Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • br Materials and methods br Results br Discussion Neointimal

    2020-06-01


    Materials and methods
    Results
    Discussion Neointimal VSMC accumulation contributes considerably to vessel occlusion observed in both autologous vein graft degeneration [1], [2] and in-stent restenosis [26], [27]. In this study, the potential for retarding VSMC proliferation and hence ameliorating pathophysiological neointima formation via manipulation of the CK2-PRH signalling axis was evaluated. Utilising appropriate strategies for delivery of synthetic CK2 inhibitors may be of benefit to patients receiving vein grafts or drug-eluting stents. Induction of VSMC proliferation with PDGF-BB and bFGF and culture of saphenous vein for 14days led to enhanced CK2 activity (detected with anti-phosphorylated CK2 substrate antibody). Pharmacological inhibition of protein kinase CK2 with the synthetic compounds TBB and K66 significantly reduced proliferation in isolated VSMCs. The K66 compound was favoured for its putative low promiscuity and hence selected for the remainder of this study. The specificity of the K66 effect was supported by the observation that siRNA-induced silencing of the catalytic subunits of CK2 displayed comparable efficacy. Inhibition of CK2 activity via administration of K66 did not affect either VSMC apoptosis or migration. Importantly, K66 reduced levels of phosphorylated PRH protein and siRNA-mediated knockdown of PRH in K66-treated cultured VSMCs demonstrated that the anti-proliferative action of K66 required the presence of PRH. This suggests that phosphorylation and degradation of PRH are prerequisites for the growth-permissive effects of protein kinase CK2, as observed in human myeloid leukaemia K562 cells [19]. The multifunctional transcription factor PRH has been previously identified as a critical modulator of NKY 80 progression, differentiation and development [14], but its role in regulation of VSMC proliferation and neointima formation was not fully elucidated. Sekiguchi et al. postulated that PRH may operate as a pathogenic factor by facilitating VSMC de-differentiation as its expression was induced with intimal thickening in conjunction with a molecular marker for de-differentiated, synthetic VSMCs [21]. In spite of this accumulation of PRH protein within the intima in vivo, our in vitro studies indicated that PRH did not facilitate cell cycle progression, but in fact revealed an anti-proliferative role for PRH. Most interestingly, the S163C:S177C PRH mutant displayed a prolonged anti-mitotic effect with respect to wild-type PRH. To clarify, mutation of the Ser163 and Ser177 residues within the PRH homeodomain is preventative of CK2-dependent phosphorylation, where phosphorylation abrogates DNA-binding potential and transcriptional regulation activity, reduces nuclear retention and decreases stability via proteolysis [19], [20]. Consequently, the S163C:S177C PRH mutant possesses enhanced stability [19], which is most likely the cause of the increased longevity of its anti-proliferative effect. As demonstrated by Soyombo and colleagues [28], neointima formation is a phenomenon that can be successfully replicated in ex vivo human saphenous vein organ cultures, which is therefore a valuable technique for the testing of novel therapeutic interventions. Utilising this model, we demonstrated that K66 and adenovirus-mediated gene transfer of S163C:S177C PRH significantly retarded neointima formation. In both cases, this was exclusively attributed to their anti-proliferative activity as migration NKY 80 and cell viability were unaffected. Moreover, as expected, inhibition of protein kinase CK2 with K66 was associated with elevated PRH protein but decreased phosphorylated PRH levels in the saphenous vein organ cultures. This provides further support that CK2 regulates PRH protein levels and activity. Regarding further translational aspirations, as protein kinase CK2 is ubiquitously expressed, systemic delivery of K66 would most likely result in undesirable off target side effects. Perivascular drug delivery systems may be employed for localised, sustained release of the K66 compound to a vascular graft. Sanders and colleagues [29], reported that delivery of anti-proliferative agents such as sunitinib – a multi-target receptor tyrosine kinase inhibitor – to porcine jugular veins could be achieved through application of a biocompatible hyaluronic acid-based hydrogel contained in a polyactide-co-glycolide perivascular wrap to impede drug diffusion into non-target extravascular tissue. Alternative drug delivery strategies include the use of drug-eluting nanoparticles or even drug-linked antibodies for direct administration of a compound to a target tissue [30]. The approaches discussed may all be considered for localised, sustained delivery of K66 to a grafted vasculature to prolong its patency.