Intercellular Adhesion Molecule 1 Regulates Left Ventricular Leukocyte Infiltration, Cardiac Remodeling, and Function in Pressure Overload-Induced Heart Failure

Abstract

Background: Left ventricular dysfunction and heart failure are strongly associated in humans with increased circulating levels of proinflammatory cytokines, T cells, and soluble intercellular cell adhesion molecule 1 (ICAM1). In mice, infiltration of T cells into the left ventricle contributes to pathological cardiac remodeling, but the mechanisms regulating their recruitment to the heart are unclear. We hypothesized that ICAM1 regulates cardiac inflammation and pathological cardiac remodeling by mediating left ventricular T-cell recruitment and thus contributing to cardiac dysfunction and heart failure.

Methods and results: In a mouse model of pressure overload-induced heart failure, intramyocardial endothelial ICAM1 increased within 48 hours in response to thoracic aortic constriction and remained upregulated as heart failure progressed. ICAM1-deficient mice had decreased T-cell and proinflammatory monocyte infiltration in the left ventricle in response to thoracic aortic constriction, despite having numbers of circulating T cells and activated T cells in the heart-draining lymph nodes that were similar to those of wild-type mice. ICAM1-deficient mice did not develop cardiac fibrosis or systolic and diastolic dysfunction in response to thoracic aortic constriction. Exploration of the mechanisms regulating ICAM1 expression revealed that endothelial ICAM1 upregulation and T-cell infiltration were not mediated by endothelial mineralocorticoid receptor signaling, as demonstrated in thoracic aortic constriction studies in mice with endothelial mineralocorticoid receptor deficiency, but rather were induced by the cardiac cytokines interleukin 1β and 6.

Conclusions: ICAM1 regulates pathological cardiac remodeling by mediating proinflammatory leukocyte infiltration in the left ventricle and cardiac fibrosis and dysfunction and thus represents a novel target for treatment of heart failure.

Keywords: adhesion molecules; heart failure; inflammation; remodeling.

Figure 1

Upregulation of ICAM1 mRNA expression and ICAM1 protein in the LV, specifically in the vascular endothelium, in response to TAC. A, ICAM1 mRNA expression in the LV of WT mice determined by quantitative reverse transcription polymerase chain reaction represented as fold change for TAC vs sham at 48 hours and at 2 and 4 weeks after surgery. Statistical analysis used the nonparametric Mann–Whitney test. Data shown as median (interquartile range): at 48 hours: WT sham 1.044 (0.754–1.307) and WT TAC 1.483 (1.374–1.841); at 2 weeks: WT sham 1.115 (0.735–1.250) and WT TAC 6.916 (1.633–13.30); at 4 weeks: WT sham 1.035 (0.582–1.770) and WT TAC 3.957 (1.519–28.71). *P<0.05. B, Representative immunohistochemistry staining for ICAM1 and PECAM1 (used as positive control for endothelial cell staining in the same sections) in sham‐operated and TAC mice at 48 hours and at 2 and 4 weeks after surgery. Scale bar=50 μm. n=4 sham, n=6 to 9 TAC. h indicates hours; ICAM1, intercellular cell adhesion molecule 1; LV, left ventricle; PECAM1, platelet/endothelial cell adhesion molecule 1; TAC, transverse aortic constriction; w, weeks; WT, wild type.

Figure 2

ICAM1‐mediated regulation of CD45.2⁺, CD3⁺, CD4⁺, and Ly6C⁺ leukocyte infiltration into the LV in response to TAC. A through C, Representative FACS plots of (A) LV CD45.2⁺ leukocyte, (B) CD3⁺ T‐cell, and (C) Ly6C⁺ monocyte infiltration in response to TAC or sham operation at 4 weeks in WT and ICAM1^−/− mice. Bar graph on the right represents fold‐change quantification of the indicated leukocytes normalized to sham controls. D, Representative FACS histogram for Ly6C⁺ low and high subpopulations, gated on CD45.2⁺ CD11b⁺ myeloid cells infiltrated into the LV of sham‐ and TAC‐operated WT and ICAM1^−/− mice. Ly6C^neg cells represent nonmonocyte myeloid cells, Ly6C^low represents patrolling monocytes, and Ly6C^high represents proinflammatory monocytes. E, Representative pictures (×40 magnification) of immunohistochemistry staining for CD4⁺ T helper lymphocytes infiltrated into the LV in sham‐ and TAC‐operated WT and ICAM1^−/− mice. Quantification of CD4⁺ cells per 6‐μm LV cross‐sections is represented. Scale bar=10 μm. n=5 to 7 sham, n=11 TAC. Statistical analysis used the unpaired t test. *P<0.05; **P<0.01. FACS indicates fluorescence‐activated cell sorting; ICAM1, intercellular cell adhesion molecule 1; LV, left ventricle; TAC, transverse aortic constriction; WT, wild type.

Figure 3

Lymphocyte activation in the heart‐draining mediastinal lymph nodes and circulating CD4⁺ T cells in WT and ICAM1^−/− mice in response to TAC. A, Diagram indicating T‐cell subpopulations using T‐cell activation markers. B, Representative FACS plots showing naïve, memory, and activated T lymphocytes in the mediastinal lymph node lymphocyte gate at 4 weeks after TAC or sham surgery. Quantification of activated T lymphocytes (CD44^high CD62L^low) is shown in the right bar graph with values normalized to sham control. C, Representative FACS plots showing blood‐circulating CD4⁺ T lymphocytes at 4 weeks after TAC or sham surgery. CD4⁺ cells are represented as percentages in the bar graph at right. n=5 sham, n=8 TAC. Statistical analysis used the unpaired t test. *P<0.05; **P<0.01. FACS indicates fluorescence‐activated cell sorting; ICAM1, intercellular cell adhesion molecule 1; TAC, transverse aortic constriction; WT, wild type.

Figure 4

LV weight, CM dimensions, and expression of natriuretic peptides and MHC isoforms in response to TAC in ICAM1^−/− mice. A, LV weight of WT and ICAM1^−/− (n=6–9 sham, n=10–14 TAC) at 4 weeks after TAC, normalized to the tibia length. B, Dimensions of CMs isolated from WT and ICAM1^−/− mice at 4 weeks after TAC or sham surgery. n=100 to 200 cells per group, from 2 independent CM isolations. Representative pictures are shown (magnification ×20). Scale bar=50 μm. Statistical analysis used the unpaired t test. C through E, Quantitative reverse transcription polymerase chain reaction of the hypertrophy markers (C) BNP and (D) ANP and the (E) ratio of MHCβ and MHCα. mRNA expression normalized to sham in the LV in response to TAC (n=7) or sham (n=5) surgery at 4 weeks. Statistical analysis used the nonparametric Mann–Whitney test. Data are shown as median (interquartile range): for BNP: WT sham 1.190 (0.508–1.876) and WT TAC 18.60 (6.985–91.82), ICAM1^−/− sham 0.682 (0.502–2.921) and ICAM1^−/− TAC 1.516 (1.168–2.236); for ANP: WT sham 1.268 (0.519–1.709) and WT TAC 14.33 (5.885–23.92), ICAM1^−/− sham 1.273 (0.264–4.664) and ICAM1^−/− TAC 0.6279 (0.509–1.797); for MHCβ/MHCα: WT sham 0.557 (0.416–2.395) and WT TAC 10.87 (8.446–14.01), ICAM1^−/− sham 1.168 (0.600–1.521) and ICAM1^−/− TAC 1.454 (1.045–3.935). *P<0.05, **P<0.01, ***P<0.005. ANP indicates atrial natriuretic peptide; BNP, brain natriuretic peptide; CM, cardiac myocyte; ICAM1, intercellular cell adhesion molecule 1; LV, left ventricle; ns, not significant; MHC, myosin heavy chain; TAC, transverse aortic constriction; w, weeks; WT, wild type.

Figure 5

Absence of fibrosis in ICAM1^−/− in response to TAC. A, Representative photomicrographs (×40 magnification) and quantification (bar graph on the right) of myocardial interstitial fibrosis evaluated by picrosirius red staining of left ventricle sections from sham and TAC mice at 4 weeks. B, Representative pictograms (×20 magnification) shown on the left and percentage of perivascular fibrosis on the right. Scale bar=50 μm. n=3 to 5 sham, n=3 to 8 TAC. Statistical analysis used the unpaired t test. *P<0.05, **P<0.01, ***P<0.005. ICAM1 indicates intercellular cell adhesion molecule 1; TAC, transverse aortic constriction; WT, wild type.

Figure 6

Absence of fibrosis mRNA marker upregulation in ICAM1^−/− in response to TAC despite equal capacity for M2 profibrotic and myofibroblast differentiation in vitro. A through C, Quantitative reverse transcription polymerase chain reaction in the left ventricle for the fibrosis markers (A) TGF‐β, (B) collagen I, and (C) α‐SMA. mRNA expression normalized to sham. n=3 to 4 sham, n=6 to 9 TAC. Statistical analysis used the nonparametric Mann–Whitney test. Data shown as median (interquartile range): for TGF‐β: WT sham 1.594 (0.297–3.180) and WT TAC 7.209 (5.212–80.73), ICAM1^−/− sham 0.2096 (0.186–3.964) and ICAM1^−/− TAC 0.472 (0.263–1.768); for collagen I: WT sham 0.758 (0.615–2.431) and WT TAC 12.51 (3.596–109.6), ICAM1^−/− sham 1.318 (0.321–3.388) and ICAM1^−/− TAC 1.048 (0.840–1.610); for α‐SMA: WT sham 0.642 (0.552–2.824) and WT TAC 7.838 (0.441–37.80), ICAM1^−/− sham 2.008 (0.240–3.953) and ICAM1^−/− TAC 0.601 (0.233–1.704). D and E, WT and ICAM1^−/− bone marrow–derived monocytes polarized to M2 and M1 macrophages in the presence of 20 ng/mL IL‐4 or 1 μg/mL LPS, respectively, for 3 days. Polarization was determined by mRNA expression of (D) arginase 1 for M2 and (E) inducible nitric oxide synthase for M1 macrophages. n=3 untreated, n=3 to 4 treated with IL‐4 or LPS. Statistical analysis used the unpaired t test. F, WT and ICAM1^−/− cardiac fibroblast polarized to myofibroblast in the presence of 100 ng/mL TGF‐β for 16 hours. Polarization determined by mRNA expression of α‐SMA, Magnification ×20 representative pictures of each condition are shown on the left. n=4 to 6 untreated, n=6 to 7 TGF‐β treated. Statistical analysis used the unpaired t test.*P<0.05, **P<0.01, ***P<0.005. α‐SMA, α‐smooth muscle actin; ICAM1 indicates intercellular cell adhesion molecule 1; IL, interleukin; TAC, transverse aortic constriction; TGF‐β, transforming growth factor β; Untr., untreated; WT, wild type.

Figure 7

Improved cardiac function in response to TAC‐induced heart failure in ICAM1^−/− mice. A through C, Invasive hemodynamic measurements in WT and ICAM1^−/− mice measured at 4 weeks after TAC and sham surgery (n=3 WT, n=6–12 ICAM1^−/−), (A) end‐diastolic pressure, (B) maximum pressure (C) contractile function (dP/dt_max), and (D) relaxation function (dP/dt_min). E, Fractional shortening at 4 weeks after sham or TAC surgery in WT and ICAM1^−/− mice (n=3–6 sham, n=5–10 TAC). Statistical analysis used the unpaired t test. *P<0.05, **P<0.01, ***P<0.005. FS indicates fractional shortening; ICAM1 indicates intercellular cell adhesion molecule 1; TAC, transverse aortic constriction; WT, wild type.

Figure 8

ICAM1 expression, LV T‐cell recruitment, and LV cytokine expression at 4 weeks after TAC are independent of EC‐MR signaling. A and B, Representative immunohistochemistry staining (×20 magnification) for ICAM1 (A) and CD4 (B) in sham, EC‐MR ^+/+ and EC‐MR ^−/− TAC mice at 4 weeks after surgery. On the right, quantification of CD4⁺ T cells per LV section after TAC (B). Scale bar=50 μm. C, mRNA expression of the cytokines IL‐1β and IL‐6 on the LV of EC‐MR ^−/− and EC‐MR ^+/+ littermates in response to TAC at 4 weeks normalized to sham. n=3 sham, n=4 TAC. Statistic analysis used the unpaired t test. D, mRNA expression of the cytokines IL‐1β and IL‐6 on the LV of WT mice in response to TAC at 48 hours and at 2 and 4 weeks, normalized to sham. n=4 to 7 sham, n=4 to 9 TAC. Statistic analysis used the nonparametric Mann–Whitney test. Data shown as median (interquartile range): for IL‐1β: at 48 hours, WT sham 1.045 (0.369–4.534) and WT TAC 7.105 (4.022–34.63); at 2 weeks, WT sham 0.898 (0.580–2.110) and WT TAC 3.537 (1.807–10.40); at 4 weeks, WT sham 0.702 (0.592–2.465) and WT TAC 3.960 (1.240–18.51); for IL‐6: at 48 hours, WT sham 1.249 (0.544–1.802) and WT TAC 11.92 (8.317–17.87); at 2 weeks, WT sham 0.952 (0.538–2.777) and WT TAC 19.16 (6.427–31.26); at 4 weeks, WT sham 0.962 (0.365–3.022) and WT TAC 3.808 (2.429–9.796). *P<0.05, **P<0.01. EC‐MR indicates endothelial cell–specific mineralocorticoid receptor; h, hours; ICAM1 indicates intercellular cell adhesion molecule 1; IL, interleukin; LV, left ventricle; TAC, transverse aortic constriction; w, weeks; WT, wild type.