Angiotensin II receptor I blockade prevents stenosis of tissue engineered vascular grafts

Abstract

We previously developed a tissue-engineered vascular graft (TEVG) made by seeding autologous cells onto a biodegradable tubular scaffold, in an attempt to create a living vascular graft with growth potential for use in children undergoing congenital heart surgery. Results of our clinical trial showed that the TEVG possesses growth capacity but that its widespread clinical use is not yet advisable due to the high incidence of TEVG stenosis. In animal models, TEVG stenosis is caused by increased monocytic cell recruitment and its classic ("M1") activation. Here, we report on the source and regulation of these monocytes. TEVGs were implanted in wild-type, CCR2 knockout ( Ccr2^-/-), splenectomized, and spleen graft recipient mice. We found that bone marrow-derived Ly6C^+hi monocytes released from sequestration by the spleen are the source of mononuclear cells infiltrating the TEVG during the acute phase of neovessel formation. Furthermore, short-term administration of losartan (0.6 g/L, 2 wk), an angiotensin II type 1 receptor antagonist, significantly reduced the macrophage populations (Ly6C^+/-/F480⁺) in the scaffolds and improved long-term patency in TEVGs. Notably, the combined effect of bone marrow-derived mononuclear cell seeding with short-term losartan treatment completely prevented the development of TEVG stenosis. Our results provide support for pharmacologic treatment with losartan as a strategy to modulate monocyte infiltration into the grafts and thus prevent TEVG stenosis.-Ruiz-Rosado, J. D. D., Lee, Y.-U., Mahler, N., Yi, T., Robledo-Avila, F., Martinez-Saucedo, D., Lee, A. Y., Shoji, T., Heuer, E., Yates, A. R., Pober, J. S., Shinoka, T., Partida-Sanchez, S., Breuer, C. K. Angiotensin II receptor I blockade prevents stenosis of tissue engineered vascular grafts.

Keywords: Losartan; macrophages; monocytes.

Figure 1

BM-derived monocytes populate the TEVGs. A) Gating strategy from flow cytometric analysis in harvested cells from TEVGs at 3 d postimplantation (DPI). Newly recruited monocytes were identified as LD⁻/CD45⁺/CD11b⁺/Ly-6C^+hi/Ly-6G⁻ cells. B, C) Representative dot plots (B) and absolute numbers (C) of Ly-6C^+hi monocytes in TEVGs from WT and Ccr2^−/− mice at 0 and 3 DPI. D, E) Representative dot plots (D) and absolute numbers (E) of Ly-6C^+hi monocytes in spleen from WT and Ccr2^−/− mice at 3 DPI. Measn ± sem; n = 6/group. *P < 0.05, **P < 0.01 ***P < 0.001.

Figure 2

The spleen (SPL) is the primary reservoir of infiltrating monocytes in the TEVGs. A, B) Representative dot plots (A) and absolute numbers (B) of Ly-6C^+hi monocytes in TEVGs from WT (SPL^+/+) and splenectomized (SPL^−/−) animals. C, D) Population of Ly-6C^+hi monocytes (C) and their absolute numbers (D) in spleens from CD45.1 CX3CR1^gfp/gfp mice before transplantation (SPL control), and 0 or 3 d postimplantation (DPI). E, F) Dot plots (E) and total numbers (F) of Ly6C^+hi/CXC3CR1⁺ monocytes in TEVGs from CD45.2 WT, CD45.1 CX3CR1^gfp/gfp, and CD45.2 WT mice receiving a CD45.1 CX3CR1^gfp/gfp spleen. Means ± sem; n = 6/group. **P < 0.01, ***P < 0.001.

Figure 3

Effect of losartan in MNC infiltration and patency in the TEVG. A, B) Absolute numbers of macrophages Ly6C⁻/F480⁺ (A) and Ly6C⁺/F480⁺ (B) in native vein and TEVGs from untreated and short-term losartan-treated mice at 2 wk postimplantation (n = 8/group). C) Representative histologic images of TEVGs from untreated control, long-term losartan-treated (6 mo, 0.6 g/L), and short-term losartan-treated (2 wk, 0.6 g/L) mice. D, E) Comparison of the patency rate between untreated, short-term, and long-term losartan-treated groups. F) Percent survival at either 6 mo or 1 yr post–TEVG implantation; n = 25/group. *P < 0.05, **P < 0.01.

Figure 4

Low-dose and 1-time high-dose losartan treatments on TEVG patency and their physiologic effects in mice. A, B) Comparison of TEVG patency between the untreated control and either low-dose losartan treatment (2–wk, 0.1 g/L) (A) or 1-time high-dose losartan (0.6 g/L) (B). C) Comparison of losartan concentration in plasma between the low-dose and high-dose losartan groups. D) Weight change comparison among the control, low-dose, and high-dose losartan groups. E) Mean arterial pressure comparison among the control, low-dose, and high-dose losartan groups. F) Cardiac output comparison among the control, low-dose, and high-dose losartan groups. Untreated control and 1-time high-dose losartan groups (n = 25); short-term low-dose losartan group (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Combined effect of BM-MNC seeding and losartan treatment. A) Comparison of the patency rate among the untreated control (6 mo), seeded control (6 mo), and short-term losartan with BM-MNC seeding (1 yr) groups. B) Comparison of luminal diameter between the native vein and short-term losartan with BM-MNC seeding. C) Representative histologic images of the seeded control, short-term losartan treatment with BM-MNC seeding, and native vein. Untreated control and seeded control (n = 25); short-term losartan with BM-MNC seeding (n = 21). **P < 0.01, ***P < 0.001.