C-Type Natriuretic Peptide (CNP) Inhibition of Interferon-γ-Mediated Gene Expression in Human Endothelial Cells In Vitro

Abstract

Cardiovascular diseases, including atherosclerosis, now account for more deaths in the Western world than from any other cause. Atherosclerosis has a chronic inflammatory component involving Th1 pro-inflammatory cytokines such as IFN-γ, which is known to induce endothelial cell inflammatory responses. On the other hand CNP, which acts via its receptors to elevate intracellular cGMP, is produced by endothelium and endocardium and is upregulated in atherosclerosis. It is believed to be protective, however its role in vascular inflammation is not well understood. The aim of this study was to investigate the effects of CNP on human endothelial cell inflammatory responses following IFN-γ stimulation. Human umbilical vein endothelial cells were treated with either IFN-γ (10 ng/mL) or CNP (100 nm), or both in combination, followed by analysis by flow cytometry for expression of MHC class I and ICAM-1. IFN-γ significantly increased expression of both molecules, which was significantly inhibited by CNP or the cGMP donor 8-Bromoguanosine 3',5'-cyclic monophosphate (1 µm). CNP also reduced IFN-γ mediated kynurenine generation by the IFN-γ regulated enzyme indoleamine-2,3-deoxygenase (IDO). We conclude that CNP downmodulates IFN-γ induced pro-inflammatory gene expression in human endothelial cells via a cGMP-mediated pathway. Thus, CNP may have a protective role in vascular inflammation and novel therapeutic strategies for CVD based on upregulation of endothelial CNP expression could reduce chronic EC inflammation.

Keywords: C-type natriuretic peptide; cardiovascular; endothelium; indoleamine-2,3-dioxygenase; inflammation; interferon gamma.

Figure 1

ICAM-1 expression in HUVEC after incubation with IFN-γ alone or in combinations with CNP for up to 72 h. (A) Representative flow cytometry histograms for ICAM-1 after incubation of HUVEC alone (Red) or with IFN-γ (Blue), CNP (Orange) or IFN-γ and CNP (Green) for the indicated times. (B–D) Mean Fluorescence Intensity (expressed as fold increase over MFI of untreated cells, which range from 820 to 3089; mean ± SEM) for ICAM-1 on untreated HUVEC or after 24 h (B), 48 h (C) or 72 h (D) treatment with IFN-γ alone or in combination with CNP; n = 3 HUVEC isolates (* p < 0.05, ** p < 0.01, significantly different from IFN-γ alone).

Figure 2

MHC class I expression in HUVEC after incubation with IFN-γ alone or in combinations with CNP for up to 72 h. (A) Representative flow cytometry histograms for MHC-I after incubation of HUVEC alone (Red) or with IFN-γ (Blue), CNP (Orange) or IFN-γ and CNP (Green) for the indicated times. (B–D) Mean Fluorescence Intensity (expressed as fold increase over MFI of untreated cells, which ranged from 2079 to 6549; mean ± SEM) for MHC-I on untreated HUVEC or after 24 h (B), 48 h (C) or 72 h (D) treatment with IFN-γ alone or in combination with CNP; n = 3 HUVEC isolates (* p < 0.05, ** p < 0.01, significantly different from IFN-γ alone).

Figure 3

MHC class II expression in HUVEC after incubation with IFN-γ alone or in combinations with CNP for up to 72 h. (A) Representative flow cytometry histograms for MHC-II after incubation of HUVEC alone (Red) or with IFN-γ (Blue), CNP (Orange) or IFN-γ and CNP (Green) for the indicated times. (B–D) Mean Fluorescence Intensity (expressed as fold increase over MFI of untreated cells, which ranged from 187 to 216; mean ± SEM) for MHC-II on untreated HUVEC or after 24 h (B), 48 h (C) or 72 h (D) treatment with IFN-γ alone or in combination with CNP; n = 3 HUVEC isolates.

Figure 4

cGMP mediated effects on cell surface receptor expression in HUVEC. (A) CNP-stimulated cGMP accumulation in HUVEC isolates, after 30 min stimulation with 100 nm CNP in the presence of 1 mm IBMX. Data shown are means ± SEM pooled from 6 independent isolates (n = 6), each performed in duplicate (* p = 0.015, significantly different from Basal). (B–D) Effect of 8-bromo-cGMP on IFN-γ mediated ICAM-1 (B), MHC-I (C), and MHC-II (D) expressed as fold increase over MFI of untreated cells expression after 48hr treatment (which ranged from 572 to 819, 1313 to 2012, 176 to 230, for ICAM-1, MHC-I and MHC-II, respectively). n = 3 HUVEC isolates (* p < 0.05, *** p < 0.001, significantly different from IFN-γ alone).

Figure 5

CNP downregulates IFN-γ-mediated tryptophan metabolism in HUVEC. (A) Representative chromatogram of kynurenine production following IFN-γ treatment. (B) Kynurenine production from untreated HUVEC or after 24 h and 48 h treatment with IFN-γ alone or in combination with CNP. Data shown are representative from a single HUVEC isolate, expressed as % IFN-γ -stimulated kyunurenine production), and performed in duplicate. (** p < 0.01, significantly different from IFN-γ alone).