Toll-like receptor 4 mutation suppresses hyperhomocysteinemia-induced hypertension

Abstract

Hyperhomocysteinemia (HHcy) has been observed to promote hypertension, but the mechanisms are unclear. Toll-like receptor 4 (TLR-4) is a cellular membrane protein that is ubiquitously expressed in all cell types of the vasculature. TLR-4 activation has been known to promote inflammation that has been associated with the pathogenesis of hypertension. In this study we hypothesize that HHcy induces hypertension by TLR-4 activation, which promotes inflammatory cytokine (IL-1β, IL-6, and TNF-α) upregulation and initiation of mitochondria-dependent apoptosis, leading to cell death and chronic vascular inflammation. To test this hypothesis, we used C57BL/6J (WT) mice, cystathionine β-synthase (CBS)-deficient (CBS^+/-) mice with genetic mild HHcy, C3H/HeJ (C3H) mice with TLR-4 mutation, and mice with combined genetic HHcy and TLR-4 mutation (CBS^+/-/C3H). Ultrasonography of the superior mesenteric artery (SMA) detected an increase in wall-to-lumen ratio, resistive index (RI), and pulsatility index (PI). Tail cuff blood pressure (BP) measurement revealed elevated BP in CBS^+/- mice. RI, PI, and wall-to-lumen ratio of the SMA in CBS^+/-/C3H mice were similar to the control group, and BP was significantly alleviated. TLR-4, IL-1β, IL-6, and TNF-α expression were upregulated in the SMA of CBS^+/- mice and reduced in the SMA of CBS^+/-/C3H mice. Molecules involved in the mitochondria-mediated cell death pathway (BAX, caspase-9, and caspase-3) were upregulated in CBS^+/- mice and attenuated in CBS^+/-/C3H mice. We conclude that HHcy promotes TLR-4-driven chronic vascular inflammation and mitochondria-mediated cell death, inducing hypertension. TLR-4 mutation attenuates vascular inflammation and cell death, which suppress hypertension.

Keywords: homocysteine; inward vascular remodeling; mitochondria-mediated cell death; peripheral resistance; vascular inflammation.

Fig. 1.

A: genotyping for the cystathionine β-synthase (CBS) gene. CBS^+/− and CBS^+/−/C3H mice had 2 bands, at 450 and 308 bp; wild-type (WT) mice had 1 band, at 308 bp. B: restriction fragment length polymorphism-PCR for the Toll-like receptor 4 (TLR-4) gene. TLR-4 mutant (C3H and CBS^+/−/C3H) mice had 2 bands, at 96 and 108 bp; nonmutant (WT and CBS^+/−) mice had 1 band, at 204 bp.

Fig. 2.

Systolic, mean, and diastolic blood pressure (BP). Hyperhomocysteinemic (HHcy) mice had significantly higher systolic, mean, and diastolic BP and mice with combined genetic HHcy and TLR-4 mutation (CBS^+/−/C3H) had significantly lower systolic, mean, and diastolic BP. Values are means ± SE (n = 10). *P < 0.05, WT vs. CBS^+/−. #P < 0.05, WT vs. C3H. §P < 0.05, CBS^+/− vs. CBS^+/−/C3H.

Fig. 3.

A: wall-to-lumen ratio. To assess structural changes in the superior mesenteric artery (SMA), wall thickness and lumen diameter were measured and the SMA wall-to-lumen ratio was calculated. The SMA wall-to-lumen ratio was increased in CBS^+/− mice compared with WT and C3H mice. The SMA wall-to-lumen ratio of CBS^+/−/C3H mice was similar to the control group. Values are means ± SE (n = 5). *P < 0.05 vs. WT. #P < 0.05 vs. CBS^+/−. B: pulsatility index (PI) and resistive index (RI) of the SMA. PI and RI are calculated from blood flow velocities in the SMA during the cardiac cycle and used to determine a peripheral resistance. PI and RI of the SMA were increased in CBS^+/− mice compared with WT and C3H mice. SMA PI and RI were similar to controls in mice with combined genetic HHcy and TLR-4 mutation. Values are means ± SE (n = 5). *P < 0.05 vs. WT. #P < 0.05 vs. CBS^+/−.

Fig. 4.

A and B: immunohistochemistry (IHC) for TLR-4 and TNF-α. IHC showed increased intensity of TLR-4 and TNF-α in the SMA of CBS^+/− mice. TLR-4 and TNF-α intensities were reduced in mice with combined genetic HHcy and TLR-4 mutation. Values are means ± SE (n = 4). *P < 0.05 vs. WT. #P < 0.05 vs. CBS^+/−.

Fig. 5.

A and B: quantitative RT-PCR for IL-1β (n = 5) and IL-6 (n = 4) and TNF-α (n = 5). IL-1β, IL-6, and TNF-α mRNA expression were elevated in the SMA of CBS^+/− mice and reduced in the SMA of CBS^+/−/C3H mice. Values are means ± SE. *P < 0.05 vs. WT. #P < 0.05 vs. CBS^+/−.

Fig. 6.

A: quantitative RT-PCR for the BAX gene. BAX mRNA expression was significantly upregulated in CBS^+/− mice compared with other groups. BAX mRNA expression was reduced in CBS-deficient mice with TLR-4 mutation (CBS^+/−/C3H) compared with CBS^+/− mice. Values are means ± SE (n = 4). *P < 0.05 vs. WT. #P < 0.05 vs. CBS^+/−. B: Western blotting for BAX protein expression. BAX protein expression was increased in CBS^+/− mice compared with the other groups. BAX protein expression was reduced in CBS-deficient mice with TLR-4 mutation (CBS^+/−/C3H) compared with CBS^+/− mice. C: Western blot for caspase-9 protein expression. Caspase-9 protein expression was increased in HHcy mice (CBS^+/−) and CBS^+/−/C3H mice compared with WT and TLR-4 mutant (C3H) mice. Values are means ± SE (n = 6). *P < 0.05, CBS^+/− vs. C3H. #P < 0.05, C3H vs. CBS^+/−/C3H. D: IHC showed elevated levels of cleaved caspase-3 in the SMA of CBS^+/− mice. Intensity of cleaved caspase-3 was reduced in the SMA of CBS^+/−/C3H mice compared with CBS^+/− mice. Values are means ± SE (n = 4). *P < 0.05 vs. WT. #P < 0.05 vs. CBS^+/−.

Fig. 7.

TLR-4 mutation mitigates HHcy-induced DNA fragmentation. TUNEL assay was used to evaluate DNA fragmentation (arrows) in the SMA. DNA fragmentation was significantly augmented in the SMA of CBS^+/− mice compared with WT and C3H mice. TUNEL-positive cell count was reduced in the SMA of CBS^+/−/C3H mice compared with CBS^+/− mice. ctr, Control. Values are means ± SE (n = 4). *P < 0.05 vs. WT. #P < 0.05 vs. CBS^+/−.

Fig. 8.

Schematic representation of the hypothesis. HHcy induces hypertension by TLR-4 activation followed by inflammatory cytokine (IL-1β, IL-6, and TNF-α) elevation and initiation of mitochondria-dependent apoptosis, which lead to cell death and chronic vascular inflammation. TLR-4 mutation attenuates vascular inflammation and cell death, which suppress hypertension. ΔΨ_m, mitochondrial membrane potential; Apaf-1, apoptotic protease-activating factor 1.