Simultaneous transforming growth factor beta-tumor necrosis factor activation and cross-talk cause aberrant remodeling response and myocardial fibrosis in Timp3-deficient heart

Abstract

The pleiotropic cytokines, transforming growth factor beta1 (TGFbeta1), and tumor necrosis factor (TNF) play critical roles in tissue homeostasis in response to injury and are implicated in multiple human diseases and cancer. We reported that the loss of Timp3 (tissue inhibitor of metalloproteinase 3) leads to abnormal TNF signaling and cardiovascular function. Here we show that parallel deregulation of TGFbeta1 and TNF signaling in Timp3(-/-) mice amplifies their cross-talk at the onset of cardiac response to mechanical stress (pressure overload), resulting in fibrosis and early heart failure. Microarray analysis showed a distinct gene expression profile in Timp3(-/-) hearts, highlighting activation of TGFbeta1 signaling as a potential mechanism underlying fibrosis. Neonatal cardiomyocyte-cardiofibroblast co-cultures were established to measure fibrogenic response to agonists known to be induced following mechanical stress in vivo. A stronger response occurred in neonatal Timp3(-/-) co-cultures, as determined by increased Smad signaling and collagen expression, due to increased TNF processing and precocious proteolytic maturation of TGFbeta1 to its active form. The relationship between TGFbeta1 and TNF was dissected using genetic and pharmacological manipulations. Timp3(-/-)/Tnf(-/-) mice had lower TGFbeta1 than Timp3(-/-), and anti-TGFbeta1 antibody (1D11) negated the abnormal TNF response, indicating their reciprocal stimulatory effects, with each manipulation abolishing fibrosis and improving heart function. Thus, TIMP3 is a common innate regulator of TGFbeta1 and TNF in tissue response to injury. The matrix-bound TIMP3 balances the anti-inflammatory and proinflammatory processes toward constructive tissue remodeling.

FIGURE 1.

Excessive interstitial fibrosis in aortic banded Timp3-deficient hearts. A, trichrome staining of sham-operated or AB WT, Timp3^−/− and Timp3^+/− hearts. The arrowheads indicate the areas of fibrosis. Scale bar, 100 μm. B, fibrillar collagen was visualized by PSR staining and confocal microscopy in WT and Timp3^−/− hearts at 3 and 6 weeks (wk) post-AB. Scale bar, 50 μm. C, collagen content is presented as collagen volume fraction calculated from PSR-stained sections. D, TaqMan quantification of RNA level of collagen type I, type III, and TIMP1 normalized to 18 S in WT and Timp3^−/− hearts in sham and at 3 weeks post-aortic banding. E, trichrome and α-smooth muscle actin (α-SMA) staining show co-localization of collagen deposit (in blue) and fibroblast activation (in brown), respectively, into fibrotic areas in Timp3^−/− hearts after 3 weeks post-AB. In contrast, WT tissue shows a limited α-smooth muscle actin immunostaining around blood vessels, as shown by arrows, and absence of tissue fibrosis. Scale bar, 400 μm. *, p < 0.05 compared with the corresponding sham groups; ‡, p < 0.05 compared with WT-AB.

FIGURE 2.

Up-regulation of TGFβ signaling in Timp3^−/−-AB hearts. Gene set enrichment analysis indicated TGFβ signaling pathway to be significantly up-regulated at 6 h after aortic banding. This was visualized by GenMAPP. Genes labeled in red were up-regulated, whereas those labeled in green were down-regulated in Timp3^−/− hearts.

FIGURE 3.

Timp3 deficiency enhances the fibrogenic response, which requires cardiomyocyte-cardiofibroblast interaction. A, schematic for isolation of neonatal mouse ventricular myocytes, fibroblasts, or their co-culture. Differential adhesion generates purified single cultures (see “Experimental Procedures”) that are verified by cell-specific markers (supplemental Fig. 1). All cultures were treated with Ang II (1 μm) or PE (1 μm) for 24 h. Collagen type I and type III RNA levels were measured by qPCR in the indicated mono or co-culture setting (B–D). Total collagenase and gelatinase activities are expressed as relative fluorescent units (RFU) (E). Shown are qPCR measurements of RNA for MMP2, -9, and -13 and MT1-MMP (F) and for TNF and TGFβ1 (G) in the neonatal co-cultures. RNA values were normalized to 18 S and are expressed as relative units as described under “Experimental Procedures.” *, p < 0.05 compared with the corresponding control cultures; ‡, p < 0.05 compared with WT. RA, right auricle; LA, left auricle; RV, right ventricle; LV, left ventricle. Con, control.

FIGURE 4.

Enhanced proteolytic processing of TGFβ1 and TNF, and activation of Smad signaling in Timp3^−/− cultures. A, detection of mature and cleaved TGFβ1, total and phosphorylated Smad2/3, full-length and cleaved TNF, and the corresponding β-actin as the loading control, in Ang II (1 μm)-treated or PE (1 μm)-treated (24 h) co-cultures of the indicated genotypes. The dotted line indicates different exposures of the same blot. B, ELISA was performed to measure active TGFβ1 protein released into the conditioned media of neonatal cardiomyocytes, cardiofibroblasts, or co-cultures. C, in vivo levels of cleaved TGFβ1 protein measured by ELISA in tissue homogenates of WT and Timp3^−/− hearts, 6 h after aortic banding. *, p < 0.05 compared with the corresponding sham groups; ‡, p < 0.05 compared with WT-AB.

FIGURE 5.

Treatment with TGFβ-neutralizing antibody (1D11) prevents fibrosis and improves heart function in aortic banded Timp3^−/− mice. A, trichrome staining of Timp3^−/− hearts after 6 weeks (wk) post-AB, with or without 1D11 treatment. Scale bar, 100 μm. B, collagen volume fraction as a measure of myocardial fibrosis. C, qPCR of RNA for collagen types I and III, normalized to 18 S. D, transverse cross-sections of sham and aortic banded hearts 6 weeks after AB. E, heart function measurements performed as fractional shortening (FS) and LV end-diastolic dimension (LVEDD) in sham and 1, 3, and 6 weeks after aortic banding. F, heart weight-to-tibial length ratio (HW/TL). G, qPCR of RNA for molecular markers of hypertrophy, brain natriuretic peptide (BNP), β-myosin heavy chain (β-MHC), and α-skeletal actin in sham or aortic banded WT, Timp3^−/−, and 1D11-treated hearts after 6 weeks. *, p < 0.05 compared with the 1D11-treated group. ‡, p < 0.05 compared with WT-AB.

FIGURE 6.

Dissecting TGFβ1-TNF link through genetic and biochemical manipulations. A, RNA expression of TNF and TGFβ in cardiomyocyte-cardiofibroblast cultures derived from WT and Timp3^−/− hearts treated with recombinant TGFβ1 (10 ng/ml) or recombinant TNF (10 ng/ml) for 1 h. Changes in TGFβ1 expression as measured by qPCR in mouse hearts of the indicated genotypes after aortic banding (B) and in TNF RNA in Timp3^−/−-AB hearts treated with or without 1D11 (C). D, qPCR measurement of RNA for MMP2, MMP13, and MT1-MMP at 6 weeks (wk) post-AB in 1D11-treated Timp3^−/− mice compared with untreated Timp3^−/− and WT mice. *, p < 0.05 compared with their corresponding sham; ‡, p < 0.05 compared with WT-AB.