Plasminogen activator inhibitor-1 (PAI-1) is cardioprotective in mice by maintaining microvascular integrity and cardiac architecture

Abstract

Although the involvement of plasminogen activator inhibitor-1 (PAI-1) in fibrotic diseases is well documented, its role in cardiac fibrosis remains controversial. The goal of this study was to determine the effect of a PAI-1 deficiency (PAI-1(-/-)) on the spontaneous development of cardiac fibrosis. PAI-1(-/-) mice developed pervasive cardiac fibrosis spontaneously with aging, and these mice displayed progressively distorted cardiac architecture and markedly reduced cardiac function. To mechanistically elucidate the role of PAI-1 in cardiac fibrosis, 12-week-old mice were chosen to study the biologic events leading to fibrosis. Although fibrosis was not observed at this early age, PAI-1(-/-) hearts presented with enhanced inflammation, along with increased microvascular permeability and hemorrhage. A potent fibrogenic cytokine, transforming growth factor-beta (TGF-beta), was markedly enhanced in PAI-1(-/-) heart tissue. Furthermore, the expression levels of several relevant proteases associated with tissue remodeling were significantly enhanced in PAI-1(-/-) hearts. These results suggest that PAI-1 is cardioprotective, and functions in maintaining normal microvasculature integrity. Microvascular leakage in PAI-1(-/-) hearts may provoke inflammation, and predispose these mice to cardiac fibrosis. Therefore, a PAI-1 deficiency contributes to the development of cardiac fibrosis by increasing vascular permeability, exacerbating local inflammation, and increasing extracellular matrix remodeling, an environment conducive to accelerated fibrosis.

Figure 1

PAI-1 deficiency causes spontaneous cardiac fibrosis formation. (A-F) Macroscopic views of hearts from WT and PAI-1^−/− mice. (A-C) WT hearts at 24, 36, and 48 weeks, respectively. None of the WT hearts at any time point examined developed fibrosis. (D-F) PAI-1^−/− hearts at 24, 36, and 48 weeks, respectively. PAI-1^−/− mice at 36 and 48 weeks developed obvious cardiac fibrosis. The fibrotic patches (red arrows) show a loss of normal architecture, paucity of stromal cells, and replacement of essential parenchymal structures by dense, pale, and increasingly rigid tissue. (G) Cardiac fibrosis formation caused an increase in the heart–body weight ratio (mg/g) in PAI-1^−/− mice with aging, compared with WT mice. (H) PAI-1 antigen levels in plasma. n ≥ 8 for each age group; *P < .05.

Figure 2

Histochemical characterization of interstitial cardiac fibrosis in hearts of PAI-1^−/− mice. H&E staining of WT hearts at (A) 12 and (B) 48 weeks, and PAI-1^−/− hearts at (C) 12 and (D) 48 weeks. At 48 weeks, replacement of cardiomyocytes by connective tissue was observed in PAI-1^−/− hearts (black arrow). Masson trichrome staining of WT hearts at (E) 12 and (F) 48 weeks, and PAI-1^−/− hearts at (G) 12 and (H) 48 weeks revealed extensive interstitial collagen deposition (white arrow, blue staining) in 48-week PAI-1^−/− hearts. Although 12-, 24-, 36-, and 48-week hearts were examined, only 12- and 48-week hearts are shown in this figure. Global view of 48-week PAI-1^−/− hearts with H&E staining (I) and Masson trichrome staining (J) revealed that fibrosis formation (black arrow) was rather pervasive throughout the entire heart. (K) Quantification of collagen by Masson trichrome staining of hearts. Data points represent individual hearts, and bars are group means. n ≥ 4 for each age group; *P < .05.

Figure 3

Echocardiographic evaluation of cardiac anatomy and cardiac function in WT and PAI-1^−/− mice. Representative M-mode images of the short-axis view of the 48-week WT (A) and PAI-1^−/− (B) LVs. Substantial LV wall motion irregularities were found in 48-week-old PAI-1^−/− mice, demonstrated by severe reductions and odd movements in both systolic and diastolic LV posterior and anterior wall movements. AW indicates anterior wall; and PW, posterior wall. The overall left ventricular function, measured by left ventricle ejection fraction (LVEF; C) and left ventricle fractioning shortening (LVFS; D), was diminished by a PAI-1 deficiency, especially after 36 weeks of age. The internal diameters of LV at diastole and systole were decreased in PAI-1^−/− mice, especially after 36 weeks (E-F). The LV posterior wall diameter was increased in PAI-1^−/− mice at both systole and diastole (G-H). n ≥ 6 for each age group; *P < .05.

Figure 4

Hemodynamic parameters of WT and PAI^−/− mice. The central arterial blood pressures and heart rates in 12- and 48-week-old WT and PAI-1^−/− mice were monitored continuously for at least 3 days by radiotelemetry. The means of systolic pressure (A), diastolic pressure (B), and pulse pressure (C) were then calculated. Heart rates (D) were also monitored in each individual WT and PAI-1^−/− mouse. The data represent means of at least 4 mice per group.

Figure 5

Spontaneous bleeding and increased vascular permeability in PAI-1^−/− hearts. Prussian blue staining of 12- and 48-week WT (A-B) and PAI-1^−/− (C-D) hearts revealed hemosiderin deposition in 12-week PAI-1^−/− hearts (C, ↑), indicating bleeding before fibrosis formation. Hemosiderin deposition was overt in 48-week PAI-1^−/− hearts (D). An in vivo vascular permeability assay revealed increased cardiac vascular leakage in 12-week PAI-1^−/− hearts (E). The plasma levels of EB were the same between WT and PAI-1^−/− mice (F). Gross photograph of PAI-1^−/− hearts at 48 weeks is shown (G), where hemorrhage spots are observable macroscopically (↑). n ≥ 5 for each age group; *P < .05.

Figure 6

Assessments of inflammation in 12-week WT and PAI-1^−/− hearts. An elevated inflammatory response, indicated by iNOS (A), iCAM (B), and KC (C) mRNA levels, was observed in 12-week PAI-1^−/− hearts. Compared with WT heart (D), CD45 staining revealed enhanced local leukocyte infiltration in cardiac tissue of 12-week-old PAI-1^−/− mice (E). Leukocyte infiltration and recruitment after peritoneal stimulation with thioglycollate were significantly enhanced in PAI-1^−/− mice compared with WT mice (F). Comparison of peritoneal leukocyte viabilities indicated that the differences in leukocyte infiltration were not due to different rates of cell death (G). n ≥ 5 for each age group; *P < .05.

Figure 7

Elevated level of cardiac ECM remodeling in PAI-1^−/− mice. Both the mRNA level (A) and the protein level (B) of TGF-β in 12-week PAI-1^−/− hearts were significantly higher than in WT hearts. Q-RT-PCR analysis of transcripts of proteins associated with tissue remodeling and fibrosis in 12-week WT and PAI-1^−/− hearts, included MMP-2 (C), MMP-9 (D), TIMP-2 (E), and uPA (F). n ≥ 5 for each group; *P < .05.