This project is intended to utilize an immobilized bio-active first generation fusion constructed cytokine inducing in receptive cell lines guided vasculogenic development. This research through the assembly, expression and purification of a bio-active molecule the CaM-VEGF120 fusion construct permitted the creation of a first generation smart-gel platform. Cell culture bringing together HUVECs or cBOECs with soluble or immobilized CaM-VEGF120 coupled with a type-I collagen platform are the main components intended to induce guided vascular sprouting. Purification of the CaM-VEGF120 was achieved utilizing HIC coupled with size exclusion chromotography. Mass Spectrometry and cellular augmentation noted by survivability and proliferation suggests the correct CaM-VEGF120 properties were achieved. Cell culture interactive changes were recorded utilizing fluorescent and phase microscopy. The 66 KDa dimeric CaM-VEGF120 was able to phosphorylate the cytoplasmic Tyr1175 localized to the C-terminal portion of the transmembrane VEGFR2. GNP immobilized CaM-VEGF120 induced VEGFR2 expressing cell lines as were imaged over a week’s period recording vascular pseudo-tube formation. These events resulting from contact with the immobilized CaM-VEGF120 and VEGFR2 induced activity thus presenting in vitro guided vascular pseudo-tube development. This research is being pursued utilizing HUVEC and cBOECs as guided vascular pseudo-tube structural formation is possible. This successful model implies a first generation model for physiological vascular development having therapeutic applications.

This project is intended to utilize an immobilized bio-active first generation fusion constructed cytokine inducing in receptive cell lines guided vasculogenic development. This research through the assembly, expression and purification of a bio-active molecule the CaM-VEGF120 fusion construct permitted the creation of a first generation smart-gel platform. Cell culture bringing together HUVECs or cBOECs with soluble or immobilized CaM-VEGF120 coupled with a type-I collagen platform are the main components intended to induce guided vascular sprouting. Purification of the CaM-VEGF120 was achieved utilizing HIC coupled with size exclusion chromotography. Mass Spectrometry and cellular augmentation noted by survivability and proliferation suggests the correct CaM-VEGF120 properties were achieved. Cell culture interactive changes were recorded utilizing fluorescent and phase microscopy. The 66 KDa dimeric CaM-VEGF120 was able to phosphorylate the cytoplasmic Tyr1175 localized to the C-terminal portion of the transmembrane VEGFR2. GNP immobilized CaM-VEGF120 induced VEGFR2 expressing cell lines as were imaged over a week’s period recording vascular pseudo-tube formation. These events resulting from contact with the immobilized CaM-VEGF120 and VEGFR2 induced activity thus presenting in vitro guided vascular pseudo-tube development. This research is being pursued utilizing HUVEC and cBOECs as guided vascular pseudo-tube structural formation is possible. This successful model implies a first generation model for physiological vascular development having therapeutic applications.

No description available.

Each step of vascular development needs to be carefully regulated; endothelial precursors must be specified, these cells then proliferate and coalesce to form vascular cords, and finally they lumenate, undergo angiogenic branching and remodeling, and recruit smooth muscle cells to establish a mature vessel. An aberration at any of these steps during embryonic development is incompatible with life, and vascular pathologies in the adult are associated with numerous diseases including stroke, arteriosclerosis, diabetic retinopathies and cancer progression. My work has aimed to understand how endothelial precursors are specified, and more precisely the cell-signaling pathways and transcriptional networks that guide their fate. This work leads us to conclude the following: (1) blood island precursor cells in the Xenopus embryo can give rise to either blood or endothelial cells, and it is BMP-mediated activation of the erythroid transcriptional program that regulates cell fate, (2) endothelial specification requires the Ets transcription factor Etv2. Persistence of Etv2 expression in blood/endothelial cell precursors allows these cells to develop into endothelium, and overexpression of Etv2 in any of the three germ layers causes activation of every endothelial marker examined. Along the way we have characterized a number of small-molecule inhibitors that should be useful to the Xenopus community and applicable to other model systems.

Indiana University-Purdue University Indianapolis (IUPUI) / 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.

Indiana University-Purdue University Indianapolis (IUPUI) / Fracture 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.

<p>The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.</p> / Dissertation

The formation of the embryonic vasculature is essential for life. The components driving this process are well conserved across vertebrate species. At the core of vascular development is the specification of endothelial precursor cells from nascent mesoderm. Transcription factors of the ETS family are important regulators of endothelial specification. In this document we characterize the role of the ETS transcription factors, ETV2, during embryonic vascular development.Expression analysis shows that Etv2 is highly expressed in hematopoietic and endothelial precursor cells in the Xenopus embryo. In gain-of-function experiments, ETV2 is sufficient to activate ectopic expression of vascular endothelial markers. In addition, ETV2 activated expression of hematopoietic genes representing the myeloid but not the erythroid lineage. Loss-of-function studies indicate that ETV2 is required for expression of all endothelial markers examined. However, knockdown of ETV2 has no detectable effects on expression of either myeloid or erythroid markers. This contrasts with studies in mouse and zebrafish where ETV2 is required for development of the myeloid lineage. Our studies confirm an essential role for ETV2 in endothelial development, but also reveal important differences in hematopoietic development between organisms.Although ETV2 is a pivotal molecule in development it remains unidentified in the chicken genome. We hypothesize that chicken Etv2 is expressed in the early Gallus embryo, and is necessary for endothelial specification consistent with its role in other species. To test this hypothesis we attempted to amplify Etv2 transcripts from Gallus embryos using degenerate PCR. Disappointingly this strategy did not reveal a putative Etv2 candidate. However, some important findings were uncovered, including the cloning of a previously uncharacterized Gallus ETS protein, SPDEF. Additionally the identification of an annotation error mis-identifying Ets gene "Erf" as "Etv3" (also an Ets gene) provided details on gene arrangement previously unknown. The workflow described could be used in future studies for the identification of other members of gene families that exhibit gaps, keeping in mind the goal of the study and the limitations of each technology.

Vasculogenic cell-based therapy combined with tissue engineering is a promising revascularization strategy for patients with hibernating myocardium, a common clinical condition. We used a clinically relevant swine model of hibernating myocardium to examine the benefits of biopolymer-supported delivery of circulating angiogenic cells (CACs) in this context. Twenty-five swine underwent placement of an ameroid constrictor on the left circumflex artery (LCx). After 2 weeks, positron emission tomography measures of myocardial blood flow (MBF) and myocardial flow reserve (MFR) were reduced in the affected region (both p<0.001). Hibernation (mismatch) was specific to the LCx territory. Swine were randomized to receive intramyocardial injections of PBS control (n=10), CACs (n=8), or CACs + a collagen-based matrix (n=7). At follow-up, stress MBF and MFR were increased only in the cells+matrix group (p<0.01), and mismatch was lower in the cells+matrix treated animals (p=0.02) compared to controls. Similar results were found using microsphere-measured MBF. Wall motion abnormalities and ejection fraction improved only in the cells+matrix group. This preclinical swine model demonstrated ischemia and hibernation, which was improved by the combined delivery of CACs and a collagen-based matrix. To our knowledge, this is the first demonstration of the mechanisms and effects of combining progenitor cells and biopolymers in the setting of myocardial hibernation, a common clinical condition in patients with advanced coronary artery disease.

Vasculogenic cell-based therapy combined with tissue engineering is a promising revascularization strategy for patients with hibernating myocardium, a common clinical condition. We used a clinically relevant swine model of hibernating myocardium to examine the benefits of biopolymer-supported delivery of circulating angiogenic cells (CACs) in this context. Twenty-five swine underwent placement of an ameroid constrictor on the left circumflex artery (LCx). After 2 weeks, positron emission tomography measures of myocardial blood flow (MBF) and myocardial flow reserve (MFR) were reduced in the affected region (both p<0.001). Hibernation (mismatch) was specific to the LCx territory. Swine were randomized to receive intramyocardial injections of PBS control (n=10), CACs (n=8), or CACs + a collagen-based matrix (n=7). At follow-up, stress MBF and MFR were increased only in the cells+matrix group (p<0.01), and mismatch was lower in the cells+matrix treated animals (p=0.02) compared to controls. Similar results were found using microsphere-measured MBF. Wall motion abnormalities and ejection fraction improved only in the cells+matrix group. This preclinical swine model demonstrated ischemia and hibernation, which was improved by the combined delivery of CACs and a collagen-based matrix. To our knowledge, this is the first demonstration of the mechanisms and effects of combining progenitor cells and biopolymers in the setting of myocardial hibernation, a common clinical condition in patients with advanced coronary artery disease.