Neutralization of Angiostatin to Promote Therapeutic Angiogenesis

• Author / Creator

  Govindasamy, Natasha P

• Within diseases such as coronary artery disease (CAD) and peripheral artery disease (PAD) ischemia is induced in tissues due to arterial accumulation of atherosclerotic plaque. A novel therapeutic approach to restore blood flow is to neutralize anti-angiogenic factors that could limit ischemia-induced angiogenesis. Angiostatin is a potent anti-angiogenic factor that reduces endothelial cell migration, proliferation, and survival due to inhibition of an adenosine triphosphate (ATP) synthase localized on the surface of endothelial cells. Importantly, angiostatin inhibits lung endothelial cell expression of matrix metalloproteinase (MMP)-2 and membrane-bound MMP, MT1-MMP specifically during hypoxia; these MMPs are important mediators of endothelial cell migration and angiogenesis. Angiostatin may also contribute to endothelial dysfunction and impaired angiogenic responses by decreasing endothelial nitric oxide synthase (eNOS) as observed in hypoxic lung endothelial cells. Overall, the anti-angiogenic effects of angiostatin can create a poor microenvironment for neovascularization of hypoxic tissues, thereby potentially contributing to the pathology of ischemic vascular diseases. The hypoxic-specific effects of angiostatin on cardiac-derived endothelial cells were investigated in vitro, while its effect on angiogenesis in vivo was investigated in a hind limb ischemia (HLI) model. Finally, a study to selectively neutralize angiostatin to restore the MMP-mediated angiogenic response of cardiac-derived cells was performed. Aims: 1. To investigate if angiostatin inhibits MMP-2 and eNOS levels in cardiac-derived human microvascular endothelial cells (HMVEC-C), cells were incubated for 48 hours in normoxic or hypoxic (95% N2, 5% CO2) conditions. Gelatin zymography and western blot were used to analyze MMP-2 and eNOS protein levels, respectively. 2. To investigate if administering angiostatin (30μg) intraperitoneally in transgenic eNOS-GFP mice will impair angiogenesis in a HLI model. 3. To determine if neutralizing angiostatin (600nM) using ATP synthase α and β subunits (3μM) will restore MMP-2 levels and HMVEC-C migration during hypoxia. MMP-2 protein expression was analyzed via gelatin zymography. A modified Boyden chamber assay was utilized to measure HMVEC-C migration. 4. To determine if the ATP synthase subunits interfere with fibrinolysis in vitro, clot formation and breakdown was induced in human platelet-poor plasma via incubation with thrombin (1U/ml) and tissue plasminogen activator (1g/ml) in the presence of the ATP synthase α, β and δ subunits (3μM). Results: Angiostatin decreased MMP-2 and eNOS protein expression in hypoxic HMVEC-C. In a HLI model, angiostatin-administrated mice exhibited significantly reduced blood perfusion recovery at day 14, and reduced eNOS-GFP and MMP-2 expression in ischemic tissue in comparison to control mice. Hypoxic HMVEC-C co-treated with angiostatin and the ATP synthase α subunit exhibited increased MMP-2 expression compared to angiostatin alone. Furthermore, hypoxic HMVEC-C co-treated with angiostatin and the ATP synthase α subunit exhibited an average increase in MMP-dependent migration compared to angiostatin alone. Lastly, at concentrations used to neutralize angiostatin (3M), the ATP synthase subunits did not interfere with fibrinolysis in vitro. Conclusions: During hypoxia, angiostatin reduced HMVEC-C expression of angiogenic mediators, MMP-2 and eNOS, which may be how it exerts its anti-angiogenic effects within an ischemic environment. In a murine HLI model, angiostatin impaired angiogenesis. Finally, neutralizing angiostatin using the ATP synthase α subunit increased MMP-2 expression in HMVEC-C, and resulted in an increase in endothelial cell migration all at a concentration that does not interfere with fibrinolysis. Therefore, using the ATP synthase α subunit to neutralize angiostatin could be a potential novel strategy to promote therapeutic angiogenesis.

• Subjects / Keywords

  • endothelial nitric oxide synthase
  • atp synthase
  • matrix metalloproteinases
  • angiostatin
  • angiogenesis

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3VQ2SS5V

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.