppression in radiation survivors in the DEARE phase via promoting hematopoietic stem cell differentiation towards to the lymphoid lineage. This is the first report of an effective MCM for H-ARS which also targets long-term thymic involution and lymphoid lineage reconstitution.
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    The Fanconi anemia signaling network regulates the mitotic spindle assembly checkpoint
    (2014) Enzor, Rikki S.; Clapp, D. Wade; Broxmeyer, Hal E.; Haneline, Laura S.; Srour, Edward F.
    Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity.
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    Fetal hyperglycemia and a high fat diet contribute to aberrant glucose tolerance and hematopoiesis in adulthood
    (Nature Publishing Group, 2015-02) Blue, Emily K.; Ballman, Kimberly; Boyle, Frances; Oh, Eunjin; Kono, Tatsuyoshi; Quinney, Sara K.; Thurmond, Debbie C.; Evans-Molina, Carmella; Haneline, Laura S.; Department of Pediatrics, IU School of Medicine
    Background Children exposed to gestational diabetes mellitus (GDM) during pregnancy are at increased risk of obesity, diabetes, and hypertension. Our goal was to identify metabolic and hematopoietic alterations after intrauterine exposure to maternal hyperglycemia that may contribute to the pathogenesis of chronic morbidities. Methods Streptozotocin treatment induced maternal hyperglycemia during the last third of gestation in rat dams. Offspring of control mothers (OCM) and diabetic mothers (ODM) were evaluated for weight, glucose tolerance, insulin tolerance, and hematopoiesis defects. The effects of aging were examined in normal and high fat diet (HFD)-fed young (8-week-old) and aged (11-month-old) OCM and ODM rats. Results Young adult ODM males on a normal diet, but not females, displayed improved glucose tolerance due to increased insulin levels. Aged ODM males and females gained more weight than OCM on a HFD and had worse glucose tolerance. Aged ODM males fed a HFD were also neutrophilic. Increases in bone marrow cellularity and myeloid progenitors preceded neutrophilia in ODM males fed a HFD. Conclusion When combined with other risk factors like HFD and aging, changes in glucose metabolism and hematopoiesis may contribute to the increased risk of obesity, type 2 diabetes, and hypertension observed in children of GDM mothers.