Osteopontin modulates angiotensin II- induced fibrosis in the intact murine heart
Alan R. Collins, PhD*,
Janet Schnee, MD*,
Wei Wang, MD*,
Sarah Kim, BS*,
Michael C. Fishbein, MD ,
Dennis Bruemmer, MD*,
Ronald E. Law, PhD*,
Susanne Nicholas, MD, PhD*,
Robert S. Ross, MD, FACC and
Willa A. Hsueh, MD*,*
* Department of Medicine, Division of Endocrinology, Diabetes and Hypertension, Los Angeles, CaliforniaUSA
Department of Pathology, The David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, USA
Departments of Medicine, VA Healthcare System-San Diego, and University of California at San Diego, San Diego, California, USA
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Figure 1 Blood pressures at baseline and after three weeks of angiotensin II (AngII) infusion in wild-type (WT) and osteopontin-null (OPN–/–) mice. Basal blood pressure (BP) is lower in OPN–/– mice versus WT mice (xp < 0.01). Systolic blood pressure (SBP) of each group increased significantly versus untreated mice (*p < 0.05 WT; p < 0.05 OPN–/–) when infused with AngII at 2.5 µg/kg/min. Infusion of AngII at 3 µg/kg/min increased BP in OPN–/– mice versus OPN–/– basal values, and to a higher absolute systolic pressure versus either WT or OPN–/– mice infused at the lower 2.5 µg/kg/min dosing. 2.5 AngII = AngII at 2.5 µg/kg/min; 3 AngII = AngII at 3 µg/kg/min. Mean change in SBP of WT and OPN–/– mice from sham-infused and after three weeks' treatment with AngII at 2.5 µg/kg/min was identical. The SBP in OPN–/– mice treated with AngII at 3 µg/kg/min was significantly higher than in OPN–/– mice or WT mice treated with AngII at 2.5 µg/kg/min (+p < 0.001 vs. 2.5 µg/kg/min AngII-infused WT or OPN–/– mice).
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Figure 2 Heart weight/body weight indices of WT and OPN–/– mice at baseline and after three weeks of AngII infusion. Angiotensin II causes hypertrophy as detected by increased heart/body weight ratio in control but not in OPN–/– mice. Heart-to-body-weight ratio of WT is significantly increased in the AngII-treated group compared with the control sham-infused group (*p < 0.001), whereas heart-to-body-weight ratio is not significantly increased in either of the treated OPN–/– groups, even when 3 µg/kg/min AngII infusion affected BPs greater in both absolute value and percent change versus baseline compared with WT control animals. White bars = control; hatched bars = AngII, 2.5 µg/kg/min; black bars = 3 µg/kg/min. Abbreviations as in Figure 1.
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Figure 3 Cardiac ankyrin repeat protein (CARP), atrial natriuretic factor (ANF) and OPN are molecular markers of cardiac hypertrophy increase after AngII infusion. (A) Ventricular CARP and ANF expression are upregulated in both WT and OPN–/– groups after four days of AngII (2.5 µg/kg/min) infusion. (B) Ventricular CARP returns to baseline but ANF remains upregulated after three weeks of AngII (2.5 µg/kg/min) infusion in both WT and OPN–/– groups. (C) Ventricular OPN messenger ribonucleic acid expression in WT mice at four days after AngII infusion returns toward baseline by three weeks post-infusion. Other abbreviations as in Figure 1.
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Figure 4 Absence of OPN leads to lessened myocardial fibrosis after AngII infusion. Trichrome stained myocardial sections demonstrate fibrosis. Wild-type mice demonstrated a 10-fold increase in interstitial fibrosis (blue staining) in response to AngII (2.5 µg/kg/min) (0.7 ± 0.16% to 8.0 ± 1.37%; n = 12, p < 0.01) as compared to a more modest four-fold response in the OPN–/– (four-fold increase, 0.4 ± 2.4% to 1.6 ± 0.73%) mice whether infused with 2.5 µg/kg/min (n = 15) or 3 µg/kg/min AngII (n = 8), a dose that caused a greater absolute BP and change from baseline than that measured in the WT animals. (A) WT after three weeks of sham infusion; (B) WT after three weeks of AngII infusion at 2.5 µg/kg/min; (C) OPN–/– after three weeks of AngII infusion at 2.5 µg/kg/min; (D) OPN–/– after three weeks of AngII infusion at 3 µg/kg/min; (E) Quantitation of interstitial fibrosis, determined via analysis of digitized image of 20x magnified trichrome-stained section, expressed as percent of total myocardial cross-sectional area. Interstitial fibrosis was significantly increased in the treated WT group but was not increased in the treated OPN–/– groups (p < 0.001). No significant difference was detected between any of the sham-infused groups. White bars = control; hatched bars = AngII, 2.5 µg/kg/min; black bars = 3 µg/kg/min. Abbreviations as in Figure 1.
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Figure 5 Angiotensin II increases ventricular fibronectin (FN), collagen I, transforming growth factor-beta, and integrin beta-1 transcript expression similarly in WT mice (A) versus OPN–/– mice (B). Ribonucleic acid was isolated from ventricular samples at baseline or after three weeks of AngII infusion. Increases in all transcripts were detected after AngII infusion in both the WT and OPN–/– mice, yet no differences were detected between the two treated groups. CHO-B was used as a loading control. Abbreviations as in Figure 1.
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Figure 6 Adhesion of OPN–/– cardiac fibroblasts to a panel of ECM substrates is reduced versus WT cells and can be returned toward WT levels with addition of recombinant OPN. The figure shows a comparison of adhesion of neonatal cardiac fibroblasts from WT, untreated OPN–/– versus OPN–/– cells grown in the presence of recombinant OPN (OPN–/– + recOPN) to a panel of ECM components, as indicated along the x-axis (BSA = bovine serum albumin, COL = collagen I, FN = fibronectin, LN = laminin, and VN = vitronectin). Overall adhesion of WT-derived cells is greater than that of OPN–/–derived cells (*p <0.001 for all substrates). Growth of OPN-derived fibroblasts in the presence of recombinant OPN increased their adhesion towards normal WT values (p < 0.01 for all substrates). White bars = wild-type (WT); hatched bars = OPN–/–; black bars = OPN–/– + recombinant OPN. BSA = bovine serum albumin, COL = collagen I, FN = fibronectin, LN = laminin, and VN = vitronectin. Other abbreviations as in Figure 1.
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Figure 7 Proliferation of OPN–/–-derived fibroblasts is blunted versus WT cells. Osteopontin-null cells grown in the absence of exogenous OPN had a significantly reduced proliferative response to serum stimulation versus WT-derived cells, whereas OPN–/– cells grown in the presence of exogenous OPN had proliferative capacities not significantly different from WT values. *p < 0.05 vs. WT. Abbreviations as in Figure 1.
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