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. 2007 Mar;170(3):1108-20.
doi: 10.2353/ajpath.2007.060960.

Endothelial-specific expression of mitochondrial thioredoxin improves endothelial cell function and reduces atherosclerotic lesions

Haifeng Zhang  1 , Yan Luo, Wei Zhang, Yun He, Shengchuan Dai, Rong Zhang, Yan Huang, Pascal Bernatchez, Frank J Giordano, Gerald Shadel, William C Sessa, Wang Min
Affiliations

Endothelial-specific expression of mitochondrial thioredoxin improves endothelial cell function and reduces atherosclerotic lesions

Haifeng Zhang et al. Am J Pathol. 2007 Mar.

Abstract

The function of the mitochondrial antioxidant system thioredoxin (Trx2) in vasculature is not understood. By using endothelial cell (EC)-specific transgenesis of the mitochondrial form of the thioredoxin gene in mice (Trx2 TG), we show the critical roles of Trx2 in regulating endothelium functions. Trx2 TG mice have increased total antioxidants, reduced oxidative stress, and increased nitric oxide (NO) levels in serum compared with their control littermates. Consistently, aortas from Trx2 TG mice show reduced vasoconstriction and enhanced vasodilation. By using ECs isolated from Trx2 TG mice, we further show that Trx2 increases the capacities of ECs in scavenging reactive oxygen species generated from mitochondria, resulting in increases in NO bioavailability in ECs. More importantly, Trx2 improves EC function and reduces atherosclerotic lesions in the apolipoprotein E-deficient mouse model. Our data provide the first evidence that Trx2 plays a critical role in preserving vascular EC function and prevention of atherosclerosis development, in part by reducing oxidative stress and increasing NO bioavailability.

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Figures

Figure 1
Figure 1
EC-specific expression of Trx2. a: Schematic diagram for EC-specific transgenic construct in which expression of the HA-tagged Trx2 transgene is driven by a promoter sequence from the VE-cadherin gene. The native mitochondrial signal peptide (msp) on Trx2, and the primers for genotyping are indicated. b: Trx2 TG founders. Tails from the Trx2 transgenic founders (four males and four females based on genotyping with specific primers) were further analyzed for Trx2 expression by Western blot with anti-HA antibody. A nontransgenic tail was used as a control. c: Tissue distribution of the Trx2 transgene. Expression of the Trx2 transgene in different tissues was determined by a real-time RT-PCR using primers recognizing both human and murine Trx2. 18S rRNA was used for normalization. Data presented are fold increase in Trx2 mRNA in Trx2 TG lines (lines 1 and 3) compared with WT mice (n = 3). d: Expression of Trx2 protein in aorta. Trx2 protein in aorta was determined by Western blot with anti-HA or anti-Trx2 antibody. Trx1 was also detected by Western blot with anti-Trx1 antibody as a control. β-Tubulin was used as protein loading control. e--g: Trx2 transgene is expressed in the aortic endothelium. The Trx2 transgene in WT and Trx2 TG (line 3) aortic sections were detected by immunohistochemistry with anti-HA antibody. e: Endothelium and media are indicated. f: The Trx2 transgene in aortic sections were detected by indirect immunofluorescence microscopy with anti-HA antibody followed by Alexa Fluor 488 (green)-conjugated anti-mouse secondary antibody. Trx2 TG aorta were co-stained with anti-HA antibody followed by Alexa Fluor 488 (green)-conjugated anti-mouse secondary antibody and anti-CD31 antibody followed by Alexa Fluor 594 (red)-conjugated anti-goat secondary antibody. g: The merged picture is shown on the right.
Figure 2
Figure 2
Hemodynamics in Trx2 TG mice. Serum from WT and Trx2 TG mice (both lines 1 and 3) were collected. a: Total antioxidant levels in the serum were measured by the Total Anti-Oxidant Status assay kit (Calbiochem) b: 8-Isoprostane, a systemic oxidative marker in serum, was determined by the enzyme-linked immunosorbent assay kit (Calbiochem). c: Serum NO was measured as NOx for both nitrite and nitrate (Cayman). d: Basal heart rate (minutes) was determined by echocardiography. e–g: End diastolic pressure, end systolic pressure, and mean artery pressure were determined at baseline and after administration of NO inhibitor l-NAME (0.1 mg/10 g body weight for 30 minutes). The number of mice in each group is shown in parentheses. *P < 0.05, statistical significance between WT and Trx2 TG groups, n = 9 in each group.
Figure 3
Figure 3
Trx2 transgene expression increases basal NO release in aortic endothelium. a: Aortas from Trx2 TG show an attenuated response to PE. Aortic rings were contracted with PE at a full range of doses (10−9 to 10−4 mol/L). Constriction force (mN) is shown. b: Basal NO is increased in Trx2 TG. Aortic rings were incubated with the NOS inhibitor l-NAME to remove basal NO synthesis and then contracted with PE as in a. c: Ratio of EC50 to PE in the presence versus absence of l-NAME (100 μmol/L). d: Aortas from Trx2 TG alter the response to ACh. Aortic rings were precontracted with PE and then relaxed with ACh at a full range of doses (10−9 to 10−4 mol/L). Percentage of relaxation is shown. e: Trx2 expression had no effects on vessel constriction in response to KCl. Aortic rings were contracted with 50 mmol/L KCl. f: Trx2 expression had no effects on vessel relaxation to the NO donor drug SNP. Aortic rings were incubated with a NOS inhibitor l-NAME to remove basal NO synthesis followed by a precontraction with PE as in b and were then relaxed with SNP at a full range of doses (10−9 to 10−6 mol/L). Data are presented are mean ± SEM, with n = 5 animals and eight aortic rings per animal. *P < 0.05 indicates statistical significance by comparing WT versus TG at a dose, which causes 50% of constriction (PE) or relaxation (ACh).
Figure 4
Figure 4
Trx2 increases NO bioavailability in ECs. Mouse aortic ECs (MECs) were isolated from WT and Trx2 TG mice. a: Trx2 mRNA was determined by qRT-PCR, and 18S rRNA was used for normalization. Data presented are fold increases in Trx2 MECs by taking WT MECs as 1.0. b: Trx2 expression had no effects on protein levels of TrxR2 or Prx3, components of the Trx2 antioxidant system. Trx2, TrxR2, and Prx3 proteins were determined by Western blot with anti-Trx2, anti-TrxR2, and anti-Prx3 antibody, respectively. β-Tubulin was used for protein normalization. c: Trx2 expression had no effects on protein levels or distributions of other ROS-scavenging enzymes. Cytoplasmic and mitochondrial fractions from WT and Trx2 TG MECs were isolated as described in Materials and Methods. Cytoplasmic and mitochondrial fractions were used for protein analyses. Cytosolic antioxidant enzymes (catalase and Trx1) and mitochondrial enzymes (Trx2 and MnSOD) were determined by Western blot with respective antibodies. Cytochrome c in mitochondria was used for protein normalization. d–f: Effects of Trx2 transgene on basal and VEGF-induced eNOS activity in MECs isolated from WT and Trx2 TG mice. MECs were cultured in media-deprived EC growth factors overnight followed by treatment with VEGF (50 ng/ml) in the absence or presence of eNOS inhibitor l-NAME (100 μmol/L) for 15 minutes (d) or 60 minutes (e–f). d: NO release was determined by measuring nitrite in the media. e: Phospho-(pSer1179) and total eNOS were determined by Western blot with respective antibodies. f: eNOS enzymatic activity was determined based on the conversion of [3H]l-arginine to [3H]l-citrulline according to the protocol from the manufacturer (Calbiochem). All eNOS activity and NO data presented are mean ± SEM of triplicates from three independent experiments. *P < 0.05.
Figure 5
Figure 5
Trx2 increases EC capacity in scavenging ROS. a and b: Trx2 blocks TNF-induced mitochondrial ROS production. WT and Trx2 TG MECs were untreated or treated with TNF (10 ng/ml for 2 hours). Mitochondrial ROS was determined by flow cytometry with a specific probe DHR123 (a) or MitoTracker Red CM-H2XROS (MitoROS) (b). Mean fluorescence intensity (MFI, mean ± SEM) from three independent experiments are indicated. c: Trx2 blocks paraquat-induced endogenous superoxide production in MECs. WT and Trx2 TG MECs were treated with paraquat overnight at indicated doses. d: Trx2 increases EC capacity in scavenging exogenous H2O2. MECs were treated with various concentrations of H2O2 (0 to 1 mmol/L for 30 minutes). Intracellular level of ROS was determined by flow cytometry analyses with DHR123 probe. Data presented are mean ± SEM from three independent experiments. *P < 0.05.
Figure 6
Figure 6
Trx2 prevents hypercholesterolemia-induced EC dysfunction in ApoE-deficient mice. WT, Trx2 TG, ApoE-deficient (ApoE), and ApoE-deficient/Trx2 TG mice (ApoE/TG) were fed with a Western-type diet for 4 weeks and vascular reactivity of aortic rings was determined as described in Figure 3. Only ApoE and ApoE/TG are shown in a–e. For comparison of WT and ApoE, see Supplemental Figure S4 at http://ajp.amjpathol.org. a: Aortic rings were contracted with PE at a full range of doses (10−9 to 10−4 mol/L). Constriction force (mN) is shown. b: Aortic rings were incubated with a NOS inhibitor l-NAME to remove basal NO synthesis and then contracted with PE as in a. c: Ratio of EC50 to PE in the presence versus absence of l-NAME (100 μmol/L). d: Aortic rings were contracted with ACh at a full range of doses (10−9 to 10−4 mol/L). Percentage of relaxation is shown. e: Aortic rings were contracted with 50 mmol/L of KCl. f: Aortic rings were incubated with a NOS inhibitor l-NAME to remove basal NO synthesis followed by a precontraction with PE as in b and were then relaxed with SNP at a full range of doses (10−9 to 10−6 mol/L). g: Comparison of the PE responses among WT, ApoE, and ApoE/TG. Aortic rings were contracted with PE at a full range of doses (10−9 to 10−4 mol/L). Constriction force is shown. All data are presented are mean ± SEM, with n = 4 animals and eight aortic rings per animal. *P < 0.05.
Figure 7
Figure 7
Trx2 reduces hypercholesterolemia-induced atherosclerotic progression in ApoE-deficient mice. WT, Trx2, ApoE-deficient (ApoE), and ApoE-deficient/Trx2 TG mice (ApoE/TG) (n = 10 for each group) were fed with a Western-type diet for 8 weeks. a: Serum cholesterol was measured enzymatically. b: Serum NO was measured as NOx for both nitrite and nitrate (Cayman). c: Whole aortas were harvested, opened longitudinally, and stained en face with Oil Red O. Percentage of lesion area was quantified in d. e: Paraffin sections from aortic arches (10 sections from each aorta) were stained with Oil Red O. Percentage of lesion area was quantified in f. Data are presented are mean ± SEM. *P < 0.05.
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