Iterleukin 1 alpha is a marker of endothelial cellular senescent

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 [9].

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 [10].

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 [2].

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.

On culture plates, HUVEC grow until they form a perfect monolayer. At this stage, cells stop growing and become quiescent. This pattern correlates with the arrest of thymidine incorporation (not shown). Since IL-1α inhibits endothelial proliferation, we evaluated whether IL-1α was modulated in actively proliferating versus quiescent HUVEC, both at PD 20. PolyA⁺ RNA was separated by electrophoresis and Northern blot was performed. We detected no modulation of IL-1α RNA in quiescent vs proliferating cells (Fig. 1A). Accordingly, the protein levels of IL-1 were comparable in quiescent and proliferating cells (Fig. 1B). Similar results were obtained in HUVEC cells when quiescence was reached by growth factor withdrawal (not shown).

Since cellular senescence correlates with the decline of the proliferating rate and an antisense against IL-1α extends endothelial lifespan [2], we evaluated IL-1α levels in HUVEC at different PD. As shown in figure 1A, a gradual increase of IL-1α mRNA paralleled the increase of PD, as detected by Northern blot performed on polyA⁺ RNA. Accordingly, we detected higher amounts of IL-1α in senescent (PD 45) than in young (PD 15) cells by western analysis (Fig. 2).

Because i) IL-1α shares many activities with IL-1β and ii) their activities are antagonized by the IL-1 receptor antagonist (IL-1ra), we determined the expression of the members of the IL-1 family, i.e. IL-1α, IL-1β and IL-1ra by RT-PCR in HUVEC at different PDs. To provide a better quantification, the PCR reaction was stopped after 15 or 30 cycles. In both conditions, we found no significant modulation of the total amounts of IL-1β and IL-1ra in young and senescent endothelial cells, while we confirmed the induction of IL-1α expression (Fig. 3).

To determine whether the relationship between IL-1s and senescence occurred also in fibroblasts, we examined young and senescent human dermal fibroblasts for their expression of IL-1α by Northern on polyA⁺ RNA and western blot and found no differences (Fig. 4A and 4B). Progeria is a rare premature aging syndrome which is characterized by external stigmata of aging and death within the fierst two decades of life [11]. We therefore evaluated IL-1α expression in dermal fibroblasts derived from progeric individuals. Figure 4A and 4B show that IL-1α mRNA and protein levels are not significantly modulated in progeric vs age-matched dermal fibroblasts.

By RT-PCR, we show that no modulation occurs in the steady state levels of IL-1β and IL-1ra (Fig. 5) in senescent and progeric fibroblasts.