Connective tissue growth factor promotes fibrosis downstream of TGFbeta and IL-6 in chronic cardiac allograft rejection

Abstract

Cardiac transplantation is an effective treatment for multiple types of heart failure refractive to therapy. Although immunosuppressive therapeutics have increased survival rates within the first year posttransplant, chronic rejection (CR) remains a significant barrier to long-term graft survival. Indicators of CR include patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Multiple factors have been implicated in the onset and progression of CR, including TGFbeta, IL-6 and connective tissue growth factor (CTGF). While associated with CR, the role of CTGF in CR and the factors necessary for CTGF induction in vivo are not understood. To this end, we utilized forced expression and neutralizing antibody approaches. Transduction of allografts with CTGF significantly increased fibrotic tissue development, though not to levels observed with TGFbeta transduction. Further, intragraft CTGF expression was inhibited by IL-6 neutralization whereas TGFbeta expression remained unchanged, indicating that IL-6 effects may potentiate TGFbeta-mediated induction of CTGF. Finally, neutralizing CTGF significantly reduced graft fibrosis without reducing TGFbeta and IL-6 expression levels. These findings indicate that CTGF functions as a downstream mediator of fibrosis in CR, and that CTGF neutralization may ameliorate fibrosis and hypertrophy associated with CR.

Figure 1. Elevated intragraft expression of TGFβ, IL-6, and CTGF in cardiac allografts undergoing CR

TGFβ, IL-6, and CTGF message levels were determined at day 30 post transplant using quantitative real time PCR in syngeneic cardiac grafts, cardiac allografts from recipients treated with anti-CD40L mAb therapy (Anti-CD40L), or cardiac allografts whose recipients were transiently depleted of CD4+ cells (Anti-CD4). Bars represent mean + S.E.M. of 4–9 grafts with expression relative to GAPDH normalized to the syngeneic group.

Figure 2. Forced expression of TGFβ or CTGF promotes allograft fibrosis

(A) Morphometric analysis of Masson’s trichrome staining at day 30 post transplant in cardiac grafts that were left untransduced or transduced with adenoviral vectors encoding βgal (Adβgal), CTGF (AdCTGF), or TGFβ (AdTGFβ) prior to grafting into syngeneic recipients or allogeneic recipients treated with anti-CD40L. Bars represent the combined mean + S.E.M. of fibrotic (blue) area of 10–12 frames of view per heart taken from 5 to 12 different cardiac grafts per group. (B) Intragraft IL-6 message levels were determined at day 30 post transplant using quantitative real time PCR in groups from (A). Bars represent mean + S.E.M. of at least 4 hearts per group with expression relative to GAPDH normalized to naïve, untransplanted BALB/c hearts. (C) Intragraft IL-17 message levels were determined using quantitative real time PCR in syngeneic grafts transduced with AdTGFβ or allogeneic grafts transduced with AdTGFβ whose recipients received anti-CD40L treatment. Bars represent mean + S.E.M. of at least 5 independent hearts per group with expression relative to GAPDH normalized to the naïve BALB/c group.

Figure 3. IL-6 neutralization reduces expression of IL-6, IL-17, and CTGF but not TGFβ in cardiac allografts undergoing CR

Intragraft IL-6, IL-17, CTGF, and TGFβ message levels were determined at day 30 post transplant using quantitative real time PCR in cardiac allograft recipients that were transiently depleted of CD4+ cells and received either neutralizing anti-IL-6 (Anti-IL-6) or control rat IgG (rIgG). Bars represent mean + S.E.M. of 6–8 grafts per group with expression relative to GAPDH normalized against rIgG-treated contols.

Figure 4. CTGF neutralization ameliorates fibrosis

(A) Representative sections of Masson’s trichrome stains, in which fibrotic tissue stains blue, of cardiac allografts from recipients transiently depleted of CD4+ cells (Anti-CD4) at day 30 post transplant in recipients treated with control IgG or neutralizing anti-CTGF mAb (200X magnification). (B) Morphometric analysis of trichrome staining of groups in (A). Bars represent mean + S.E.M. of 10–12 frames of view from each of 6 to 9 hearts. (C) TGFβ, IL-6, and CTGF message levels were determined at day 30 post transplant using quantitative real time PCR in cardiac allografts described in (A). Bars represent mean + S.E.M. of samples taken from 8–12 different cardiac grafts with expression relative to GAPDH normalized against hIgG-treated controls.

Figure 5. CTGF neutralization ameliorates cardiac hypertrophy in CR grafts

(A) Cardiomyocyte area was quantified from H&E stains of day 30 post transplant cardiac allografts taken from recipients transiently depleted of CD4+ cells (Anti-CD4) and receiving CTGF neutralizing mAb (Anti-CTGF) or control antibodies (hIgG), recipients treated with Anti-CD40L mAb, or naïve, untransplanted BALB/c hearts. Bars represent mean + S.E.M. of area measurements taken from ≥100 cardiomyocytes per heart from 5 (naïve BALB/c and Anti-CD40L), 8 (Anti-CD4+hIgG), or 10 (Anti-CD4+Anti-CTGF) different hearts per group. (B) Intragraft message levels of atrial natriuretic peptide (ANP), a marker of cardiac hypertrophy, were quantified with real time PCR in cardiac grafts from groups in (A) at day 30 post transplant. Bars represent mean + S.E.M. of 8–12 grafts per experimental group (Anti-CD4+hIgG or Anti-CTGF) and 4 grafts per control group (Anti-CD40L and naïve BALB/c) with expression relative to GAPDH normalized against the naïve BALB/c hearts.

Figure 6. CTGF neutralization limits graft infiltration by T cells in CR grafts

(A) Representative H&E stains of day 30 post transplant cardiac allografts taken from recipients transiently depleted of CD4+ cells (Anti-CD4) and receiving CTGF neutralizing mAb (Anti-CTGF) or control antibodies (hIgG). Stains suggest a reduction in perivascular infiltrate density in grafts treated with neutralizing Anti-CTGF. (B) Intragraft message levels of T cell receptor β constant region (TCRβ) were quantified at day 30 post transplant with real time PCR as a measure of T cell infiltration of allografts in recipients transiently depleted of CD4+ cells (Anti-CD4) and receiving anti-CTGF mAb or control hIgG antibodies, recipients treated with Anti-CD40L mAb, or naïve BALB/c hearts. Bars represent mean + S.E.M. of 8–12 grafts per group with expression relative to GAPDH normalized against the hIgG group. (C) Repopulation of CD4+ cells in the periphery at day 30 post transplant was determined by flow cytometric analysis of splenocytes isolated from graft recipients. Bars represent mean + S.E.M. of the percentage CD4+ cells of the gated cell population in 5 to 7 recipients tested.

Figure 7. Proposed model of cytokine interactions in chronic rejection

(A) In cardiac allografts, TGFβ and IL-6 contribute to CTGF production. IL-6 and CTGF are both known to promote hypertrophy in cardiac myocytes, which in turn can produce CTGF. CTGF functions as a downstream mediator of fibrosis. (B) Induction of CTGF downstream of TGFβ and IL-6 could be explained by the respective presence of a consensus SMAD binding element and a STAT3 response element in 5′ region upstream of the CTGF promoter. For expanded explanations, please see text.