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Item Angiopoietin-like protein 2 regulates endothelial colony forming cell vasculogenesis(Springer, 2014-07) Richardson, Matthew R.; Robbins, Emilie P.; Vemula, Sasidhar; Critser, Paul J.; Whittington, Catherine; Voytik-Harbin, Sherry L.; Yoder, Mervin C.; Department of Pediatrics, IU School of MedicineAngiopoietin-like 2 (ANGPTL2) has been reported to induce sprouting angiogenesis; however, its role in vasculogenesis, the de novo lumenization of endothelial cells (EC), remains unexplored. We sought to investigate the potential role of ANGPTL2 in regulating human cord blood derived endothelial colony forming cell (ECFC) vasculogenesis through siRNA mediated inhibition of ANGPTL2 gene expression. We found that ECFCs in which ANGPTL2 was diminished displayed a threefold decrease in in vitro lumenal area whereas addition of exogenous ANGPTL2 protein domains to ECFCs lead to increased lumen formation within a 3 dimensional (3D) collagen assay of vasculogenesis. ECFC migration was attenuated by 36 % via ANGPTL2 knockdown (KD) although proliferation and apoptosis were not affected. We subsequently found that c-Jun NH2-terminal kinase (JNK), but not ERK1/2, phosphorylation was decreased upon ANGPTL2 KD, and expression of membrane type 1 matrix metalloproteinase (MT1-MMP), known to be regulated by JNK and a critical regulator of EC migration and 3D lumen formation, was decreased in lumenized structures in vitro derived from ANGPTL2 silenced ECFCs. Treatment of ECFCs in 3D collagen matrices with either a JNK inhibitor or exogenous rhTIMP-3 (an inhibitor of MT1-MMP activity) resulted in a similar phenotype of decreased vascular lumen formation as observed with ANGPTL2 KD, whereas stimulation of JNK activity increased vasculogenesis. Based on gene silencing, pharmacologic, cellular, and biochemical approaches, we conclude that ANGPTL2 positively regulates ECFC vascular lumen formation likely through its effects on migration and in part by activating JNK and increasing MT1-MMP expression.Item Characterization and Assessment of Lung and Bone Marrow Derived Endothelial Cells and their Bone Regenerative Potential(2021-12) Valuch, Conner R.; Li, Jiliang; Kacena, Melissa; Marrs, JamesFracture repair is costly and difficult to treat. One of the main causations of nonunion is a lack of essential blood supply. The needed blood is supplied by the growth of new blood vessels, a process known as angiogenesis, that invade the damaged tissue early in the healing process. We proposed using bone tissue engineering as an effective therapy. This therapy uses stem cells to aid in tissue regeneration. Endothelial progenitor cells (EPCs) were selected due to their ability to form tube-like networks in vitro. EPCs were isolated from murine bone marrow and lung tissue. We tested EPC’s tube forming, proliferative, and wound migration ability in vitro. To test their ability in vivo we created a femoral fracture in young and old mice. EPCs were seeded to the fracture site upon a collagen scaffold. The in vitro studies displayed that the bone marrow and lung-derived endothelial cells presented EPC traits. In the mouse fracture model bone marrow, endothelial cells did not significantly improve the healing process. In the future, we want to improve our cell extraction and purification method, as well as test a new stem cell delivery biomaterial. We also want to select and use a growth factor (GF) that can help to promote bone regeneration in tandem with the EPCs.Item Damaging effects of cigarette smoke on organs and stem/progenitor cells and the restorative potential of cell therapy(2017-06-23) Barwinska, Daria; March, Keith L.; Basile, David P.; Broxmeyer, Hal; Clauss, Matthias; Traktuev, Dmitry O.Cigarette smoking (CS) continues to be a significant modifiable factor contributing to a variety of diseases including cardiovascular, pulmonary and renal pathologies. It was suggested that smoking have detrimental effect of the body’s progenitor cells of bone marrow and peripheral organs. Since the concept of cell therapy that utilizes adipose stem/stromal cells (ASC) is gaining momentum it becomes critical to assess the therapeutic activities of the progenitors isolated from smokers. This study has revealed that CS negatively impacts the vasculogenic potential of ASC, in vitro, as well as weakening their therapeutic activity in vivo when tested in mouse model of hindlimb ischemia. We hypothesized that the decrease in vasculogenic activity of ASC is attributed to a higher level of expression of an angiostatic factor Activin A by ASC from CS donors. These findings clearly suggest that smokers should be evaluated for potential exclusion from early clinical trials of autologous cell therapies, or assessed as a separate cohort. The donor’s health status should be considered when choosing between autologous vs allogeneic cell therapies. We then examined the effect of CS on development of kidney pathology in mice. CS exposure led to decrease in kidney weights, capillary rarefaction, and cortical blood perfusion, and in parallel led to increase in kidney fibrosis and iron deposition. Interestingly, infusion of healthy ASC to the mice following CSexposure reversed CS-induced damages. This strongly support the notion that ASC-based therapy may provide rejuvenation effect. In the other subset of studies, we hypothesized that CS-induced lung emphysematous changes are preceded by suppression of bone marrow (BM) hematopoietic progenitor cells (HPC). We have revealed that intermittent BM mobilization with AMD3100 may mitigate the CS-induced myelo-suppression and deterioration of lung function and morphology. We observed that treatment of mice with AMD3100, while exposed to CS, preserves HPC at the levels of healthy control mice. Furthermore, AMD3100 treatment preserved lung parenchyma from pathological changes. These data suggest that while CS has a myelo-suppressive effect, administration of AMD3100 preserved BM-HPC and ameliorated lung damage.Item Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices(Elsevier, 2015-09) Kim, Hyojin; Prasain, Nutan; Vemula, Sasidhar; Ferkowicz, Michael J.; Yoshimoto, Momoko; Voytik-Harbin, Sherry L.; Yoder, Mervin C.; Department of Pediatrics, IU School of MedicineHuman cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC.Item 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 MedicineVasculogenesis 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.Item 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 MedicineVasculogenesis 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.Item Kinetic vasculogenic analyses of endothelial colony forming cells exposed to intrauterine diabetes(2017-05-11) Varberg, Kaela Margaret; Haneline, Laura S.; Clauss, Matthias A.; Day, Richard N.; Harrington, Maureen A.; Srour, Edward F.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, including a tissue cytometry approach to quantify the frequency and localization of dividing ECFCs within cell networks. Additionally, FIJI TrackMate was used to quantify ECFC displacement and speed at the single cell level during network formation. These novel approaches were then applied to determine how intrauterine exposure to maternal type 2 diabetes mellitus (T2DM) impairs fetal ECFC vasculogenesis, and whether increased Transgelin 1 (TAGLN) expression in ECFCs from pregnancies complicated by gestational diabetes (GDM) was sufficient to impair vasculogenesis. Fetal ECFCs exposed to maternal T2DM formed fewer initial network structures, which were not stable over time. Correlation analyses identified that ECFC samples with greater division in branches formed fewer closed network structures and that reductions in ECFC movement decreased structural connectivity. To identify specific cellular mechanisms and signaling pathways altered in ECFCs following intrauterine GDM exposure, these new techniques were also applied in TAGLN expression studies. Similarly, ECFCs from GDM pregnancies and ECFCs overexpressing TAGLN exhibited impaired vasculogenesis and decreased migration. Both ECFCs from GDM pregnancies as well as ECFCs over expressing TAGLN exhibited increased phosphorylation of myosin light chain. Reduction of myosin light chain phosphorylation via Rho kinase inhibition increased ECFC migration; therefore, increased TAGLN was sufficient to impair ECFC vasculogenic function. Overall, identification of these novel phenotypes provides evidence for the molecular mechanisms contributing to aberrant ECFC vasculogenesis. Determining how intrauterine exposure to maternal T2DM and GDM alters fetal ECFC function will enable greater understanding of the chronic vascular pathologies observed in children from pregnancies complicated by diabetes mellitus.Item Mesenchyme homeobox 2 regulation of fetal endothelial progenitor cell function(2017-06-19) Gohn, Cassandra Rebekah; Haneline, Laura S.; Elmendorf, Jeffrey S.; Herring, B. Paul; Tune, Johnathan D.In the United States, 10% of pregnancies are complicated by diabetes mellitus (DM). Intrauterine DM exposure can have long-lasting implications for the fetus, including cardiovascular morbidity. Previously, we showed that fetal endothelial colony forming cells (ECFCs) from DM pregnancies have decreased vessel-forming ability and increased senescence. However, the molecular mechanisms responsible for this dysfunction remain largely unknown. The objective of this thesis was to understand how Mesenchyme Homeobox 2 (MEOX2) interacts with pathways that regulate cell cycle progression and migration, and how this interaction results in impaired vasculogenesis in DM exposed ECFCs. We tested the hypothesis that upregulated MEOX2 in DM-exposed ECFCs decreases network formation through impairments in senescence, cell cycle progression, migration, and adhesion. MEOX2 is a transcription factor which inhibits angiogenesis by upregulating cyclin dependent kinase inhibitors. Here, data show that nuclear MEOX2 is increased in DM-exposed ECFCs. Lentiviral-mediated overexpression of MEOX2 in control ECFCs increased network formation, altered cell cycle progression, increased senescence, and enhanced migration. In contrast, MEOX2-knockdown in DM-exposed ECFCs decreased network formation and migration, while cell cycle progression and senescence were unchanged. Adhesion and integrin expression defects were evaluated as mechanisms by which MEOX2 altered ECFC migration. While MEOX2-overexpression did not alter adhesion or cell surface integrin levels in control cells, MEOX2 overexpression in DM-exposed ECFCs resulted in an increase in α6 integrin surface expression. Similarly, MEOX2-knockdown in DM-exposed ECFCs did not alter adhesion, though did reduce α6 integrin surface expression and total cellular α6 mRNA and protein levels. Together, these data suggest that alterations in cell cycle progression and senescence are not responsible for the disrupted vasculogenesis of DM-exposed ECFCs. Importantly, these data suggest that altered migration may be a key mechanism involved and that altered cell surface levels of the α6 integrin may modify migratory capacity. Moreover, these data suggest that the α6 integrin may be a previously unrecognized transcriptional target of MEOX2. Ultimately, while initially believed to be maladaptive, these data suggest that increased nuclear MEOX2 in DM-exposed ECFCs may serve a protective role, enabling vessel formation despite exposure to a DM intrauterine environment.Item 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 MedicineFetal 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.