Established neointimal hyperplasia in vein grafts expands via TGF-beta-mediated progressive fibrosis - PubMed (original) (raw)

Established neointimal hyperplasia in vein grafts expands via TGF-beta-mediated progressive fibrosis

Zhihua Jiang et al. Am J Physiol Heart Circ Physiol. 2009 Oct.

Abstract

In weeks to months following implantation, neointimal hyperplasia (NIH) in vein grafts (VGs) transitions from a cellularized to a decellularized phenotype. The inhibition of early cellular proliferation failed to improve long-term VG patency. We have previously demonstrated that transforming growth factor-beta(1) (TGF-beta(1))/connective tissue growth factor (CTGF) pathways mediate a conversion of fibroblasts to myofibroblasts in the early VG (<2 wk). We hypothesize that these similar pathways drive fibrosis observed in the late VG lesion. Within rabbit VGs, real-time RT-PCR, Western blot analysis, ELISA, and immunohistochemistry were used to examine TGF-beta/CTGF pathways in late (1-6 mo) NIH. All VGs exhibited a steady NIH growth (P = 0.006) with significant reduction in cellularity (P = 0.01) over time. Substantial TGF-beta profibrotic activities, as evidenced by enhanced TGF-beta(1) activation, TGF-beta receptor types I (activin receptor-like kinase 5)-to-II receptor ratio, SMAD2/3 phosphorylation, and CTGF production, persisted throughout the observation period. An increased matrix synthesis was accompanied by a temporal reduction of matrix metalloproteinase-2 (P = 0.001) and -9 (P < 0.001) activity. VG NIH is characterized by a conversion from a proproliferative to a profibrotic morphology. An enhanced signaling via TGF-beta/CTGF coupled with reduced matrix metalloproteinase activities promotes progressive fibrotic NIH expansion. The modulation of late TGF-beta/CTGF signaling may offer a novel therapeutic strategy to improve the long-term VG durability.

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Figures

Fig. 1.

Fig. 1.

Morphological analyses of vein grafts 1 to 6 mo following implantation. With one-way ANOVA, significant differences were detected among time points for lumen cross-sectional area (A), area within the internal elastic lamina (IEL; A), and neointimal area (B). *P = 0.02; #P = 0.01; $P = 0.006 (one-way ANOVA). Bottom: representative images of Masson's trichrome stain for vein grafts harvested at 1, 3, and 6 mo following implantation.

Fig. 2.

Fig. 2.

Temporal changes in cell proliferation (A), content of proteoglycans (B), and cellular density (C) during late vein graft neointimal hyperplasia (NIH). Significant reductions in cell proliferation (A) and nuclear density (C) along with dramatic increases in the content of proteoglycans within the neointima are observed in the 1- to 6- mo time frame. *P = 0.01 (one-way ANOVA). Brdu, bromodeoxyuridine. Bottom: representative images of Movat's stain for vein grafts harvested at 1, 3, and 6 mo following implantation.

Fig. 3.

Fig. 3.

Transforming growth factor-β1 (TGF-β1) production during late vein graft remodeling. TGF-β1 mRNA (A) and total and activated TGF-β1 protein content (B) are significantly elevated from baseline. *P < 0.001 for each data point vs. baseline level. Cells with the most intensive TGF-β1 immunogenicity (arrowheads) are located at luminal surface of the graft wall (C, 3-mo vein graft). IHC, immunohistochemistry. D: negative control using isotype antibody.

Fig. 4.

Fig. 4.

Western blot analyses for the production of TGF-β receptors types I [activin receptor-like kinase 5 (Alk5)] and TβRII (A) and the phosphorylation of Smad2/3 (B). Late vein graft neointimal expansion is associated with a progressive shift in the stoichiometry of Alk5 and TβRII (P < 0.001) and marked reduction of Smad2/3 phosphorylation (P = 0.001). *P = 0.001 (one-way ANOVA). pSmad2/3, phosphorylated Smad2/3.

Fig. 5.

Fig. 5.

IHC assay for Smad1 phosphorylation. pSmad1 protein presented in the majority of the neointimal nuclei during late neointimal expansion. Whereas the absolute number of pSmad1-positive cells decreases, the averaged density of nuclear stain exhibits a significant increase a month after graft implantation. *P = 0.02 (one-way ANOVA).

Fig. 6.

Fig. 6.

Connective tissue growth factor (CTGF) production during late vein graft remodeling. CTGF mRNA levels (A) are significantly elevated from the baseline. *P = 0.01 (one-way ANOVA). Cells lining the luminal surface (arrowheads) are negative for CTGF, whereas the majority of neointimal cells display intensive CTGF immunogenicity (B). Negative control using nonimmunized serum yielded completely negative staining (C).

Fig. 7.

Fig. 7.

Zymographic analysis of gelatinolytic activity of matrix metalloproteinase (MMP)-2 and -9. Significant reduction in pro-MMP-2 (P < 0.001), active (Act) MMP-2 (P < 0.001), and active MMP-9 (P = 0.036) activities were detected when growth of the matrix-dominant neointima is most pronounced.

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