Vein Graft Intimal Hyperplasia Research Articles

Nano-based perivascular intervention sustains a nine-month long-term suppression of intimal hyperplasia in vein grafts

Open vascular reconstructions (OVR), including bypass grafts and dialysis access, are standard treatments for cardiovascular and renal diseases. Unfortunately, OVR often fail largely due to intimal hyperplasia (IH), and there are no clinical methods to prevent this complication. Perivascular drug administration during OVR presents a promising strategy for IH suppression. However, durations of drug release from carriers are generally short whereas sustained efficacy is essential for clinical success. This raises a critical question in clinical translation: can IH suppression be realistically maintained long-term (e.g., over 6 months) with short-term perivascular interventions? To address this question, we modified a rat vein-graft model to prolong IH progression. We then applied Pericelle, a nanoparticle/hydrogel hybrid system that we developed for perivascular delivery of rapamycin, an established IH-inhibitory drug. Surprisingly, despite short (∼3-month) drug release, Pericelle demonstrated IH suppression throughout 3, 6, and 9 months with IH reduced from 115.58 ± 27.89 to 40.34 ± 5.18 at 9 months (P < 0.05, n = 6 rats), as indicated by morphometric analysis. Live animal ultrasonography showed the same trend. Consistently, histone-3 lysine-27 trimethylation, an epigenetic mark associated with IH progression, was decreased at 6 months after Pericelle treatment. Moreover, Pericelle exhibited promising efficacy in mitigating IH in a porcine model of arteriovenous fistula that mimics dialysis access. These results suggest that Pericelle-mediated suppression of IH in rat vein-grafts extends much beyond drug release, offering potential solutions to longstanding translational challenges in reducing OVR failure.

Platelet Membrane-Encapsulated Poly(lactic-co-glycolic acid) Nanoparticles Loaded with Sildenafil for Targeted Therapy of Vein Graft Intimal Hyperplasia

Autologous vein grafts have attracted widespread attention for their high transplantation success rate and low risk of immune rejection. However, this technique is limited by the postoperative neointimal hyperplasia, recurrent stenosis and vein graft occlusion. Hence, we propose the platelet membrane-coated Poly(lactic-co-glycolic acid) (PLGA) containing sildenafil (PPS). Platelet membrane (PM) is characterised by actively targeting damaged blood vessels. The PPS can effectively target the vein grafts and then slowly release sildenafil to treat intimal hyperplasia in the vein grafts, thereby preventing the progression of vein graft restenosis. PPS effectively inhibits the proliferation and migration of vascular smooth muscle cell (VSMCs) and promotes the migration and vascularisation of human umbilical vein endothelial cells (HUVECs). In a New Zealand rabbit model of intimal hyperplasia in vein grafts, the PPS significantly suppressed vascular stenosis and intimal hyperplasia at 14 and 28 days after surgery. Thus, PPS represents a nanomedicine with therapeutic potential for treating intimal hyperplasia of vein grafts.

Ginger protects against vein graft remodeling by precisely modulating ferroptotic stress in vascular smooth muscle cell dedifferentiation

Vein graft failure (VGF) is associated with vein graft (VG) intimal hyperplasia, which is characterized by abnormal accumulation of vascular smooth muscle cells (VSMCs). Most neointimal VSMCs are derived from pre-existing VSMCs via a process of VSMC phenotypic transition, also known as dedifferentiation. There is increasing evidence to suggest that ginger or its bioactive ingredients may block VSMC dedifferentiation, exerting vasoprotective functions; however, the precise mechanisms have not been fully characterized. Therefore, we investigated the effect of ginger on VSMC phenotypic transition in VG remodeling after transplantation. Ginger significantly inhibited neointimal hyperplasia and promoted lumen opening in a 3-month VG, which was primarily achieved by reducing ferroptotic stress. Ferroptotic stress is a pro-ferroptotic state. Contractile VSMCs did not die but instead gained a proliferative capacity and switched to the secretory type, forming neointima after vein transplantation. Ginger and its two main vasoprotective ingredients (6-gingerol and 6-shogaol) inhibit VSMC dedifferentiation by reducing ferroptotic stress. Network pharmacology analysis revealed that 6-gingerol inhibits ferroptotic stress by targeting P53 while 6-shogaol inhibits ferroptotic stress by targeting 5-lipoxygenase (Alox5), both promoting ferroptosis. Furthermore, both ingredients co-target peroxisome proliferator-activated receptor gamma (PPARγ), decreasing PPARγ mediated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1) expression. Nox1 promotes intracellular reactive oxygen species (ROS) production and directly induces VSMC dedifferentiation. In addition, Nox1 is a ferroptosis-promoting gene that encourages ferroptotic stress production, indirectly leading to VSMC dedifferentiation. Ginger, a natural multi-targeted ferroptotic stress inhibitor, finely and effectively prevents VSMC phenotypic transition and protects against venous injury remodeling.

Abstract 2111: Nano-based Perivascular Intervention Sustains A Nine-month Long-term Suppression Of Intimal Hyperplasia In Vein Grafts

Introduction: Open vascular reconstructions (OVR), such as bypass grafts and dialysis access, are commonly performed to treat cardiovascular and renal diseases. Unfortunately, OVR often fail, largely due to intimal hyperplasia (IH), and there are no clinical methods to prevent the failure. OVR provide accessibility to the graft surface, hence an opportunity for perivascular drug delivery to suppress IH. However, drug release generally lasts for limited time whereas long-term efficacy is important for clinical success. Goals: We addressed a prominent question in clinical translation: can IH suppression be realistically sustained for a long term (longer than 6 months) via shorter-term perivascular interventions? Methods: We applied Pericelle, a nanoparticle+hydrogel system for perivascular delivery of the IH-mitigating drug rapamycin, to a modified rat vein-graft model that exhibits long-term IH progression. Results: IH was reduced throughout 3, 6, and 9 months, as indicated by morphometry (115.58±27.89 to 40.34±5.18 at 9 months) and ultrasonography (Fig 1), although rapamycin release was estimated to be around 3 months. Pericelle also mitigated IH in porcine arteriovenous fistulas (Fig 2). Conclusions: Suppression of vein-graft IH can be sustained much beyond drug release; Pericelle provides a potential strategy for reducing OVR failure. We thank Dr. Keith Ozaki and Dr. Prabir Roy-Chaudhury for instructions on models.

Osteopontin Activation and Microcalcification in Venous Grafts Can Be Modulated by Dexamethasone.

Osteopontin has been implicated in vascular calcification formation and vein graft intimal hyperplasia, and its expression can be triggered by pro-inflammatory activation of cells. The role of osteopontin and the temporal formation of microcalcification in vein grafts is poorly understood with a lack of understanding of the interaction between haemodynamic changes and the activation of osteopontin. We used a porcine model of vein interposition grafts, and human long saphenous veins exposed to ex vivo perfusion, to study the activation of osteopontin using polymerase chain reaction, immunostaining, and 18F-sodium fluoride autoradiography. The porcine model showed that osteopontin is active in grafts within 1 week following surgery and demonstrated the presence of microcalcification. A brief pretreatment of long saphenous veins with dexamethasone can suppress osteopontin activation. Prolonged culture of veins after exposure to acute arterial haemodynamics resulted in the formation of microcalcification but this was suppressed by pretreatment with dexamethasone. 18F-sodium fluoride uptake was significantly increased as early as 1 week in both models, and the pretreatment of long saphenous veins with dexamethasone was able to abolish its uptake. Osteopontin is activated in vein grafts and is associated with microcalcification formation. A brief pretreatment of veins ex vivo with dexamethasone can suppress its activation and associated microcalcification.

CAR-T cells inhibit vascular smooth muscle phenotypic switching and reduce intimal hyperplasia in vein grafts

Abstract Background Intimal hyperplasia (IH) is a prevalent cause of vein graft stenosis after coronary artery bypass grafting (CABG) in the middle and long term. Vascular smooth muscle cell (VSMC) phenotypic switching from a contractile phenotype to a synthetic phenotype is a hallmark of this process, along with the degradation of extracellular matrix (ECM) and smooth muscle migration. Chimeric antigen receptor (CAR)-T cell therapy is a targeted cellular immunotherapy that uses genetically engineered T cells to specifically eliminate the antigen-bearing cells. There is a potential to apply CAR-T therapy to cardiovascular diseases. Purpose In this study, we aim to design a novel CAR-T cell therapy to eliminate synthetic vascular smooth muscle cells in intimal hyperplasia, inhibit phenotypic switching, in order to reduce vein graft stenosis. Methods and results Using bulk RNA-seq dataset from the GEO database, we have identified human organic cation transporter (OCT1) as a key marker for synthetic vascular smooth muscle cell in vein grafts (Fig.1A, B). We then engineered switchable fourth-generation CAR-T cells incorporating CD28 and CD3 ζ costimulatory domains (Fig.1C, E). In addition, these CAR-T cells secrete tissue inhibitor of metalloproteinase-3 (TIMP3), a protein that had been shown to inhibit matrix metalloproteinases (MMPs) activity and the ERK1/2 pathway (Fig. 1F). Targeting FITC instead of OCT1 itself allows a controllable and switchable working process for the CAR-T cells, as they only start to identify synthetic VSMCs in combination with a FITC-conjugated OCT1 antibody (Fig.1D). Immunostaining of PDGF-induced synthetic VSMCs co-cultured with CAR-T cells and FITC-conjugated OCT1 antibody revealed increased expression of contractile markers, and reduced expression of synthetic markers (Fig1.G), along with decreased mitochondrial membrane potentials, which could be reverted after pre-treatment with a pan caspase inhibitor (Fig.1H),. In vivo studies using mice vein graft model showed reduced intimal thickness, increased intimal/medial ratio in cross-sections, in addition to increased peak velocity and inner wall diameter on ultrasound after CAR-T cell therapy (Fig.2). Conclusion Switchable CAR-T cell therapy targeting synthetic vascular smooth muscle cells might be a promising approach to alleviate intimal hyperplasia in vein graft stenosis.

Oscillatory shear stress promotes vein graft intimal hyperplasia via NADPH oxidase-related pathways.

Uncontrolled intimal hyperplasia (IH) after autologous saphenous vein grafting triggers a high restenosis rate; however, its association with the activation of NADPH oxidase (NOX)-related pathways is unclear. Here, we investigated the effects and mechanism of oscillatory shear stress (OSS) on grafted vein IH. Thirty male New Zealand rabbits were randomly divided into control, high-OSS (HOSS), and low-OSS (LOSS) groups, and the vein grafts were harvested after 4 weeks. Hematoxylin and eosin staining and Masson staining assays were used to observe morphological and structural changes. Immunohistochemical staining was used to detect α-SMA, PCNA, MMP-2, and MMP-9 expression. Immunofluorescence staining was used to observe reactive oxygen species (ROS) production in the tissues. Western blotting was used to determine the expression levels of pathway-related proteins (NOX1, NOX2, AKT, p-AKT, and BIRC5), PCNA, BCL-2, BAX, and caspase-3/cleaved caspase-3 in tissues. Blood flow velocity was lower in the LOSS group than in the HOSS group, while vessel diameter did not change significantly. Shear rate was elevated in both HOSS and LOSS groups but was higher in the HOSS group. Additionally, vessel diameter increased with time in the HOSS and LOSS groups, whereas flow velocity did not. Intimal hyperplasia was significantly lower in the LOSS group than in the HOSS group. IH was dominated by smooth muscle fibers in the grafted veins and collagen fibers in the media. OSS restriction significantly reduced the α-SMA, PCNA, MMP-2, and MMP-9 levels. Moreover, ROS production and the expression of NOX1, NOX2, p-AKT, BIRC5, PCNA, BCL-2, BAX, and cleaved caspase-3 were phase-reduced in LOSS compared to the levels in the HOSS group. Total AKT was not differentially expressed among the three groups. OSS promotes the proliferation, migration, and survival of subendothelial vascular smooth muscle cells in grafted veins, which may be related to the regulation of downstream p-AKT/BIRC5 levels through the increased production of ROS by NOX. Drugs inhibiting this pathway might be used to prolong vein graft survival time.

The effect of lncRNA MIR155HG-modified MSCs and exosome delivery to synergistically attenuate vein graft intimal hyperplasia

BackgroundThe mesenchymal stem cells (MSCs) were used to repair tissue injury. However, the treatment effect was not satisfactory. We investigated whether lncRNA MIR155HG could promote survival and migration of MSCs under oxidative stress, which mimics in vivo environments. Furthermore, we studied the protective effect of exosomes secreted by MSCs transfected with MIR155HG on endothelial cells. This study aimed to determine whether exploiting MSCs and exosomes modified with lncRNA MIR155HG would exert synergistic therapeutic effect to attenuate vein graft intimal hyperplasia more effectively.MethodsLentivirus containing lncRNA MIR155HG overexpressing vector was packaged and used to infect MSCs. Then, CCK-8 assay, flow cytometry, Transwell assay, and Elisa assay were used to assess the functional changes of MSCs with overexpressed MIR155HG (OE-MSCs). Furthermore, the associated pathways were screened by Western blot. MIR155HG-MSCs-derived exosomes (OE-exo) were collected and co-cultured with human umbilicus vein endothelial cell (HUVEC). We validated the protective effect of OE-exo on HUVEC. In vivo, both MSCs and exosomes modified with MIR155HG were injected into a vein graft rat model via tail vein. We observed MSCs homing and intimal hyperplasia of vein graft using a fluorescent microscope and histological stain.ResultsOur study found that lncRNA MIR155HG promoted proliferation, migration, and anti-apoptosis of MSCs. NF-κB pathway took part in the regulation process induced by MIR155HG. OE-exo could enhance the activity and healing ability of HUVEC and reduce apoptosis. In vivo, OE-MSCs had a higher rate of homing to vascular endothelium. The combined treatment with OE-MSCs and OE-exo protected vascular endothelial integrity, reduced inflammatory cell proliferation, and significantly attenuated intimal hyperplasia of vein graft.ConclusionLncRNA MIR155HG could promote the survival and activity of MSCs, and reduce the apoptosis of HUVECs using exosome delivery. Exploiting MSCs and exosomes modified with MIR155HG could attenuate vein graft intimal hyperplasia more effectively and maximize the surgical effect.

Acute arterial haemodynamics activation of endothelial to mesenchymal transition in long saphenous veins. Impact on vein graft disease

Abstract Introduction The long saphenous vein (LSV) is frequently used in cardiac surgery; however, its use is complicated by late stenosis or occlusion due to the development of intimal hyperplasia (IH). TGF-β has been implicated in the process of IH however the impact of acute haemodynamic changes on the activation of TGF-β endothelial-to mesenchymal transition (EndMT) has not been assessed and it's the focus of this study. Purpose To assess the role of acute haemodynamics changes in veins implanted into arterial circulation on EndMT. Methods Surplus LSV were exposed to acute arterial haemodynamics using a perfusion bioreactor. Changes in EndMT markers at the RNA and protein level evaluated by quantitative real time PCR, RNAScope and immunofluorescence. Results The acute exposure of veins to arterial haemodynamics ex-vivo induced significant increase in inflammatory marker IL-8 expression and transcription factor TWIST1 in LSV (both p≤0.01, Figure 1A) endothelium. Furthermore, immunostaining demonstrated the activation of pSMAD (p≤0.01, Figure 1B) acutely in endothelium after 45 minutes of exposure to arterial haemodynamics. This was followed by significant increase of SMC related markers (Vimentin and α-SMA; Figure 1C, both p≤0.001) and the suppression of endothelial cell related marker CD31 after 4 hours of LSV exposure to acute arterial haemodynamics. RNAScope and IHC results showed localisation of TWIST1 RNA and protein in CD31+ (p≤0.001) and VECAD+ (p≤0.001) cells respectively following exposure to acute arterial haemodynamics (Figure 1D). Furthermore, TGF-β pathway phosphorylation array identified the activation of Smad1 (p≤0.05) but not TAK1 in LSV endothelial cells indicating that acute arterial haemodynamics activates the TGF-β–SMAD pathway specifically (Figure 1E). Conclusion The exposure of LSV to acute arterial haemodynamics is associated with the activation of TGF-β–SMAD pathway leading to EndMT changes in the endothelium of vein grafts ex-vivo. This contributes to our understanding of the changes that occur in veins after implantation into arterial circulation and that the acute changes in the endothelium and suggests that strategies to modulate TGF-β–SMAD can be utilised to modulate IH in vein grafts. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Van Geest Foundation Heart and Cardiovascular Diseases Research Fund

The Etiology and Molecular Mechanism Underlying Smooth Muscle Phenotype Switching in Intimal Hyperplasia of Vein Graft and the Regulatory Role of microRNAs.

Mounting evidence suggests that the phenotypic transformation of venous smooth muscle cells (SMCs) from differentiated (contractile) to dedifferentiated (proliferative and migratory) phenotypes causes excessive proliferation and further migration to the intima leading to intimal hyperplasia, which represents one of the key pathophysiological mechanisms of vein graft restenosis. In recent years, numerous miRNAs have been identified as specific phenotypic regulators of vascular SMCs (VSMCs), which play a vital role in intimal hyperplasia in vein grafts. The review sought to provide a comprehensive overview of the etiology of intimal hyperplasia, factors affecting the phenotypic transformation of VSMCs in vein graft, and molecular mechanisms of miRNAs involved in SMCs phenotypic modulation in intimal hyperplasia of vein graft reported in recent years.

Poly (ɛ-Caprolactone-co-l,l-Lactide) Vascular External Sheath Carrying Prednisone for Improving Patency Rate of the Vein Graft.

Coronary artery bypass graft (CABG) surgery is an impactful treatment for coronary heart disease. Intimal hyperplasia is the central reason for the restenosis of vein grafts (VGs) after CABG. The introduction of external vascular sheaths around VGs can effectively inhibit intimal hyperplasia and ensure the patency of VGs. In this study, the well-known biodegradable copolymer poly (ɛ-caprolactone-co-l,l-lactide) (PLCL) was electrospun into high porosity external sheaths. The prednisone loaded in the PLCL sheath was slowly released during the degradation process of PLCL. Under the combined effects of sheath and prednisone, intimal hyperplasia was inhibited. For the cell experiments, all sheaths show low cytotoxicity to L929 cells at different concentrations at different time intervals. The ultrasonography and histological results showed prominent dilation and intimal hyperplasia of VG without sheath after 2 months of surgery. But there was no dilation in PLCL and PLCLPrednisone groups. Of note, the prednisone-loaded sheath group exhibited efficacy in inhibiting intimal hyperplasia and ensured graft patency. Impact statement To inhibit intimal hyperplasia after coronary artery bypass graft, the use of external vascular sheaths can prevent vein graft (VG) dilatation, then reduce turbulent blood flow shear stress to vessel wall, and lower the stimulation of shear stress to smooth muscle cells (SMCs), so as to prevent the proliferation and migration of vascular SMC. We provide a biodegradable sheath electrospun by poly (ɛ-caprolactone-co-l,l-lactide) (PLCL) loading prednisone and utilize it around VG in animal models. Vascular ultrasound examinations show strong evidence of vascular patency. The histological alterations of VGs in PLCLPrednisone group gave a narrower intima layer owing to the inhibition effect of prednisone.

Abstract P178: A Novel Subset Of Adventitial Vascular Stem Cells Paracrine Control Media Smooth Muscle Cell Dedifferentiation Leading To Intimal Hyperplasia In Vein Grafts

Vein graft failure (VGF) is associated with vein graft (VG) intimal hyperplasia, which is characterized by abnormal accumulation of vascular smooth muscle cells (SMCs). The majority of neointimal SMCs are derived from pre-existing vascular SMCs via a process of vascular SMC dedifferentiation; however, the underlying mechanisms remain poorly understood. Here, we tracked down the fate of vascular stem cells (VSCs) expressing stem cell antigen 1 (SCA1) in VG remodeling. After transplantation, most of the donor venous cells including endothelial cells, SMCs, and VSCs died within 3 days, and the recipient arterial SCA1+ VSCs were recruited to repopulate the adventitia and the intima but did not differentiate into neointimal SMCs in VGs. However, ablation of the SCA1+ VSCs ameliorated intimal hyperplasia in VGs. Single-Cell RNA sequencing (scRNA-seq) analysis revealed a unique subset of VSCs expressing Sca1 and cyclin-dependent kinase 8 (Cdk8) in the artery. Notably, the number of SCA1+CDK8+ cells was increased in the adventitia prior to dramatic proliferation of SMCs in the media and neointima in VGs. Inactivation of CDK8 intensified SCA1+ VSC naïve stemness but suppressed SCA1+ VSC proliferation, migration, and exosome release for paracrine enforcement of vascular SMC dedifferentiation. A short-time perivascular delivery of CDK8 inhibitors ameliorated adventitial SCA1+ VSC accumulation associated with a long-term efficacy in suppressing intimal hyperplasia in VGs. There findings uncover that a novel subset of adventitial VSCs paracrine control media SMC dedifferentiation for intimal hyperplasia in VGs toward VGF.

External stenting and disease progression in saphenous vein grafts two years after coronary artery bypass grafting: A multicenter randomized trial

ObjectivesLittle data exist regarding the potential of external stents to mitigate long-term disease progression in saphenous vein grafts. We investigated the effect of external stents on the progression of saphenous vein graft disease. MethodsA total of 184 patients undergoing isolated coronary artery bypass grafting, using an internal thoracic artery graft and at least 2 additional saphenous vein grafts, were enrolled in 14 European centers. One saphenous vein graft was randomized to an external stent, and 1 nonstented saphenous vein graft served as the control. The primary end point was the saphenous vein graft Fitzgibbon patency scale assessed by angiography, and the secondary end point was saphenous vein graft intimal hyperplasia assessed by intravascular ultrasound in a prespecified subgroup at 2 years. ResultsAngiography was completed in 128 patients and intravascular ultrasound in the entire prespecified cohort (n = 51) at 2 years. Overall patency rates were similar between stented and nonstented saphenous vein grafts (78.3% vs 82.2%, P = .43). However, the Fitzgibbon patency scale was significantly improved in stented versus nonstented saphenous vein grafts, with Fitzgibbon patency scale I, II, and III rates of 66.7% versus 54.9%, 27.8% versus 34.3%, and 5.5% versus 10.8%, respectively (odds ratio, 2.02; P = .03). Fitzgibbon patency scale was inversely related to saphenous vein graft minimal lumen diameter, with Fitzgibbon patency scale I, II, and III saphenous vein grafts having an average minimal lumen diameter of 2.62 mm, 1.98 mm, and 1.32 mm, respectively (P < .05). Externally stented saphenous vein grafts also showed significant reductions in mean intimal hyperplasia area (22.5%; P < .001) and thickness (23.5%; P < .001). ConclusionsTwo years after coronary artery bypass grafting, external stenting improves Fitzgibbon patency scales of saphenous vein grafts and significantly reduces intimal hyperplasia area and thickness. Whether this will eventually lead to improved long-term patency is still unknown.

MicroRNA-92a -mediated endothelial to mesenchymal transition controls vein graft neointimal lesion formation

PurposeLong-term failure of vein grafts due to neointimal hyperplasia remains an important problem in coronary artery bypass graft surgery. Endothelial to mesenchymal transition (EndMT) contributes to vein graft vascular remodeling. However, there is little study on microRNA-mediated EndMT contributions to neointimal formation in vein graft. We hypothesized that microRNA-92a (miR-92a) might play an important role in determining EndMT contributions to neointimal formation. MethodsmiR-92a and EndMT-related proteins detected by qRT-PCR and Western blot in vitro and in vivo. Adeno-associated virus 6 (AAV6) delivery gene therapy was used to inhibit neointimal formation in vivo. The intimal hyperplasia of vein grafts was measured by HE staining, the expression of EndMT-related protein in vein grafts was measured by immunofluorescence. Immunohistochemistry and luciferase assay were used to detect potential targets of miR-92a. ResultsThe expression of miR-92a was found to be upregulated in neointimal hyperplasic lesions after vein grafting. Using cultured human umbilical vein endothelial cells (HUVECs), we show that TGF-β1 treatment of HUVECs significantly increased miR-92a expression and induced EndMT, characterized by suppression of endothelial-specific markers (CD31 and VE-cadherin) and an increase in mesenchymal-specific markers (a-SMA and vimentin), while inhibition of miR-92a expression blunted EndMT in cultured HUVECs. Furthermore, AAV6 mediated miR-92a suppression gene therapy effectively resulted in decreased EndMT and less neointimal formation in vein grafts in vivo. We further identified that integrin alpha 5 (ITGA5) is a potential target gene involved in the development of neointima formation in these vein grafts. ConclusionThis data suggests that neointimal formation does not solely rely on vascular smooth muscle cell phenotypic switching but is also related to EndMT, and miR-92a-mediated EndMT is an important mechanism underlying neointimal formation in vein grafts.

MiRNA-126-3p carried by human umbilical cord mesenchymal stem cell enhances endothelial function through exosome-mediated mechanisms in vitro and attenuates vein graft neointimal formation in vivo

BackgroundThe aim of this study was to determine whether the combination of MSC implantation with miRNA-126-3p overexpression would further improve the surgical results after vein grafting.Methodshuman umbilical cord MSCs (hucMSCs) and human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords and characterized by a series of experiments. Lentivirus vector encoding miRNA-126-3p was transfected into hucMSCs and verified by PCR. We analyzed the miRNA-126-3p-hucMSC function in vascular endothelial cells by using a series of co-culture experiments. miRNA-126-3p-hucMSCs-exosomes were separated from cell culture supernatants and identified by WB and TEM. We validated the role of miRNA-126-3p-hucMSCs-exosomes on HUVECs proliferative and migratory and angiogenic activities by using a series of function experiments. We further performed co-culture experiments to detect downstream target genes and signaling pathways of miRNA-126-3p-hucMSCs in HUVECs. We established a rat vein grafting model, CM-Dil-labeled hucMSCs were injected intravenously into rats, and the transplanted cells homing to the vein grafts were detected by fluorescent microscopy. We performed historical and immunohistochemical experiments to exam miRNA-126-3p-hucMSC transplantation on vein graft neointimal formation and reendothelialization in vitro.ResultsWe successfully isolated and identified primary hucMSCs and HUVECs. Primary hucMSCs were transfected with lentiviral vectors carrying miRNA-126-3p at a MOI 75. Co-culture studies indicated that overexpression of miRNA-126-3p in hucMSCs enhanced HUVECs proliferation, migration, and tube formation in vivo. We successfully separated hucMSCs-exosomes and found that miRNA-126-3p-hucMSCs-exosomes can strengthen the proliferative, migratory, and tube formation capacities of HUVECs. Further PCR and WB analysis indicated that, SPRED-1/PIK3R2/AKT/ERK1/2 pathways are involved in this process. In the rat vein arterialization model, reendothelialization analysis showed that transplantation with hucMSCs modified with miRNA-126-3p had a higher reendothelialization of the vein grafts. The subsequent historical and immunohistochemical examination revealed that delivery with miRNA-126-3p overexpressed hucMSCs significantly reduced vein graft intimal hyperplasia in rats.ConclusionThese results suggest hucMSC-based miRNA-126-3p gene therapy may be a novel option for the treatment of vein graft disease after CABG.

A Soft, Conductive External Stent Inhibits Intimal Hyperplasia in Vein Grafts by Electroporation and Mechanical Restriction.

Intimal hyperplasia (IH) in vein grafts (VGs) is a major issue in coronary artery bypass grafting (CABG) surgery. Although external stents can attenuate IH of VGs to some extent, none of the existing external stents have shown satisfactory clinical outcomes. Here we develop a flexible, biodegradable, and conductive external metal-polymer conductor stent (MPCS) that can electroporate the vessel wall and produce a protein that prevents IH. We designed the plasmid DNA encoding the tissue inhibitor of metalloproteinases-3 (TIMP-3) and lyophilized it on the inner surface of the MPCS to deliver into the adventitia and the middle layer of VGs for gene therapy. Coupled with its continuous mechanical support to prevent dilation after implanting, the MPCS can inhibit the IH of VGs significantly in the rabbit model. This proof-of-concept demonstration may aid the development of other implantable bioelectronics for electroporation gene therapy.

Mature Vascular Smooth Muscle Cells, but Not Endothelial Cells, Serve as the Major Cellular Source of Intimal Hyperplasia in Vein Grafts.

Neointima formation is a primary cause of intermediate to late vein graft (VG) failure. However, the precise source of neointima cells in VGs remains unclear. Approach and Results: Herein we clarify the relative contributions of mature vascular smooth muscle cells (SMCs) and endothelial cells (ECs) to neointima formation in a mouse model of VG remodeling via the genetic-inducible fate mapping approaches. Regardless of the magnitude of neointima formation, the recipient arterial and the donor venous SMCs contributed ≈55% of the neointima cells at the anastomotic regions, whereas only donor venous SMCs donated ≈68% of the neointima cells at the middle bodies. A small portion of the SMC-derived cells became non-SMC cells, most likely vascular stem cells, and constituted 2% to 11% of the cells in each major layer of VGs. In addition, the recipient arterial ECs were the major cellular source of re-endothelialization but did not contribute to neointima formation. The donor venous ECs donated ≈17% neointima cells in the VGs with mild neointima formation and conditional media from ECs after endothelial-to-mesenchymal transition suppressed vascular SMC dedifferentiation. The recipient arterial and donor venous mature SMCs dominate but contribute distinctly to intimal hyperplasia at the anastomosis and the middle body regions of VGs. The recipient arterial ECs are the major cellular source of re-endothelialization but do not donate neointima formation in VGs. Only the donor venous ECs undergo endothelial-to-mesenchymal transition. Endothelial-to-mesenchymal transition is marginal for generating neointima cells but is likely required for controlling the quality of VG remodeling.

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure.

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

Exosomes derived from human umbilical cord mesenchymal stem cells inhibit vein graft intimal hyperplasia and accelerate reendothelialization by enhancing endothelial function

BackgroundIn our previous research, we found that mesenchymal stem cell (MSC) transplantation therapy can inhibit intimal hyperplasia and enhance endothelial function in arterialized vein grafts in rats. However, whether MSC-derived exosomes (MSC-exosomes) can reduce neointimal formation and its possible mechanism is still unclear.MethodsThe primary human umbilical cord MSCs (hucMSCs) and human umbilical vein endothelial cells (HUVECs) were isolated and characterized by flow cytometry and immunofluorescence. The exosomes derived from hucMSCs (hucMSC-exosomes) were identified by transmission electron microscopy and western blots. hucMSC-exosomes were intravenously injected into a rat model of vein grafting, and its effect on vein grafts reendothelialization and intimal hyperplasia was assessed by physical, histological, immunohistochemistry, and immunofluorescence examinations. The effects of hucMSC-exosomes on endothelial cells were evaluated by integrated experiment, EdU staining, scratch assay, and Transwell assay. The expression levels of key gene and pathways associated with the biological activity of vascular endothelial cells were evaluated following the stimulation of hucMSC-exosomes.ResultsWe successfully isolated and characterized primary hucMSCs and hucMSC-exosomes and primary HUVECs. We verified that the systemic administration of hucMSC-exosomes accelerates reendothelialization and decreases intimal hyperplasia of autologous vein graft in a rat model. We also identified that hucMSC-exosomes can be uptaken by endothelial cells to stimulate cell proliferative and migratory activity in vitro. Furthermore, we detected that vascular endothelial growth factor (VEGF) plays an important part in hucMSC-exosome-mediated proliferation and migration in HUVECs. In addition, we also provided evidence that the signalling pathways of PI3K/AKT and MAPK/ERK1/2 take part in hucMSC-exosome-induced VEGF regulation.ConclusionOur data suggest that hucMSC-exosomes exert a vasculoprotective role in the setting of vein graft disease, which may provide a new clue to protect against vein graft failure in the future.

Effects of the different-sized external stents on vein graft intimal hyperplasia and inflammation.

The poor long-term patency ratio of vein grafts prevents patients from benefiting from coronary artery bypass graft (CABG). It is reported that external venous stents have notably improved the patency ratio of stented vein grafts in animal models. The most crucial influence on stented grafts' fate is the size of the stents. This study aims to investigate the effects on intimal hyperplasia and inflammation of vein graft by using different sizes of stents and explore the potential mechanism. Two different sizes of external stents were fabricated through 3D printing technology. Male SD rats were divided into three groups. In the control group rat's autologous left jugular vein was grafted on the ipsilateral artery directly. In the stent groups, grafts were surrounded by two different-sized stents before anastomosing with arteries. The patency ratio and diameter of the grafts were examined by ultrasound. Masson staining was used to characterize intimal hyperplasia. The expression of inflammatory factors was detected by immunohistochemical staining. Moreover, TUNEL staining was used to label apoptotic cells. The two sizes of external stents were fabricated by 3D printing technology. In the control group, the intima area and wall thickness dramatically increased 8 weeks after implantation. While in the stent groups, these data only slightly increased, especially in the 1.5 mm-stent group. The expressions of inflammatory factors in TNF signaling were more remarkable than in the control group. On the contrary, the expressions were rarely detected in the stent groups. Similarly, the number of TUNEL positive cells dramatically decreased by using the appropriate-sized stent. In this study, we concluded that the appropriate sizes of external stents could effectively inhibit vein graft neointima formation, attenuate inflammatory reaction and reduce cell apoptosis, which might improve the long-term patency ratio of vein grafts.