Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction

Abstract

Objective: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-C_C156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4⁺ and CD8⁺ T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI.

Conclusions: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-C_C156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.

Keywords: heart failure; inflammation; interferon; lymphangiogenesis; macrophages.

Figure 1.

Adeno-associated viral (AAV)-VEGF (vascular endothelial growth factor)-CC156S therapy stimulates cardiac lymphangiogenesis postmyocardial infarction (MI). Mice were treated with AAV-hVEGF (human VEGF)-C_C156S gene therapy (red circles, n=8), sVEGFR3 (vascular endothelial growth factor receptor 3; AAV-sVEGFR3, blue triangles, n=5–7), or AAV-scrambled virus (MI controls, black circles, n=8), and sham-operated mice (white circles, n=7–9) served as healthy controls. Cardiac gene expression of lymphangiogenic factors at 21 days post-MI (A). Lymphangiogenesis in the viable left ventricular (LV) wall bordering the infarct was evaluated as % proliferating lymphatic vessels (B), lymphatic densities (C), and lymphatic area at 7 or 21 days post-MI (D). Examples of light sheet imaging (E, left) of Lyve1⁺ (lymphatic vessel endothelial receptor 1) cardiac lymphatics in healthy mice, in AAV-MI controls, and VEGFC-treated (AAV–VEGF-C_C156S) mice at 21 d post-MI. Infarct outlined by white dashed lines. Scale bar=1 mm; ×0.8. Confocal imaging (E, right) of viable LV areas in the same samples. Cardiac precollectors indicated by white asterisk. Scale bar=50 µm; ×25. Relative cardiac expression of lymphatic-related genes in sham (n=8), MI controls (AAV-scrambled virus, n=8), and VEGFC-treated (AAV-hVEGF-C_C156S, n=5) at 21 days post-MI (F). Kruskal-Wallis followed by Dunn posthoc test. *P<0.05 vs sham; #P<0.05; ##P<0.01; ###P<0.001 vs AAV-MI controls. con indicates control.

Figure 2.

Adeno-associated viral (AAV)-sVEGFR3 (soluble vascular endothelial growth factor receptor 3) therapy inhibits infarct lymphangiogenesis postmyocardial infarction (MI). Mice were treated with AAV-hVEGF (human VEGF)-C_C156S gene therapy (red circles, n=8), sVEGFR3 (AAV-sVEGFR3, blue triangles, n=5–7), or AAV-scrambled virus (MI controls, black circles, n=8), and sham-operated mice (white circle, n=7–9) served as healthy controls. Lymphangiogenesis in the infarct scar was evaluated as % proliferating lymphatic vessels (A), open lymphatic densities (B), and lymphatic area at 7 or 21 days post-MI (C). Examples (D) at 21 days post-MI of infarct zone left ventricular lymphatics (Lyve1 [lymphatic vessel endothelial receptor 1], red), macrophages (F4-80, gray), and cell nuclei (DAPI, blue); ×20 magnification, scale bar=50 µm. Note LYVE1-expressing macrophages close to lymphatic vessels. con indicates control.

Figure 3.

Adeno-associated viral (AAV) delivery of VEGF (vascular endothelial growth factor)-CC156S reduces cardiac inflammation and improves cardiac function, while AAV-sVEGFR3 (soluble VEGF receptor 3) therapy reduces infarct scar T-cell levels and wall thinning leading to improved cardiac function in mice postmyocardial infarction (MI). Cardiac densities in the viable left ventricular (LV) of CD3⁺ T cells (A) and M1 pro-inflammatory macrophages (CD68⁺/CD206⁻ cells), (B) were determined by immunohistochemistry in MI controls (AAV-scramble, black circles, n=8), and VEGFC (AAV–VEGF-C_C156S, red circles, n=7–8) or sVEGFR3 (AAV-sVEGFR3, blue triangles, n=5–7) treated mice at 7 and 21 d post-MI. Healthy sham levels (n=7–9) are indicated by white circles / bars. Cardiac function and remodeling were analyzed by echocardiography at 21 days post-MI to determine LV fractional shortening (C) and LV anterior wall thickness in end-systole (AWT ES, D). Infarct densities of CD3⁺ total T cells (E) and CD8⁺ T-cell subpopulation (F). Examples of CD8⁺ T-cell density in the infarct at 7 d post-MI (G). CD3 (red), CD8 (green), DAPI (blue), ×20 magnification, scale bar=50 µm. Infarct scar remodeling evaluated in Sirius red-stained histological sections as absolute infarct area (H) at 21 days post-MI in MI controls (AAV-scramble, n=8), VEGFC-(AAV-hVEGF-C_C156S, n=7–8) and sVEGFR3-(AAV-sVEGFR3, n=5–7) treated mice. Examples of infarct scar remodeling. Scale bar=1 mm (I). LV hypertrophy/dilatation index was calculated from echocardiographic parameters at 21 days post-MI (J). Kruskal-Wallis, Dunn posthoc test. *P<0.05; **P<0.01; ***P<0.001 vs sham; #P<0.05; ##P<0.01 vs MI control. con indicates control.

Figure 4.

Adeno-associated viral (AAV)-sVEGFR3 (soluble vascular endothelial growth factor receptor 3) therapy suppresses infarct lymphangiogenesis and improves cardiac function and remodeling in rats postmyocardial infarction (MI). Lymphatic remodeling was investigated in cardiac sections from MI controls (AAV-scramble, black circles, n=8) and sVEGFR3-treated (AAV-sVEGFR3, blue triangles, n=9) rats. Examples of blood vessels and lymphatics in the infarct zone at 28 d post-MI (A): middle panel: lymphatics (VEGFR3, red), and blood vessels (RECA1, green); bottom panel: lymphatics (Lyve1 [lymphatic vessel endothelial receptor 1], green; Prox1 [homeobox gene homologue Drosophilia prospero gene], red). ×20, scale bar=50 µm. White dashed lines outline infarct epicardium. Quantification of VEGFR3⁺ lymphatic area (B) and RECA1⁺ blood vascular area (C) in the infarct at 28 days post-MI. Comparison by Student 2-tailed t test: ***P<0.001 vs MI control. Cardiac function was investigated by serial echocardiography in healthy sham rats (open circles, n=4), MI controls (AAV-scramble, black circles, n=13), and sVEGFR3-treated (AAV-sVEGFR3, blue triangles, n=10) MI rats. Left ventricular (LV) fractional shortening (D), end-systolic diameter (ESD, E), systolic interventricular septal wall thickness (IVSTs, F) were measured, and LV hypertrophy/dilatation index (G) calculated. Two-way ANOVA, followed by Bonferroni posthoc: *P<0.05; **P<0.01; ***P<0.001 vs sham; #P<0.05, ##P<0.01 vs MI control. RECA indicates rat endothelial cell antigen.

Figure 5.

Cardiac-infiltrating T cells suppress lymphangiogenesis in mice postmyocardial infarction (MI). Circulating T-cell levels (A and B) were determined by flow cytometry in saline-injected MI controls (black circles, n=4–5) and Fingolimod-treated wild-type (wt) MI mice (yellow triangles, n=4–5) at 3 and 7 days post-MI and healthy sham mice (white circles, n=4). Cardiac sections were analyzed to determine densities of CD3⁺ total T cells (C), M1 proinflammatory macrophages (D), lymphatic vessels (E), and open lymphatic vessels (F) in the viable left ventricular (LV) of saline-injected MI controls (black circles, n=4–13) and Fingolimod-treated MI mice (yellow triangles, n=7–9) at 3, 7 and 21 days post-MI. Healthy sham levels (n=5–10) indicated by white circle. One-way ANOVA, Dunn posthoc test. *P<0.05; **P<0.01; ***P<0.001 vs sham; #P<0.05; ##P<0.01 vs MI controls. Examples of cardiac lymphatics (Lyve1 [lymphatic vessel endothelial receptor 1], red) in the viable LV at 7 days post-MI (G). ×20, scale bar=50 µm. Note peri-lymphatic macrophages (CD68, green). Analysis of lymphatic remodeling in wt (black, n=7) vs MHC (major histocompatibility complex) II^Δ/Δ CD4⁺ T cell-deficient (yellow, n=6) mice at 7 days post-MI assessed as densities of total lymphatic vessel (H) and open lymphatic vessels (I and J) in the infarct zone. Kruskal-Wallis followed by Dunn posthoc test. #P<0.05 vs wt MI controls. Examples of Lyve1⁺ (green) lymphatics and CD31⁺ blood vessels (red) in the infarct zone at 7 days post-MI (J). ×10, scale bar=50 µm. APC indicates antigen-presenting cell.

Figure 6.

Depletion of CD4 and CD8, but not NKT, T-cell subpopulations prevents rarefaction of cardiac lymphatics acutely postmyocardial infarction (MI). Assessment by flow cytometry at 8 days post-MI of circulating T-cell populations (A and B) in control MI mice (black, n=2), CD4-depleted mice (yellow, n=6), CD8-depleted mice (blue, n=6), and mice treated with an IFN (interferon) γ–neutralizing antibody (red, n=6). Evaluation by immunohistochemistry of viable left ventricular (LV) at 8 days post-MI of CD4⁺ and CD8⁺ cardiac T-cell densities (C) and total lymphatic density (E) and lymphatic area (F) in healthy sham mice (white, n=3–5) control MI mice (black, n=10), CD4-depleted mice (yellow, n=6), CD8-depleted mice (blue, n=6), and anti-IFNγ–treated mice (red, n=7). Examples of cardiac T-cell densities (D) and lymphatic densities (G) in viable LV at 8 days post-MI. CD4⁺ cells indicated by arrow, CD8⁺ cells by arrowhead, and open lymphatics indicated by asterisk. ×20, scale bar=50 µm. Kruskal-Wallis followed by Dunn posthoc test. *P<0.05; ***P<0.001 vs sham; #P<0.05; ###P<0.001 vs MI controls.