Antirestenotic mechanisms of everolimus on human coronary artery smooth muscle cells: inhibition of human coronary artery smooth muscle cell proliferation, but not migration.

Everolimus, a pharmaceutical component of drug-eluting stents, inhibits coronary vessel restenosis, but the antirestenotic mechanisms of action remain unclear. Here, we describe the effects of everolimus on key contributors to vessel restenosis, smooth muscle cell proliferation, and migration. In a dose-dependent fashion, everolimus reduced human coronary artery smooth muscle cell (HCASMC) proliferation without toxicity in a bimodal fashion, with accentuated potency occurring at 10 µM. Everolimus arrested the majority of HCASMCs in G1-phase, whereas it reduced the fraction of cells in S-phase at doses that inhibited DNA synthesis (bromodeoxyuridine incorporation). Consistent with this, Western blotting demonstrated that everolimus reduced activation and expression of G1-phase cell cycle progression factors, including p70S6K and cyclin D, respectively, decreased levels of proliferating cell nuclear antigen, and attenuated growth factor/serum-induced phosphorylation of the cell cycle phase transition intermediate, retinoblastoma protein. Everolimus did not, however, affect HCASMC migration. These observations suggest that everolimus acts as an antiproliferative, but not antimigratory, compound to account for at least some of the clinical efficacy exhibited by this drug as an antirestenotic agent. Moreover, everolimus-induced inhibition of the mammalian target of rapamycin complex 1 and regulation of cyclin-mediated cell cycle progression actions likely account for the antiproliferative effects of this compound on HCASMCs.

Role of lysyl oxidase propeptide in secretion and enzyme activity.

Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX-PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full-length LOX cDNA (pre-pro-LOX), the N-glycosylation null pre-[N/Q]pro-LOX cDNA and the deletion mutant pre-LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N-terminal propeptide sequence. The results show that glycosylation of the LOX-PP is not required for secretion and extracellular processing of pro-LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX-PP for pro-LOX exit from the ER and is the first to highlight the influence of LOX-PP glycosylation on LOX enzyme activity.

Impact of stent surface on thrombogenicity and vascular healing: a comparative analysis of metallic and polymeric surfaces.

BACKGROUND: Emerging drug-eluting stent technologies are evolving toward the elimination of polymeric component used as the method for modulating drug delivery. Although this technological approach seems to be biologically appealing, the impact of durable polymers and metallic stent surfaces on vascular healing remains unclear. In the present study, we aimed to compare the independent effect of a durable polymer and a metallic stent surface on thrombogenicity and endothelial cell coverage using different in vitro and in vivo experimental models. METHODS AND RESULTS: Platinum chromium (PtCr) and polyvinylidene fluoride-co-hexafluoropropene (PVDF-HFP)-coated surfaces were evaluated in this study. Thrombogenicity was assessed by exposing all surfaces to human blood under shear flow conditions. The inflammatory potential of the material was evaluated by measuring cytokine release from THP-1 cells exposed to all surfaces for 24 hours. Endothelial cell coverage was evaluated by detection of CD31 after the stents were exposed to human coronary artery endothelial cells for ≤ 14 days. Platelet adhesion (P<0.01) and activation (P=0.03) on PVDF-HFP were greater than on PtCr. In vivo, PVDF-HFP revealed more neointimal area (P<0.01) and residual parastrut fibrin (P=0.01) at 30 days compared with PtCr. PtCr displayed higher endothelialization rates and higher vascular endothelial-cadherin expression at 7 and 14 days (P=0.02) compared with PVDF-HFP. CONCLUSIONS: Thrombogenicity and vascular healing differ among metallic and polymeric stent surfaces. PVDF-HFP exhibits higher degrees of platelet activation-adhesion and thrombus accumulation in vivo compared with PtCr. PtCr displayed higher degrees of endothelial surface coverage compared with PVDF-HFP surfaces.

Lysyl oxidase oxidizes cell membrane proteins and enhances the chemotactic response of vascular smooth muscle cells.

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.