Application of bioactive compounds on small-caliber vascular graft for prevention of intimal hyperplasia

Abstract

Intimal hyperplasia is an excessive tissue ingrowths and a chronic structural lesion that is observed at a site of atherosclerotic lesion formation, arterial angioplasty, and vascular graft anastomoses etc. It is a result of the vessel wall’s response to injury, and characterized by formation of a neointiml consisting of mainly attributable vascular smooth muscle cell (VSMC) migration and proliferation from the media to the intima and extracellular matrix remodeling. Dedifferented VSMCs of the media layers are phenotypically modulated from contractile state to the active synthetic state and induce proliferation and migration of VSMC from the media to the intima and extracellular matrix (ECM) protein deposition. Epigallocatechin 3-O-gallate (EGCG) and resveratrol have been exported to have antioxidant, anti-proliferative and anti-thrombogenic effect. In this study, we investigate the effects of natural polyphenol, such as EGCG and resveratrol, on dedifferentiation, and the intracellular signal transduction pathway of PDGF-bb in rat aortic vascular smooth muscle cell and demonstrate the prevention mechanism of VSMC on intimal hyperplasia. Additionally, we prepare the polyphenol eluting biodegradable PLGA sheet for local delivery of polyphenol and investigate the effect of external application of polyphenol eluting biodegradable sheet on intimal hyperplasia after balloon injury of abdominal aorta in an experimental rabbit model. 10 ng/ml PDGF-bb induced a RAOSMC proliferation and cell vyvle progression. 10 ng/ml PDGF-bb treatments to serum starved RAOSMC induced phosphorylation of PDGFR-β after 10min incubation and maintained over 4h of stimulation on RAOSMC and led to a complete 42/44 MAPK phosphorylation which peaked within 30 min and returned to baseline levels at 120 min. Moreover, other intracellular signal pathways, phosphorylation of p38 MAPK and Akt, was activated by PDGF-bb stimulation. Treatment of EGCG with PDGF-bb significantly inhibited the proliferation of RAOSMC stimulated by dose dependently. Therefore, RAOSMC synchronized with EGCG inhibited proliferation stimulated by PDGF-bb stimulation. Treatment of 50 μM EGCG inhibited almost completely the growth of ROASMC. Also, cell cycle progression and MMP release in RAOSMC was inhibited by EGCG treatment whether stimulated with EGCG and PDGF-bb or stimulated with PDGF-bb after EGCG pretreatment. However, PDGF-bb stimulation on RAOSMC starved with 50 μM EGCG was inhibited, as shown by decreased phosphorylation of PDGFR-β and intracellular signal cascade. Therefore, PDGF-bb stimulation with 50 μM EGCG on serum starved RAOSMC completely inactivated PDGFR-β phosphorylation. According to inactivation of PDGFR-β by EGCG, phosphorylation of 42/44 MAPK, p38 MAPK, and Akt was suppressed to baseline levels as in serum starved RAOSMC. In conclusion, EGCG inhibited RAOSMC dedifferentiation by interruption of PDGF-bb signal pathway, probably by directly binding with PDGF-bb and preventing PDGFR-b phosphorylation by incorporation into cell membrane. Treatment of RAOSMC with resveratrol were significantly inhibited the PDGF-bb stimulated proliferation dose dependently. Also, treatment of 100 μM resveratrol inhibited almost completely the growth. In phenotype exchange in PDGF stimulation, serum starved RAOSMC maintained spindle morphology and aligned arrangement of actin filament, whereas PDGF-bb stimulated RAOSMC change form spindle to polygonal morphology and disassembled distribution of actin filaments. However, resveratrol treatment inhibited the phenotype change and disassembly of actin filament, and maintained the expression of contractile phenotype related protein such as calponin and smooth muscle actin–alpha. In addition, morphology of PDGF-bb stimulated RAOSMC exhibited greater circularity and area, compared with serum starved RAOSMC. Resveratrol inhibited the change of circularity and area stimulated by PDGF-bb. Phosphorylation of PDGFR-b decreased at 10min incubation with 10 ng/ml PDGF-bb and 100 μM resveratrol, however it was similar with 10min incubation with only PDGF-bb stimulation. Similarly, resveratrol slightly inhibited phosphorylation of 42/44 MAPK and p38 MAPK. However, Akt and mTOR phosphorylation upon PDGF stimulation elicited a strong and detectable signal for several hours, while PDGF stimulation with resveratrol induced only weakly Akt phosphorylation. In conclusion, RAOSMC dedifferentiation, phenotype and proliferation rate were inhibited by resveratrol by interrupting the balance of the Akt and 42/44MAPK and p38MAPK pathway activation stimulated by PDGF-bb. The electrospun PLGA fibers had typical fiber mat and a bead-free fibrous structure with wide range of fiber diameters whether in the presence or absence of EGCG or resveratrol. EGCG and resveratrol was encapsulated in PLGA fiber. EGCG and resveratrol eluting PLGA sheet appeared a red and yellow color, respectively. From the FTIR spectra, distinctive peaks of EGCG and resveratrol were observed from spectra of their respective EGCG or resveratrol eluting PLGA sheet. This indicated that EGCG or resveratrol were well loaded and dispersed into PLGA sheets. The EGCG and resveratrol loading efficiencies on EGCG and resveratrol eluting PLGA sheet were found to be 80~90 % of input amount for sheet fabrication. Each polyphenol release profiles are shown to be different for EGCG and resveratrol. In case of EGCG there was burst diffusion at 1 day followed by sustained release up to 40 days from EGCG eluting PLGA sheets. Resveratrol was released in a logarithmic manner up to 20 days after which the release rate decreased with time. We investigated the effect of externally wrap with EGCG and resveratrol eluting PLGA sheet in balloon injury aorta of rabbit. Morphometric analysis revealed significantly decreased intima/media (I/M) ratio in EGCG eluting electrospun PLGA fiber sheet. We found a significantly (p<0.05) 30% and 50% reduction of intimal area in wrapped aorta with EGCG and resveratrol. The medial area was not significantly by EGCG and resveratrol treatment in comparison with vehicle PLGA control. From the therapeutic point of view, these results suggest that EGCG and resveratrol can be a potential agent for the prevention of VSMC dedifferentiation and their local release from electrospun PLGA fiber sheet can be applicable as a method to prevent intimal hyperplasia in stent, catheter, and vascular bypass and graft.