Acceleration of cardiovascular disease by a dysfunctional prostacyclin receptor mutation: potential implications for cyclooxygenase-2 inhibition

Abstract

Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 inhibition led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major end product of cyclooxygenase-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein-coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant (R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in 3 separate white cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort (n=980), with no association in the low-risk cohort (n=2293). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared with age- and risk factor-matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent on the presence of existing atherosclerosis or injury (high risk factors), analogous to what has been observed in the cyclooxygenase-2 inhibition studies or prostacyclin receptor knockout mice studies. Combining both biochemical and clinical approaches, we conclude that diminished prostacyclin receptor signaling may contribute, in part, to the underlying adverse cardiovascular outcomes observed with cyclooxygenase-2 inhibition.

Figures

Figure 1. Detection, localization and function of hIP receptor mutant R212C

PANEL A: Amino acid sequence of the human prostacyclin receptor placed in a secondary structure (snake-plot) format with the seven transmembrane (TM) helices. The N-terminus is located extracellular (EC), with two sites of glycosylation indicated by pentagonal chains. The dual disulfide bonds are shown by the dashed line. The C-terminus is intracellular (IC) with putative sites of palmitoylation-isoprenylation indicated (serrated lines). The position of the R212C is highlighted with a black arrow. PANEL B: Chromatogram from genomic DNA sequencing of cardiology patients showing the nucleotide changes at position 212 wild-type codon (CGC - Panel I), heterozygote mutation (T/CGC - Panel II) and homozygote mutation (TGC - Panel III). PANEL C: Computer derived 3D-model (energy-minimized) of hIP receptor, showing TM helices (red) and EC and IC loops (gray). Enlarged region highlights third intracellular loop and location of Arg residue at position 212, at the C-terminal end of the putative alpha-helix; the position disrupted upon conversion to a cysteine (R212C).

Figure 1. Detection, localization and function of hIP receptor mutant R212C

PANEL A: Amino acid sequence of the human prostacyclin receptor placed in a secondary structure (snake-plot) format with the seven transmembrane (TM) helices. The N-terminus is located extracellular (EC), with two sites of glycosylation indicated by pentagonal chains. The dual disulfide bonds are shown by the dashed line. The C-terminus is intracellular (IC) with putative sites of palmitoylation-isoprenylation indicated (serrated lines). The position of the R212C is highlighted with a black arrow. PANEL B: Chromatogram from genomic DNA sequencing of cardiology patients showing the nucleotide changes at position 212 wild-type codon (CGC - Panel I), heterozygote mutation (T/CGC - Panel II) and homozygote mutation (TGC - Panel III). PANEL C: Computer derived 3D-model (energy-minimized) of hIP receptor, showing TM helices (red) and EC and IC loops (gray). Enlarged region highlights third intracellular loop and location of Arg residue at position 212, at the C-terminal end of the putative alpha-helix; the position disrupted upon conversion to a cysteine (R212C).

Figure 2. Patient blood analysis

PANEL A: Representative saturation binding curves on patient bloods for 3 individual patients DHMC 918 (wild-type), DHMC 826 (heterozygote R212C) and DHMC 726 (homozygote R212C) to demonstrate changes in binding and expression. [3H] iloprost was used for detection of the human prostacyclin receptor (hIP) and [3H]-SQ 29548 for the human thromboxane receptor (hTP). Arrows indicate relative changes in receptor numbers comparing hIP to hTP. PANEL B: Cyclic-AMP determination from the patient samples described in Panel B. Picomoles cAMP were corrected for changes in receptor numbers. PANEL C: Cyclic-AMP determination from COS-1 cell overexpression experiments for WT and R212C constructs. A dose response for iloprost was established (1μM, 0.1μM and 0.01μM).

Figure 3. R212C expression in a COS-1 system

PANEL A: Results of saturation binding and western analysis performed on COS-1 membrane preparations containing R212C and wild type constructs. PANEL B: Corresponding confocal microscopy overlay images (63 x resolution) showing predominant membrane trafficking only for wild-type protein. The hIP receptors both wild type and mutants are labeled red (1D4 monoclonal antibody). The endoplasmic reticulum are labeled green (anti-calnexin antibody) and the overlay picture additionally has blue nuclear staining (DAPI) and a phase contrast microscopic image of the cell to localize the cells perimeter. White arrows are used to localize areas of cell surface membrane.

Figure 4. Platelet cAMP and inhibition of aggregation

Plot of percent (%) inhibition of aggregation versus platelet cAMP production (pM). Reduced cAMP promotes aggregation (observed with R212C) whereas increased cAMP inhibits aggregation (observed with wild type).

Figure 5. Clinical analysis of three Caucasian Cohorts

A graph representing the R212C frequencies in the 3 Caucasian cohorts, DHMC, NHS and the HPFS. The white bars represent no coronary heart disease (No CHD) and the shaded bars the coronary heart disease groups (CHD). The odds ratios (OR) are described and confidence limit observed in Online Table 2.

Figure 6. R212C association with accelerated disease

PANEL A: A comparison of disease severity (vessels with significant obstruction, left anterior descending, left circumflex and right coronary artery) in the cardiology cohort versus the R212C (both heterozygote and homozygote) patients. PANEL B: An analysis of disease severity in the cardiology control cohort versus the R212C cohort focusing on percent of cohort with the number of vessels obstructed. PANEL C: Total cardiovascular adverse events recorded in the risk-matched cardiology control patients (n=20) and the R212C patients (n=20). PANEL D: Number of cardiovascular events recorded for patients from either the risk-matched control patients or the R212C patients. Events were recorded for three years from the initial hospital visit/admission for a cardiovascular event.