Hyperhomocysteinemia promotes vascular remodeling in vein graph in mice

Abstract

This study investigated the role and mechanism of Hyperhomocysteinemia (HHcy) on vascular remodeling in mice. We assessed the effect of HHcy on vascular remodeling using a carotid arterial vein patch model in mice with the gene deletion of cystathionine-beta-synthase (Cbs). Vein grafts were harvested 4 weeks after surgery. Cross sections were analyzed using Verhoeff-van Gieson staining, Masson`s Trichrome staining, and immunostaining for morphological analysis and protein level assessment. The effect of Hcy on collagen secretion was examined in cultured rat aortic smooth muscle cells (RASMC). We found that Cbs-/- mice with severe HHcy exhibited thicker neointima and a higher percentage of luminal narrowing in vein grafts. In addition, severe HHcy increased elastin and collagen deposition in the neointima. Further, severe HHcy increases CD45 positive cells and proliferative cells in vein grafts. Finally, Hcy increases collagen secretion in RASMC. These results demonstrate that HHcy increases neointima formation, elastin and collagen deposition following a carotid arterial vein patch. The capacity of Hcy to promote vascular fibrosis and inflammation may contribute to the development of vascular remodeling.

Figure 1. Mouse carotid arterial vein patch model

The mouse vein graft surgery procedure was performed on Cbs mice at the age of 12 weeks. The left external jugular vein from a C57 mouse was dissected from the main trunk. A segment of the jugular vein (≈5 mm long) was sutured into place to repair a longitudinal defect (about the same length as the jugular vein patch) in the left carotid artery from Cbs mouse. The grafts, together with a short segment of the native carotid artery, were harvested at 4 weeks after surgery by cutting at the center of the graft. One portion was processed for paraffin embedding and the other for cryostat sectioning. A. Schema of the vein graft and cross-section of the composite vessel at the center of the graft. B. Verhoeff-van Gieson (VVG) staining in paraffin-embedded cross-section from center of the graft of mice at 4 weeks post-operation. 3–4 layers of elastic lamina mark the artery. The vein is greatly enlarged in response to arterial blood pressure. C. α-smooth muscle actin (α-SMA) immunostaining in vein graft cryostat section from Cbs^−/− mice at 4 weeks post-operation. Vascular smooth muscle cell appears red. D. Verhoeff-van Gieson (VVG) staining in paraffin embedded cross-section of aorta from normal mice. Notice that normal artery has well-organized and circumferentially-oriented elastin.

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.

Figure 3. Severe HHcy increases total collagen and type 1 collage 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 grafts, together with a short segment of the native carotid artery, were harvested at 4 weeks after surgery by cutting at the center of the graft. One portion was processed for paraffin embedding and the other for cryostat sectioning. A. Photomicrographs of collagen staining. Paraffin-embedded cross sections of the vein graft stained for total collagen by Masson’s trichrome method and cryostat sections immunostained with antibody against type 1 collagen. Collagen shows as blue and type 1 collagen as brown. B&C. Total collagen and type 1 collagen quantification. Total collagen and type 1 collagen area were analyzed by computerized planimetry. Notice that total collagen deposition is increased in the adventitia, and type 1 collagen deposition is increased in the neointima in Cbs^−/− mice. Values represent mean±SEM, n=9, p values from independent t test * p<0.05 versus Cbs^+/+ group.

Figure 4. Severe HHcy increases CD45 positive cells and proliferative cells in cross section of 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 grafts, together with a short segment of the native carotid artery, were harvested at 4 weeks after surgery by cutting at the centre of the graft. One portion was processed for paraffin embedding and the other for cryostat sectioning. A. Photomicrographs of infiltrated leukocyte and proliferative cells. Cryostat sections were stained with antibodies against mouse CD45, a common antigen of leukocyte and anti-proliferating cell nuclear antigen (PCNA), marker of proliferative cells. CD45 positive nuclei and proliferative cell nuclei were showed as dark brown. B. Quantitative analysis of CD45 positive nuclei. C. Quantitative analysis of PCNA positive nuclei. CD45 and PCNA positive nuclei were analyzed using a computer program. The percentage of positive nuclei was calculated as 100x [positive nuclei ÷ (positive nuclei + negative nuclei)] in the cross section. HHcy significantly increased the ratio of CD45 positive cells and of PCNA positive cells in the cross section. Values represent mean±SEM, n=9, p values from independent t test * p<0.05 versus Cbs^+/+ group.

Figure 5. Hcy increases collagen secretion in cultured rat aortic smooth muscle cells (RASMC)

Confluent RASMC at passage 6^th were cultured in M199 medium with 10% CS and treated DL-Hcy at indicated concentrations for additional 3 weeks. Supernatant was collected and used for Western blotting analysis with rabbit anti-rat type 1 collagen monoclonal antibody. A. Representative blot. B. Quantitative analysis of type 1 collagen. Data are representative of 3 independent experiments, and expressed as mean±SEM, † p<0.05 versus no Hcy control.