Thrombospondin-1 (TSP-1) is a multifunctional extracellular matrix glycoprotein that is released from platelets α-granule to regulate angiogenesis process. TSP-1 is well-known as an inhibitory factor of angiogenesis that binds to angiogenesis stimulating factors, for example fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF) and hepatocyte growth factor/scatter factor (HGF/SF), to inhibit angiogenesis. We have cloned TSP-1 domains separately to allow studying of their function and effect on proliferation of human umbilical vein endothelial cells (HUVECs). We used an Escherichia coli expressionsvektor including poly histidin-tags and lac-promoter for induction of the seven successfully cloned domains by IPTG and arabinose. Our result shows that we have very low expression and induction of our protein in the E.coli by IPTG and arabinose, which is most likely due to complications associated with expressing a human protein in a prokaryotic system.

The development of a functional vascular system is impinged upon the restructuring of a primitive vasculature into a more complex and mature vessel network via a process known as angiogenesis. Of particular importance to this vascular remodeling process is the function of the Tek/Tie-2 receptor tyrosine kinase. Mouse gene-targeting studies have shown that Tie-2 deficient embryos succumb to embryonic death at embryonic day 9.5 due to insufficient sprouting and remodeling of the primary capillary plexus. Over the years, the functions and the signaling pathways downstream of Tie-2 receptor have been elucidated; however, the repertoire of genes controlled by Tie-2 signaling leading to angiogenesis had not been studied. To identify the underlying genetic mechanisms, transcriptomes from Tie-2 wild-type (WT) and knockout (KO) embryonic day 8.5 yolk sac tissues were quantitatively analyzed using a gene expression profiling technique called Serial Analysis of Gene Expression (SAGE). Tie-2 WT and KO SAGE libraries were constructed, sequenced and compared to identify genes that were differentially expressed. A list of candidate genes was selected for further validation using semi-quantitative PCR that included 4933402E13Rik, a novel transcript encoding a protein product containing the melanoma-associated antigen (MAGE) domain. Initial characterization of 4933402E13Rik suggested a murine-specific expression profile restricted to the yolk sac, embryo, placenta, testis, endothelial and embryonic stem cells. The expression of 4933402E13Rik in mouse endothelial cells was found to be regulated by Tie-2 signaling since down-regulation of Tie-2 level via siRNA knockdown resulted in decreased 4933402E13Rik mRNA expression. In contrast, stimulation of Tie-2 in mouse endothelial cells using its ligand, Angiopoietin-1, increased 4933402E13Rik mRNA levels. Additionally, 4933402E13Rik expression was found to be modulated through epigenetics especially by histone deacetylation. Mouse endothelial cells treated with Trichostatin A, a potent inhibitor of histone deacetylase, led to an increase in the expression of 4933402E13Rik. Taken together, the results of this study shed new insight on the repertoire of genes implicated in Tie-2 signaling. The identification of 4933402E13Rik as a novel gene modulated by Tie-2 provides a new avenue of research on Tie-2 signaling that may contribute further to our understanding of vascular development.

Treatment of cardiac diseases involves transplantation of donor hearts, since the damaged heart has limited self-regeneration potential. An alternative treatment option has emerged as engineered cardiac tissues, grown in vitro by cultivation of cardiac cells on biomaterials, have comparable properties to native myocardium and can be implanted for cardiac repair. Major current limitations are a viable cell source and adequate vascularization to support cell survival. In this thesis, two proangiogenic biomaterials, a scaffold and a hydrogel, were developed to achieve vascularization in vitro and in vivo for cardiac repair. Scaffold patches are suitable for repairing congestive heart failure or congenital malformations, while injectable biomaterials allow minimally-invasive treatment post-myocardial infarction (MI). In the first aim, a collagen scaffold with covalently immobilized vascular endothelial growth factor (VEGF) was developed, and improved cell mobilization, survival and proliferation when used for free wall repair in adult rats. This increased angiogenesis, which aided in retaining the biomaterial size to allow tissue growth. In the second aim, a collagen-chitosan hydrogel with encapsulated thymosin β4 (Tβ4) was developed to 1) recruit cells from the heart epicardium for repair post-MI in vivo, and 2) guide capillary outgrowths from arteries and veins to form oriented capillary structure for in vitro cardiac tissue engineering. Results showed that the encapsulation of Tβ4 into collagen-chitosan hydrogels led to cell outgrowths from rat or mouse cardiac explants in vitro. A portion of the recruited cells were CD31-positive endothelial cells (ECs) that formed tubes. The hydrogel was injected in vivo to increase vascularization and number of cardiomyocytes within the infarct area post-MI, which improved left ventricular wall thickness. Tβ4-hydrogel also promoted the outgrowth of capillaries from vascular explants that followed the direction of the hydrogel-coated grooves of a micropatterned polydimethylsiloxane (PDMS) substrate. These capillary outgrowths eventually formed a vascular bed for engineering vascularized cardiac tissues. This thesis presents two bioinstructive biomaterials with sustained and localized delivery of angiogenic molecules to be used for in situ cardiac repair based on improved vascularization. The use of cell-free bioactive materials overcomes limitations of cell isolation and expansion as required for cell therapies or implantation of engineered tissues.

Endogenously, VEGF initiates angiogenesis, then later Angiopoietin (Ang)-1 matures vessels. We hypothesized that multigene therapy of VEGF before Ang1 to ischemic hindlimb tissue would result in persistent angiogenesis. At 2, 4 and 8 wks after inducing ischemia, blood flow was assessed by contrast-enhanced ultrasound. Animals were treated with VEGF at 2 wks, VEGF/Ang1 at 2 wks, or VEGF at 2 wks and Ang1 at 4 wks. In untreated controls, blood flow remained reduced. After VEGF delivery, resting flow and vessel density increased; however, flow reserve remained reduced, and vasculature was capillary-rich and eventually regressed. After VEGF/Ang1 co-delivery, flow increased marginally, flow reserve improved and vascular architecture remained normal. After separated VEGF and Ang1 delivery, flow, vessel density and flow reserve increased and were sustained, while vascular architecture remained normal. In conclusion, temporally separated VEGF and Ang1 delivery promotes sustained angiogenesis and improved vessel functionality.

Endogenously, VEGF initiates angiogenesis, then later Angiopoietin (Ang)-1 matures vessels. We hypothesized that multigene therapy of VEGF before Ang1 to ischemic hindlimb tissue would result in persistent angiogenesis. At 2, 4 and 8 wks after inducing ischemia, blood flow was assessed by contrast-enhanced ultrasound. Animals were treated with VEGF at 2 wks, VEGF/Ang1 at 2 wks, or VEGF at 2 wks and Ang1 at 4 wks. In untreated controls, blood flow remained reduced. After VEGF delivery, resting flow and vessel density increased; however, flow reserve remained reduced, and vasculature was capillary-rich and eventually regressed. After VEGF/Ang1 co-delivery, flow increased marginally, flow reserve improved and vascular architecture remained normal. After separated VEGF and Ang1 delivery, flow, vessel density and flow reserve increased and were sustained, while vascular architecture remained normal. In conclusion, temporally separated VEGF and Ang1 delivery promotes sustained angiogenesis and improved vessel functionality.

The development of a functional vascular system is impinged upon the restructuring of a primitive vasculature into a more complex and mature vessel network via a process known as angiogenesis. Of particular importance to this vascular remodeling process is the function of the Tek/Tie-2 receptor tyrosine kinase. Mouse gene-targeting studies have shown that Tie-2 deficient embryos succumb to embryonic death at embryonic day 9.5 due to insufficient sprouting and remodeling of the primary capillary plexus. Over the years, the functions and the signaling pathways downstream of Tie-2 receptor have been elucidated; however, the repertoire of genes controlled by Tie-2 signaling leading to angiogenesis had not been studied. To identify the underlying genetic mechanisms, transcriptomes from Tie-2 wild-type (WT) and knockout (KO) embryonic day 8.5 yolk sac tissues were quantitatively analyzed using a gene expression profiling technique called Serial Analysis of Gene Expression (SAGE). Tie-2 WT and KO SAGE libraries were constructed, sequenced and compared to identify genes that were differentially expressed. A list of candidate genes was selected for further validation using semi-quantitative PCR that included 4933402E13Rik, a novel transcript encoding a protein product containing the melanoma-associated antigen (MAGE) domain. Initial characterization of 4933402E13Rik suggested a murine-specific expression profile restricted to the yolk sac, embryo, placenta, testis, endothelial and embryonic stem cells. The expression of 4933402E13Rik in mouse endothelial cells was found to be regulated by Tie-2 signaling since down-regulation of Tie-2 level via siRNA knockdown resulted in decreased 4933402E13Rik mRNA expression. In contrast, stimulation of Tie-2 in mouse endothelial cells using its ligand, Angiopoietin-1, increased 4933402E13Rik mRNA levels. Additionally, 4933402E13Rik expression was found to be modulated through epigenetics especially by histone deacetylation. Mouse endothelial cells treated with Trichostatin A, a potent inhibitor of histone deacetylase, led to an increase in the expression of 4933402E13Rik. Taken together, the results of this study shed new insight on the repertoire of genes implicated in Tie-2 signaling. The identification of 4933402E13Rik as a novel gene modulated by Tie-2 provides a new avenue of research on Tie-2 signaling that may contribute further to our understanding of vascular development.

Damage to peripheral nerves following trauma or disease has a number of consequences including burning pain, muscle wasting, paralysis, or organ dysfunction. The most common form of neuropathy is that associated with metabolic abnormality, notably diabetes. Many diabetics, especially those with poor blood sugar control, ultimately develop a distal symmetrical and painful neuropathy that initially affects the longest peripheral axons, but with time spreads proximally. Deficiency in neurotrophic support has been proposed to contribute to the development of diabetic neuropathy. Recently, peripheral gene delivery of vascular endothelial growth factor (VEGF), neurotrophin-3 (NT-3), NGF, BDNF or hepatocyte growth factor (HGF) has been shown to facilitate the continuous production of neurotrophic factors and alleviate the diabetic neuropathy. The role of glial cell-derived neurotrophic factor (GDNF) in the pathogenesis and therapeutics of diabetic neuropathy is not well defined. The main objectives of this research sought to inspect the protective effect of GDNF peripheral gene delivery during hyperglycemia- or constriction- induced sciatic nerve injury in rats. In present proposal, we propose to investigate the change in organization and expressions of GDNF signaling complex in the sciatic nerve following injury in the initial stage. Subsequently, the recombinant adenovirus was used gene delivery system for GDNF to evaluate the potential of intramuscular administration of gene delivery for prevent nerve degeneration, and the molecular mechanism of GDNF to ameliorate neuropathy will be clarified. The above study would enable us to test the hypothesis that the topical gene delivery might be a suitable strategy for the treatment of diabetic neuropathy and other disorders in peripheral nerve. Furthermore, the results of animal studies might be extrapolated for future clinical application.

Angiogenesis is a fundamental process in reproduction and wound healing. Angiogenesis is also indispensable for solid tumor growth and metastasis, and also associated with angiogenic diseases. Beta-endorphin (£]-EP), derived from its precursor pro-opiomelancortin (POMC), is well known for its role in nociception and immune regulation. However, the function of morphine and £]-EP during angiogenesis remains characterization. One previous study indicated that morphine inhibited the proliferation and hypoxia-induced vascular endothelial growth factor (VEGF) release of endothelial cells. Contrastingly, another report found that morphine via Ras/PI3k/MAPK/ERK signaling promotes the survival and angiogenesis in endothelial cells. Besides, endogenous opioid peptides stimulated angiogenesis in chicken allantoic membrane assay through opioid receptors. Thus, the function and mechanism of £]-EP and opioid receptors in angiogenesis are controversial. This study evaluated the culture effects of £]-EP and morphine on angiogenesis . It was found that £]-EP stimulated the proliferation, migration, and tube formation of endothelial cells in a dose-dependent manner. Morphine at a high dose inhibited the proliferation, migration, and tube formation of endothelial cells. In the ex vivo rat aortic ring assay, £]-EP enhanced, whereas morphine perturbed, the microvessel sprouting. We also confirmed the expression of MOR¡ADOR¡AKOR opioid receptor in endothelial cells. Application of naloxone, a selective opioid antagonist, and neutralizing antibodies of MOR abolished the angiogenic effect of £]-EP and morphine. Thus £]-EP and morphine exert the pro- and anti-angiogenic effect via MOR, respectively .Besides, £]-EP can be regarded as a novel angiogenic factor.

Angiogenesis is not only essential for tumor growth but is also implicated in invasion of the cancer cells into the circulation, and growth of dormant micro-metastases into frank metastatic lesions. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis as well as in solid tumors. It also has a role for VEGF in hematopoietic neoplasms; although has not been fully elucidated. This study will examine the VEGF secretary activity of malignant cells in the patients with hematologic malignancies. Supernatants of cell culture after 72 hours & 7th day were analyzed for VEGF value by ELISA method. The purposes of this study are to assay the VEGF activity and its correlation with disease prognosis in various hematological disorders; and to detect the VEGF autocrine activity of tumor cells in sequential culture without any stimulation in vitro. Results: our research samples were 75 specimens. The VEGF value was low in 13 cases of benign diseases, no obvious auto-secretary activity of those cells. There was no significant correlation between VEGF value and disease status in acute lymphoblastic leukemia; however, the cases were too small to had exact predict value (only 7 cases). In 17 Cases of malignant lymphoma, low D0 VEGF value (<300 pg) had good prognosis; those cases relapsed after treatment had high auto-secretary activity (high VEGF value of 7th culture day) and had bad disease prognosis, but there was no statistic significance. In 14 Cases of acute myelogenous leukemia, high D0 VEGF value (>150 pg) and high VEGF value of 7th culture day both presented bad disease outcome. In 16 cases of chronic myeloid leukemia (CML), low plasma VEGF level (D0<300pg) was all in chronic phase; all cases in accelerated phase (3 cases) and acute blastic crisis (5 cases) presented with high plasma VEGF level (>300pg). Patients with high plasma VEGF level had 70 % (7of 10) in worse disease status (P=0.010). Patients in chronic phase of CML had low VEGF auto-secretary activity and most of the blastic crisis patients were with high VEGF auto-secretary activity and also had bad prognosis. (P=0.248) Conclusion: although our study is a primary result, study cases are varied, but it still provide important information that VEGF has an important role in hematological malignancies. We will process further research of single and specific disease in the future to analyze the exact correlation of VEGF and hematological diseases.

The growth and metastasis of solid tumors are dependent on angiogenesis. An endogenous angiogenesis inhibitor, vasostatin, is the proteolytic fragment derived from the N-terminal 180 residues of calreticulin. Previous studies indicated that vasostatin specifically inhibits endothelial cell proliferation, angiogenesis and tumor growth. However, continuous administration of vasostatin is difficult and expensive to facilitate, thereby underscoring the need to develop gene delivery approach. Adenovirus vectors possess advantages for gene delivery including high titer, high infection efficiency and broad host range. The aim of the present study was to generate and characterize recombinant adenovirus vectors encoding vasostatin (Ad-VS) or Igk-fused vasostatin (Ad-Igk-VS), thereby to evaluate the efficacy of anti-angiogenesis gene therapy for tumor suppression. Recombinant Ad-VS and Ad-Igk-VS were generated and verified by PCR and western blot analysis. In addition, adenovirus encoding angiostatin was also produced as positive control for angiogenesis assays. Adenovirus-mediated vasostatin gene delivery specifically inhibited the proliferation of bovine aortic endothelial cells (BAEC), but not non-endothelial cells such as Hela or NIH3T3 cells. Moreover, vasostatin gene delivery potently inhibited the proliferation, migration and tube formation, but not secretion of matrix metalloproteinases (MMPs), in endothelial cells. Flow cytometry analysis indicated that vasostatin gene delivery induced apoptosis in BAEC. Using western blot analysis, it was revealed that gene delivery of vasostatin increased the levels of Fas and FADD in BAEC. In conclusion, adenovirus-mediated vasostatin gene delivery inhibited various angiogenesis processes at least via induction of Fas/FasL pathway and may hold potential for cancer therapy.