Background: Femoral artery (FA) is large conducting vessel build at the first trimester of embryonal development. Together with its important role for adequate nutrient and oxygen supply of lower extremities, in recent years the percutaneous access through the common femoral artery has become most common in vascular surgery for both diagnostic and therapeutic vascular procedures. This shift from traditional open surgery to endovascular interventions also raises the question whether the safety of those diagnostic and surgery methods could be meliorated by matching them with the contractile state of common FA. Here, we investigate whether senescence alters vascular reactivity in isolated ring preparation from young and senescent of murine FA used as a model.

Methods: The reactivity of ring preparation from young (12-17 weeks) and senescent (> 104 weeks) FAs was measured by wire myography. Protein phosphorylation of the regulatory myosin light chain (MLC₂₀) and targeting subunit of myosin-phosphatase, MYPT1 were studied by western blotting.

Results:  Passive length-tension-relationships were not altered by senescence and the values of calculated internal diameter of FA was ~400 µm in both age groups. Initial tone after stretching and 20 minutes equilibration was 4.3±0.5 mN in yFA vs. 7.1±0.9 mN in sFA (n=5). Inhibition of endogenous NO-release by 100 µM L-NAME raised tone additionally to 12.6±4.8 mN in sFA while this treatment had a negligible effect of tone in in yFA (5.1±1.1 mN). In line with this the reactivity to NO-donor, DEA-NONOate of submaximally preconstricted sFA was augmented compared to those of yFA (-4.5±0.3 in yFA vs. 5.2±0.1 in FA of senescent mice; n=4-5). No significant difference in concentration-response relationships to the tromboxaneA2 analogue U46619 was observed in both age groups. Senescence reduced maximal relaxation to acetylcholine (48.1±7.1 in sFA vs. 62.4±3.1% in yFA; n=5), but had no significant effect on its pD2 values (-7.2±0.08 vs. -6.7±0.09). At basal conditions (no stimulus applied), pMLC₂₀-S19 immunoreactivity normalized to immunoreactivity obtained in presence of 0.1 µM phosphatase inhibitor, CalyculinA was 19.2±5.8% in yFA compared to 49.2±12.6% in sFA (n=6). In yFA treatment with the fairly specific inhibitor of RhoA-associated kinase, Y27632 (3 µM) had no effect of MLC₂₀-S19 (18.1±9.5%), but reduced it to 24.8±3.5% in sFA (n=6). The phosphorylation of the RhoA-kinase sensitive regulatory threonine site of myosin phosphatse, MYPPT1-T696 decreased by ~30% in senescent FAs, while the compound was virtually ineffective in yFA. No difference in other ROK site of MYPT1, T853 as well as the PKG-site of MYPT1, S668 have been observed in both age groups.

Conclusions: The present work provides evidence that senescent murine FA undergo vascular remodeling associated with increases in stretch-activated contractility. Our study supports the view that the underling mechanism of altered contractility involves increase in MLC₂₀-phosphorylation and change in the reactivity of RhoA-kinase sensitive site of targeting subunit of myosin-phosphatase, MYPT1-T696.