Angiogenesis is a normal and vital process that occurs during growth and development. Repair of bony defects, whether in the craniofacial complex or the alveolus, require an alloplastic or xenoplastic bone graft with angiogenic potential. This angiogenic potential is derived from existing blood vessels adjacent to the graft site. Improving the endogenous angiogenic potential with a molecule would drastically improve the survival rate of the bone graft material. This study was conducted to test the hypothesis that specific stem cell lines treated with erythropoietin, a positive promoter of angiogenesis, may increase the erythropoietin receptor expression in-vitro. In addition, this study also evaluated the vascular branching in vitro of human umbilical vein-derived endothelial cells treated with erythropoietin in the matrigel assay. Human umbilical vein-derived endothelial cells were treated for seven days with four concentrations of erythropoietin and cellular branching was evaluated in the matrigel assay. human bone marrow-derived mesenchymal stem cells and multi-potent cord blood derived unrestricted stromal stem cells were treated for seven days with erythropoietin and erythropoietin receptor expression was evaluated via reverse transcriptase real time polymerase chain reaction and real time polymerase chain reaction assays. The results of this study indicate that: erythropoietin had no effect on human umbilical vein-derived endothelial cells in the matrigel assay from a qualitative perspective, after treating multi-potent cord blood derived unrestricted stromal stem cells cells for 7 days with erythropoietin, there was no statistically significant difference between treatment groups when compared to control, and after treating human bone marrow-derived mesenchymal stem cells cells for 7 days with erythropoietin, the 20 U/ml treatment group showed a statistically significant reduction of the erythropoietin receptor as compared to the control group. / Biology

Our lab has recently shown that IL-19 is expressed in angiogenic ECs, opening the possibility for its use as a medicine to increase perfusion in patients with PAD. The first aim of the current study is to show IL-19’s ability to increase perfusion in vivo using C57BL/6 wild type and IL-19 KO mice in the hindlimb ischemia (HLI) model. Wild-type mice injected with 10ng/g/day of rmIL-19 after being subject to hindlimb ischemia showed significantly greater levels of perfusion than PBS injected littermates. Immunohistochemistry of harvested gastrocnemius muscle showed a greater level of capillary density in IL-19 injected mice as well. IL-19-/- mice also showed a slower recovery of perfusion in a ligated limb in addition to less CD31 positive cells in gastrocnemius muscle when compared to C57BL/6 wild type mice. IL-19 -/- mice also showed increased perfusion when injected with rmIL-19. The second aim of the study is to show more precisely if IL-19 increases angiogenesis by increasing angiogenic cytokine production, polarizing macrophage phenotype, or by influencing angiogenic and anti-angiogenic factors. Spleen, serum, and bone marrow derived macrophage (BMDM) from mouse models used in Aim 1 showed increased levels of angiogenic cytokines, decreased anti-angiogenic cytokines, and markers of M2 macrophage polarization when IL-19 was injected i.p. or present genetically. The third aim of the study examines whether or not IL-19 can increase perfusion within an atherosclerotic background. It also addresses whether IL-19 can both simultaneously reduce atherosclerosis and increase perfusion. This aim also uses mice lacking LDLR-/- genes to further evaluate these questions. LDLR-/- mice fed a high fat diet for 12 weeks underwent HLI and had perfusion levels measured using Doppler imaging in addition to four weeks of 10ng/g/day of IL-19 or PBS injections. Upon sacrifice mice also had their aortas harvested and stained for plaque measurement. This experiment seeks to demonstrate if IL-19 can increase perfusion on an atherosclerotic background. Additionally, a second set of experiments addresses if LDLR-/- mice injected with recombinant mouse IL-19 (rmIL-19) or PBS for 16 weeks on a HFD in addition to HLI being performed at week 12 showed decreased levels of plaque and increased levels of hindlimb perfusion. These experiments seek to demonstrate if IL-19 can simultaneously reduce atherosclerosis while increasing perfusion. A third set of experiments attempts to evaluate the hypothesis that double knock out mice (DKO) lacking both LDLR and IL-19 genes will have increased plaque after being fed a HFD for 16 weeks. These aims all support the overall hypothesis that IL-19 can increase angiogenesis while additionally proving to be anti-inflammatory and anti-atherogenic in vivo / Physiology

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease (CVD). We previously demonstrated that homocysteine (Hcy) suppresses endothelial cell (EC) proliferation, migration, and post-injury EC repair, but the molecular mechanism underlying Hcy-induced EC injury is unclear. In this study, we identified a novel gene, Carom, which mediates Hcy-induced suppression of EC migration and angiogenesis. We identified FCH and double SH3 domains 2 (FCHSD2), a novel gene, as an Hcy-responsive gene through Differential Display in Hcy (50µM)-treated human umbilical vein endothelial cells (HUVEC). FCHSD2 was initially named as Carom, based on the identification of this molecule as an interacting protein of calcium/calmodulin-dependent serine protein kinase (CASK) and membrane associated guanylate kinase, WW and PDZ domain containing 1 (MAGI1). In this thesis, we describe this gene as Carom. Carom belongs to the Fes/CIP4 homology and Bin/amphiphysin/Rvs (F-BAR) protein family, which is a group of multivalent adaptors linking plasma membrane and cytoskeleton, involved in endocytosis and cell migration. However, Carom's function is poorly characterized. Based on the findings that CASK and MAGI1 inhibit cell migration and growth, and the role of F-BAR proteins in cell migration, we hypothesize that Hcy up-regulates Carom to inhibit EC growth and/or migration, finally leading to CVD. We confirmed the significant induction of Carom mRNA expression in Hcy-treated HUVECs or human aortic endothelial cells (HAEC) by Northern blot and Real-time PCR. In addition, we found that Carom protein expressions were significantly increased both in Hcy-treated HAECs and lung ECs isolated from HHcy mice by Western blot using our homemade rabbit antibody against Carom. These data indicate that Hcy increases endothelial expression of Carom both in vitro and in vivo. Furthermore, in order to characterize Carom function in EC, we generated recombinant adenovirus Adv-Carom to transduce Carom for gain-of-function study and Adv-Carom-shRNA to express Carom shRNA for loss-of-function study. We found that neither adenovirus-transduced Carom expression nor adenoviral Carom shRNA had any impact on HUVEC proliferation by using [3H]-thymidine incorporation. Interestingly, we demonstrated that Adv-Carom inhibited HAEC migration, while Hcy-induced HEAC migration inhibition could be rescued by Adv-Carom-shRNA. These data suggest that Carom may inhibit angiogenesis via a cell proliferation-independent mechanism. Furthermore, we found that Hcy significantly increased the intracellular level of S-adenosyl homocysteine (SAH) but not S-adenosyl methionine (SAM), and decreased the SAM/SAH ratio, an indicator of cellular methylation, in HAECs, by using High-performance liquid chromatography/electrospray tandem mass spectrometry (HPLC-MS) to measure SAH and SAM levels. Meanwhile, Carom protein expression was significantly induced by azacytidine (AZC), a DNA methyltransferse inhibitor, in a dose-dependent manner in HAECs. Based on these data, we speculated that Hcy-induced hypomethylation could associate with Carom up-regulation. Thus we used bisulfite deep sequencing to profile methylation status of Carom gene in Hcy-treated HUVECs and found that Carom promoter was hypomethylated by Hcy. In addition, eight transcriptional factor binding sites on Carom were hypomethylated by Hcy. These data suggest that Hcy may induce Carom via a DNA hypomethylation-dependent mechanism. Moreover, we found that adenovirus-transduced Carom expression significantly increased the secretions of two anti-angiogenic chemokines, CXCL10 and CXCL11 in HAECs by using human cytokine array. Similarly, Hcy also significantly increased mRNA expressions of CXCL10 and CXCL11, while Adv-Carom-shRNA blocked down the inductions of CXCL10 and CXCL11 by Hcy. We further demonstrated that adenovirus-transduced Carom expression inhibited angiogenesis by performing tube formation assay of HAECs, whereas Hcy-induced angiogenesis suppression were rescued by Adv-Carom-shRNA as well as the neutralizing antibodies of CXCL10 and CXCL11. These data suggest that Hcy induces Carom to trigger CXCL10 and CXCL11 downstream to inhibit angiogenesis. In conclusion, Hcy induces Carom expression through DNA hypomethylation to inhibit EC migration and angiogenesis. / Pharmacology

Radiation therapy has been most commonly used modality in the treatment of brain tumors. About 200,000 patients with brain tumors are treated with either partial large field or whole brain irradiation every year in the United States. The use of radiation therapy for treatment of brain tumor, however, can subsequently lead to devastating functional deficits several months to years after treatment. Unfortunately, there are no known successful treatments and effective strategies for mitigating radiation-induced brain injury. In addition, the specific mechanisms by which irradiation causes brain injury in normal tissues are not fully understood. A deeper understanding of the molecular mechanisms underlying these phenomena could enable the development of more effective therapies to contribute to long-term disease suppression or even cure. Therefore,the primary goal of this research project was to determine the molecular mechanisms responsible for radiation-induced brain injury in normal tissues. In the first study, the effects of whole brain irradiation on pro-inflammatory pathways in the brain were examined. Results demonstrated that brain irradiation induces regionally specific alterations in pro-inflammatory environments through activation of pro-inflammatory transcription factors (e.g., activator protein-1 (AP-1),nuclear factor-κB (NF-κB), and cAMP response element-binding protein (CREB)) and overexpression of pro-inflammatory mediators (e.g., tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1)) in brain. This study provides evidence for a differential induction of pro-inflammatory mediators in specific brain regions that have importance for the neurological/neuropathological consequences of irradiation. In the second study, a mathematical model describing radiation-induced mRNA and protein expression kinetics of TNF-α in hippocampus was reconstructed. This study demonstrated that the reaction kinetic model could predict protein expression levels of TNF-α in cortex, suggesting that this model could be used to predict protein expression levels of pro-inflammatory mediators in other parts of the brain. In the third study, the effects of aging on radiation-mediated impairment of immune responses in brain were examined. Results showed that radiation-induced acute inflammatory responses, such as overexpression of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6),adhesion molecules (e.g., intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin), chemokine MCP-1, and matrix metalloproteinase-9 (MMP-9), are significantly impaired in aged brain. This study suggests that reduced production of pro-inflammatory mediators in response to irradiation compromises the normal host defense mechanisms in damaged brain tissue and subsequently leads to impaired repair/remodeling responses in old individuals. In the fourth study, the effects of irradiation on MMPs/tissue inhibitor of metalloproteinases (TIMPs) and extracellular matrix (ECM) degradation in brain were examined. Results demonstrated that whole brain irradiation induces an imbalance between MMPs and TIMPs expression, increases gelatinase activity, and degrades collagen type IV in the brain. This study suggests that a radiation-induced imbalance between MMP-2 and TIMP-2 expression may have an important role in the pathogenesis of brain injury by degrading ECM components of the blood-brain barrier (BBB) basement membrane. In the fifth study, the effects of irradiation on angiogenic factors and vessel rarefaction in brain were examined. Results demonstrated that whole brain irradiation decreases endothelial cell (EC) proliferation, increases EC apoptosis, and differentially regulates the expression of angiogenic factors such as angiopoietin-1 (Ang-1), Ang-2, Tie-2, and vascular endothelial growth factor (VEGF) in brain. This study suggests that radiation-induced differential regulation of angiogenic factors may be responsible for vessel rarefaction. In summary, the results from these studies demonstrated that whole brain irradiation induces brain injury by triggering pro-inflammatory pathways, degrading extracellular matrix, and altering physiologic angiogenesis. Therefore, this work may be beneficial in defining a new cellular and molecular basis responsible for radiation-induced brain injury. Furthermore, it may provide new opportunities for prevention and treatment of brain tumor patients who are undergoing radiotherapy. / Ph. D.

The inability to accurately reproduce the complexities of the in vivo tumor microenvironment with reductionist-based two-dimensional in vitro cell culture models has been a notable deterrent in identifying therapeutic agents that reliably translate to in vivo animal and human clinical trials. In an effort to address this, a growing number of three-dimensional (3D) in vitro tumor models capable of mimicking specific tumorigenic processes have emerged within the last decade. This concept stems from the understanding that cells cultured within 3D in vitro matrices have the ability to acquire phenotypes representative of the in vivo microenvironment. The objective of this project was to apply a tissue engineering approach towards developing a 3D in vitro tumor angiogenesis model. Initially, different scaffolds were investigated for supporting 3D tumor growth, including bacterial cellulose, electrospun polycaprolactone/collagen I, and highly porous electrospun poly(L-lactic acid). However, cancer cells cultured on these scaffolds demonstrated poor adhesion, sufficient adhesion with poor infiltration, and increased but still inadequate infiltration, respectively. Collagen I hydrogels were chosen as an appropriate scaffold for facilitating 3D in vitro tumor growth for two reasons -- cell-mediated degradation and immediate 3D cell growth. It was hypothesized that cancer cells cultured within collagen I hydrogels could be encouraged to recapitulate key characteristics of in vivo tumor progression. MDA-MB-231 human breast cancer cells were shown to experience hypoxia and undergo necrosis in response to limitations in oxygen diffusion and competition for nutrients. Upregulation of hypoxia-inducible factor-1" resulted in a significant increase in vascular endothelial growth factor gene expression. To capitalize on this endogenous angiogenic potential, microvascular endothelial cells were cultured on the surface of the designated "bioengineered tumors." It was hypothesized that paracrine signaling between tumor and endothelial cells co-cultured within this system would be sufficient for inducing an angiogenic response in the absence of exogenous pro-angiogenic growth factors. Endothelial cells in the co-culture group were shown to invasively sprout into the underlying collagen matrix, forming a capillary-like tubule network. This project culminated with the establishment of an improved in vitro tumor model that can be used as a tool for accurate evaluation and refinement of cancer therapies. / Ph. D.

Yes / Objectives Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-β and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. Methods P EDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast–endothelial cell cocultures and matrigel assays were employed to assess angiogenesis. Results P EDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-β in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-β receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. Conclusions O ur data reveal a new pathway by which TGF-β suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc. / NIHR CDF; EULAR ODP

Yes / Glioblastoma multiforme (GBM) is a grade IV brain tumor characterized by a heterogeneous population of cells that are highly infiltrative, angiogenic and resistant to chemotherapy. The current standard of care, comprised of surgical resection followed by radiation and the chemotherapeutic agent temozolomide, only provides patients with a 12–14 month survival period post-diagnosis. Long-term survival for GBM patients remains uncommon as cells with intrinsic or acquired resistance to treatment repopulate the tumor. In this review we will describe the mechanisms of resistance, and how they may be overcome to improve the survival of GBM patients by implementing novel chemotherapy drugs, new drug combinations and new approaches relating to DNA damage, angiogenesis and autophagy.

Yes / It is now well established that the tumor microenvironment plays an essential role in the survival, growth, invasion, and spread of cancer through the regulation of angiogenesis and localized immune responses. This review examines the role of the HOX genes, which encode a family of homeodomain-containing transcription factors, in the interaction between prostate tumors and their microenvironment. Previous studies have established that HOX genes have an important function in prostate cancer cell survival in vitro and in vivo, but there is also evidence that HOX proteins regulate the expression of genes in the cancer cell that influence the tumor microenvironment, and that cells in the microenvironment likewise express HOX genes that confer a tumor-supportive function. Here we provide an overview of these studies that, taken together, indicate that the HOX genes help mediate cross talk between prostate tumors and their microenvironment.

Yes / The IL-1 family member cytokine IL-36γ is recognised as key mediator in the immunopathology of psoriasis, hallmarks of which involve the activation of both resident and infiltrating inflammatory myeloid cells and aberrant angiogenesis. This research demonstrates a role for IL-36γ in both myeloid activation and angiogenesis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23 and TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. IL-36γ was also demonstrated to induce endothelial tube formation and branching, in a VEGF-A-dependent manner. Furthermore, IL-36γ-stimulated macrophages potently activated endothelial cells and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, psoriasis monocytes showed increased adherence to both the stimulated and unstimulated endothelium when compared with monocytes from healthy individuals. Collectively, these findings show that IL-36γ has the potential to enhance endothelium directed leucocyte infiltration into the skin and strengthen the IL-23/IL-17 pathway adding to the growing evidence of pathogenetic roles for IL-36γ in psoriatic responses. Our findings also point to a cellular response, which could potentially explain cardiovascular comorbidities in psoriasis in the form of endothelial activation and increased monocyte adherence. / Faculty of Life Sciences, University of Bradford. MRC, Grant/Award Number: MR/M01942X/1; British Skin Foundation, Grant/Award Number: BSF 5035.

Yes / Extracellular-signal-regulated kinase 5 (ERK5) is critical for normal cardiovascular development. Previous studies have defined a canonical pathway for ERK5 activation, showing that ligand stimulation leads to MEK5 activation resulting in dual phosphorylation of ERK5 on Thr218/Tyr220 residues within the activation loop. ERK5 then undergoes a conformational change, facilitating phosphorylation on residues in the C-terminal domain and translocation to the nucleus where it regulates MEF2 transcriptional activity. Our previous research into the importance of ERK5 in endothelial cells highlighted its role in VEGF-mediated tubular morphogenesis and cell survival, suggesting that ERK5 played a unique role in endothelial cells. Our current data show that in contrast to EGF-stimulated HeLa cells, VEGF-mediated ERK5 activation in human dermal microvascular endothelial cells (HDMECs) does not result in C-terminal phosphorylation of ERK5 and translocation to the nucleus, but instead to a more plasma membrane/cytoplasmic localisation. Furthermore, the use of small-molecule inhibitors to MEK5 and ERK5 shows that instead of regulating MEF2 activity, VEGF-mediated ERK5 is important for regulating AKT activity. Our data define a novel pathway for ERK5 activation in endothelial cells leading to cell survival. / This research was funded by grants from: North West Cancer Research (NWCR): M.J.C. and A.K.M.; Medical Research Council (MRC DiMeN PhD): M.J.C. and K.A.; Biotechnology and Biological Sciences Research Council (BBSRC DTG Studentship): M.J.C., C.E.P.G., B.W. and G.N.N.; and Wellcome Trust Institutional Strategic Fund: M.J.C. and A.K.M.