Date of Award
Open Access Thesis
Medical Doctor (MD)
Vein graft adaptation to the arterial circulation entails many changes at the cellular/molecular levels, including both cell proliferation and extracellular matrix (ECM) deposition/remodeling, resulting in thickening of the vein wall, and in excessive cases, pathological neointimal hyperplasia. Eph-B4 is a receptor tyrosine kinase and embryonic determinant of veins that, when stimulated, has been shown by our lab to decrease vein graft wall thickening. We hypothesize that among other mechanisms, Eph-B4 exerts this effect through modulating extracellular matrix production and/or degradation, and that Eph-B4 interacts directly with eNOS to mediate some of its downstream effects.
Mouse Embryonic Fibroblasts (MEFs) were stimulated with 2 ug/ml Ephrin-B2/Fc, the stimulatory ligand for Eph-B4. To determine mRNA changes, cell lysates were collected after various stimulation periods, followed by mRNA purification and RT-PCR for each specified gene of interest. Similarly, mRNA was also extracted from human saphenous veins in culture. For Western Blot and Zymography, both cell lysates and conditioned media (secreted proteins) were collected after various periods of stimulation. In vivo, 4 week old vein graft sections from WT and Eph-B4 knockout mice, or from Ephrin-B2/Fc stimulated or unstimulated mice, were stained with Masson's Trichrome, and the total area of blue staining surrounding the vein graft was quantified to determine graft wall collagen content. For eNOS experiments, either Eph-B4 or Y774F mutant Eph-B4 was transfected into COS cells, followed by Ephrin-B2/Fc stimulation for 1-60 minutes and determination of eNOS phosphorylation utilizing Western Blot.
mRNA expression of Collagen I, Collagen III, Matrix Metalloproteinase 2 (MMP2), MMP9, MMP14, elastin, Tissue Inhibitor of Metalloproteinases 1 (TIMP1), and TIMP2 were not affected by Eph-B4 stimulation. mRNA expression of Collagen 1 in human saphenous veins in culture was not affected by Eph-B4 stimulation. Eph-B4 had no effect on MMP2 and MMP9 cellular protein levels as detected by Western Blot, and no effect on MMP2 protein activity as detected by zymography in cell lysates and conditioned media. Masson's Trichrome staining of mice vein graft sections revealed that most of the collagen within the vein graft was within the adventitia. There were no differences in either collagen content or lumen circumference between vein grafts from WT and Eph-B4 KO mice, and between control mice and mice stimulated with Ephrin-B2/Fc. Dual transfection of cells with endothelial nitric oxide synthase (eNOS) and Eph-B4 resulted in increased eNOS phosphorylation; Eph-B4 stimulation further increased eNOS phosphorylation. Y774 is not crucial to Eph-B4's effects on eNOS.
Eph-B4 likely does not exert its effects on decreasing vein graft thickening through directly modulating ECM deposition and remodeling. Eph-B4 likely exerts its effects through other mechanisms such as modulating cellular proliferation within the vein graft wall. Eph-B4 likely mediates at least some of its downstream effects through a direct interaction with eNOS.
Lu, Daniel, "Mechanism For Eph-B4 Regulation Of Vein Graft Adaptation" (2014). Yale Medicine Thesis Digital Library. 1900.