Objectives Abundant epidemiological and clinical studies have revealed the close relationship between hyperhomocysteinemia (HHcy) and the increased frequency of CVD. During the last decade, we and others have demonstrated that HHcy can inhibit endothelial cell growth and postinjury reendothelialisation, stimulate vascular smooth muscle cell proliferation, and inhibit high-density lipoprotein biosynthesis. However, the fundamental basis of HHcy-impaired angiogenesis remains unknown. In order to verify the mechanism of HHcy impaird angiogenesis, we use several classic angiogenesis models.
Methods 1. Cystathionine-synthase (CBS) is a key enzyme in the transsulfuration pathway that catabolizes Hcy. Kruger’s laboratory (Fox Chase Cancer Center, Philadelphia, PA) created a transgenic mouse (Tg-hCBS) in which the human CBS cDNA is under the control of a zinc-inducible metallothionein promoter. Tg-hCBS was able to rescue the neonatal lethal phenotype. 2. Verify the influence of Hcy on mouse retinal vascular plexus angiogenesis. Mouse eyeballs were dissected from postnatal day 1, 3, 7, 14 mice and 8-12 weeks old Tg-hCBS+Cbs+/+, Tg-hCBS+Cbs+/-, and Tg-hCBS+Cbs-/- mice. Whole-mount of mouse retinals was staining of Isolectin B4 (endothelial cell). Images were acquired by 2-laser Nikon confocal microscopy. 3. Aortic ring assay. Sacrifice the 8-12 month old mice and excise thoracic aorta. Section arteries into 1-1.5 mm long cross sections, and place them on the Matrigel-coated wells. Take images of aortic rings everyday. 4. Tube formation. The HAEC/HCMEC were seeded on growth factor-reduced Matrigel and visualised 8-12 h later, in control conditions, or in the presence of 100/200/500μm DL-Hcy. Compare the tube length and tube loop number between two groups.5. The Human angiogenesis RT2ProfilerTMPCR Array profiles the expression of 84 key genes involved in modulating the biological processes of angiogenesis. 6. Extract RNA and protein from HAEC/mouse aorta/mouse heart tissue, Real-time PCR and western blot to confirm the down-regulate of Hepatocyte Growth Factor (HGF) under Hcy treat or in HHcy mice. 7. Mouse HGF Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of mouse HGF in serum. 8. HGF repair Hcy impaird angiogenesis. Add HGF back into tube formation and aorta ring assay, check tube length and micro-vessel growth.
Results 1. HHcy impairs mouse angiogenesis in the retinal of hCBS+Cbs-/- mice. Retinal vasculature plexus length of both postnatal and adult (12 weeks old) hCBS+Cbs-/- mice are all decreased. 2. Hcy suppresses mice aortic ring micro-vessel growth in a concentration-dependent manner. 3.Hcy impaird HAEC/HCMEC in vitro tube formation process in a concentration- dependent manner. 4. Angiogenesis RT2ProfilerTMPCR Array revealed up-and down-regulated genes in HAEC. Genes encoding the Collagen, type IV, Hepatocyte growth factor, Interferon were down-regulated. 5. HAEC/Aorta/Heart of hCBS+Cbs+/- and hCBS+Cbs-/- mice were collected at age of 8-12 weeks. Quantitative results are shown that Hcy impair HAEC/Aorta/Heart HGF expression. 6. HGF level in mouse serum was detected by Enzyme-Linked Immunosorbent Assay. HGF decreased in hCBS+Cbs-/- mice serum and also decreased in hCBS+Cbs-/- + MI mice serum. 7. HGF repair Hcy inhibited tube formation and aorta ring assay. Tube length and micro-vessel are all increased after HGF treat.
Conclusions HHcy impair angiogenesis via HGF inhibition.