Iterleukin 1 alpha is a marker of endothelial cellular senescent
© Mariotti et al; licensee BioMed Central Ltd. 2006
Received: 28 October 2005
Accepted: 06 April 2006
Published: 06 April 2006
The functional changes associated with endothelial senescence may be involved in human aging and age-related vascular disorders. Since the inflammatory cytokine interleukin (IL-)1 inhibits endothelial growth, we evaluated the expression of IL-1α, IL-1β and their antagonist, the IL-1 receptor antagonist (IL-1ra), in endothelial in vitro senescence and quiescence. We also examined the expression of IL-1α in human senescent and progeric fibroblasts.
We found that the overexpression of IL-1α specifically characterizes endothelial senescence. No modulation of this cytokine was observed in endothelial quiescence and in senescent or progeric human fibroblasts. The expression of IL-1β and IL-1ra was also assessed and found not to be affected by senescence.
Our results indicate that a dysfunction of the cytokine network associates with aging and point to a specific role of IL-1α in endothelial senescence.
The endothelium is a dynamic, heterogeneous, disseminated organ that possesses vital secretory, synthetic, metabolic and immunologic functions. In vivo endothelial cells (EC) represent a large population of quiescent cells lining the vessels. Macrovascular EC rarely divide with a turnover rate of approximately once every three years , although replication is increased under conditions that favor atherogenesis, such as hypertension, high cholesterol levels and anatomical branch points. In vitro, EC have a finite number of cell replication reaching replicative senescence . This cessation of cell division is accompanied by a specific set of changes in cell function, morphology and gene expression [3, 1] that may contribute to age-associated diseases, including atherosclerosis. Interestingly, vascular endothelial cells with senescence-associated phenotypes have been detected in the atherosclerotic regions of human aorta  and coronary arteries . Accordingly, multiple baloon endothelial denudation in non-atheromatous rabbit carotid arteries promoted the accumulation of senescent cells in the arterial wall .
Endothelial senescence is modulated in part by the inflammatory cytokine interleukin (IL-)1α [2, 7]. IL-1 and its family members are expressed in human atherosclerotic vessels, mainly in the endothelium . It is noteworthy that EC replicative senescence and IL-1 have been associated with atherosclerosis.
Although senescence and quiescence have a common denominator represented by the inhibition of cell growth, the two processes are different, because senescence is characterized by an irreversible growth arrest as well as by a specific gene expression profile .
This paper addresses the relation between IL-1α, IL-1β and IL-1ra expression and macrovascular endothelial cell quiescence and senescence. We also examined the expression of IL-1α in human senescent and progeric fibroblasts. We conclude that the overexpression of IL-1α is a specific marker of in vitro endothelial senescence.
Human umbilical vein EC (HUVEC) were cultured in M199 containing 10% fetal calf serum (FCS), Endothelial Cell Growth Supplement (150 μg/ml) and heparin (5 U/ml) on 2% gelatin coated dishes. All culture reagents were from Gibco. Human fibroblasts from progeric individuals (GM0498, 3 year-old male; GM2037, 13 year old male) and age-matched controls (AG6917, 3 year-old male; AG3513, 13 year old male) were from ATCC and cultured in D-MEM containing 20% FCS. Human dermal fibroblasts were isolated and propagated in D-MEM with 10% FCS until they reached cellular senescence .
The population doublings (PD) were calculated as log2 (number of cells at time of subculture/number of cells plated). The senescent phenotype was assessed by evaluating the senescence-associated (SA)-beta galactosidase activity as described .
Northern blot and RT-PCR analysis
HUVEC were rinsed with phosphate buffered saline and lyzed in RNAzol (Gibco). PolyA+ RNA was purified on oligodT columns, electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde, capillary blotted onto nylon membranes and UV crosslinked. IL-1α and GAPDH cDNAs were labelled with a random primer labeling kit (Ambion). Filters were hybridized in 0.5 M sodium phosphate (pH 7.2) containing 7% SDS, 1 mM EDTA and 20% formamide at 65°C for 20 h and extensively washed at high stringency before autoradiography. The results were quantitated by densitometry. To establish whether comparable amounts of RNA had been loaded, the ratio GAPDH/IL-1α was evaluated. For RT-PCR, 1 μg of total RNA was reverse transcribed and PCR amplification was carried out using 1/50 of the final RT reaction. Each amplification cycle consisted of 30 sec at 95°C, 30 sec at 52°C and 1 min at 72°C using 30 pmol of each primer. The reaction was stopped after 15 or 30 cycles. One fifth of the reaction mix was separated on a 1% agarose gel. The primers used to amplify IL-1β are the following: 5'-GACTTGTTCTTTGAAGTCGAT-3' (sense) and 5'-TAGAGTGGGCTTATCATCTTT-3' (reverse). The primers for IL-1ra are: 5'-ATGGAAATCTGCAGAGGCCTCCGCAGT-3' (sense) and 5'-CTGGTCAGCTTCCATCGCTGTGCAGAGGAA-3' (antisense). The sequence of the GAPDH primers has been published .
HUVEC were lysed in 10 mM Tris-HCl (pH 7.4) containing 3 mM MgCl2, 10 mM NaCl, 0,1% SDS, 0,1% Triton X-100, 0,5 mM EDTA and protein inhibitors, separated on 15% SDS-PAGE and transferred to nitrocellulose sheets. Western blot analysis was performed using polyclonal goat antibodies against IL-1α (Santa Cruz-TebuBio). Secondary antibodies were labeled with horseradish peroxidase (Amersham Pharmacia Biotech). The SuperSignal chemiluminescence kit (Pierce) was used to detect immunoreactive proteins following the manufacturer's instructions. The blots were stripped and incubated with an anti-actin antibody (Santa Cruz – Tebu-bio) to show that comparable amounts of protein were loaded per lane. Densitometric analysis was performed to better quantitate the results. All the western blots have been repeated at least three times on cell extracts from different experiments.
IL-1α levels in proliferating vs quiescent HUVEC
IL-1s expression in HUVEC cells at various PD
IL-1s in young, senescent and progeric human fibroblasts
The results of the present study indicate that the overexpression of IL-1α specifically characterizes endothelial senescence. No modulation of this cytokine was observed in endothelial quiescence and in senescent or progeric human fibroblasts.
IL-1α shares many activity with IL-1β, since they act by binding to a common receptor, the type I IL-1 receptor . A third member of the family, the IL-1 receptor antagonist (IL-1ra), also binds to the type I IL-1 receptor and blocks the receptor, preventing the action of the agonist IL-1s . In senescent endothelial cells we did not detect any modulation of the mRNA levels for IL-1β and IL-1ra. We therefore propose that IL-1α could be used as a marker of endothelial senescence. IL-1α causes multiple responses in vascular endothelial cells including inhibition of cell proliferation , induction of adhesion molecules which bind leukocytes  and promotion of thrombus formation . Indeed, in vitro, IL-1α overexpression has been linked to endothelial lifespan and to several dysfunctions . It is noteworthy that senescent endothelial cells are found on the surface of atherosclerotic plaques  and that IL-1 is produced by endothelium on aged coronary arteries .
Increased vascular production of pro-inflammatory cytokines may contribute to the increased plasma levels of these mediators in aging [16, 17]. We propose that the upregulation of IL-1 can be linked to the activation of various pathophysiological programs that underlie the complex biological phenomenon described as "vascular aging". Proinflammatory status of aged arteries may shift the intravascular environment from a hemodynamically stable state to a pro-coagulant, pro-oxidant state which may favour an exaggerated response to vessel injury, promoting the development of ischemic heart disease in the elderly.
IL-1ra allelic polymorphisms affect replicative lifespan of human EC . The polymorphism IL-1RN*2*2, which decreases the levels of IL-1ra, was associated with increased numbers of senescent endothelial cells and an inhibition of proliferation, while the addition of IL-1ra restored the proliferative potential of the cells and extended their lifespan . Because endothelial turnover is enhanced under conditions that favour atherogenesis thus leading to a senescent phenotype, it is noteworthy that the IL-1RN*2 allele is associated with atherosclerotic coronary disease . All together, these data suggest that IL-1ra may prevent the senescent-promoting effects of IL-1 in the endothelium.
We did not detect any modulation of the steady state levels of IL-1s in in vitro aged human dermal and in progeric fibroblasts. This is in agreement with a previous study performed on IMR-90 cells, demonstrating that the human diploid fibroblast senescence pathway is independent of IL-1α mRNA levels . On the contrary, expression of IL-1β and IL-1ra was induced in senescent mouse embryonic fibroblasts . Interestingly, the IL-1ra-/- mice presented early mortality compared to wild-type mice and accelerated senescence was observed in IL-1ra deficient fibroblasts . All together these data indicate that a dysfunction of the cytokine network associates with aging and point to a specific role of IL-1α in endothelial senescence.
receptor antagonist IL-1ra
- EC HUVEC:
human umbilical vein
reverse transcription- polymerase chain reaction
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