Sex-Dependent Impairment of Endothelium-Dependent Relaxation in Aorta of Mice with Overexpression of Hyaluronan in Tunica Media

Figures

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  Figure A1. The contractile response to high physiological potassium solution (KPSS). The results are presented as the means ± s.e. # p < 0.05 versus the aortas of male mice. The female aortas exhibited a higher contractile response than the male aortas (p = 0.003, n = 5 F, n = 13 M). We found no genotype-dependent differences following a two-way ANOVA.

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  Figure A2. Male and female concentration–response curves for ACH and SNP with and without the NO synthase inhibitor L-Gnitro-arginine (L-NOARG). All graphs on left are control curves of aortas incubated with L-NOARG for 30 min prior to CRC, and all graphs on right are those of mice incubated without L-NOARG; they are presented in the article. (a) Responses of male aorta segments to ACh after L-NOARG incubation. No vasodilation was observed. (b) The corresponding experiment without L-NOARG. The difference between the myographs with and without L-NOARG was statistically significant (p < 0.0001). (c) Responses of female aorta segments to ACH after L-NOARG incubation. No vasodilation was observed. (d) The corresponding experiment without L-NOARG. The difference between the myographs with and without L-NOARG was statistically significant (p < 0.0001). (e) Responses of male aorta segments to SNP after L-NOARG incubation. The vasodilation was enhanced compared to (f), which shows the corresponding experiment without L-NOARG. The difference between the myographs with and without L-NOARG was statistically significant (p < 0.0001, n = 11/group). (g) Responses of female aortas to SNP after L-NOARG…

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  Figure A3. Representative immunoblot of TG2, located at approximately 75 kDa, in the aortas of wild-type (WT) mice and those overexpressing hyaluronan synthase 2 (HAS-2) (left), and average TG2 expression in WT and HAS-2 female (F) and male (M) mice. # p < 0.05 compared to female WT and † p < 0.05 compared to male HAS-2 according to unpaired t-test. Two-way ANOVA was not appropriate due to significant interaction between variables.

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  Figure 1. Response of aortas from WT and HAS-2 mice to phenylephrine, presented as mN contraction per mm aorta. Average contractions measured as an increase in tension (N/m) induced in the aortas of (a) male (n = 13/group) and (b) female (n = 5/group) wild-type (WT) mice and hyaluronan synthase (HAS-2) transgenic mice. (c) Graph of the contraction response of male WT and HAS-2 aortas. We analyzed the data via two-way ANOVA, and we detected no significant interactions or differences between the WT and the HAS-2 aortas (p = 0.47, n = 13/group). (d) Graph of the contraction response of female WT and HAS-2 aortas to phenylephrine. We analyzed the data via two-way ANOVA and detected no significant interaction, but the HAS-2 aortas contracted slightly less than the WT aortas (p = 0.025, n = 5/group). Results are presented as the means ± s.e. * p < 0.05 versus the control curve. # p < 0.05 versus the response in the aortas of female mice (two-way ANOVA).

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  Figure 2. Sex-specific differences in acetylcholine and sodium nitroprusside relaxation in the aortas of wild-type (C57Bl6J) mice. Average concentration–response curves for (a) acetylcholine (ACh) and (b) sodium nitroprusside (SNP) in phenylephrine (PhE)-contracted aortas of male (M) and female (F) mice. The curves were obtained in the absence and the presence of an inhibitor of nitric oxide (NO) synthase (L-NOARG) (3 × 10−4 M). Results are presented as the means ± s.e. * p < 0.05 according to two-way ANOVA.

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  Figure 3. Blunted acetylcholine and sodium nitroprusside relaxation in the aortas of male mice overexpressing hyaluronan synthase 2 (HAS-2). (a,b) Traces representative of relaxation induced by increasing concentrations of acetylcholine (ACh) in the aortas of wild-type (WT) (a) and HAS-2 (b) male mice. (c,d) Averaged concentration–response curves for ACh in WT and HAS-2 male (c) (n = 11) and female (d) (n = 5) mice (n = 11). (e,f) Averaged concentration–response curves for sodium nitroprusside (SNP) in aorta segments from male (e) (n = 11) and female (f) (n = 5) mice. The results are presented as the means ± s.e. * p < 0.05 according to two-way ANOVA.

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  Figure 4. Nitric oxide (NO) levels were decreased in the aortas of male mice overexpressing hyaluronan synthase 2 (HAS-2). Average diaminofluorescence (DAF) in aortas of male (a) (n = 7–15) and female (b) (n = 7) mice measured at baseline level (BASAL) and in response to phenylephrine (PhE) and acetylcholine (ACh), and normalized to tissue weight. The results are presented as the means ± s.e. * p < 0.05 compared to the aortas of WT mice according to two-way ANOVA.

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  Figure 5. Expression of endothelial nitric oxide synthase (eNOS) and eNOS-Ser1177 phosphorylation (P-eNOS). (a) Representative immunoblot of P-eNOS, located at approximately 150 kDa, and eNOS, located at approximately 130-140 kDa, in aortas of wild-type (WT) and mice overexpressing hyaluronan synthase 2 (HAS-2). (b) Average eNOS expression in WT and HAS-2 female (F) and male (M) mice. (c) Average p-eNOS/eNOS ratio in HAS-2 mice and their WT littermates. (d) Average dimer/monomer eNOS ratio in HAS-2 and WT mice. ** p < 0.01, *** p < 0.001, and **** p < 0.0001 according to two-way ANOVA with Šídák’s multiple comparisons test. # p < 0.05 compared to female mice according to two-way ANOVA.

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  Figure 6. HAS-2 overexpression increases O2- production in the aorta. Representative vessel from each group showing dihydroethidium (DHE) staining for O2− production. (a) Female wild-type (WT) mice. (b) Female transgenic mice overexpressing hyaluronan synthase 2 (HAS-2). (c) DHE staining after polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) pretreatment of the aortas of female HAS-2 transgenic mice. (d) Male WT mice. (e) male HAS-2 transgenic mice. Calibration bar: 100 μm. (f) Average values for DHE staining in the aortas are presented as the means ± s.e. Comparisons were made according to two-way ANOVA. * p < 0.05 versus wild-type aorta.

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  Figure 7. Extracellular superoxide dismutase (EcSOD) is downregulated in the aortas of mice overexpressing hyaluronan synthase 2 (HAS-2). Representative section of an ecSOD Western blot and the corresponding average results normalized to total protein. * p < 0.05 according to two-way ANOVA.

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  Figure 8. Endothelial cell–cell distance increases in HAS-2 mice. (a) Electron micrograph image showing how distance measurements were made. The blue dotted line indicates the border between endothelial cells (ECs). Red arrowheads indicate some of the spots where distance was measured. The red square is a magnification of one of those spots. (b) Average distance between ECs from wild-type mice (WT) and transgenic mice overexpressing hyaluronan synthase 2 (HAS-2). Each point represents the mean of ~329 measurements. * p < 0.05 according to two-way ANOVA. Graphs depict the distribution of measurements in the WT vs. HAS-2 females (c) and males (d). * p < 0.05 at the indicated distances compared WT.

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  Figure 9. Male mice overexpressing hyaluronan synthase 2 (HAS-2) showed increased expression of the low-density lipoprotein receptor (LDL-R). Representative immunoblots (left) and average protein expression of (a) occludin, (b) vascular endothelial cadherin (VE cadherin), and (c) LDL-R. Immunoblotting for occludin in the aortas of male (M) and female (F) wild-type transgenic mice and mice overexpressing HAS-2. Arrows indicate the quantified band, which in the case of LDL-R, is based on the band for the positive control. The data are presented as the means ± s.e. * p < 0.05 versus WT mice according to two-way ANOVA with Šídák’s multiple comparisons tests.