Interleukin-1 Receptor Antagonist (IL-1RN) Genotype Modulates the Replicative Capacity of Human Endothelial Cells
Endothelial cells (ECs) undergo a finite number of cell divisions before growth arrest or replicative senescence, modulated in part by the proinflammatory cytokine, interleukin-1 (IL-1). IL-1 and its family members are expressed in human atherosclerotic vessels, mainly in the endothelium. EC replicative senescence and IL-1 have been associated with atherosclerosis. Genetic variants at the IL-1 locus have been associated with a variety of coronary phenotypes. In this study, we examined the relationship between the interleukin-1 receptor antagonist variable number tandem repeat allele 2 (IL-1RN*2*2) and EC replicative capacity. A significant decrease in EC cumulative population doublings (CPDs) was associated with the rare allele (IL-1RN*2*2) at IL-1RN, 8.56±0.97 (n=7) versus 13.14±1.00 (IL-1RN*1*1, n=20), P=0.0118. Proliferation of IL-1RN*2*2 ECs detected by Ki67 expression was also significantly reduced particularly at later passage, passage 6: 21.76±0.93% (n=6) versus 48.10±8.81% (IL-1RN*1*1, n=7) (P=0.0323) and passage 8: 22.48±3.08% (n=6) versus 42.29±3.06% (IL-1RN*1*1, n=7) (P=0.0028). IL-1RN*2 carriage was associated with increased numbers of senescent ECs. Basal apoptosis, telomerase activity, and telomere length were not different with respect to IL-1RN genotype. Addition of exogenous IL-1ra (1 ng/mL) increased CPDs in a number of human umbilical vein endothelial cell cultures and increased proliferating cells from 12.11±1.21% to 27.82±2.82% (P=0.0216, IL-1RN*2*2, passage 8, n=2). These data suggest genetic control of EC proliferation and life span by the IL-1 locus and imply that IL-1ra may have a function connected with EC growth.
Coronary artery disease (CAD) has a substantial inflammatory component,1 which is in part genetic.2 Arterial inflammation, in the context of atherosclerosis, may be partially mediated by interleukin-1 (IL-1) and IL-1–related cytokines. IL-1 causes multiple responses within the vessel wall including inhibition of EC proliferation3 and induction of adhesion molecule expression promoting leukocyte infiltration.4 IL-1 exerts its effects via the type I IL-1 receptor, which is blocked by the endogenous, nonsignaling receptor antagonist (IL-1ra).
A gene variant at IL-1RN has been associated with chronic inflammatory disorders including angiographic single vessel coronary disease2 and protection from restenosis after percutaneous transluminal coronary angioplasty5 and stenting.6
The functional consequences of the IL-1RN gene variant appear complex. The less common allele (2) is associated with decreased levels of intracellular (ic) IL-1ra in endothelial cells (ECs).7
IL-1 has been linked with EC life span in culture. As ECs age, IL-1 accumulates, and antisense oligonucleotides to IL-1α extend EC life span.8,9 We speculate that ECs with lower amounts of the natural antagonist of IL-1 might have an attenuated life span in culture.
Materials and Methods
Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as described.7 Population doublings were calculated at each passage until growth arrest: log10 cells harvested−log10 cells plated/log10 2.
Exogenous secreted IL-1ra (1 ng/mL or 100 ng/mL) (Amgen) was added to paired HUVEC cultures at 2- to 3-day intervals from onset of culture; proliferation and cumulative population doublings (CPDs) were calculated as described.
DNA Isolation and IL-1RN Genotyping
Genomic DNAs isolated from HUVECs were genotyped at IL-1RN (86 bp, variable number tandem repeat, intron 2) as described.2 The common 4 repeat polymorphism was designated allele 1 (*1); the less common 2 repeat, allele 2 (*2).
Ki67 Proliferation Marker
Ki67, a proliferation-related antigen, was used to determine the proliferating fraction of ECs as detailed.10 Ki67-positive cells were counted in 5 random fields of view.
Detection of Senescence and Apoptotic HUVECs
Senescence-associated (SA) β-galactosidase (β-Gal) staining11 and terminal transferase-mediated dUTP nick-end labeling (TUNEL) were used to detect senescent and apoptotic HUVECs, respectively.
Telomere Length and Telomerase Activity Assay
Genomic DNAs (5 μg) digested overnight at 37°C with RsaI/HinfI (Promega) were separated on a 0.6% agarose gel, transferred to a Hybond N+ membrane (Amersham) and hybridized with 32P-labeled 5′(TTAGGG)6 at 65°C. Bound oligonucleotide was detected by exposure to XAR-Omat film (Kodak).
The telomerase assay is detailed in the online data supplement (available online at http://www.circresaha.org).
Data are expressed as the mean (±SEM). Statistical analyses were performed using 2 sample t tests and one-way ANOVA, with significance at P<0.05.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
EC length of life in culture, assessed as CPDs, calculated from the onset of culture until growth arrest, showed significant association with IL-1RN genotype: any carriage *2, 10.19±0.86 (n=24) versus *1*1, 13.14±1.00, (n=20), P=0.0259. Homozygosity for *2 was associated with mean significant attenuation of life span: *2*2, 8.56±0.97 (n=7) versus *1*1, 13.14±1.00 (n=20), P=0.0118 (Figure 1A). No association was apparent with CPDs and other polymorphisms of the IL-1 gene cluster (data not shown). HUVECs conformed to the characteristic senescent phenotype described by Maciag et al12 with increasing passage.
Decreased expression of Ki67 was associated with IL-1RN genotype at passage 6: 21.76±0.93% (*2*2, n=6) versus 48.10±8.81% (*1*1, n=7) (P=0.0323) and passage 8: 22.48±3.08% (*2*2, n=6) versus 42.29±3.06% (*1*1, n=7) (P=0.0028) (Figure 1B). SA–β-Gal staining was not statistically associated with IL-1RN*2 carriage (n=17) compared with wild type (n=10); there was a trend at passages 6 and 7, P6 55.32±10.00% (*2 carriage) versus 42.89% (1*1*) and P7, 59.45±10.30% versus 32.36% (P=NS) (online Figure 1, see online data supplement).
An index of apoptotic nuclei showed no association with IL-1RN*2*2 (n=6, P=NS) (online Figure 2).
We investigated whether a change in telomere length was associated with reduced CPDs at the end of life in ECs of different genotypes. Importantly, the IL-1RN genotypes of HUVECs (*1,*1 and *2,*2) had the same telomere length at the start of culture, and although there was a gradual reduction in length with passage, we observed no association with genotype (n=2, Figure 2). Since telomerase activity may abrogate telomere loss, we investigated telomerase activity in IL-1RN*2*2 HUVECs and showed no apparent reduction in activity (n=3, online Figure 2). These data suggest a telomerase-independent mechanism of EC cell survival under the conditions studied.
Since the only known function of IL-1ra is to block IL-1–mediated events, the replicative capacity of ECs in response to continuous addition of exogenous IL-1ra (1 ng/mL) throughout EC life span was examined. Proliferation (Ki67 expression) was increased in HUVECs (IL-1RN*2*2) continually treated with IL-1ra: passage 7, no IL-1ra 12.39±3.95% versus +IL-1ra 22.29±6.37%), P=0.0763 (n=2); passage 8, no IL-1ra 12.11±1.21% versus +IL-1ra 27.82±2.82%, P=0.0216 (n=2) (Figure 3). Mean CPDs were increased in some HUVEC cultures in response to 1 ng/mL IL-1ra (n=7) 25.51±2.03 versus untreated controls 20.56±2.03 (P=0.018); this increase was not observed with 100 ng/mL IL-1ra (n=7).
The results of the present study suggest a link between IL-1RN genotype and the growth dynamics of HUVECs in vitro with a reduction in EC growth potential associated with IL-1RN*2. The rare allele *2 at IL-1RN is known to be associated with lower production of icIL-1ra protein in HUVECs.7 A similar observation was made for columnar epithelial cells in ulcerative colitis.13
We report that carriage of allele 2 (IL-1RN*2) at IL-1RN is associated with decreased CPDs and proliferation in HUVECs. Addition of exogenous IL-1ra restores the proliferative potential of HUVECs with IL-1RN*2*2 to levels observed with IL-1RN*1*1 and extends CPDs in selected HUVEC cultures. It is known that IL-1 inhibits EC proliferation; we propose that IL-1 released after senescence binds available IL-1Rs, thus decreasing proliferation. Addition of exogenous IL-1ra may block IL-1 signaling and extend EC life span. This mechanism could partly explain the change in proliferation observed at later passages in IL-1RN*2*2 (Figure 3).
These data suggest that decreased proliferation seen in IL-1RN*2*2 HUVECs occurs, in part, as a result of an increase in senescence rather than apoptosis. EC senescence has previously been shown to be associated with reduced telomere length and telomerase activity14,15; these did not appear to be associated with IL-1RN genotype in this study.
Although the specific cellular mechanism for reduction of EC proliferation in relation to genotype remains to be determined, we suggest that IL-1RN*2*2 genotype leads to low levels of icIL-1ra in ECs causing an imbalance of IL-1 family members favoring IL-1 agonistic activity. This may regulate the activity of cell cycle components including p21.16 These data also demonstrate telomere-independent control of EC proliferative capacity and life span, by genetic variation at the IL-1 locus.
These data may have important implications for the role of IL-1 in vessel wall homeostasis and atherosclerotic and inflammatory diseases.
EC turnover is increased under conditions that favor atherogenesis (hypertension, high cholesterol levels, and anatomical branch points). The accelerated development of a dysfunctional or senescent phenotype under these conditions is plausibly associated with atherogenesis or the clinical presentation of atherosclerosis. Of relevance to this are the data that the IL-1RN*2 allele is associated with atherosclerotic coronary disease.
Although we favor the hypothesis that allele 2 of IL-1RN, in association with reduced IL-1ra production, is a central mechanism for the observed cell biological association reported, it is acknowledged that the mechanism of reduced IL-1ra production under these conditions remains unclear and requires further study.
In summary, it appears that the IL-1RN*2 allele is at the very least a marker for an EC phenotype plausibly associated with vascular disease.
This work was funded by the British Heart Foundation.
Original received March 5, 2003; resubmission received April 22, 2003; revised resubmission received May 9, 2003; accepted May 9, 2003.
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