Endothelium-specific overexpression of human IC53 downregulates endothelial nitric oxide synthase activity and elevates systolic blood pressure in mice

Abstract

Aims: Hypertension is one of the major risk factors for cardiovascular diseases. Endothelial cells (ECs) exert important functions in the regulation of blood pressure. A novel gene, IC53, as an isoform of the cyclin-dependent kinase (CDK)-binding protein gene C53, is mainly expressed in vascular ECs and is upregulated in the failing heart of rats. Overexpression of IC53 promotes proliferation of ECs. To examine whether IC53 plays a role in the regulation of vascular tone and blood pressure, we constructed a transgenic (tg) mouse model of the IC53 gene and studied its phenotypes relevant to vascular function.

Methods and results: IC53 cDNA was cloned from a human aorta cDNA library. Using the endothelium-specific VE-cadherin promoter, we constructed tg mice in which IC53 was specifically overexpressed in vascular endothelia and found that the tg mice exhibit elevated systolic blood pressure (SBP) in contrast to the wild-type (wt) controls. Further studies revealed impaired endothelium-dependent vasodilation, reduced nitric oxide (NO) production and decreased endothelial NO synthase (eNOS) expression, and activity in the tg mice. Inhibition of IC53 in human umbilical vein ECs induces upregulation of eNOS activity.

Conclusion: Our results indicate that IC53 participates in the regulation of vascular homeostasis. Endothelium-specific overexpression of IC53 is associated with elevated SBP, which may be in part attributed to the downregulation of eNOS signalling.

Figure 1

(A) The LacZ ORF of the PBSmVELacZ plasmid was replaced by the IC53 cDNA, which was isolated from the PGEMIC53 plasmid by digestion with SalI/NotI. The newly cloned PBSmVEIC53 plasmid was digested with BspHI/SalI; the 4.1 kb DNA fragment was recovered from electrophoresis gel and purified for microinjection. (B) Identification of the transcription of IC53 in tissue (top panels). The GAPDH is used as control for cDNA quality test (bottom panels). The DNA marker is DL2000 (TAKARA, Japan). (C) Localization of IC53 transcripts in the cardiac tissue sections. Positive staining signals were detected in the endothelium layer of endocardium (b) and small vessels (d) of tg mice but not in those of wt mice (a and c). Scale ×80 in a and b, ×320 in c and d.

Figure 2

Haemodynamic parameters, including the systolic blood pressure (SBP), diastolic blood pressure (DBP), mean artery pressure (MAP), and pulse pressure (PP), are recorded. (A) Results of the conscious mice. n = 14 per group; *P < 0.05 vs. wt. (B) Results of the anaesthetized mice. n = 12 per group; #P < 0.05 vs. wt.

Figure 3

(A) The dose–response curves of aortic rings to ACh in tg and wt mice. (B) The dose–response curves of aortic rings to SNP in mice. Magnitudes of relaxation are mean ± SEM of percentage reversal of PE-induced contractile responses. n = 12 in 12 mice per group. *P < 0.05 vs. wt.

Figure 4

(A) Serum ET-1 concentrations in tg and wt mice. (B) Serum NO concentrations in tg and wt mice. (C) Aortic eNOS activities in tg and wt mice. One unit of the eNOS activity is defined as the ability to create 1 nmol NO within 1 min by 1 mg tissue protein. (D) Aortic eNOS mRNA levels in tg and wt mice. The values are the relative expression levels of samples comparing with β-actin. (E) Aortic eNOS protein levels in tg and wt mice, with molecular weight marked (KDa). (F) Quantification of the eNOS protein levels (relative to β-actin) by measuring the density of the bands in (E). (G) Endogenous IC53 mRNA levels in tg and wt mice aorta, relative to β-actin. (H) Intracellular eNOS activity in shRNA-treated and control HUVECs. n = 6 per group; *P < 0.05 vs. wt.