Reduced vasorin enhances angiotensin II signaling within the aging arterial wall

Figures

• 

  Figure 1. Vasorin expression decreases in arterial walls with aging (A) Aortic vasorin mRNA determined by RT-PCR. Data shown as mean ± SEM (n= 4 rats/group). T-test, ***= p<0.001. (B) Immunohistostaining of vasorin (brown color, 400X) in aortic walls. All micrographs were taken at the same exposure time. Data of staining vasorin area/arterial area ratio (%, lower panel) shown as mean ± SEM (n= 4 rats/group). T-test, * =p<0.05. L=lumen; M=media. Bar scale, 10 μm. (C) Representative western blots of aortic vasorin. Data (right panel) shown as mean ± SEM (n= 3 rats/group). Each column represents a normalized ratio (fold-changes) to tubulin and to 8 mo. T-test, *=p<0.05.

• 

  Figure 2. Vasorin expression decreases in VSMCs with aging (A) VSMC vasorinm RNA determined by RT-PCR. Data shown as mean ± SEM (n= 6 rats/group). T-test, *=p<0.05. (B) Representative immunohistostaining (X400) of vasorin (green) and nuclei (blue) in VSMCs captured by confocal microscopy. All micrographs were taken at the same exposure time. Bar scale, 20 μm. (C) Representative western blots of VSMC vasorin. Data (right panel) shown as mean ± SEM (n=3 independent experiments from n=3 rats/group). Each column represents a normalized ratio (fold-change) to tubulin and to 8 mo. T-test, *=p<0.05.

• 

  Figure 3. Age decreases vasorin expression in aortic wall or VSMCs via Ang II signaling (A) Representative immunohistostaining of vasorin (brown, X400) of carotid arteries harvested from young rats infused with either Ang II (Ang II infusion) or vehicle saline animals (control). All micrographs were taken at the same exposure time. Data of relative vasorin staining area (%) (right panel) shown as mean ± SEM (n= 5 rats/group). T-test, *=p<0.05. L=lumen; M=media. Bar scale, 10 μm. (B) Representative western blots of VSMC vasorin. Cells have been pre-treated for 1 hour with Los, and then exposed for 24 hours to a medium containing both Ang II and Los. Data (right panel) shown as mean ± SEM (n=4 independent experiments from n=4 rats/group). One-way ANOVA followed Bonferroni post hoc test, *p<0.05, **=p<0.01; and ***=p<0.001. Ang II = Angiotensin II; Los=Losartan.; and M=Molar concentration. (C) VSMC vasorin mRNA determined by RT-PCR. Cells have been treated for 24 hours in medium containing Ang II. Data shown as mean ± SEM (n= 6 rats/group). T-test, n.s = no significant.

• 

  Figure 4. Vasorin is an MMP-2 substrate (A) Representative western blots of vasorin showing that the purified human vasorin was cleaved by the activated MMP-2. Recombinant human vasorin (400 ng/ml) was incubated in a developing buffer containing activated MMP-2 (50 nM) with or without an MMP inhibitor GM6001 (20 nM) for 24 hours at 37°C (n= 4 independent experiments). Western blots were probed with vasorin antibody, which detected the intact vasorin (∼100 kDa) and a soluble fragment (∼75 kDa). (B) Representative western blots of vasorin showing that the monkey aortic homogenate containing vasorin was cleaved step-wisely by MMP-2. Monkey aortic lysates (25 μg/ml) were incubated in a developing buffer containing activated MMP-2 (50 nM) with or without an MMP inhibitor GM6001 (20 nM) for 24 hours at 37°C (n= 3 independent experiments from 3 monkeys). Western blots were probed with vasorin antibody, which detected the intact vasorin (∼100 kDa) and three soluble fragments (∼75 kDa, 50 kDa, and 37 kDa) showing intact arterial vasorin was processed by activated MMP-2. (C) Representative immunohistostaining of vasorin (brown, X400) of aortae harvested from adult young rats administrated with…

• 

  Figure 5. The Interaction of vasorin and TGF-β1 in VSMCs with aging (A) Co-immunoprecipitation of TGF-β1 and vasorin in VSMCs. Cell lysates were immunoprecipitated with an anti- TGF-β1 antibody and then the western blotting against both vasorin and TGF-β1 were performed. Data (right panel) shown as mean ± SEM (n= 4 independent experiments/group). Each column represents a normalized ratio (fold-changes) to tubulin and to 8 mo. T-test, *=p<0.05. (B) Dual labelling (X630) of vasorin (green) and TGF-β1 (red) in VSMCs. All micrographs were taken at the same exposure time. Data of the relative colocalized area (%, right panel) shown as mean ± SEM (n= 4 independent experiments/group). T-test, *=p<0.05. T-test, ***=p<0.001. Bar scale, 10 μm.

• 

  Figure 6. Ang II, MMP-2 and TGF-β1 activation in VSMCs (A) Representative zymogram of the medium from early passage cultured VSMCs treated with either Angiotensin II (Ang II) Losartan (Los) (upper panel). Cells have been pre-treated for 1 hour with Los (5X10-6 M), and then exposed for 24 hours to a medium containing both Ang II (5X10-6 M) and Los (5X10-6 M). Data (lower panel) shown as mean ± SEM (n=4 independent/group). One-way ANOVA followed Bonferroni post hoc test, *=p<0.05, and **=p<0.01. (B) Representative western blots of TGF-β1 from lysates from early passage cultured VSMCs treated with either Ang II or Los (upper panel). Cells have been pre-treated for 1 hour with Losartan (5X10-6 M), and then exposed for 24 hours to a medium containing both Ang II (5X10-6 M) and Los (5X10-6 M). Data (lower panel) shown as mean ± SEM (n=4 independent/group). Each column represents a normalized ratio (fold-changes) to tubulin and to untreated 8 mo VSMCs. One-way ANOVA followed Bonferroni post hoc test, *=p<0.05, and **=p<0.01.

• 

  Figure 7. Age increases TGF-β1 downstream profibrotic molecules in VSMCs mediated by over-expressing vasorin (A) Representative western blots for p-SMAD-2/3 and total SMAD-2/3 of VSMCs transfected with either control DsRed or DsRed containing human vasorin cDNA (DsRed HV) plasmid and treated with or without Ang II (5X10-6 M) for 24 hours. Data (right panel) shown as mean ± SEM (n=6 independent/group). Each column represents a normalized ratio (fold-changes) to tubulin and to 8 mo + DsRed. One-way ANOVA followed Bonferroni post hoc test, *=p<0.05, and **=p<0.01. (B) Representative western blots for collagen Type I (Col I) of VSMCs transfected with either control DsRed or DsRed containing human vasorin cDNA (DsRed HV) plasmid and treated with or without Ang II (5X10-6 M) for 24 hours. Data (right panel) shown as mean ± SEM (n=6 independent/group). Each column represents a normalized ratio (fold-change) to tublin and to 8 mo + DsRed. One-way ANOVA followed Bonferroni post hoc test, *=p<0.05, and **=p<0.01.

• 

  Figure 8. TGF-β1 downstream profibrotic effects in old VSMCs regulated by recombinant human vasorin proteins (A) Representative western blots of TGF-β1, p-SMAD-2/3, SMAD-2/3, Coll I, β-actin, and Zymogram from lysates of early passage cultured VSMCs treated with or without recombinant human vasorin proteins at the indicated doses. (B) Data shown as mean ± SEM (n=3 independent/group). Each column represents a normalized ratio (fold-change) to β-actin and to untreated control (0). One-way ANOVA followed Bonferroni post hoc test, *=p<0.05, **=p<0.01 and ***=p<0.001.

• 

  Figure 9. The Ang II associated MMP-2 activation with aging mediated by vasorin (A) Representative zymogram from VSMCs transfected with either control DsRed or DsRed containing human vasorin cDNA (DsRed HV) plasmid and treated with or without Ang II (5X10-6M) for 24 hours. Data (right panel) shown as mean ± SEM (n=4 independent experiments/group). One-way ANOVA followed Bonferroni post hoc test, *=p<0.05 and **=p<0.01. STD=standard: activated MMP2 protein. (B) Representative zymogram from VSMCs treated with Ang II (5X10-6M) with or without recombinant human vasorin protein (100 ng/ml). Data (right panel) shown as mean ± SEM (n=3 independent experiments/group). One-way ANOVA followed Bonferroni post hoc test, ***=p<0.001.

• 

  Figure 10. Vasorin decreases both Ang II- and aging-associated phenotypic shift of VSMCs into invasiveness (A) Invasion assay of VSMCs transfected with either control DsRed or DsRed containing human vasorin cDNA (DsRed HV) plasmid and treated with or without Ang II (5X10-6 M) for 4 hours using modified Boyden chamber. Data shown as mean ± SEM (n=3 independent experiments/group). One-way ANOVA followed Bonferroni post hoc test, **=p<0.01 and **=p<0.001. HPF= high power field. (B) Invasion assay of VSMCs treated with without Ang II and human recombinant vasorin protein for 4 hours using modified Boyden chamber. Data shown as mean ± SEM (n=3 independent experiments/group). One-way ANOVA followed Bonferroni post hoc test, *=p<0.5 and **p<0.001. HPF= high power field.

• 

  Figure 11. Schematic diagram showing the Ang II-associated TGF-β1 signaling affected by vasorin cleavage due to MMP-2 activation in VSMCs or arterial walls with aging Ang II=angiotensin II, Los=losartan, MMP-2=matrix metalloproteinase type II, TGF-β1=tissue transforming growth factor-beta1, VSMC=vascular smooth muscle cells.