Angiopoietin-1 prevents hypertension and target organ damage through its interaction with endothelial Tie2 receptor

Abstract

Aims: The endothelium has emerged recently as a therapeutic target in the treatment of hypertension because endothelial dysfunction and subsequent vascular rarefaction cause target organ damage and further elevate blood pressure (BP). It led us to hypothesize that one of the endothelial survival factors, a potent derivative of angiopoietin-1 (cartilage oligomeric matrix protein, COMP-Ang-1), could be a novel class of antihypertensive agents that maintain endothelial integrity and function, thereby preventing the development of hypertension and target organ damage.

Methods and results: To study the role of COMP-Ang-1 in preventing hypertension and target organ damage, a COMP-Ang-1 plasmid was electroporated into adductor muscles of 6 weeks old, pre-hypertensive, spontaneously hypertensive rats (SHRs), and the secretion of its expressed protein into the bloodstream was confirmed by western blotting. In comparison with sham and reporter gene transfer, COMP-Ang-1 gene transfer significantly prevented increases in systolic BP and reduced microvascular rarefaction and tissue damage in the heart and kidney. However, overexpression of soluble Tie2 receptor completely abolished these beneficial effects of COMP-Ang-1 gene transfer on SHRs, indicating that expressed COMP-Ang-1 protein has antihypertensive effects in SHRs by binding Tie2 receptors on the vascular endothelium. In particular, COMP-Ang-1 gene-transferred SHRs had significantly higher plasma levels of nitrite than other controls, which was found to be due to that expressed COMP-Ang-1 protein promoted nitrite synthesis by activating endothelial nitric oxide synthase, one of the Tie2 downstream-signalling molecules.

Conclusion: The present study suggests a new potential of endothelial survival factor, COMP-Ang-1, as an antihypertensive agent that effectively reduces the hypertension-associated cardiovascular and renal damage, as well as prevents the further elevation of BP.

Figure 1

COMP-Ang-1 gene transfer prevents the blood pressure (BP) elevation in spontaneously hypertensive rats (SHRs). (A) COMP-Ang-1 protein secreted into the bloodstream was quantified by western blotting using IgG against the FLAG sequence (DYKDDDDK) tagged at the N-terminus of COMP-Ang-1. As a positive control, 100 ng of COMP-Ang-1 protein was loaded with 7 μL of blood serum from spontaneously hypertensive rats. (B) Systolic BP (in mmHg) of each group was measured using the tail-cuff method in the third and fifth weeks after the first gene transfer. **P < 0.01 vs. pLacZ-transferred or untreated spontaneously hypertensive rats (n = 6).

Figure 2

COMP-Ang-1 gene transfer reduces myocardial damage in SHRs. (A) Representative images of H&E-stained heart. (B) Inflammatory infiltrates shown in H&E sections were quantified as a percentage of the total tissue area. (C) Interventricular septal thickness was measured on H&E-stained heart images using ImagePro. *P < 0.05 vs. untreated or pLacZ (n = 6). Scale bar is 20 µm.

Figure 3

COMP-Ang-1 gene transfer reduces renal damage in SHRs. (A) Representative images of H&E-stained kidney. (B) Inflammatory infiltrates shown in H&E sections were quantified as a percentage of the total tissue area. (C) The amount of protein in urine collected at the end of the experiment. *P < 0.05 vs. pLacZ-transferred or untreated SHRs (n = 6). Scale bar is 20 µm.

Figure 4

COMP-Ang-1 gene transfer attenuates the microvascular rarefaction and tissue ischaemia in the heart and kidney of SHRs. The microvasculature of the heart (A) [arrowheads in (A) indicate the absence of capillaries], (B) and kidney (C) was visualized and quantified by immunohistochemical staining with CD31 IgG on frozen sections or RECA IgG on paraffin sections, respectively. In addition, the extent of hypoxia in the heart and kidney was visualized by immunohistochemical staining with Hydroxyprobe-1. (D) Dermal blood flow on the tibialis muscle of SHRs was evaluated using laser Doppler imager. The blood perfusion signal is displayed in colour codes ranging from dark blue (0) to white (1000). Representative laser Doppler images of each group and its blood perfusion percentage, which was normalized to the blood perfusion extent of the normotensive control, Wistar Kyoto rats. *P < 0.05, **P < 0.01 vs. pLacZ-transferred or untreated SHRs (n = 6). Scale bars on CD31- or Hydroxyprobe-1-stained figures are 20 and 10 µm, respectively.

Figure 5

COMP-Ang-1 protein induces beneficial effects in SHRs via Tie2 receptors on endothelium. (A) Tie2 phosphorylation in the lung tissues of pLacZ- or pCOMP-Ang-1-transferred SHRs sacrificed 3 weeks after the gene transfer. One day before the first gene transfer of pLacZ or pCOMP-Ang-1, SHRs were intravenously injected with 1 × 10⁹ pfu of adenoviral soluble Tie2 (sTie2). Then their systolic BP (B) capillary density (C) [arrowheads in (C) indicate the absence of blood vessels], and tissue damage (D) in the heart and kidney were evaluated as described earlier. *P < 0.05 vs. pLacZ or pLacZ+sTie2; ^#P < 0.05 vs. pCOMP-Ang-1 (n = 3). Scale bar is 20 µm.

Figure 6

COMP-Ang-1 enhances nitric oxide production by activating endothelial nitric oxide synthase (eNOS). (A) Nitrite levels in peripheral bloods from SHRs. (B) Nitrite levels in human umbilical vein endothelial cells (HUVEC) after the incubation with serum obtained from SHRs. Of note, pre-treatment with soluble Tie2 abolished the increase of nitrite production in HUVEC treated with serum from pCOMP-Ang-1 transferred SHRs. *P < 0.05, ***P < 0.001 vs. untreated or pLacZ; ^#P < 0.001 vs. pCOMP-Ang-1 (A, n = 5; B, n = 4). (C) eNOS phosphorylation in the lung tissues of pLacZ- or pCOMP-Ang-1-transferred SHRs sacrificed 3 weeks after the gene transfer. (D) eNOS phosphorylation in HUVEC after 60 min incubation with COMP-Ang-1 protein (100 ng/mL). (E) Nitrite production in HUVEC after the treatment with COMP-Ang-1 protein (100 ng/mL). *P < 0.05 vs. PBS (n = 3).