IU Indianapolis ScholarWorks :: Browsing by Author "Varberg, Kaela M."

Browsing by Author "Varberg, Kaela M."

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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.
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.
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.
Kinetic analyses of vasculogenesis inform mechanistic studies
(American Physiological Society, 2017-04-01) Varberg, Kaela M.; Winfree, Seth; Chu, Chenghao; Tu, Wanzhu; Blue, Emily K.; Gohn, Cassandra R.; Dunn, Kenneth W.; Haneline, Laura S.; Cellular and Integrative Physiology, School of Medicine
Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess in vitro endothelial colony-forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts. A tissue cytometry approach was established to quantify the frequency and localization of dividing ECFCs. Additionally, Fiji TrackMate was used to quantify ECFC displacement and speed at the single-cell level during network formation. These novel approaches were then implemented to identify how intrauterine exposure to maternal diabetes mellitus (DM) impairs fetal ECFC vasculogenesis. Fetal ECFCs exposed to maternal DM form fewer initial network structures, which are not stable over time. Correlation analyses demonstrated that ECFC samples with greater division in branches form fewer closed network structures. Additionally, reductions in average ECFC movement over time decrease structural connectivity. Identification of these novel phenotypes utilizing the newly established methodologies provides evidence for the cellular mechanisms contributing to aberrant ECFC vasculogenesis.
Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro
(JoVE, 2018-01-31) Varberg, Kaela M.; Winfree, Seth; Dunn, Kenneth W.; Haneline, Laura S.; Cellular and Integrative Physiology, School of Medicine
Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.
Mesenchyme Homeobox 2 Enhances Migration of Endothelial Colony Forming Cells Exposed to Intrauterine Diabetes Mellitus
(Wiley, 2017-07) Gohn, Cassandra R.; Blue, Emily K.; Sheehan, BreAnn M.; Varberg, Kaela M.; Haneline, Laura S.; Cellular and Integrative Physiology, School of Medicine
Diabetes mellitus (DM) during pregnancy has long-lasting implications for the fetus, including cardiovascular morbidity. Previously, we showed that endothelial colony forming cells (ECFCs) from DM human pregnancies have decreased vasculogenic potential. Here, we evaluate whether the molecular mechanism responsible for this phenotype involves the transcription factor, Mesenchyme Homeobox 2 (MEOX2). In human umbilical vein endothelial cells, MEOX2 upregulates cyclin-dependent kinase inhibitor expression, resulting in increased senescence and decreased proliferation. We hypothesized that dysregulated MEOX2 expression in neonatal ECFCs from DM pregnancies decreases network formation through increased senescence and altered cell cycle progression. Our studies show that nuclear MEOX2 is increased in ECFCs from DM pregnancies. To determine if MEOX2 is sufficient and/or required to induce impaired network formation, MEOX2 was overexpressed and depleted in ECFCs from control and DM pregnancies, respectively. Surprisingly, MEOX2 overexpression in control ECFCs resulted in increased network formation, altered cell cycle progression, and increased senescence. In contrast, MEOX2 knockdown in ECFCs from DM pregnancies led to decreased network formation, while cell cycle progression and senescence were unaffected. Importantly, migration studies demonstrated that MEOX2 overexpression increased migration, while MEOX2 knockdown decreased migration. Taken together, these data suggest that altered migration may be mediating the impaired vasculogenesis of ECFCs from DM pregnancies. While initially believed to be maladaptive, these data suggest that MEOX2 may serve a protective role, enabling increased vessel formation despite exposure to a DM intrauterine environment. J. Cell. Physiol. 232: 1885-1892, 2017.
Transgelin Induces Dysfunction of Fetal Endothelial Colony-Forming Cells From Gestational Diabetic Pregnancies
(American Physiological Society, 2018-10-01) Varberg, Kaela M.; Garretson, Rashell O.; Blue, Emily K.; Chu, Chenghao; Gohn, Cassandra R.; Tu, Wanzhu; Haneline, Laura S.; Cellular and Integrative Physiology, School of Medicine
Fetal exposure to gestational diabetes mellitus (GDM) predisposes children to future health complications including hypertension and cardiovascular disease. A key mechanism by which these complications occur is through the functional impairment of vascular progenitor cells, including endothelial colony-forming cells (ECFCs). Previously, we showed that fetal ECFCs exposed to GDM have decreased vasculogenic potential and altered gene expression. In this study, we evaluate whether transgelin (TAGLN), which is increased in GDM-exposed ECFCs, contributes to vasculogenic dysfunction. TAGLN is an actin-binding protein involved in the regulation of cytoskeletal rearrangement. We hypothesized that increased TAGLN expression in GDM-exposed fetal ECFCs decreases network formation by impairing cytoskeletal rearrangement resulting in reduced cell migration. To determine if TAGLN is required and/or sufficient to impair ECFC network formation, TAGLN was reduced and overexpressed in ECFCs from GDM and uncomplicated pregnancies, respectively. Decreasing TAGLN expression in GDM-exposed ECFCs improved network formation and stability as well as increased migration. In contrast, overexpressing TAGLN in ECFCs from uncomplicated pregnancies decreased network formation, network stability, migration, and alignment to laminar flow. Overall, these data suggest that increased TAGLN likely contributes to the vasculogenic dysfunction observed in GDM-exposed ECFCs, as it impairs ECFC migration, cell alignment, and network formation. Identifying the molecular mechanisms underlying fetal ECFC dysfunction following GDM exposure is key to ascertain mechanistically the basis for cardiovascular disease predisposition later in life.

Browsing by Author "Varberg, Kaela M."

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