Coronary artery remodeling in a model of left ventricular pressure overload is influenced by platelets and inflammatory cells

Abstract

Left ventricular hypertrophy (LVH) is usually accompanied by intensive interstitial and perivascular fibrosis, which may contribute to arrhythmogenic sudden cardiac death. The mechanisms underlying the development of cardiac fibrosis are incompletely understood. To investigate the role of perivascular inflammation in coronary artery remodeling and cardiac fibrosis during hypertrophic ventricular remodeling, we used a well-established mouse model of LVH (transverse aortic constriction [TAC]). Three days after pressure overload, macrophages and T lymphocytes accumulated around and along left coronary arteries in association with luminal platelet deposition. Consistent with these histological findings, cardiac expression of IL-10 was upregulated and in the systemic circulation, platelet white blood cell aggregates tended to be higher in TAC animals compared to sham controls. Since platelets can dynamically modulate perivascular inflammation, we investigated the impact of thrombocytopenia on the response to TAC. Immunodepletion of platelets decreased early perivascular T lymphocytes' accumulation and altered subsequent coronary artery remodeling. The contribution of lymphocytes were examined in Rag1(-/-) mice, which displayed significantly more intimal hyperplasia and perivascular fibrosis compared to wild-type mice following TAC. Collectively, our studies support a role of early perivascular accumulation of platelets and T lymphocytes in pressure overload-induced inflammation.

Figure 1. TAC-induced LV pressure overload stimulates left coronary remodeling and dysfunction in wild-type C57BL/6 male mice.

Figure 2. TAC elicits an early inflammatory response in wild-type (C57BL/6) male mice.

A) Immunohistochemical staining of inflammatory cells and adhesion molecules in LV sections from wild-type male mice subjected to sham or TAC surgery and sacrificed at different time points (D = days). Positive staining is in red-brown (mag. 40×; Bar = 70 µm T lymphocytes are indicated by CD90.2 and CD8; CD19 is a B lymphocyte marker. VCAM is an endothelial inflammatory marker and myeloperoxidase (MPO) an enzyme stored and released by neutrophils and macrophages. B) and C) Quantification of the accumulation of macrophage (CD68) and T lymphocyte (CD90.2), as measured by area of positive staining in and around the coronary arteries, at different time points following sham (open circles) and TAC (closed circles) surgery. Values are presented as mean ± sem. The images are representative of those obtained in the following numbers. For macrophages: TAC day one, n = 5; TAC day three, n = 7; TAC day seven, n = 9; sham day one, n = 3; sham day three, n = 4; sham day seven, n = 5. For lymphocytes: TAC day one, n = 7; TAC day three, n = 6; TAC day seven, n = 6; sham day one, n = 4; sham day three; n = 5; sham day seven, n = 5. *P<0.05 versus same time point in sham.

Figure 3. Endothelial disruption at three and seven days after TAC surgery.

Confocal (left and middle panel) and IHC (right panel) images of PECAM (CD31) staining along the lumen of left coronary arteries at three days after sham surgery (sham D3) and at three (TAC D3) and seven days (TAC 7D) after TAC surgery. CD31 staining appears continuous along the luminal side in sham mice and discontinuous along similar vessels in TAC mice. Bar = 70 µm.

Figure 4. Upregulation of inflammatory markers after TAC in wild-type mice.

Levels of MCP-1 (A) and VEGF (B) were measured in homogenized LV at seven days after sham (n = 6) or TAC surgery (n = 8) by Luminex assay. Protein expression (pg/ml) in the homogenate is reported for each individual mouse, and boxes indicated median and 95% CI. (C) RNA was isolated from LV apex at one to seven days after TAC (n = 3–7 per time point). IL-10 mRNA levels were measured by qPCR and values normalized to the sham value at day one, which was set at 100% expression, and presented as mean ± SD. *P<0.05 versus sham.

Figure 5. Platelet accumulation and effects on perivascular inflammation.

A) Immunohistochemical staining for platelets three days after TAC (mag. 40×). B) Platelet deposition co-localized with macrophages in coronary arteries at seven days after TAC (TAC D7). Serial sections through coronary arteries were stained with antibodies to platelets (top), CD68 (middle) and CD31 (bottom). (mag. 40×; Bar = 70 µm). Area of positive perivascular staining for C) macrophages (vessel number = n, anti-GPIbα IgG n = 23, non-immune IgG n = 18) and D) T lymphocytes (vessel number = n, anti-GPIbα IgG n = 26, non-immune IgG n = 18) three days after TAC. *P = 0.05. E) Expression of inflammation markers in LV apex at three days post-TAC was measured by qPCR (mice number = n, anti-GPIbα IgG n = 13, non-immune IgG n = 10) presented as mean ± SD.

Figure 6. Thrombocytopenia promotes coronary vessel remodeling and perivascular fibrosis after TAC.

A) Representative images (mag. 40×) and B) quantification of α-smooth muscle actin staining in coronary arteries of thrombocytopenic (anti-GPIbα IgG treated) and control (non-immune IgG injected) mice five weeks after TAC surgery (vessel number = n, anti-GPIbα IgG n = 11, non-immune IgG n = 5). *P<0.05. C) Area of pericoronary fibrosis thrombocytopenic and control mice five weeks after TAC (vessel number = n, anti-GPIbα IgG n = 10, non-immune IgG n = 5). Fibrosis was identified by Masson's Trichrome stain and reported as area mean ± sem.

Figure 7. The effects of platelet releasate on myocardial fibroblasts.

qPCR was performed to detect the differences of mRNA expression in cardiac fibroblasts exposed to platelet releasate (dark bars) or buffer (open bars) for six (left) or 24 (right) hours. The values were normalized to the buffer-treatment group at the same time point, which was set at fold increase and presented as mean ± SD.

Figure 8. Enhanced TAC-induced coronary remodeling in the absence of lymphocytes.

A) H&E staining (mag. 20×) and B) measurement of vessel area inside the external elastic laminar in wild-type (WT) and Rag1^−/− male mice five weeks after sham or TAC surgery (TAC: WT n = 10, Rag1^−/− n = 17. Sham: WT n = 9, Rag1^−/− n = 6). C) Masson's Trichrome staining and D) quantification of pericoronary fibrosis formation in WT and Rag1^−/− male mice five weeks after surgery reported as area mean ± sem (TAC: WT n = 10, Rag1^−/− n = 13. Sham: WT n = 9, Rag1^−/− n = 6). E) Picrosirius red staining and F) quantification of pericoronary collagen deposition in WT and Rag1^−/− male mice five weeks after surgery reported as area mean ± sem (TAC: WT n = 9, Rag1^−/− n = 19. Sham: WT n = 7, Rag1^−/− n = 7). *P<0.05. Bar = 70 µm.

Figure 9. TAC-induced coronary remodeling in the absence of IL-10.

A) Representative sections stained with Masson's Trichrome (MT) and visualized at 40× mag. five weeks after sham or TAC surgery (left) and measurements of perivascular fibrosis area (sham n = 5; TAC n = 5). B) SMC α-actin staining five weeks after sham or TAC surgery (left) and measurements of vessel area occupied by SMC α-actin (right). *P<0.05. Bar = 70 µm.

Figure 10. Model of TAC-induced early inflammatory response in which platelets and inflammatory cells are recruited and contribute to the subsequent development of intimal hyperplasia and fibrosis.