The mesenchyme homeobox genes, MEOX1 and MEOX2, encode homeodomain transcription factors. Studies of Meox1/Meox2 knockout mice established that these proteins are partially redundant during development, suggesting that they may regulate common target genes. In the adult vasculature, MEOX2 is expressed in vascular smooth muscle and endothelial cells. MEOX2 has been demonstrated to: i) inhibit proliferation, ii) activate apoptosis and iii) induce senescence. In contrast, the role of MEOX1 has not been studied in the vasculature. Currently, there are two known target genes of MEOX2: cyclin-dependent kinase inhibitor 1A (CDKN1A/p21CIP1/WAF1) and cyclin-dependent kinase inhibitor 2A (CDKN2A/p16INK4a), which regulate transient (quiescent) and permanent (senescent) cell cycle arrest. Senescence is postulated to contribute to the development of atherosclerotic vascular disease by promoting endothelial dysfunction. We hypothesized that MEOX1 and MEOX2 would activate both p21CIP1/WAF1 and p16INK4a expression, as well as induce apoptosis, cell cycle arrest and senescence in endothelial cells. Furthermore, we postulated that the majority of newly identified MEOX target genes in endothelial cells would be regulated by both MEOX1 and MEOX2. MEOX proteins were expressed in human endothelial cells via adenoviral transduction. Levels of target gene expression were measured by luciferase reporter gene assays, western blot and quantitative real-time PCR. Electrophoretic mobility shift assays were used to demonstrate MEOX binding to DNA. Cellular proliferation, senescence, and apoptosis were evaluated. For the identification of novel target genes, microarrays were used to compare levels of gene expression in endothelial cells transduced with MEOX constructs or control virus. Both MEOX1 and MEOX2 activated p21CIP1/WAF1 and p16INK4a gene transcription, inhibited proliferation and induced apoptosis and senescence in endothelial cells. MEOX activation of p21CIP1/WAF1 transcription occurs via a DNA-binding independent mechanism that requires the SP1 transcription factor. In contrast, MEOX activation of p16INK4a transcription is dependent upon DNA-binding. Microarray analysis revealed that both MEOX1 and MEOX2 increased the expression of intercellular adhesion molecule 1 (ICAM-1) and decreased the expression of nitric oxide synthase 3 (NOS3/eNOS). Taken together, we conclude that MEOX1 and MEOX2 have similar target genes in endothelial cells including p21CIP1/WAF1, p16INK4a and eNOS. As increased endothelial senescence and decreased nitric oxide production are hallmarks of endothelial dysfunction, this study proposes a role for the MEOX proteins in the progression of atherosclerotic vascular disease.

The objective of this study was to develop a fluorescent near-infrared endothelial cell binding conjugate using Lycopersicon esculentum lectin and Cy5.5 N-hydroxysuccinimide ester for the purpose of imaging the microvascular network in mouse hearts under in vivo and ex vivo conditions. Cy5.5-lectin conjugate was synthesized with a dye/protein ratio of 2.90 ± 1.54 (n=6). Mouse hearts were successfully labelled in both in vivo and ex vivo and showed similar labelling patterns. Cy5.5-lectin labelling patterns and that of ICAM2 and FITC-lectin co-localized, indicating binding to endothelial cells. Finally, it was shown that Cy5.5-lectin is capable of visualizing, in real-time, areas of normal and abnormal heart perfusion at resolutions of 76.8 pixels/mm. Areas of the heart that were not perfused post-ligation displayed no Cy5.5 staining on histological sections and during real-time cardiac imaging of intact hearts showed minimal fluorescent signal (~35 a.u.) compared to areas where normal perfusion occurred (~150 a.u.).

The mesenchyme homeobox genes, MEOX1 and MEOX2, encode homeodomain transcription factors. Studies of Meox1/Meox2 knockout mice established that these proteins are partially redundant during development, suggesting that they may regulate common target genes. In the adult vasculature, MEOX2 is expressed in vascular smooth muscle and endothelial cells. MEOX2 has been demonstrated to: i) inhibit proliferation, ii) activate apoptosis and iii) induce senescence. In contrast, the role of MEOX1 has not been studied in the vasculature. Currently, there are two known target genes of MEOX2: cyclin-dependent kinase inhibitor 1A (CDKN1A/p21CIP1/WAF1) and cyclin-dependent kinase inhibitor 2A (CDKN2A/p16INK4a), which regulate transient (quiescent) and permanent (senescent) cell cycle arrest. Senescence is postulated to contribute to the development of atherosclerotic vascular disease by promoting endothelial dysfunction. We hypothesized that MEOX1 and MEOX2 would activate both p21CIP1/WAF1 and p16INK4a expression, as well as induce apoptosis, cell cycle arrest and senescence in endothelial cells. Furthermore, we postulated that the majority of newly identified MEOX target genes in endothelial cells would be regulated by both MEOX1 and MEOX2. MEOX proteins were expressed in human endothelial cells via adenoviral transduction. Levels of target gene expression were measured by luciferase reporter gene assays, western blot and quantitative real-time PCR. Electrophoretic mobility shift assays were used to demonstrate MEOX binding to DNA. Cellular proliferation, senescence, and apoptosis were evaluated. For the identification of novel target genes, microarrays were used to compare levels of gene expression in endothelial cells transduced with MEOX constructs or control virus. Both MEOX1 and MEOX2 activated p21CIP1/WAF1 and p16INK4a gene transcription, inhibited proliferation and induced apoptosis and senescence in endothelial cells. MEOX activation of p21CIP1/WAF1 transcription occurs via a DNA-binding independent mechanism that requires the SP1 transcription factor. In contrast, MEOX activation of p16INK4a transcription is dependent upon DNA-binding. Microarray analysis revealed that both MEOX1 and MEOX2 increased the expression of intercellular adhesion molecule 1 (ICAM-1) and decreased the expression of nitric oxide synthase 3 (NOS3/eNOS). Taken together, we conclude that MEOX1 and MEOX2 have similar target genes in endothelial cells including p21CIP1/WAF1, p16INK4a and eNOS. As increased endothelial senescence and decreased nitric oxide production are hallmarks of endothelial dysfunction, this study proposes a role for the MEOX proteins in the progression of atherosclerotic vascular disease.

In normal human somatic cells, the length of telomeres (chromosomal end structures) decreases with each cell division until reaching a critically short length, which halts cell proliferation and induces senescence. The enzyme telomerase, which functions to maintain telomeres at a length that is permissive for cell division, is expressed in approximately 85% of cancer cells and some stem and progenitor cells, including haematopoietic progenitor cells (HPCs), but not most other normal somatic cells. Previous investigations have demonstrated that ectopic expression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity, resulting in telomere elongation in some normal human cell types. However, similar experiments performed in HPCs and endothelial cells have demonstrated a dissociation between the expression of telomerase activity and telomere lengthening. This thesis is focussed on further investigating telomerase-mediated telomere length regulation in HPCs and endothelial cells. Short telomeres in bone marrow and blood leukocytes are associated with the development of disorders linked to bone marrow failure. However, to date a relationship between telomere length and myeloid cell proliferative potential has not been demonstrated. In the current investigations, the telomere length and proliferative potential of 31 cord blood-derived HPCs was determined. Regression analysis revealed a significant correlation between mean telomere length and erythroid cell expansion, but not expansion of other myeloid lineage cells. Another novel finding was that telomerase activity was upregulated in lineage-committed CD34- erythroid cells that were positive for the erythroid-specific lineage marker glycophorin A. It was also functionally demonstrated that telomerase activity facilitates the maximum expansion of erythroid cells. To address the dissociation between telomerase activity and telomere maintenance in BMECs, a dominant negative mutant of the telomere binding protein TRF1, which functions to regulate telomere accessibility, was over-expressed in hTERT-transduced BMECs. These studies showed that telomere access, as well as oncogene expression and exposure to oxidative stress, contribute to telomere length regulation in BMECs. Overall, the results from these investigations demonstrate for the first time the functional significance of telomere length and telomerase activity in ex vivo expansion of erythroid cells, and provide novel insight to the molecular complexity of telomere length maintenance in endothelial cells.

Thesis (Ph. D.)--Cornell University, January, 2008. / Vita. Includes bibliographical references (leaves 96-108).

Endothelial cells (ECs) are part of almost every human organ, and disturbance in their function can cause several human diseases such as pulmonary arterial hypertension. We sought to develop an in vitro model to derive ECs from human induced pluripotent stem cells (hiPSCs) in an effort to develop future regenerative therapies or disease models. By stimulating signaling pathways gleaned from prior studies of the early embryonic development of endothelial cells and their precursor germlayer, mesoderm, hiPSCs were differentiated in vitro using a media containing 10ng/ml of Activin, FGF2, VEGF and BMP4 to induce mesoderm over 4 days, followed by VEGF and FGF2 to subsequently specify ECs. By day 12, putative endothelial cells identified by the co-expression of CD31, CD144 and KDR, emerged and could be sorted and re-plated in endothelial maintenance media for expansion over several passages. Time series characterization of mRNA and protein gene expression indicates that to form endothelial cells, hiPSCs developmentally differentiate first into a posterior primitive streak-like stage (indicated by high T and low FOXA2 expression), then into a heterogeneous population of mesodermal subsets (day 4). Subsequently, lateral plate mesoderm cells predominate over intermediate or paraxial mesoderm. To understand the role of BMP4 in this process, each factor was withheld from various stages of the protocol. Results indicate that before day 4, BMP4 alone is necessary and sufficient to direct cells through posterior primitive streak into mesodermal progenitors that upon subsequent VEGF/FGF2 exposure can give rise to CD31+CD144+KDR+ endothelial-like cells. In contrast, BMP4 is dispensable after day4, whereas VEGF and FGF2 are dispensable in the first 4 days of the protocol. To develop a disease model hiPSCs from patients with BMPR2 mutations associated with pulmonary hypertension were generated by reprogramming their archived fibroblasts. By differentiating these patient-specific iPSCs into mesodermal progenitors and ECs using our protocol, preliminary results indicate that the induction of mesodermal cells by day 4 was diminished in cells carrying BMPR2 mutations,, but whether their efficiency of differentiation into ECs is affected by these mutations remains in question. It can be concluded from this project, that we develop an in vitro differentiation model that replicates the developmental pathways of mesodermal and EC derivation in embryos, and suggests that BMP4 is necessary and sufficient to derive mesodermal subsets with EC competence but is dispensable for subsequent endothelial lineage specification. / 2018-09-26T00:00:00Z

Physiological electrical fields (EFs) can direct some important angiogenic responses of endothelial cells such as directional migration, orientation and proliferation. It has been reported that human umbilical vein endothelial cells (HUVEC) and human microvasculature endothelial cells (HMEC) migrate in opposite directions; to anode and cathode, respectively. Although, in the present study both cell types migrated toward the cathode, HUVEC directedness started at 50mV/mm while HMEC directedness started at 100mV/mm. These results suggest that EFs can promote wound healing by directing endothelial cells to the wound site since EFs of 40 to 100 mV/mm are present in normal healing wounds. EFs also increased cell proliferation and orientated the cleavage plane of dividing cells perpendicular to the EF vector in both endothelial cell lines. The present study showed for the first the time that EFs upregulated the expression of the chemokine receptors CXCR4 and CXCR2 as well as upregulating the levels of phosphorylation of both chemokines in HUVEC and HMEC. It also showed differences of chemokine receptors used by HUVEC and HMEC cells in the early stages of electrotaxis. Ionizing radiation has been shown to directly phosphorylate VEGF receptors in the absence of its ligand VEGF. A question was raised: in the absence of the ligands are EFs able to directly phosphorylate the chemokine receptors? Results showed that in starved HUVEC cells EFs had no effect on the phosphorylation levels of CXCR4 and CXCR2 however in starved HMEC cells an EF may have a direct effect on the phosphorylation levels of CXCR4 and CXCR2. Therefore, EFs represent a physical stimulus that could directly phosphorylate proteins in the absence of its ligand. This work substantiate the importance of endogenous EFs in directing endothelial cells and suggests that EFs might be developed as a component in the clinic to control angiogenesis.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontics (RE) is a treatment alternative for the infected immature tooth to establish an environment in the canal that enables continued root development and the growth of pulp or pulp-like tissue within the canal. A scaffold created in the canal encourages the formation of vital tissue. The porcine sub-intestinal-submucosa (SIS) membrane, Dynamatrix®, has the potential to serve as an endodontic scaffold. Research at Indiana University School of Dentistry (IUSD) has shown that Dynamatrix® can support the growth of human dental pulp stem cells (HDPSC) and human pulp fibroblasts (HPF). Positive angiogenic cytokine profiles were seen after these cells were seeded on Dynamatrix®. Endothelial cells play an important role in the formation of blood vessels and are a source of angiogenic cytokines. Exposure of these cells to DynaMatrix® may result in a positive angiogenic profile for both cytokines and matrix metalloproteinases (MMPs). Objective: The aim of this in-vitro study was to investigate if the exposure of human endothelial cells to the DynaMatrix® membrane would result in differences in the expression of cytokines and MMPs that play roles in angiogenesis. Materials and Methods: Human endothelial cells (HUVECs) were obtained from American Type Culture Collection (ATTC, Manassas, VA) and used in this study. Groups were established as follows: (a) Group 1: HUVECs seeded in culture media only, (b) Group 2: DynaMatrix® membrane incubated alone in the serum-media without any cells, and (c) Group 3: HUVECs seeded on DynaMatrix® membranes. After 72 hours of incubation, the conditioned media were collected and analyzed for the expression of 20 angiogenic cytokines and MMPs utilizing cytokine and MMP protein arrays. The density of each cytokine and MMP expressed was measured, averaged, and statistically analyzed by ANOVA. Results: Exposure of human umbilical vein endothelial cells (HUVECs) to the DynaMatrix® membrane resulted in a positive angiogenic profile for both cytokines and MMPs. Conclusion: This work furthers the evidence for the potential of DynaMatrix® to serve as a more predictable scaffold in RE.

No description available.