Endothelial cell-specific aryl hydrocarbon receptor knockout mice exhibit hypotension mediated, in part, by an attenuated angiotensin II responsiveness

Abstract

Hypotension in aryl hydrocarbon receptor knockout mice (ahr(-/-)) is mediated, in part, by a reduced contribution of angiotensin (Ang) II to basal blood pressure (BP). Since AHR is highly expressed in endothelial cells (EC), we hypothesized that EC-specific ahr(-/-) (ECahr(-/-)) mice would exhibit a similar phenotype. We generated ECahr(-/-) mice by crossing AHR floxed mice (ahr(fx/fx)) to mice expressing Cre recombinase driven by an EC-specific promoter. BP was assessed by radiotelemetry prior to and following an acute injection of Ang II or chronic treatment with an angiotensin converting enzyme inhibitor (ACEi). ECahr(-/-) mice were hypotensive (ECahr(+/+): 116.1±1.4; ECahr(-/-): 107.4±2.0 mmHg, n=11, p<0.05) and exhibited significantly different responses to Ang II and ACEi. While Ang II increased BP in both genotypes, the increase was sustained in ECahr(+/+), whereas the increase in ECahr(-/-) mice steadily declined. Area under the curve analysis showed that Ang II-induced increase in diastolic BP (DBP) over 30 min was significantly lower in ECahr(-/-) mice (ECahr(+/+) 1297±223 mmHg/30 min; ECahr(-/-)(AUC): 504±138 mmHg/30 min, p<0.05). In contrast, while ACEi decreased BP in both genotypes, the subsequent rise in DBP after treatment was significantly delayed in the ECahr(-/-) mice. ECahr(-/-) mice also exhibited reduced vascular and adipose Ang II type 1 receptor (AT1R) expression, and reduced aortic Ang II-dependent vasoconstriction in the presence of vascular adipose. Taken together these data suggest that hypotension in ECahr(-/-) mice results from reduced vascular responsiveness to Ang II that is influenced by AT1R expression and adipose.

Figure 1

Cre^Tek-mediated excision of the ahr floxed allele (ahr^fx/fx). (A) Excision by Cre^Tek was determined by genotyping for both the unexcised (140 bp) and excised (180 bp) alleles of ahr^fx/fx in genomic DNA isolated from liver, kidney, heart, aorta and mesenteric arterioles obtained from ahr^fx/fxCre⁻ (ECahr^+/+) and ahr^fx/fxCre⁺ (ECahr^−/−) mice. (B) Representative sections of aorta from ECahr^−/− and ECahr^+/+ mice stained with primary AHR antibody. Positive horseradish peroxidase activity (arrows) can be seen in the endothelium of ECahr^+/+, but absent in the ECahr^−/− mice.

Figure 2

Loss of ahr alleles in endothelial cells (EC) decreases systolic and diastolic blood pressure. (A) Systolic and diastolic blood pressure, (B) heart rate, (C) hourly mean arterial pressure (MAP) over a 24 hr period (light and dark cycle), and (D) activity of ECahr^+/+ and ECahr^−/− mice, as measured by radiotelemetry (n=12/genotype). Data represent mean ± SEM and were analyzed by Student’s t-test; *p < 0.05, compared to ECahr^+/+ (A and B) or by two-way, repeated measures ANOVA, using post hoc Holm-Sidak comparisons; *p < 0.05, compared to ECahr^+/+ mice (C and D).

Figure 3

ECahr^−/− mice exhibit normal responses to NOS inhibition by LNNA in vivo. (A) Change in MAP after treatment with 250 mg/L LNNA in drinking water of male ECahr^−/− and ECahr^+/+ mice. (B) Percent change in MAP after treatment with 250 mg/L LNNA. Data represent mean ± SEM and were analyzed by two-way, repeated measures ANOVA, using post hoc Holm-Sidak comparisons; *p < 0.05, compared to ECahr^+/+ (A), and by Student’s t-test (B) (n=8/genotype for all experiments).

Figure 4

Loss of ahr in endothelium attenuates RAS responsiveness in vivo. (A) MAP response, (B) change in systolic, (C) change in diastolic blood pressure, and (D) area under curve analysis, for 30 min following i.p. injection of Ang II (30 µg/kg). Blood pressure was recorded starting after 5 mins of Ang II administration to exclude handling as a confounding factor. Data represent mean ± SEM and were analyzed by two-way, repeated measures ANOVA, using post hoc Holm-Sidak comparisons; *p < 0.05 compared to ECahr^+/+ (Fig. A, B, and C). Data in panel (D) were analyzed by student t-test *p < 0.05 compared to ECahr^+/+ (n=4/genotype).

Figure 5

Loss of ahr in endothelium attenuates the contribution of Ang II to basal blood pressure. (A) MAP response, (B) change in diastolic, and (C) change in systolic blood pressure, following treatment with 4 mg/kg ACEi, captopril, in drinking water for 5 d, followed by a 4 d washout. (D) Area under the curve analysis of systolic and diastolic blood pressure response during washout. Data represent mean ± SEM and were analyzed by repeated measures two-way ANOVA, using post hoc Holm-Sidak comparisons; *p < 0.05, compared to ECahr^+/+ (Fig. A, B and C). Data in panel (D) were analyzed by student’s t-test *p < 0.05, compared to ECahr^+/+ (n=4/genotype).

Figure 6

Loss of ahr in endothelium alters mRNA expression of RAS components in adipose. mRNA quantification of (A) Agt, (B) renin, and (C) AT1R from visceral white adipose, aortic PVAT, and aorta free of PVAT. Data represent mean ± SEM and were analyzed by Student’s t-test; *P < 0.05, compared to ECahr^+/+.

Figure 7

Loss of ahr in endothelium diminishes AT1R expression in aorta. (A) Representative western blot of abdominal aortic AT1R protein expression. (B) Quantification of AT1R protein expression relative to GAPDH. Data represent mean ± SEM and were analyzed by Student’s t-test; *P < 0.05, compared to ECahr^+/+.

Figure 8

Loss of ahr in endothelium reduces abdominal aortic reactivity to Ang II in the presence of perivascular adipose tissue (PVAT). (A) Ang II-induced contraction (% KCl) in absence of PVAT. (B) Ang II-induced contraction (% KCl) in the presence of PVAT. (ECahr^−/− n=12; and ECahr^+/+ n=11). Data represent the mean ± SEM and were analyzed by Student’s t-test; *P < 0.05, compared to ECahr^+/+.