Angiotensin II (AT(1)) receptor blockade reduces vascular tissue factor in angiotensin II-induced cardiac vasculopathy

Abstract

Tissue factor (TF), a main initiator of clotting, is up-regulated in vasculopathy. We tested the hypothesis that chronic in vivo angiotensin (ANG) II receptor AT(1) receptor blockade inhibits TF expression in a model of ANG II-induced cardiac vasculopathy. Furthermore, we explored the mechanisms by examining transcription factor activation and analyzing the TF promoter. Untreated transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) feature hypertension and severe left ventricular hypertrophy with focal areas of necrosis, and die at age 7 weeks. Plasma and cardiac ANG II was three- to fivefold increased compared to Sprague-Dawley rats. Chronic treatment with valsartan normalized blood pressure and coronary resistance completely, and ameliorated cardiac hypertrophy (P < 0.001). Valsartan prevented monocyte/macrophage infiltration, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) activation, and c-fos expression in dTGR hearts. NF-kappaB subunit p65 and TF expression was increased in the endothelium and media of cardiac vessels and markedly reduced by valsartan treatment. To analyze the mechanism of TF transcription, we then transfected human coronary artery smooth muscle cells and Chinese hamster ovary cells overexpressing the AT(1) receptor with plasmids containing the human TF promoter and the luciferase reporter gene. ANG II induced the full-length TF promoter in both transfected cell lines. TF transcription was abolished by AT(1) receptor blockade. Deletion of both AP-1 and NF-kappaB sites reduced ANG II-induced TF gene transcription completely, whereas the deletion of AP-1 sites reduced transcription. Thus, the present study clearly shows an aberrant TF expression in the endothelium and media in rats with ANG II-induced vasculopathy. The beneficial effects of AT(1) receptor blockade in this model are mediated via the inhibition of NF-kappaB and AP-1 activation, thereby preventing TF expression, cardiac vasculopathy, and microinfarctions.

Figure 1.

A and B: Goldner-Trichrome panel, C and D: van Gieson-stained section from dTGR- and valsartan-treated hearts. Section of myocardium from dTGR show hemorrhages and patchy areas of necrosis, as well as an interstitial fibroblastic reaction. Valsartan treatment prevented cardiac damage. Untreated dTGR vessel (C) shows damaged lamina elastica interna and intimal proliferation (arrow). However, valsartan treatment prevented vascular damage.

Figure 2.

A and B: Systolic blood pressure and coronary resistance in dTGR, dTGR treated with valsartan, and SD rats. Valsartan normalized blood pressure (177 ± 5 vs. 103 ± 5 vs. 106 ± 2 mmHg, P < 0.0001, dTGR vs. dTGR + valsartan vs. SD rats, respectively) and coronary resistance (6.0 ± 0.2 vs. 4.2 ± 0.2 vs. 3.9 ± 0.1 mmHg* ml/minute) and prevented the development of cardiac hypertrophy (5.7 ± 0.2 vs. 3.6 ± 0.1 vs. 3.4 ± 0.1 mg/g, C). Results are expressed as mean ± SE of 8 to 13 animals per group. *P < 0.0001.

Figure 3.

Semiquantitative scoring of ED-1-positive (A) and VLA-4-positive (B) cells in hearts of dTGR, valsartan-treated dTGR, and SD rats was performed using a computerized cell count program. Ten different areas of each heart were analyzed. Results are expressed as mean ± SE of 5 animals per group. *P < 0.001.

Figure 4.

Activation of DNA-binding nuclear factors by ANG II. EMSA for the detection of NF-κB and AP-1 shows a higher binding activity of dTGR heart extracts compared with SD rats. Valsartan treatment reduced levels of NF-κB and AP-1 in the heart. EMSA was performed 3 times independently with similar results.

Figure 5.

Untreated dTGR show significantly increased c-fos (A) and TF (B) mRNA levels. Valsartan reduced both c-fos (*P < 0.05) and TF expression (P = 0.055). mRNA levels of the target genes were normalized for the housekeeping gene GAPDH. Results are expressed as mean ± SE of 5 to 8 animals per group.

Figure 6.

Immunohistochemical analysis (phase contrast resolution) shows the localization of the subunit p65 of NF-κB in a cardiac vessel. NF-κB activity was increased in the endothelium and smooth muscle cells of damaged dTGR vessels (A−C). The antibody only recognizes the active form of NF-κB after dissociation of its inhibitor I-κBα. D−F: Representative immunohistochemical photomicrographs of TF in dTGR cardiac vessels, valsartan-treated dTGR, and SD rats. TF expression was increased in the endothelium and smooth muscles. The staining pattern of TF resembles the localization of p65 in the vessel wall of dTGR. AT₁ receptor blockade markedly reduced ANG II-induced TF expression. Nontransgenic SD rats were used as negative controls (C and F).

Figure 7.

Representative immunohistochemical photomicrographs of TF in the heart of dTGR (A), valsartan-treated dTGR (B), and SD rats (C). TF expression was increased in the endothelium, smooth muscle cells, adventitia, and in infiltrating cells. TF expression was markedly reduced by valsartan.

Figure 8.

TF promoter activity induced by ANG II in human coronary smooth muscle cells (VSMC). A shows various deletion mutants of the human TF promoter. B shows the average fold induction of luciferase activity expressed by each plasmid in response to 10⁻ mol/L ANG II with and without valsartan preincubation. The average fold induction of luciferase activity is expressed as percentage of unstimulated experiment. The measurements were performed in duplicate. Results are expressed as mean ± SE of 3 to 5 independent transfections.

Figure 9.

TF promoter activity induced by ANG II in CHO cells overexpressing the AT₁ receptor (CHO-AT₁) and the corresponding wild-type cell line (CHO-WT). A shows various deletion mutants of the human TF promoter. B shows the average fold induction of luciferase activity expressed by each plasmid in response to 10⁻ mol/L ANG II with and without valsartan preincubation. The average fold induction of luciferase activity is expressed as percentage of unstimulated experiment. The measurements were performed in duplicate. Results are expressed as mean ± SE of 3 to 5 independent transfections.