Apoptosis, cell proliferation and modulation of cyclin‐dependent kinase inhibitor p21^cip1 in vascular remodelling during vein arterialization in the rat

Abstract

Neo-intima development and atherosclerosis limit long-term vein graft use for revascularization of ischaemic tissues. Using a rat model, which is technically less challenging than smaller rodents, we provide evidence that the temporal morphological, cellular, and key molecular events during vein arterialization resemble the human vein graft adaptation. Right jugular vein was surgically connected to carotid artery and observed up to 90 days. Morphometry demonstrated gradual thickening of the medial layer and important formation of neo-intima with deposition of smooth muscle cells (SMC) in the subendothelial layer from day 7 onwards. Transmission electron microscopy showed that SMCs switch from the contractile to synthetic phenotype on day 3 and new elastic lamellae formation occurs from day 7 onwards. Apoptosis markedly increased on day 1, while alpha-actin immunostaining for SMC almost disappeared by day 3. On day 7, cell proliferation reached the highest level and cellular density gradually increased until day 90. The relative magnitude of cellular changes was higher in the intima vs. the media layer (100 vs. 2 times respectively). Cyclin-dependent kinase inhibitors (CDKIs) p27(Kip1) and p16(INKA) remained unchanged, whereas p21(Cip1) was gradually downregulated, reaching the lowest levels by day 7 until day 90. Taken together, these data indicate for the first time that p21(Cip1) is the main CDKI protein modulated during the arterialization process the rat model of vein arterialization that may be useful to identify and validate new targets and interventions to improve the long-term patency of vein grafts.

The Severity of Internal Carotid Artery Stenosis is Associated with the Cyclin-Dependent Kinase Inhibitor 2A Gene Expression

“…INK4a remains unchanged during rat vein arterialization 24) . Cardenas et al demonstrated that the rate of venous thrombosis is increased in vascular injury models of p16…”

“…Cell proliferation is required for the arterial-vein graft to adjust to new hemodynamic conditions. However, an uncontrolled proliferative process can result in the occlusion of the vein graft or the development of atherosclerotic lesions 24) . Borin et al reported that p21 is the main CKI, while the level of p16…”

“…In order to determine the specificity of immunostaining, a negative control was always used in which the primary antibody was omitted. Mouse IgG (Santa Cruz Biotechnology, Santa Cruz CA, USA) was used as a negative control 23,24) .…”

“…Cell counting was performed under 400× magnification, with two researchers examining different fields at different times in a single-blind fashion. At least 10 fields per slide were evaluated for every patient sample 23,24) .…”

“…Nevertheless, this result indicates that the levels of CDKN2A and ANRIL NR_003529 transcripts are inversely correlated in carotid plaque tissues. Borin et al demonstrated that apoptosis and cell proliferation are both involved in the remodeling process induced by arterialization of the rat jugular vein 24) . In the present study, we showed that cell proliferation is increased in plaques associated with ICA stenosis of ≥ 80%, although this finding was not statistically significant.…”