Plasminogen Activator Inhibitor Type I Controls Cardiomyocyte Transforming Growth Factor-β and Cardiac Fibrosis

Abstract

Background: Fibrosis is the pathological consequence of stress-induced tissue remodeling and matrix accumulation. Increased levels of plasminogen activator inhibitor type I (PAI-1) have been shown to promote fibrosis in multiple organ systems. Paradoxically, homozygous genetic deficiency of PAI-1 is associated with spontaneous age-dependent, cardiac-selective fibrosis in mice. We have identified a novel PAI-1-dependent mechanism that regulates cardiomyocyte-derived fibrogenic signals and cardiac transcriptional pathways during injury.

Methods: Cardiac fibrosis in subjects with homozygous mutation in SERPINE-1 was evaluated with late gadolinium-enhanced cardiac magnetic resonance imaging. A murine cardiac injury model was performed by subcutaneous infusion of either saline or Angiotensin II by osmotic minipumps. We evaluated blood pressure, cardiac function (by echocardiography), fibrosis (with Masson Trichrome staining), and apoptosis (with TUNEL staining), and we performed transcriptome analysis (with RNA sequencing). We further evaluated fibrotic signaling in isolated murine primary ventricular myocytes.

Results: Cardiac fibrosis was detected in 2 otherwise healthy humans with complete PAI-1 deficiency because of a homozygous frameshift mutation in SERPINE-1. In addition to its suppressive role during spontaneous cardiac fibrosis in multiple species, we hypothesized that PAI-1 also regulates fibrosis during cardiac injury. Treatment of young PAI-1-/- mice with Angiotensin II induced extensive hypertrophy and fibrotic cardiomyopathy, with increased cardiac apoptosis and both reactive and replacement fibrosis. Although Angiotensin II-induced hypertension was blunted in PAI-1-/- mice, cardiac hypertrophy was accelerated. Furthermore, ventricular myocytes were found to be an important source of cardiac transforming growth factor-β (TGF-β) and PAI-1 regulated TGF-β synthesis by cardiomyocytes in vitro as well as in vivo during cardiac injury. Transcriptome analysis of ventricular RNA after Angiotensin II treatment confirmed that PAI-1 deficiency significantly enhanced multiple TGF-β signaling elements and transcriptional targets, including genes for extracellular matrix components, mediators of extracellular matrix remodeling, matricellular proteins, and cardiac integrins compared with wild-type mice.

Conclusions: PAI-1 is an essential repressor of cardiac fibrosis in mammals. We define a novel cardiomyocyte-specific regulatory mechanism for TGF-β production by PAI-1, which explains the paradoxical effect of PAI-1 deficiency in promoting cardiac-selective fibrosis. Thus, PAI-1 is a molecular switch that controls the cardiac TGF-β axis and its early transcriptional effects that lead to myocardial fibrosis.

Keywords: cardiac remodeling; cardiomyopathy; protease inhibitor.

© 2017 American Heart Association, Inc.

Figures

Figure 1. Spontaneous cardiac fibrosis in 2 human subjects with homozygous deficiency of plasminogen activator inhibitor type I (PAI-1)

Late gadolinium-enhanced cardiac magnetic resonance imaging short-axis views (base, mid, and apex). Multiple bright foci in the left ventricle represent cardiac scar. Normal myocardium appears black. Subepicardial enhancement (single arrowhead) and a midmyocardial stripe (double arrowhead) are evident. This pattern of fibrosis is consistent with a noncoronary distribution.

Figure 2. Angiotensin II (AngII)-mediated cardiovascular effects in wild-type (WT) and plasminogen activator inhibitor type I (PAI-1)−/− mice

A, Mice were implanted with osmotic minipumps loaded with PBS or AngII (1000 ng/kg/min) and were studied for 28 days. One group of PAI-1−/− mice also received intraperitoneal injections of BMP-7 (300 μg/kg). B, Changes in systolic blood pressure after 1 week of treatment. AngII increases blood pressure in both WT and PAI-1−/− mice compared with saline. PAI-1 deficiency confers partial protection from AngII-mediated hypertension. C, Changes in heart weight to tibial length ratio. Cardiac mass increases to a similar degree in both WT and PAI-1−/− 28 days after initiation of AngII. D, EF was quantified using 2-dimensional (2D) echocardiography. AngII treatment induced moderate to severe systolic dysfunction in PAI-1 −/− mice but did not affect the EF of WT mice. Coadministration of BMP-7 restored systolic function in PAI-1−/− mice. E, Representative short-axis M-mode images from each group. Images were obtained at similar heart rates. B through E, n=6 to 10 per group. Error bars represent SD. All statistical analyses were performed using 1-way analysis of variance followed by Tukey’s HSD test. EF indicates ejection fraction; and NS, not significant. *P<0.05; **P<0.005; ***P<0.0005.

Figure 3. Homozygous deficiency of plasminogen activator inhibitor type I (PAI-1) accelerates Angiotensin II (AngII)-mediated cardiac fibrosis, which is inhibited by exogenous BMP-7

A, Short-axis sections through the ventricles of wild-type (WT) and PAI-1−/− mice at the level of the papillary muscles. Representative images with Masson’s Trichrome stain demonstrate a prominent increase in collagen deposition (blue stain) in PAI-1−/− hearts compared with WT. Hearts of WT and PAI-1−/− mice both develop interstitial and perivascular fibrosis in response to AngII compared with PBS. In addition, PAI-1−/− hearts contain areas of replacement fibrosis in the epicardial (single arrowhead) and midmyocardial (double arrowhead) regions. Coadministration of BMP-7 with AngII significantly reduces fibrotic deposition. B, Higher magnification images from AngII-treated WT and PAI-1−/− hearts stained with Masson’s Trichrome. Interstitial and perivascular collagen deposition occurs in both WT and PAI-1−/− hearts. Replacement fibrosis only occurs in the PAI-1−/− myocardium treated with AngII alone. Bar=20 μm. C, Quantification of percent fibrotic area was performed using Image J (n=6 to 10 per group). Error bars represent SD. Statistical analysis was performed using 1-way analysis of variance followed by Tukey’s HSD test. *** P<0.0001; ****P<0.00001.

Figure 4. Homozygous deficiency of plasminogen activator inhibitor type I (PAI-1) potentiates Angiotensin II (AngII)-induced transforming growth factor-β (TGF-β) production by ventricular myocytes in vivo

A, Fluorescence microscopy images of mouse cardiac sections immunostained for TGF-β (red/555), troponin (green/488), and with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Left, TGF-β staining. Right, Staining merged with troponin and DAPI for improved tissue visualization. Hearts of wild-type (WT) and PAI-1−/− mice treated with PBS do not stain for TGF-β. AngII induces the production of TGF-β in ventricular myocytes (troponin positive, single arrowhead). Compared with WT myocardium, PAI-1−/− sections stain more intensely for TGF-β, with more positive myocytes. Interstitial areas that are troponin negative are marked with an asterisk. Images were taken with identical exposure times for each channel. Representative images from 4 experiments (4 different hearts per group). Bar=10 μm. B, Sections from PAI-1−/− hearts were treated with secondary antibody alone and imaged with identical exposure times to sections from A. Both the red/555 and green/488 controls are shown. Representative images from 4 experiments (4 different hearts per group). Bar=10 μm. C, Fluorescence microscopy images from AngII-treated mouse cardiac sections immunostained for TGF-β (green/488) and with DAPI (blue). Merged images are shown. WT myocardium stains most intensely for TGF-β in the epicardium (single arrowhead) and perivascular areas (double arrowhead). Compared with WT, PAI-1−/− myocardium more diffusely expresses TGF-β, and several waves of involvement are evident: epicardial (single arrowhead), subendocardial (asterisk), and perivascular (double arrowhead). Representative images from 4 experiments (4 different hearts per group). Bar=200 μm.

Figure 5. Homozygous deficiency of plasminogen activator inhibitor type I (PAI-1) enhances transforming growth factor-β (TGF-β) production in cardiomyocytes

A, Production of TGF-β by cardiomyocytes. Wild-type (WT) and PAI-1−/− adult mouse ventricular myocytes were isolated and cultured for 24 hours in the presence or absence of TGF-β or Angiotensin II (AngII). Cells were rinsed and solubilized in radioimmunoprecipitation assay (RIPA) buffer. Lysates were analyzed by Western blot for TGF-β, phospho-SMAD2/3, and troponin (loading control). TGF-β or AngII-induced TGF-β production in WT and PAI-1−/− cardiomyocytes. Intracellular TGF-β is increased in treated PAI-1−/− cardiomyocytes compared with WT, suggesting increased susceptibility to either TGF-β or AngII stimulation. SMAD2/3 phosphorylation suggests that TGF-β produced by the myocytes is functionally active. Representative Western blot from 3 separate experiments. B, Quantification of normalized relative intensity in A. Each sample was normalized to troponin intensity. C, Culture medium from cardiomyocytes in A. Equal volume of medium was loaded in each lane and analyzed by Western blot for TGF-β. The nonspecific (NS) band reflects loading. Medium in lane 7 contains 5ng/mL TGF-β but did not incubate with cardiomyocytes and did not react with TGF-β Ab. PAI-1−/− myocytes secrete more TGF-β into the medium than WT cardiomyocytes (red arrow). Representative Western blot from 3 separate experiments. D, Quantification of normalized relative intensity in C. Each sample was normalized to troponin intensity. E, Representative image of adult mouse ventricular myocyte preparations. All statistical analyses were performed using 1-way analysis of variance followed by Tukey’s HSD test. NS indicates not significant. *P<0.05; **P<0.005; ***P<0.0005.

Figure 6. Plasminogen activator inhibitor type I (PAI-1) regulates early Angiotensin II (AngII)-mediated cardiac hypertrophy and transcriptional events

A, Mice were implanted with osmotic minipumps loaded with PBS or AngII (1000 ng/kg/min) for 7 days before ventricular RNA isolation. B, Changes in heart weight to tibial length ratio. Cardiac hypertrophy is accelerated in PAI-1−/− mice treated with AngII compared with wild type (WT) (n=3 mice per group). Error bars represent SD. Statistical analysis was performed using 1-way analysis of variance followed by Tukey’s HSD test. *P<0.05; **P<0.005. C, RNA sequencing results. Number of significantly different genes (P<0.05) with ≥100 reads. Hearts from WT and PAI-1−/− mice treated with PBS were transcriptionally similar with the exception of 26 genes. In contrast, treatment with 7 days of AngII induced 4168 differentially expressed genes in the PAI-1−/−compared with WT (n=3 per group). Statistical analysis was performed using DESeq2 with adjustment for multiple comparisons as described in the online-only Data Supplement (false discovery rate [FDR]-adjusted P value). D, Computational analysis reveals that numerous pathways involving inflammation, matrix remodeling, and wound healing are markedly upregulated in the AngII-treated PAI-1−/− heart compared with WT (n=3 hearts per group). Pathway analysis was performed using Metascape.

Figure 7. Homozygous deficiency of plasminogen activator inhibitor type I (PAI-1) is associated with reduced cardiac SMAD6, and BMP-7 inhibits cardiomyocyte transforming growth factor-β (TGF-β) expression and release

A, Cardiac SMAD6 content. Wild-type (WT) and PAI-1−/− ventricular tissue was solubilized in RIPA buffer. Lysates were analyzed by Western blot for SMAD6 and troponin (loading control). Lysates from 3 different hearts per group are shown. B, Quantification of relative intensity in A. Statistical analysis performed by nonpaired Student’s t test. C, PAI-1−/− adult mouse ventricular myocytes were isolated and treated for 24 hours with either TGF-β, Angiotensin II (AngII), or TGF-β+BMP-7. Cells were rinsed and solubilized in RIPA buffer. Lysates were analyzed by Western blot for TGF-β, phospho-SMAD2/3, and troponin (loading control). TGF-β or AngII induced TGF-β production. Intracellular TGF-β and phospho-SMAD 2/3 are decreased in BMP-7-treated PAI-1−/− cardiomyocytes, suggesting decreased susceptibility to autocrine stimulation. Representative Western blot from 3 separate experiments. D, Quantification of normalized relative intensity in C. E, Culture medium from cardiomyocytes in C. Equal volume of medium was loaded in each lane and analyzed by Western blot for TGF-β. Medium in lane 5 contains 5ng/mL TGF-β but did not incubate with cardiomyocytes. F, Quantification of normalized relative intensity in E. Values from each sample were normalized to troponin intensity, and statistical analyses in D and F were performed using 1-way analysis of variance followed by Tukey’s HSD test. **P<0.005; ***P<0.0005. G, Model for the role of cardiomyocyte PAI-1 in TGF-β-mediated cardiac fibrosis. PAI-1 is required for feedback inhibition of early autocrine TGF-β expression and secretion by cardiomyocytes. BMP-7-induced Smad6 activation suppresses both early autocrine and late paracrine TGF-β-mediated fibrogenesis.