Figure 2. Severe HHcy promotes vascular remodeling and elastin production in the neointima in the vein graft in Cbs^−/− mice.

The mouse vein graft surgery procedure was performed on Cbs mice at the age of 12 weeks as described in Figure 1 and in the section of Methods. The vein patch grafts, together with a short segment of the native carotid artery, were harvested at 4 weeks after surgery and processed for paraffin embedding (VVG) and cryostat sections (α–actin). Mouse plasma was collected at the end of each experiment (at age 16 weeks). A. Photomicrographs of morphological analysis. Sections are oriented with a vein patch on top and artery on bottom. VVG staining shows elastin as black. Anti-α-mouse smooth muscle (α-SMA) antibody staining shows smooth muscle cell as red. B. Quantitative analysis. Neointimal area and thickness were analyzed by computerized planimetry using the updated Image-Pro Plus program. We determined the elastin positive area by computer-assisted color gated measurement (Image-Pro Plus) and expressed the data as a percentage. The neointima was defined as the region between the internal elastic lamina (IEL) and the lumen. The percentage luminal narrowing was calculated as 100 x (the difference between area inside the IEL and area of the lumen ÷ the area inside the IEL. Hcy significantly increased neointima formation, luminal narrowing and neointima elastin deposition. C. Plasma level of Hcy. Hcy concentration was measured by liquid chromatography electrospray tandem mass spectrometry methods. Values represent mean±SEM, n=9, p values from independent t test * p<0.05 versus Cbs^+/+ group, #p<0.001 versus Cbs^+/+ group. VVG, Verhoeff-van Gieson staining.