Fig 2: Stromal angiotensin signalling induces matrix production in tumours.(a) Tumour collagen and (b) hyaluronan levels in angiotensin-II-receptor-1 (AT1)-knockout mice. Orthotopic E0771 breast tumours implanted in AT1-knockout mice (Agtr1a−/−) have (a) a lower collagen I area fraction (*P=0.026, Student’s t-test) and (b) a lower hyaluronan area fraction (*P=0.011, Student’s t-test) than E0771 tumours implanted in wild-type C57BL/6 mice. (c) Tumour collagen and (d) hyaluronan levels in angiotensin-II-receptor-2 (AT2) knockout mice. Orthotopic E0771 breast tumours implanted in AT2-knockout mice (Agtr2−/−) have (c) a higher collagen I area fraction (*P=0.047, Student’s t-test) and (d) a higher hyaluronan area fraction (*P=0.012, Student’s t-test) than E0771 tumours implanted in wild-type C57BL/6 mice. Tumours were time- and size-matched at ~200 mm3. (e) Expression of AT1 (Agtr1a and Agtr1b genes) and AT2 (Agtr2 gene) mRNA in murine cancer-associated fibroblasts (CAFs) and cancer cells. CAFs isolated from AK4.4 tumours express over one order of magnitude more AT1 than either E0771 or AK4.4 cancer cells. Similarly, the CAFs express over two orders of magnitude more AT2 than either E0771 or AK4.4 cells. (f) Expression of AT1 (red) in αSMA+ CAFs (green) and cancer cells in tumours. We imaged CAFs based on αSMA+ expression in E0771 tumours. We found that some CAFs in these tumours express AT1 at high levels, whereas other cells express AT1 at low levels. Colocalization is shown in yellow. Scale bar, 25 μm. (g) Expression of AT2 (red) in αSMA+ CAFs (green) and cancer cells in tumours. Most CAFs express high levels of AT2, whereas some other cells express similar AT2 levels. Colocalization is shown in yellow. Scale bar, 25 μm. Animal numbers n=4–5. Error bars indicate s.e.m.

Fig 3: Loss of Cx37 selectively alters the expression of the AT2 receptors in the aortas of Cx37−/− mice. A, Immunofluorescence showed a similar staining for AT1R (upper panels) in the aortas of all WT and Cx37−/− mice. In contrast, the staining for AT2R was more intense in the aortas of Cx37−/− than in WT mice. Bar=20 μm. L indicates lumen; M, media. B, Western blot showed that the aortas of WT and Cx37−/− mice expressed similar levels of the AT1R protein. C, In contrast, the basal levels of the AT2R protein were higher in Cx37−/− than in WT mice, and were not increased after the 2K1C surgery. WT sham N=21; Cx37−/− sham N=17; WT 2K1C N=19; Cx37−/− 2K1C N=17. D and E, Comparable observations were made for both AT1R (D) and AT2R (E) in mice infused daily with 1 mg Ang II/kg body weight WT NaCl N=4; Cx37−/− NaCl, Cx37−/− Ang II N=3, WT Ang II N=6. Results are means+SEM. ° P≤0.05 vs WT mice, as given by 2‐way ANOVA. 2K1C indicates 2‐kidney, 1‐clip; AngII, angiotensin II; AT1R, Ang II type 1 receptors; AT2R, Ang II type 2 receptors; Cx37, Connexin37; DAPI, 4′,6‐diamidino‐2‐phenylindole; Sham, sham‐operated controls; WT, wild type.

Fig 4: Expression of mRNAs was studied by RT–PCR. Relative gene expression of AT1, AT2, MAS, and NOX4 mRNA profile in the SCG normalized to β-actin expression by the 2ΔΔCT comparative method and expressed as fold change compared with HnSD. Values are mean ± SEM. mRNA for AT2 is significantly lower in SCG of (mRen2)27 transgenic hypertensive animals compared with control (1.013 ± 0.105 vs. 0.171 ± 0.013 a.u.; n = 6; *P < 0.05).

Fig 5: The fluorescent targets and their corresponding antibody dilutions are as follows; for AT1—extracellular-ATTO Fluor-550; AT2—extracellular-ATTO Fluor-488 and MAS receptor –Fluor-488 tags (1° antibody delusion 1:50) were visualized by confocal microscopy. The 4′, 6-diamidino-2-phenylindole and F-actin merged images show the green fluorescence is specific for angiotensin receptors. Images were adjusted for brightness/contrast using the Zeiss LSM 800 image browser similar for all 3 panels. Fluorescence intensity was quantified by ImageJ software and cumulative data are presented in a bar graph. Values in each panel are mean ± SEM; *P < 0.05.