Bioscience Reports (2010) 30, 11-18 - L. Rodriguez-Menocal and others

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Bioscience Reports (2010) 30, (11–18) (Printed in Great Britain)

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Original Paper

Aging increases p16^INK4a expression in vascular smooth-muscle cells

Luis Rodriguez‑Menocal, Si M. Pham, Dania Mateu, Melissa St‑Pierre, Yuntao Wei, Ivo Pestana, Abdelouahab Aitouche¹ and Roberto I. Vazquez‑Padron²

Department of Surgery and Vascular Biology Institute, University of Miami Miller School of Medicine, Miami, FL 33136, U.S.A.

Key words: aging, aorta, cell-cycle inhibitor, p16^INK4a, restenosis, vascular smooth-muscle cell.

Abbreviations used: Arf, ADP-ribosylation factor; Cdk, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitor; DMEM, Dulbecco's modified Eagle's medium; qRT–PCR, quantitative reverse transcription–PCR; SMA, smooth-muscle actin; VSMC, vascular smooth-muscle cell; vWF, von Willebrand factor.

¹Present address: NIH (National Institutes of Health) CSR, Room 2183, MSC 7818, 6701 Rockledge Drive, Bethesda, MD 20892, U.S.A.

²To whom correspondence should be addressed, at Division of Cardiothoracic Surgery and Vascular Biology Institute, University of Miami Miller School of Medicine, 1600 NW 10th Avenue, RMSB 7147B, Miami, FL 33136, U.S.A. (email rvazquez@med.miami.edu).

Alteration of VSMC (vascular smooth-muscle cell) physiology is associated with the development of atherosclerosis and restenosis. We hypothesize that aging up-regulates the expression of p16^INK4a in VSMCs, which may increase the susceptibility of blood vessels to vascular occlusive diseases. Aortic VSMCs were obtained from young and aged mice. Cells from aged mice grew more slowly than those from their younger counterparts. Progression of cell cycle in response to serum stimulation was significantly inhibited in those cells with aging, as determined by FACS after propidium iodide staining. A significant up-regulation of p16^INK4a (2.5-fold, P=0.0012) was found in VSMC from aged animals using gene arrays. The up-regulation of this gene was further confirmed by quantitative RT–PCR (reverse transcription–PCR) and Western-blot experiments. Immunostaining for p16^INK4a confirmed that aortas from aged mice contained more p16^INK4a+ SMA (smooth-muscle cell actin)⁺ cells than aortas from young animals (26.79±2.45 versus 7.06±1.44, P=0.00027, n=4). In conclusion, we have shown that aging up-regulates the expression of p16^INK4a in VSMC in both cultures and arteries. The increase in p16^INK4a in the vasculature with aging may modify VSMC's response to post-injury stress and therefore accelerate the development of age-related cardiovascular diseases.