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The well conserved Notch signalling pathway plays a crucial role in vascular development and physiology. Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key notch ligands implicated in angiogenesis. Both ligands were shown to have opposite effects on vasculature. DLL4-Notch signalling inhibits sprouting resulting in fewer but better perfused blood vessels, promoting tumour growth. In contrast to DLL4, very little is known about JAG1- Notch signalling in tumour angiogenesis and its influence on tumour growth and progression. The overall aim of this work is to study the functional difference between DLL4 and JAG1-Notch signalling. The effects of murine DLL4 and murine JAG1 over-expression on tumour growth and angiogenesis was also investigated in a mouse U87 xenograft model. Firstly, the downstream target genes of DLL4 and JAG1-Notch signalling were established through microarray and QPCR. Angiogenic assays such as sprouting, network formation and migration assays were employed to study the functional effects of these two ligands in endothelial cells. The thesis firstly demonstrates that JAG1 has opposing effects on endothelial cells compared to DLL4 by increasing sprout coverage and network formation. JAG1 is less potent than DLL4 in stimulation of Notch target genes in primary endothelial cell (HUVEC) but both displayed equal potency in HMEC-1, an immortalised endothelial cell line. The growth of U87 cell lines which over-expressed murine DLL4 or murine JAG1 was slower compared to wild-type U87 cell line in vitro. JAG1- and DLL4- Notch signaling have different effects on vessel formation, which impacted on the tumour growth in vivo. Interestingly, tumours over-expressing mDLL4 had less but larger vessels compared to control, whereas mJAG1 produced more yet functional vessels; both tumours had significantly reduced pericyte coverage. Both U87 mDLL4 and mJAG1 over-expressing tumours showed increased resistance towards anti-VEGF therapy, compared to control tumours. Sensitivity to therapy was restored in combinational treatment with DBZ and bevacizumab. The mechanism behind the differential responsiveness of the Notch receptors to DLL4 or JAG1 ligands could either reflect modulation by fringes, a family of glycosyltransferases that regulate Notch signalling or by a positive feedback loop present for DLL4-Notch signalling only. Fringe was found to be abundantly expressed in endothelial cells and highly vascularised tumours. This work has highlighted some key novel differences between the two Notch Ligands.

Cilengitide was anticipated to be a highly promising potential anti-angiogenic small molecule drug but unfortunately it failed phase III clinical trial in early 2013 (survival rate not passed). Today, there is still not a small molecule αvβ3 integrin drug in use in the clinic, new antagonists were urgently needed to advance the treatment of the αvβ3 integrin related diseases. / Peptide-type antagonists possess unique advantages such as non-accumulative, bio-friendly etc. comparing to other types of antagonists (peptidomimetics, non-peptidic etc.). Cilengitide is one of the peptide-type antagonists and it remains the most successful drug candidate demonstrated. Reinvestigation of the binding between Cilengitide and the αvβ3 integrin is therefore still meaningful. Specifically, the present study will concentrate on revealing the factors responsible for higher binding affinity of Cilengitide, comparing to the parent compound c(RGDfV), c(RADfV) and other N-methylated derivatives of c(RGDfV). Note that c(RADfV) could be also viewed as a methylation product of c(RGDfV) (methylation of side chain of the -G- residue). It is intended that the results of this investigation could provide clear guidance towards future efforts in the design of new peptide αvβ3 integrin antagonists. / Utilizing a novel but successful computational protocol, namely an integrated docking, molecular dynamics simulation and MM_GBSA free energy calculation method, the present thesis found that the binding profile between Cilengitide and the αvβ3 integrin is unique when compared to bindings of other methylation compounds. On the one hand, structural analysis (such as RMSD, ligand structure, hydrogen bond, backbone and side chain orientation) revealed that only c(RGDfV) and Cilengitide substantially retain the binding mode of the parent compound c(RGDfV) (Cilengitide is closer than c(RGDfV)). On the other hand, calculated binding energy results revealed that only c(RGDfV) and Cilengitide bind more strongly than c(RGDfV) to the αvβ3 integrin. Since in experiments only Cilengitide binds more strongly than c(RGDfV), it is therefore believed that both structural and energetic factors are responsible for the high binding affinity of Cilengitide. / Other energetic study revealed that the high binding affinity of Cilengtide compared to c(RGDfV) originates from Coulombic interaction (sum of electrostatic interaction and polar solvation energy), while van der Waals interaction and non-polar solvation energy was not favorable for Cilengitide binding. For the residue contribution, energy changes on the -R- and -G- residues upon methylation were nearly zero. Changes on the -D- and -f- residues were favorable for Cilengitide binding, while change on the -V- residue was not. Total changes on the five residues are favorable however. Pair-wise analysis suggests that interactions of the -D- residue in Cilengitide were very important for the binding, as suggested by its sizeable coupling energies with other residues. Amongst them the coupling between -D- and Mg688 is the most important pair. / In brief, the present study provides a quantitative understanding towards the binding between Cilengitide series ligand and the αvβ3 integrin. Through comparison with bindings between the methylated analogues of c(RGDfV), important features responsible for high binding affinity of Cilengitide were revealed. Some of the results contained in the thesis are in the process of being reported in "C. Yan, S. C. F. Au-Yeung. Investigation of the 'Methyl Effect' in the Cilengitide Binding Ligand Series and Its Implications on Future Integrin Antagonist Design. J. Med. Chem., in preparation (2014)". / Cilengitide是一种曾被高度寄望成为抗血管增生小分子药物的化合物。遗憾的是，2013年初所述化合物没有通过临床三期测试 (存活率不过关)。由于至今临床上仍然没有αvβ3整合素的小分子药物在使用，新的拮抗剂需要被设计出来以推进αvβ3整合素相关疾病的治疗。 / 相比于其它类型的拮抗剂 (类肽物、非肽类等)，多肽型的拮抗剂具有其独特的优点，譬如不积聚、生物友好等。Cilengitide是一种多肽型的拮抗剂，至今为止其仍是已证明的最成功的药物候选。重新研究Cilengitide与αvβ3整合素的结合情况因此仍然具有意义。具体地，本研究将聚焦于揭示相比于母体化合物c(RGDfV)、c(RADfV) 和c(RGDfV) 其它N-甲基化衍生物Cilengitide高亲和性的因素。c(RADfV) 可视为c(RGDfV) 另一甲基化的产物（-G- 残基侧链甲基化）。本研究的结果希望能给未来新型多肽型αvβ3整合素拮抗剂的设计以清晰的指导。 / 通过采用一种新的但颇成功的计算协议，即一种分子对接、分子动力学和MM_GBSA的综合方法，本论文发现相比于上述非Cilengitide的配体，Cilengitide与 αvβ3整合素的结合模式非常独特。一方面，结构分析 (RMSD、配体结构、氢键和骨架及侧链取向) 显示只有c(RGDfV) 与Cilengitide保留了其母体化合物，即c(RGDfV) 的结合模式（Cilengitide比c(RGDfV) 更接近）。另一方面，结合能计算结果显示只有Cilengitide和c(RGDfV) 与αvβ3整合素的结合比母体化合物c(RGDfV) 更强。由于实验上只有Cilengitide的结合强于c(RGDfV)，因此有理由相信结构和能量因素均对Cilengitide高亲和性负责。 / 其它能量研究显示相比于c(RGDfV)，Cilengitide的高亲和性来源于库仑作用 (静电作用和极性溶剂化能的加和)。范德华作用和非极性溶剂化能则对Cilengitide结合不利。残基贡献方面，甲基化后 -R- 和 -G- 的能量变化基本为零；-D- 和 -f- 残基的能量变化则有利于Cilengitide的结合，而 -V- 能量变化则不利。然而这五个残基的总变化对结合是有利的。配对分析结果显示Cilengitide中 -D- 残基的作用对于结合是非常重要的，这从其与其它残基可观的耦合能可以得知。其中，-D- 与Mg688的耦合是最重要的一对。 / 简言之，本研究提供了对Cilengitide系列配体与αvβ3整合素结合的定量理解。通过c(RGDfV) 甲基化后同类物的结合相互间的比较，揭示了负责Cilengitide高亲和性的重要特征。本论文中的一些研究结果正将报道于： / “严长青，欧阳植勋。关于Cilengitide配体系列中“甲基效应”的研究及其对未来αvβ3整合素拮抗剂设计的启示。药物化学杂志，准备中(2014)” / Yan, Changqing. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 138-146). / Abstracts also in Chinese. / Title from PDF title page (viewed on 18, January, 2017). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

archives@tulane.edu / Models that mimic angiogenesis are extremely valuable for elucidating underlining mechanisms and pre-clinical development of therapies. Angiogenesis, defined as the growth of new blood vessels from preexisting vessels, is a multi-cellular process that involves the temporal and spatial coordination between endothelial cells, pericytes, nerves, growth factors, and macrophages. A need exists for biomimetic models that bridge the gap between the mechanistic control of in vitro constructs and the multi-system physiological environment of in vivo models. To meet this need our lab has introduced the rat mesentery culture model as top down approach with intact microvascular networks and a nearly two-dimensional view. Previous development of the model has proven its time-lapse, angiogenic, and lymphangiogenic capabilities. The goal of this work is to advance the model to include the maintenance of peripheral nerves in culture and develop it as a platform for aging and cell therapy studies. The first aim of this study was to expand the rat mesentery culture model to maintain nerves and the spatiotemporal relationship between nerves and blood vessels in culture. We developed a nerve culture media to prevent regression of nerves. Nerve alignment was maintained at the network feeding arteriole and capillary plexus levels. We further demonstrated the presence of neurotransmitter precursors was preserved. We demonstrate for the first time the ability to maintain adult peripheral nerves in an ex vivo model. For the second aim of this study, we developed an aging rat mesentery culture model as a basis for investigating differences in angiogenesis across age groups. We demonstrated that impaired angiogenesis associated with advanced age could be recovered to adult-like levels with serum and individual growth factor stimulation. The discovery of increased vascular island frequency in aged tissues reveals that the method of angiogenesis for older networks can differ. These results establish the rat mesentery culture model as a method for studying aging effect on angiogenesis. The objective of the third aim was to demonstrate the capability of the rat mesentery culture model to study stromal vascular fraction therapy. We developed a protocol to isolate the SVF from adipose tissue and transplant onto the mesentery. We identified unique patterns of vasculogenesis and increased vascular coverage. We confirmed that this increase in vascular area was a combination of the vasculogenesis of SVF, proangiogenic effect on host vessels, and incorporation of SVF into the growing host vessels. We used the aging model in developed in the second aim to show that adult SVF on adult tissue has the greatest therapeutic potential. These results display the investigative potential of the rat mesentery culture model in cell therapy research. This work establishes for the first time, to the best of our knowledge, an ex vivo model capable of maintaining adult peripheral nerves. We demonstrate that angiogenesis can be rescued in aging scenarios. The results, for the first time, reveal the effect that SVF therapy has on preexisting networks as well as how it integrates during microvascular remodeling. / 1 / Nicholas Hodges

archives@tulane.edu / 1 / Jessica Margaret Mary Motherwell

Peripheral artery disease (PAD) and osteoporosis have recently been shown to be associated; with the parallel occurrence suggesting that PAD related ischemia may cause or enhance the onset of osteoporosis. In order to determine the mechanism linking osteoporosis and PAD this paper will examine the effects of ischemia and mechanical stimulation on bone remodeling. An immunohistochemistry protocol for vessel marking in bone was also developed. Ischemia was induced in a mouse model to determine vascular and mechanical property changes in bone in response to hypoxia, and mechanical loading-induced remodeling was analyzed for vascular changes. Both ischemia and mechanical loading increased bone vessel density, with ischemic bone increases seen at day 7 and 14. Bone stiffness increased after induced ischemia at day 28. These results point to resultant hypoxia from ischemia drives bone mechanical property changes, likely through stimulation of bone remodeling. We also conclude that an increase in vessel density is seen after induced mechanical loading of bone. Establishing the vascular contribution to the remodeling process may reveal treatment opportunities for remodeling-dependent pathologies such as osteoporosis.

As anti-cancer agents, retinoid induce cell growth arrest and differentiation, while HDACIs cause cell differentiation, growth inhibition, death and inhibit angiogenesis in many cancer types. However, a proportion of patients respond poorly to these therapies. My studies, presented here, aimed to improve the anti-cancer effects of these agents by identifying key factors which mediate cancer cell sensitivity or resistance to their action. In this study I have found that the clinically used retinoid, 13-cis RA, exerts its anti-cancer signal in a manner similar to atRA, by modulating the transcriptional response of retinoid-regulated genes. HDACI-induced cytotoxicity is significantly enhanced when combined with IFNα in 8 out of 9 cancer cell lines from various organ origins. Sensitivity to the combination treatment correlated with an absence of basal p21 protein expression, and cell cycle arrest. Knocking-down p21 gene expression further sensitized cancer cells to the combination therapy. Moreover, IFNα and HDACI co-operatively inhibited pro-angiogenic gene expression in cancer cells, and the combination therapy decreased endothelial cell migration, invasion, and capillary tubule formation. Further experiments on p21 as a resistance factor to anti-cancer treatment demonstrated that conditioned media from breast cancer MCF-7 cells transfected with p21 siRNA, induced significantly less endothelial cell migration, invasion and vascular sprouting, compared with media from cells transfected with scrambled siRNA. LC/MS analysis of the conditioned media revealed that Trx secretion was significantly reduced after p21 knockdown. The reduction in Trx secretion following p21 knockdown was due to a direct effect of p21 siRNA on the expression of intracellular TBP2 in neuroblastoma, prostate and lung cancer cells. Consistent with this result, media from MCF7 cells transfected with TBP2-specific siRNA alone, promoted endothelial cell invasion and vascular sprouting, Trx knockdown resulted in opposite effects, and the anti-angiogenic effect of p21 siRNA was offset by simultaneous TBP2 siRNA transfection. ChIP assay revealed that p21 directly bound to an E2F1-bindng site in the TBP2 gene promoter. These data indicate that p21 promoted tumour-driven angiogenesis through transcriptional repression of TBP2. Collectively, my experiments indicate several potential treatment targets directed toward enhancing the effectiveness of HDACIs and retinoids.

Le psoriasis est une pathologie cutanée chronique fréquente qui semble déterminée par des facteurs génétiques multiples et par des facteurs environnementaux. L'expansion vasculaire est un processus précoce lors du développement des plaques de psoriasis. La lymphangiogenèse a jusqu'alors été peu étudiée dans le développement de ces lésions bien que l'expansion du réseau lymphatique y soit histologiquement décrite. Des anomalies dans les systèmes régulant ces processus pourraient faire partie de la pathogénie du psoriasis. Nous avons étudié les réseaux vasculaires et lymphatiques ainsi que lexpression de facteurs régulant leur expansion dans le psoriasis vulgaire en plaques. Nos résultats montrent une surexpression de l'ARNm du VEGF-A 121 et du VEGFR3 dans la peau non lésionnelle des patients atteints de psoriasis sans altération histologique décelable par rapport à la peau de volontaires sains. En outre, l'étude de lésions débutantes de type pinpoint en comparaison des plaques établies de psoriasis montre que l'expansion lymphatique suit l'expansion vasculaire sanguine dans le psoriasis. Les facteurs VEGFR3 et prox-1, marqueurs des vaisseaux lymphatiques, sont en effet exprimés de manière intermédiaire dans les pinpoint par rapport aux plaques et à la peau saine contrairement aux marqueurs d'angiogenèse, déjà exprimés de manière identique entre pinpoint et plaques. Le VEGF-A 189 est accru dans les lésions débutantes, ce qui suggère un rôle pour cette isoforme dans l'élongation vasculaire qui accompagne le psoriasis. En outre, la régression des lésions au cours d'un traitement montre que le réseau lymphatique régresse après le réseau sanguin. Ce travail démontre des différences dans le transcriptome vasculaire (sanguin et lymphatique) de la peau saine des patients par rapport à celle des volontaires sains et une chronologie séquentielle pour le développement sanguin et lymphatique dans le psoriasis. Il met aussi en évidence les variations d'expression de facteurs contrôlant l'angiogenèse et la lymphangiogenèse dans diverses lésions de psoriasis par rapport à la peau saine.

In recent years, research in the area of cancer stem cells has spiked tremendously. Numerous investigators have found that several types of cancers contain a subpopulation of tumor cells that display many defining characteristics of normal tissue stem cells, including multipotent differentiation potential, long-term self-renewal capacity, and expression of molecular markers of stemness. Most importantly, these cancer stem cells (CSCs) have very high tumor initiating potential, a finding that has led to the development of the cancer stem cell model for tumor progression. This model suggests that tumors are organized in a developmental hierarchy (similar to healthy tissue), with long-term tumor progression being driven by self-renewing CSCs at the top of the hierarchy. The CSC model represents a significant shift in our understanding of tumor progression, and as such, it may be possible to expand our knowledge of other aspects of tumor biology by re-examining them in the context of the CSC model. My work focuses on investigating interactions between CSCs and the tumor vasculature. Previous work has demonstrated heterogeneity in the proangiogenic potential of cells in a tumor. Considering the possibility that angiogenesis may be driven by specific subsets of tumor cells, I investigated the contribution of the CSC fraction to tumor angiogenesis. Comparing tumors with low or high CSC fractions, I have found that CSCs contribute to tumor vascular development through promotion of endothelial cell activity and recruitment of bone marrow-derived proangiogenic cells, mediated in part by vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF1). Since some tissue stem cells are known to reside in a vascular niche, I investigated the possibility that CSCs may also be supported by blood vessels in the tumor microenvironment, and that consequently CSCs may be targeted by disruption of tumor vasculature with antiangiogenic therapy. By testing multiple antiangiogenic therapeutic strategies, I have found that antiangiogenic therapy sensitizes CSCs to the effects of cytotoxic chemotherapy. Taken together, my work demonstrates a bi-directional relationship in which CSCs promote tumor vascular development, and tumor vasculature supports and protects CSCs. This work has implications for our understanding of CSC biology, tumor angiogenesis and antiangiogenic therapy, and provides insight into strategies for targeting the critical CSC population.