Fig 2: Early upregulation of myocardial CTGF and TGF-β transcript levels appeared as early as six hours after Ang-II exposure. Myocardial (a) CTGF and (b) TGF-β transcript levels after six or 24 hours of Ang-II infusion relative to saline controls. Transcript levels are reported relative to housekeeping gene ribosomal 18S.Data are expressed as means ± SEM.n = 5–6.*p < 0.05, **p ≤ 0.01 and ***p ≤ 0.001, compared to saline controls.Ang-II: Angiotensin II; CTGF: connective tissue growth factor; TGF-β: transforming growth factor-β.

Fig 3: Exogenous CTGF incubation does not appear to affect differentiation of NIH/3T3 fibroblasts into α-SMA+ myofibroblasts independently or synergistically with exogenous TGF-β incubation in vitro. NIH/3T3 fibroblasts were incubated with exogenous CTGF only, TGF-β only or CTGF plus TGF-β for 24 hours. Representative fields of view stained for α-SMA, Hoechst and phase-contrast are shown for fibroblasts in (a) media control (1% FBS/DMEM), (b) exogenous CTGF, (c) exogenous TGF-β and (d) exogenous CTGF plus TGF-β. (e) Graphical representation of α-SMA expression as measured by fluorescence intensity relative to Hoechst fluorescence intensity and represented as relative to media controls.Data are represented as means ± SEM.n = 3 independent experiments in triplicates.*p < 0.05, **p ≤ 0.01, compared to media control.α-SMA: alpha-smooth muscle actin; AU: arbitrary units; CTGF: connective tissue growth factor; DMEM: Dulbecco’s modified Eagle media; FBS: fetal bovine serum; TGF-β: transforming growth factor-β.

Fig 4: Exogenous CTGF incubation does not appear to have an effect on NIH/3T3 fibroblast production of fibrogenic CTGF, TGF-β or collagen type I transcripts independently or synergistically with exogenous TGF-β incubation in vitro. NIH/3T3 fibroblasts were incubated with exogenous CTGF only, TGF-β only, or CTGF plus TGF-β for six hours. Transcript levels of (a) CTGF, (b) TGF-β and (c) collagen type I were obtained using qPCR analyses. Transcript levels are reported relative to housekeeping genes ribosomal 18S and GAPDH.Data are represented as means ± SEM and expressed relative to media control.n = 3 independent experiments in triplicate.*p<0.05, **p ≤ 0.01, compared to media control.CTGF: connective tissue growth factor; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; qPCR: quantitative polymerase chain reaction; TGF-β: transforming growth factor-β.

Fig 5: Administration of TGF-β trap reduced levels of Ang-II-induced early upregulation of CTGF transcript levels at six hours. Myocardial CTGF and TGF-β transcript levels after (a) six hours or (b) 24 hours of Ang-II infused animals injected with TGF-β trap or PBS relative to saline controls. Transcript levels are reported relative to housekeeping gene ribosomal 18S.Data are expressed as means ± SEM.n = 5–6.*p < 0.05, compared to PBS group.Ang-II: Angiotensin II; CTGF: connective tissue growth factor; PBS: phosphate-buffered saline; Trap: TGF-β trap; TGF-β: transforming growth factor-β.