Dedicator of cytokinesis 2 silencing therapy inhibits neointima formation and improves blood flow in rat vein grafts.

OBJECTIVE: The high rate of vein graft failure due to neointimal hyperplasia is a major challenge for cardiovascular surgery. Finding novel approaches to prevent neointimal hyperplasia is important. Thus, the purpose of this study was to investigate whether dedicator of cytokinesis 2 (DOCK2) plays a role in the development of neointima formation in the vein grafts. METHODS AND RESULTS: We found that DOCK2 levels were significantly elevated in the vein grafts following grafting surgery. In addition, overexpression of DOCK2 promoted venous smooth muscle cell (SMC) proliferation and migration. Conversely, knocking-down endogenous DOCK2 expression in venous SMCs inhibited SMC proliferation and migration. Consistent with this, knocking-down DOCK2 expression in the grafted veins significantly reduced neointimal formation compared with the controls 28 days after vein transplantation. Moreover, DOCK2 silencing treatment improved hemodynamics in the vein grafts. Mechanistically, knockdown of DOCK2 significantly alleviated the vein graft-induced down regulation of SMC contractile protein expression and impeded the vein graft-induction of both Cyclin D1 and PCNA expression. In particular, to ensure high efficiency when transferring the DOCK2 short hairpin RNA (shDOCK2) into the grafted veins, a 30% poloxamer F-127 gel incorporated with 0.25% trypsin was smeared around the vein grafts to increase the adenovirus contact time and penetration. CONCLUSIONS: DOCK2 silencing gene therapy effectively attenuates neointimal hyperplasia in vein grafts. Knock-down of DOCK2 would be a potential therapeutic approach for the treatment of vein graft failure.

MicroRNA-365 promotes the contractile phenotype of venous smooth muscle cells and inhibits neointimal formation in rat vein grafts.

The high rate of autologous vein graft failure caused by neointimal hyperplasia remains an unresolved issue in the field of cardiovascular surgery; therefore, it is important to explore new methods for protecting against neointimal hyperplasia. MicroRNA-365 has been reported to inhibit the proliferation of vascular smooth muscle cells (SMCs). This study aimed to test whether adenovirus-mediated miR-365 was able to attenuate neointimal formation in rat vein grafts. We found that miR-365 expression was substantially reduced in vein grafts following engraftment. In vitro, overexpression of miR-365 promoted smooth muscle-specific gene expression and inhibited venous SMC proliferation and migration. Consistent with this, overexpression of miR-365 in a rat vein graft model significantly reduced grafting-induced neointimal formation and effectively improved the hemodynamics of the vein grafts. Mechanistically, we identified that cyclin D1 as a potential downstream target of miR-365 in vein grafts. Specially, to increase the efficiency of miR-365 gene transfection, a 30% poloxamer F-127 gel containing 0.25% trypsin was mixed with adenovirus and spread around the vein grafts to increase the adenovirus contact time and penetration. We showed that adenovirus-mediated miR-365 attenuated venous SMC proliferation and migration in vitro and effectively inhibited neointimal formation in rat vein grafts. Restoring expression of miR-365 is a potential therapeutic approach for the treatment of vein graft failure. © 2019 IUBMB Life, 2019.

MicroRNA-146a sponge therapy suppresses neointimal formation in rat vein grafts.

The long-term failure of vein grafts due to neointimal hyperplasia remains a difficult problem in cardiovascular surgery. Exploring novel approaches to prevent neointimal hyperplasia is important. MicroRNA-146a (miR-146a) plays an essential role in promoting vascular smooth muscle cell (VSMC) proliferation. Thus, the aim of the present study is to investigate whether adenovirus-mediated miR-146a sponge (Ad-miR-146a-SP) gene therapy could attenuate neointimal formation in rat vein grafts. (Ad-miR-146a-SP) was constructed to transfect cultured VSMCs and grafted veins. To improve the efficiency of transferring the miR-146a sponge gene into the grafted veins, 20% poloxamer F-127 gel incorporated with 0.25% trypsin was used to increase adenovirus contact time and penetration. miR-146a-SP transduction significantly reduced the expression of miR-146a both in cultured VSMCs and vein grafts. miR-146a sponge markedly attenuated VSMC proliferation and migration. Consistent with this, miR-146a sponge gene therapy significantly attenuated neointimal formation and also improved blood flow in the vein grafts. Mechanistically, we identified the Krüppel-like factor 4(KLF4) as a potential downstream target gene of miR-146a in vein grafts. Our data show that miR-146a sponge gene therapy could effectively reduce miR-146a activity and attenuate neointimal formation in vein grafts, suggesting its potential therapeutic application for prevention of vein graft failure. © 2018 IUBMB Life, 71(1):125-133, 2019.

Promoting Vasa Vasorum Neovascularization of Vein Grafts Extenuates Hypoxia of the Wall and Its Subsequent Influence on Intimal Hyperplasia.

BACKGROUND: The autologous saphenous vein is the most common conduit for coronary artery bypass grafting, but the vein graft disease will occur. This study used Matrigel basement membrane matrix with many different growth factors to promote vasa vasorum neovascularization and extenuate the hypoxia to improve remodeling. METHODS: This study observed the hypoxia and thickness of the vein grafts at different times. Normal veins and vein grafts with 15 min of ischemia one day postoperatively were harvested in the neck of rabbits. Paired vein grafts with 15 min ischemia bilaterally (control vs. Matrigel basement membrane matrix) were performed and harvested at 2, 6, and 12 weeks postoperatively. The rabbits were randomly divided into four postoperative groups (six rabbits in each group): Group 1, one day postoperatively; Group 2, 2 weeks postoperatively; Group 3, 6 weeks postoperatively; and Group 4, 12 weeks postoperatively. The dimensions of vessel wall were captured, and the mean thicknesses of intima, media, and adventitia were measured. The hypoxia-inducible factor (HIF)-1α and HIF-2α labeling indices of intima, media, and adventitia were also measured. RESULTS: In Group 1, the labeling index of HIF-1α was high in the normal vein and decreased significantly in the vein graft one day postoperatively (intima: 80 ± 3% vs. 12 ± 1%, P = 0.01; media: 67 ± 5% vs. 11 ± 1%, P = 0.01; adventitia: 40 ± 10% vs. 7 ± 2%, P = 0.03). The labeling index of HIF-2α had similar trend as HIF-1α (intima: 80 ± 10% vs. 10 ± 5%, P = 0.02; media: 60 ± 14% vs. 12 ± 2%, P = 0.01; adventitia: 45 ± 20% vs. 10 ± 4%, P = 0.03). Compared with the control vein grafts, vein grafts with Matrigel basement membrane matrix had lower labeling indices of HIF-1α and HIF-2α in media and adventitia at Group 2 (HIF-1α: 34 ± 5% vs. 20 ± 4%, P = 0.04 for media; 23 ± 3% vs. 11 ± 2%, P = 0.03 for adventitia; HIF-2α: 37 ± 6% vs. 21 ± 4%, P = 0.03 for media; 24 ± 4% vs. 13 ± 2%, P = 0.04 for adventitia) and Group 3 (HIF-1α: 33 ± 4% vs. 7 ± 2%, P = 0.04 for media; 13 ± 3% vs. 3 ± 1%, P = 0.02 for adventitia; HIF-2α: 27 ± 4% vs. 12 ± 3%, P = 0.02 for media; 19 ± 2% vs. 6 ± 1%, P = 0.02 for adventitia). There were no differences in mean thickness of intima, media, and adventitia between bilateral vein grafts at 2, 6, and 12 weeks postoperatively. CONCLUSIONS: This study indicated that promoting vasa vasorum neovascularization of vein grafts extenuated hypoxia, but did not influence the intimal hyperplasia of the wall.

Ascending Aortic Constriction Promotes Cardiomyocyte Proliferation in Neonatal Rats.

Adult heart suffering from increased workload will undergo myocardial hypertrophy, subsequent cardiomyocyte (CM) death, and eventually heart failure. However, the effect of increasing afterload on the neonatal heart remains unknown. We performed ascending aortic constriction (AAC) in neonatal rats 8-12 hours after birth (P0, P indicates postpartum). Seven days after surgery, in vivo heart function was evaluated using cardiac ultrasonography. Haematoxylineosin and Masson staining were used to assess CM diameter and collagen deposition. Moreover, expression of both EdU and Ki67 were evaluated to determine DNA synthesis levels, and pH3 and aurora B as markers for mitosis in CMs. CM isolation was performed by heart perfusion at P0, P3, P5, and P7, respectively. CM number on P0 was 1.01 ± 0.29 × 106. We found that CM cell cycle activation was significantly increased among constricted hearts, as demonstrated by increased Ki67, EdU, pH3, and aurora B positive cells/1000 CMs. At day 7 (P7), constriction group hearts manifested increased wall thickness (0.55 ± 0.05 mm versus 0.85 ± 0.10 mm, P < 0.01, n = 6), and improved hemodynamics as well as left ventricular ejection fraction (65.5 ± 3.7% versus 77.7 ± 4.8%, P < 0.01, n = 6). Of note, the population of CMs was also markedly increased in the constriction group (2.92 ± 0.27 × 106 versus 3.41 ± 0.40 × 106, P < 0.05, n = 6). In summary, we found that during the first week after birth significant numbers of neonatal CMs can reenter the cell cycle. Ascending aortic constriction promotes neonatal rat CM proliferation resulting in 16.7% more CMs in the heart.

Expression and purification of human ARP1 protein and rapid preparation of polyclonal antibody.

Angiopoietin-related protein 1 (ARP1) is one of the antiangiogenic factors and plays an important role in endothelial cell proliferation, migration, and blood vessel network formation. Here a rapid method to prepare ARP1 polyclonal antibody in 1 month was developed. The gene of fibrinogen homology domain (FD) for ARP1 was cloned and the protein was expressed in a soluble form of MBP-FD fused protein. The MBP-FD protein was purified using amylose affinity chromatography of maltose-binding protein. Polyclonal antibodies against MBP-FD were obtained through immunization in BALB/c mice. The titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and the antibody specificity was assessed by Western blot. The full-length ARP1 protein in stable form expressed in transfected human large lung cancer cell lines NCI-H460 was detected by immunocytochemistry (ICC) analysis using ARP1 polyclonal antibodies. The result shows that the antibody possesses good specificity and sensitivity. This work provides a substantial base for the further studies of ARP1 function and associated mechanisms. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

Influence of ischemia before vein grafting on early hyperplasia of the graft and the dynamic changes of the intima after grafting.

BACKGROUND: To investigate both the influence of ischemia before grafting on early hyperplasia of the vein grafts, and the dynamic changes of the intima after grafting in a rabbit model of vein graft disease. METHODS: We performed paired vein graft experiments under different ischemic conditions (15 vs. 60 min; 15 vs. 90 min) in the neck of the rabbits and compared the differences between the grafts. Clopidogrel, an anti-platelet agent, was administered before and after surgery. Twenty-eight days after the grafting procedure, the veins were evaluated microscopically. The dynamic changes of the intima after grafting were evaluated by scanning electron microscopy over time. RESULTS: The vein grafts subjected to 60- or 90-min ischemia exhibited no differences compared to those subjected to 15-min ischemia in terms of the mean thickness of the intimal, medial, and adventitial layers of the graft. Similarly, there was no difference in the Ki-67 labeling index (proliferation marker) between the vein grafts. Vein grafts with 15-min ischemia lost endothelial cells (ECs) but healed by 3 days post graft, whereas vein grafts with 90-min ischemia suffered serious EC loss, which was restored with new ECs during days 2 to 14 post graft. CONCLUSIONS: Ninety-minute ischemia before vein grafting can cause serious EC loss, but does not increase early intimal hyperplasia when clopidogrel is administered. Protecting the vein from ischemia and reperfusion injury preserves ECs.

Evaluation of a novel paclitaxel-eluting stent with a bioabsorbable polymeric surface coating in the porcine artery injury model.

PURPOSE: Drug-eluting stents (DES) are unique in allowing sustained release after a single short intervention. The challenge with DES still remaining is the optimal combination of a biocompatible drug-eluting matrix including an antiproliferative drug. We studied the role of a novel paclitaxel-eluting stent with a bioabsorbable polymer coating in preventing vascular restenosis in the porcine artery injury model. MATERIAL AND METHODS: Bare metal stents (BMS); polymer-coated-only stents (POLY); and polymer-based paclitaxel-eluting stents (PACL) were randomly implanted in pig femoral arteries. The dose density of paclitaxel was 1 microg/mm2 with in vitro studies demonstrating a gradual elution over a course of 6 month. RESULTS: After 1-, 3- and 6-month follow-up, respectively, the animals underwent angiographic restudy and were terminated for histomorphometrical and histopathological analyses. At 1 month, the PACL group had the lowest histological percent stenosis when compared to the BMS and POLY groups (20 +/- 4% vs 39 +/- 6% and 41 +/- 6%, respectively, P < 0.05). At 3 months, the PACL group still presents the lowest level of histological percent stenosis among the three groups (27 +/- 6% vs 50 +/- 10% and 46 +/- 5%, respectively, P < 0.01). Six months later, the PACL group showed a similar histological percent stenosis as the BMS and POLY groups (44 +/- 9% vs 56 +/- 11% and 53 +/- 9%, respectively, P = 0.145). CONCLUSIONS: This study shows favourable vascular compatibility and efficacy for a novel DES to inhibit in-stent neointima formation and preserve lumen area in the porcine artery model.

Effect of external stents on prevention of intimal hyperplasia in a canine vein graft model.

BACKGROUND: External stents have been used to reduce intimal hyperplasia of vein grafts. The aim of the present study was to define the size of an external stent appropriate for a particular graft by comparing vein grafts with different sizes of external stents. METHODS: A series of paired trials was performed to compare femoral vein grafts with different sizes of external stents, where 30 modeled canines were equally divided into three groups: 6-mm external stent vs non-stent control, 4-mm vs 6-mm external stent, and 4-mm vs 8-mm external stent. At day 3 after operation, color Doppler flow imaging (CDFI) was done to observe blood flow in the lumen. Four weeks later, CDFI was re-checked and the veins were harvested, stained and measured. RESULTS: All grafts were patent without formation of thrombosis. External stents significantly reduced intimal thickness of the vein grafts with a 6-mm external stent compared with the vein grafts without external stents (P < 0.05). The vein grafts with the 4-mm external stent had similar intimal, medial and adventitial thicknesses compared with those with the 6-mm external stent and the 8-mm external stent. CONCLUSIONS: External stents can reduce intimal hyperplasia of vein grafts. Stents of different diameters exert the similar effect on prevention of intimal hyperplasia.