Angiotensin II-Induced Cardiovascular Fibrosis Is Attenuated by NO-Sensitive Guanylyl Cyclase1

Figures

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  Figure 1. In NO-sensitive guanylyl cyclase (NO-GC)-1 KO mice, perivascular fibrosis and collagen1 mRNA content were increased by AngiotensinII (AngII) treatment. Medial area (a) and wall thickness (b) were measured in cross sections (5 µm) of abdominal aortae from non-treated and AngII-treated WT and NO-GC1 KO mice. Shown are individual values (per animal) together with means ± SD. (c) Representative cross sections (5 µm) of abdominal aortae from non-treated and AngII-treated WT and NO-GC1 KO mice stained with elastica Van Gieson (for elastin) and picrosirius red (for collagen). Scale bar, 100 µm. (d) Elastin content is expressed as percentage of medial area of aortae from non-treated and AngII-treated WT and NO-GC1 KO mice. (e) Perivascular area of aortae from non-treated and AngII-treated WT and NO-GC1 KO mice were quantified. Shown are individual values (per animal) together with means ± SD. mRNA levels of (f) collagen1 (g) collagen3, and (h) fibronectin (FN) were determined using quantitative real-time PCR. ∆∆Ct values to GAPDH and subsequently to non-treated WT were calculated and statistically analyzed. For graphical representation, 2−∆∆C are shown in semi-log plots. Shown are…

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  Figure 2. AngII induces an increase of perivascular area of cardiac vessels in NO-GC1 KO mice. (a) Media to lumen and (b) perivascular to lumen ratios of cardiac vessels were determined in picrosirius red-stained heart slices from non-treated and AngII-treated WT and NO-GC1 KO mice. Values are normalized to media to lumen ratio and perivascular area to lumen ratio of WT (in a: 0.59; in b: 0.53). Because residuals were not normally distributed, individual values were log-transformed and statistically analyzed. For graphical representation, original (not-transformed) values are shown in semi-log plots as individual values (per animal) together with geometric means ± geometric SD. (c) Representative cross sections (5 µm) of cardiac vessels from non-treated and AngII-treated WT and NO-GC1 KO mice stained with picrosirius red (for collagen). Scale bar, 20 µm.

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  Figure 3. AngII induced increase of left ventricular wall thickness and cardiomyocyte cross sectional area. (a) Left ventricular wall thickness was measured in hearts of non-treated and AngII-treated WT and NO-GC1 KO mice. Individual values (per animal) are shown together with means ± SD. (b) Cardiomyocyte cross sectional areas of WGA-stained heart slices (c) were quantified. Individual values (per animal) are shown together with means ± SD. (c) Shown are representative WGA-stained heart sections. Scale bar, 20 µm.

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  Figure 4. NO-GC-1 KOs display substantially enhanced AngII-induced interstitial fibrosis. (a) Picrosirius red-stained areas of hearts (b,c) were quantified and normalized to non-treated WT. Shown are individual values (per animal) together with means ± SD. (b) Shown are representative paraffin-embedded heart sections (5 µm) stained with picrosirius red. Scale bar, 500 µm (c) Shown are magnifications of heart sections stained with picrosirius red. Strongly red stained areas found in NO-GC1 KO hearts were not used for analysis. Scale bar, 20 µm.

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  Figure 5. Cardiac mRNA of proteins implicated in fibrosis are increased in AngII-treated NO-GC1 KO compared to WT mice. mRNA levels of (a) collagen 1 (b) periostin (c) TGFβ (d) matrix metalloproteinase 2 (e) CTGF (f) ANP (g) BNP, and (h) fibronectin were determined using quantitative real-time PCR in heart sections from WT and KO mice under non-treated and AngII-treated conditions. ∆∆Ct values to GAPDH and subsequently to non-treated WT were calculated and statistically analyzed. For graphical representation, 2−∆∆Ct values were calculated and are shown in semi-log plots. Shown are individual values (per animal) together with geometric means ± geometric SD.