Neointima Formation In Vein Grafts Research Articles

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.

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.

The BD2 domain of BRD4 is a determinant in EndoMT and vein graft neointima formation

BackgroundVein-graft bypass is commonly performed to overcome atherosclerosis but is limited by high failure rates, principally due to neointimal wall thickening. Recent studies reveal that endothelial-mesenchymal transition (EndoMT) is critical for vein-graft neointima formation. BETs are a family of Bromo/ExtraTerminal domains-containing epigenetic reader proteins (BRD2, BRD3, BRD4). They bind acetylated histones through their unique tandem bromodomains (BD1, BD2), facilitating transcriptional complex formation and cell-state transitions. The role for BETs, including individual BRDs and their unique BDs, is not well understood in EndoMT and neointimal formation. Methods and resultsRepression of BRD4 expression abrogated TGFβ1-induced EndoMT, with greater effects than BRD2 or BRD3 knockdown. An inhibitor selective for BD2 in all BETs, but not that for BD1, blocked EndoMT. Moreover, expression of a dominant-negative BRD4-specific BD2 fully abolished EndoMT. Concordantly, BRD4 knockdown repressed TGFβ1-stimulated increase of ZEB1 protein – a transcription factor integral in EndoMT. In vivo, lentiviral gene transfer of either BRD4 shRNA or dominant negative BRD4-specific BD2 mitigated neointimal development in rat jugular veins grafted to carotid arteries. ConclusionsOur data reveal the BD2 domain of BRD4 as a determinant driving EndoMT in vitro and neointimal formation in vivo. These findings provide new insight into BET biology, while offering prospects of specific BET domain targeting as an approach to limiting neointima and extending vein graft patency.

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.

Abstract 254: The Bromo and Extraterminal Domain Protein (BET) Family Drives Endothelial-To-Mesenchymal Transition and Contributes to Vein Graft Stenosis

Background: Vein graft bypass remains the most commonly used open procedure for flow-limiting cardiovascular diseases. However, vein grafts are prone to stenosis, which ultimately leads to graft failure (4-year failure rate at ~43%). Previous studies have provided strong evidence supporting that dysfunctional endothelial cells (ECs) of the vein grafts contribute significantly to neoinitma formation (and hence stenosis) by undergoing EndoMT. Our group has recently shown that BRD4, a member of the BET epigenetic reader protein family, is up-regulated in the neointima lesion of stenotic failed vein grafts. Here we have determined whether BRD4, and possibly other BET proteins, would be involved in EndoMT in vein grafts, and ultimately vein graft stenosis/failure. Method: For in vitro experiments, we treated rat primary vascular ECs with 100ng/ml TGFβ-1 (to induce EndoMT), and/or BET inhibitors (Pan inhibitor: JQ1; Domain-selective inhibitors: Olinone and RVX208), and/or siRNAs for BRD2, 3, and 4. For in vivo experiments, we used a cuff-based technique to establish jugular vein to carotid artery interposition graft in rats, and lentiviral shRNA delivery to achieve specific knockdown of BRD4 in the graft. Results: BET pan-inhibition significantly reduced EndoMT of rat primary vascular ECs in vitro. Similar effects were observed via selective inhibition of bromodomain 2, but not bromodomain 1. Knockdown of BRD4 protein expression in vitro fully recapitulated the inhibitory effect of BET pan-inhibition on EndoMT, whereas knockdown of BRD2 and BRD3 could only lead to partial and marginal abrogation of EndoMT, respectively. In the rat vein graft model, local lentiviral delivery of BRD4 shRNA led to decrease of neointima formation. Conclusion: BET proteins, especially BRD4, play an important role in EndoMT of vascular ECs and also neointima formation in vein grafts. Blockage of BRD4 protein could potentially serve as a novel strategy to achieve prolonged patency of vein graft conduits.

Adenovirus-Mediated Gene Transfer of microRNA-21 Sponge Inhibits Neointimal Hyperplasia in Rat Vein Grafts.

Background:Vein graft failure due to neointimal hyperplasia remains an important and unresolved complication of cardiovascular surgery. microRNA-21 (miR-21) plays a major role in regulating vascular smooth muscle cell (VSMC) proliferation and phenotype transformation. Thus, the purpose of this study was to determine whether adenovirus-mediated miR-21 sponge gene therapy was able to inhibit neointimal hyperplasia in rat vein grafts.Methods:Adenovirus-mediated miR-21 sponge was used to inhibit VSMC proliferation in vitro and neointimal formation in vivo. To improve efficiency of delivery gene transfer to the vein grafts, 20% poloxamer F-127 gel was used to increase virus contact time and 0.25% trypsin to increase virus penetration. Morphometric analyses and cellular proliferation were assessed for neointimal hyperplasia and VSMC proliferation.Results:miR-21 sponge can significantly decrease the expression of miR-21 and proliferation in cultured VSMCs. Cellular proliferation rates were significantly reduced in miR-21 sponge-treated grafts compared with controls at 28 days after bypass surgery (14.6±9.4 vs 34.9±10.8%, P=0.0032). miR-21 sponge gene transfer therapy reduced the intimal/media area ratio in vein grafts compared with the controls (1.38±0.08 vs. 0.6±0.10, P<0.0001). miR-21 sponge treatment also improved vein graft hemodynamics. We further identified that phosphatase and tensin homolog (PTEN) is a potential target gene that was involved in the miR-21-mediated effect on neointimal hyperplasia in vein grafts.Conclusions:Adenovirus-mediated miR-21 sponge gene therapy effectively reduced neointimal formation in vein grafts. These results suggest that there is potential for miR-21 sponge to be used to prevent vein graft failure.

Corrigendum: Role of Excessive Autophagy Induced by Mechanical Overload in Vein Graft Neointima Formation: Prediction and Prevention.

Scientific Reports 6: Article number: 22147; published online: 26 February 2016; updated: 14 November 2016 The original version of this Article contained errors in the affiliations. ‘Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan’ was incomplete, and now reads: ‘Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan’

Role of Excessive Autophagy Induced by Mechanical Overload in Vein Graft Neointima Formation: Prediction and Prevention

Role of Excessive Autophagy Induced by Mechanical Overload in Vein Graft Neointima Formation: Prediction and Prevention

Role of Excessive Autophagy Induced by Mechanical Overload in Vein Graft Neointima Formation: Prediction and Prevention.

Little is known regarding the interplays between the mechanical and molecular bases for vein graft restenosis. We elucidated the stenosis initiation using a high-frequency ultrasonic (HFU) echogenicity platform and estimated the endothelium yield stress from von-Mises stress computation to predict the damage locations in living rats over time. The venous-arterial transition induced the molecular cascades for autophagy and apoptosis in venous endothelial cells (ECs) to cause neointimal hyperplasia, which correlated with the high echogenicity in HFU images and the large mechanical stress that exceeded the yield strength. The ex vivo perfusion of arterial laminar shear stress to isolated veins further confirmed the correlation. EC damage can be rescued by inhibiting autophagy formation using 3-methyladenine (3-MA). Pretreatment of veins with 3-MA prior to grafting reduced the pathological increases of echogenicity and neointima formation in rats. Therefore, this platform provides non-invasive temporal spatial measurement and prediction of restenosis after venous-arterial transition as well as monitoring the progression of the treatments.

Extracellular Vesicles Derived from Adipose Mesenchymal Stem Cells Regulate the Phenotype of Smooth Muscle Cells to Limit Intimal Hyperplasia.

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) play important roles in the reduction of inflammation in multiple disease models. However, their role in vein graft (VG) remodeling is undefined. We aimed to investigate the effect of EVs from adipose MSCs (ADMSC-EVs) on VG intimal hyperplasia and to explore the possible mechanisms. After generation and characterization of control-EVs and ADMSC-EVs in vitro, we investigated their effect on the proliferation and migration of vascular smooth muscle cells (VSMCs) in vitro. Next, we established a mouse model of VG transplantation. Mice underwent surgery and received control-EVs or ADMSC-EVs by intraperitoneal injection every other day for 20days. VG remodeling was evaluated after 4weeks. We also assessed the effect of ADMSC-EVs on macrophage migration and inflammatory cytokine expression. Significant inhibitory effects of ADMSC-EVs on in vitro VSMC proliferation (p < 0.05) and migration (p < 0.05) were observed compared with control-EVs. The extent of intimal hyperplasia was significantly decreased in ADMSC-EV-treated mice compared with control-EV-treated mice (26 ± 8.4 vs. 45 ± 9.0μm, p < 0.05). A reduced presence of macrophages was observed in ADMSC-EV-treated mice (p < 0.05). Significantly decreased expression of inflammatory cytokines interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1) was also found in the ADMSC-EV-treated group (both p < 0.05). In addition, phosphorylation of Akt, Erk1/2, and p38 in VGs was decreased in the ADMSC-EV-treated group. We demonstrated that ADMSC-EVs exert an inhibitory effect on VG neointima formation by regulating VSMC proliferation and migration, macrophage migration, inflammatory cytokine expression, and the related signaling pathways.

Smooth muscle cells from the anastomosed artery are the major precursors for neointima formation in both artery and vein grafts.

Accumulation of smooth muscle cells (SMC) results in neointima formation in injured vessels. Two graft models consisting of vein and artery grafts were created by anastomosing common carotid arteries to donor vessels. To identify the origin of the neointima cells from anastomosed arteries, we use Wnt1-Cre/reporter mice to label and track SMCs in the common carotid artery. The contribution of SMCs in the neighboring arteries to neointima formation was studied. On evaluating the artery grafts after 1 month, >90 % of the labeled neointima cells were found to have originated from the anastomosing host arteries. Most of the neointima cells were also smooth muscle α-actin positive (SMA-α(+)) and expressed the smooth muscle myosin heavy chain (SMMHC), the SMC terminal differentiation marker. In vein grafts, about 60 % SMA-α-positive cells were from anastomosing arteries. Bone marrow cells did not contribute to neointima SMCs in vein grafts, but did co-stain with markers of inflammatory cells. Wnt1 expression was not detected in the neointima cells in the vein or artery grafts, or the injured femoral arteries. Neointima SMCs showed the synthetic phenotype and were positively labeled with BrdU in vitro and in vivo. Treatment with the IGF-1 receptor inhibitor suppressed SMC proliferation and neointima formation in vein grafts. Our results indicate that SMCs from the neighboring artery are predominantly present in the neointima formed in both vein and artery grafts and that Wnt1-Cre mice can be used to explore the role of SMCs originating from neighboring vessels in vascular remodeling.

Polymer-based delivering of shRNA to rabbit aortic smooth muscle cells suppressed the expression of IGF-1R &amp;lt;italic&amp;gt;in vitro&amp;lt;/italic&amp;gt; and &amp;lt;italic&amp;gt;in vivo&amp;lt;/italic&amp;gt;

Restenosis is one of clinical limitations for vein graft in coronary bypass graft. It has been proved that signal pathway IGF-1 and its receptor (IGF-1R) activated by hemodynamic mechanical stretch are responsible for the vascular smooth muscle cells proliferation in vein graft neointima formation. Unfortunately, there is no routinely successful method to resolve this problem. Gene delivering to vein graft possesses great therapeutic potential to prevent neointima formation. Polymer is one kind of nanoparticles, which can activate the process of endocytosis of cells. In this study, we evaluated the transfection efficiency and therapeutic potential of polymer-based transfection of plasmids expressing GFP and shRNAs targeting IGF-1R (pGFPshIGF-1Rs) to smooth muscle cells and rabbit external jugular vein graft. Results showed that polymer-based transfection provided high efficiency of transgene expression in smooth muscle cells in vitro. In vitro, IGF-1R-specific shRNA transfected by polymer inhibited IGF-1R protein expression by 52 ± 3.6%, when compared with mock transfected cells. In vivo delivering efficiency of pGFPshIGF-1R plasmid into the rabbit external jugular vein graft was significantly high in the polymer-based transfection group, when compared with negative control group. In vivo, polymer-based transfection IGF-1R-specific shRNA efficiently inhibited the expression of IGF-1R protein by 77 ± 3.6%, 65.6 ± 4.9%, and 76.7 ± 4.3% at 24, 48, and 72 h, respectively, when compared with negative control group. Our findings indicated that polymer-based transfection may be a promising technique that allows the targeting of gene therapy for vein graft restenosis.

MiRNA-21 is dysregulated in response to vein grafting in multiple models and genetic ablation in mice attenuates neointima formation

AimsThe long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation.Methods and resultsWe undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model. These profiling experiments identified a number of miRNAs which were dysregulated following engraftment. miR-21 levels were substantially elevated following engraftment and these results were confirmed by quantitative real-time PCR in mouse, pig, and human models of vein graft neointimal formation. Genetic ablation of miR-21 in mice or grafted veins dramatically reduced neointimal formation in a mouse model of vein grafting. Furthermore, pharmacological knockdown of miR-21 in human veins resulted in target gene de-repression and a significant reduction in neointimal formation.ConclusionThis is the first report demonstrating that miR-21 plays a pathological role in vein graft failure. Furthermore, we also provided evidence that knockdown of miR-21 has therapeutic potential for the prevention of pathological vein graft remodelling.

Abstract 18255: MicroRNA Analysis in Experimental Models of Vein Graft Neointima Formation Indentifies a Critical Role For Mir-21 in Neointima Formation

Background: Coronary artery bypass grafting (CABG) is an established treatment to relieve the symptoms of ischemic heart disease. However premature graft failure, in saphenous veins is a major limitation. One of the principal etiological factors responsible for vein graft failure is activation and phenotypic modulation of smooth muscle cells (SMC), resulting in accelerated rates of proliferation and migration. MicroRNAs (miRNAs) are short, single stranded, non-coding RNAs that play important roles in modulating cell phenotypes through regulation of gene expression. Methods and results: In order to assess the expression levels of miR-21 in the progression of vein graft neointimal formation we quantified miR-21 levels in three well-established models of vein graft disease. These were the in vivo porcine model of interposition graft, the isogenic mouse model, as well as the ex vivo human sapehnous vein model of neointimal formation. MiR-21 levels were up-regulated approximately 7-fold in both the porcine and murine models at 7 and 28 days following grafting (n=6). In surgically prepared HSV which were subjected to culture ex vivo we found that miR-21 levels were elevated approximately 3-fold following 7 and 14 days of culture (n=6). In situ localization in the HSV demonstrated that miR-21 was expressed most intensely in the neointimal layer of HSV which also stained positive for SMC actin. Co-localization studies in murine vein grafts revealed that miR-21 was localised to areas of the graft that stained positive for proliferating cell nuclear antigen with substantial co-staining with SMC actin. We assessed the potential functional role of miR-21 in vein graft failure using miR-21 knockout mice. Analysis of neointimal lesion size in miR-21 knockout mice revealed an 81% reduction in neointimal area compared to wild type controls (p&lt; 0.0001, n=7-10/group). Conclusion: These findings demonstrate that miR-21 is up-regulated in multiple models of vein graft disease and that genetic abolition of miR-21 levels can attenuate neointimal formation in response to grafting. Therein, modulation of miR-21 expression represents a novel therapeutic opportunity in the setting of vein graft neoinitma formation.

Abstract 18427: Dysregulation of the miR143/145 Cluster and Mir133a During Progression of Vein Graft Neointima Formation

Background: The late failure of autologus human saphenous vein bypass grafts due to neointimal formation carries a major clinical burden. Therefore, identifying novel strategies to prevent neointimal formation is desirable. The principal cell type responsible for the neointimal lesion are smooth muscle cells (SMC), which are activated in response to injury leading to accelerated rates of proliferation and migration. MicroRNAs (miRs) are small non-coding RNAs that play an important role in modulating vascular phenotypes through regulation of gene expression. Methods and results: We undertook a microarray profiling approach to identify miRs which may be modulated during the development of neointimal lesions in the setting of vein graft disease. Profiling experiments revealed several miRs (miR133a, miR143 and miR145) which have been previously implicated in SMC de-differentiation and activation. These results were validated by q-PCR which demonstrated that miR-133a and miR-143 were down-regulated 4-5 fold and miR-145 was down regulated between 7-11 fold in the human saphenous vein ex vivo culture model and the porcine interposition model of vein graft neoinitma formation (both models, n=6). Treatment of isolated SMC harvested from human saphenous veins with TGF-β 1 (10ng/ml), to induce differentiation to a more quiescent and contractile phenotype, up-regulated miR-145 by 2.2+0.2 fold (n=3) but miR-133a levels were unaffected . A wounding assay was utilised to determine whether miR levels were elevated during SMC migration. Following quiescence, SMC were subjected to multiple scratch wounds and placed in serum to induce migration for a period of 16 hours. Quantification of miR-145 levels in migrating SMC revealed a 5-fold reduction in miR-145 levels following induction of migration. Conclusion: These results suggest that the miR-143/145 cluster plays an important role in the dedifferentiation of SMCs during neointimal formation following vein grafting. We also demonstrate that TGF-β 1, an established modulator of SMC differentiation and proliferation, increased miR-143/145 levels suggesting that elevation of miR-143/145 in vein grafts may prevent SMC de-differentiation and elevated rates of SMC proliferation and migration.

Simulated Bladder Pressure Stimulates Human Bladder Smooth Muscle Cell Proliferation via the PI3K/SGK1 Signaling Pathway

A mechanical stimulus on detrusor tissue is critical to bladder outlet obstruction progression and functional bladder tissue engineering. A hypothesis is that mechanical stimulus triggers human bladder smooth muscle cell proliferation. To help better understand this relationship of bladder function to growth at the cellular level we used a novel method of applying cyclic hydrodynamic pressure that simulates the bladder cycle to cell cultures. We detected the proliferation response of human bladder smooth muscle cells (4310, ScienCell™) to different pressures as well as the signal transduction mechanism of this process. Human bladder smooth muscle cells cultured in scaffolds underwent 4 pressures (0, 100, 200 and 300 cm H(2)O) for 24 hours, as controlled by a BioDynamic® bioreactor. We then used flow cytometry to examine cell cycle distribution, and polymerase chain reaction and immunoblot to quantify SGK1 and AKT expression and activation in each group. SGK1 was silenced in human bladder smooth muscle cells using small interfering RNA to validate the role of SGK1 in mediating pressure induced cell proliferation. Compared with the 0 cm H(2)O control group, human bladder smooth muscle cells in the 200 and 300 cm H(2)O groups showed increased cell proliferation. SGK1 expression and activity were also increased while AKT, another downstream signal of PI3K, did not change significantly. SGK1 silencing abolished the increases in cell proliferation induced by pressure. To our knowledge we provide the first report of cyclic hydrodynamic pressure stimulating the proliferation of human bladder smooth muscle cells cultured in scaffolds. The signal transduction mechanism for this process is involved with the PI3K/SGK1 and not the PI3K/AKT signaling pathway.

Abstract 10: Systemic Administration of an Optimized Human Apyrase Inhibits Acute Thrombosis and Neointima Formation in Vein Grafts

Introduction. Occlusion of vein grafts (VGs) after bypass surgery is a major clinical problem. Apyrase (CD39) plays a key role in inhibiting thrombosis and vascular inflammation by scavenging ADP and ATP at sites of vascular injury. We examined the capacity of APT102, a recombinant mutant human apyrase with enhanced activity and prolonged half-life, to inhibit thrombosis and intimal hyperplasia in VGs. Methods and Results. Segments of inferior vena cava from C57BL/6J donor mice were grafted into carotid arteries of recipient mice. APT102 (1 mg/kg) or saline was delivered by intraperitoneal (IP) injection every 2 days after surgery. To determine if APT102 inhibits VG thrombosis, VGs were exposed 6 days after surgery, injured with ferric chloride, and formation of thrombi was assessed by monitoring blood flow. Within 60 minutes after injury an occlusive thrombus formed in 1 of 7 mice treated with APT102 vs. 5 of 6 mice treated with vehicle control (P&lt;0.05). No abnormal bleeding was observed in APT102-treated mice. To examine the effect of APT102 on neointima formation, mice received APT102 or vehicle control by IP injection for 2 weeks after surgery. Intimal hyperplasia in VGs was measured 2 weeks later (i.e. 4 weeks after surgery). Mean intima thickness was significantly less (P&lt;0.05) in VGs of APT102-treated mice (26±8.4 μm, n=4) than in VGs of saline-treated mice (48±9.4 μm, n=5). Conclusions. Systemic administration of a recombinant human apyrase inhibits thrombosis and intimal hyperplasia in VGs without increasing bleeding. These results suggest that APT102 has the potential to become a novel treatment strategy to prevent thrombotic occlusion and adverse remodeling of VGs.

Abstract 317: Mast Cells Do Not Contribute to a Reduction in Vein Graft Neointima in the C-kit Mutant KitW-sh/w-sh Mice

Background: Recent evidence suggests mast cells are involved in the development of vascular diseases such as atherosclerosis. The presence of mast cells in vein grafts led us to hypothesise that mast cells may regulate the extent of vein graft neointima hyperplasia. In this study, we employed the Kit W-sh/W-sh mouse strain, a widely accepted mast cell deficient mouse model induced by a spontaneous inversion mutation in the transcriptional regulatory elements of the c-kit gene. Methods: The vein graft surgery was performed by inserting the inferior thoracic vena cava from the donor mouse into the common carotid artery of the littermate recipient. Vein grafts were harvested at 4 weeks and fixed with 4% formaldehyde for histology. In some groups, Kit W-sh/W-sh mice were reconstituted with cultured mast cells either systemically or locally 9 weeks before vein graft surgery. For primary cell culture studies, aortic endothelial cells and smooth muscle cells were isolated by collagenase digestion method and cultured in standard conditions. Results: Kit w-sh/w-sh vein grafts had a significant reduction (33%; p&lt;0.05) in neointima area, compared to the wild type controls. The cellular composition of the Kit w-sh/w-sh neointima was similar to the wild type controls, most cells being smooth muscle alpha-actin positive. Neither the systemic nor local reconstitution of mast cells restored the neointima area to the wild type level. In the primary cell culture, Kit w-sh/w-sh vascular cells showed less migrating activity in the scratch wound healing test, which may account for the decrease in neointima formation. Conlusion: This study suggests that it is not the deficiency of mast cells in the Kit W-sh/W-sh mice which accounts for the reduction in neointima formation in vein grafts. Other cell types affected by the c-kit mutation may be responsible for the altered neointima formation in Kit W-sh/W-sh mice.

Sustained Reduction of Vein Graft Neointima Formation by Ex Vivo TIMP-3 Gene Therapy

Coronary artery vein graft failure, resulting from thrombosis, intimal thickening, and atherosclerosis, is a significant clinical problem, with approximately 50% of vein grafts failing within 10 years. Intimal thickening is caused by migration of vascular smooth muscle cells from the media to the intima, where they proliferate. Interventions using gene transfer to inhibit vascular smooth muscle cells proliferation and migration are attractive because ex vivo access to the graft is possible. The involvement of matrix-degrading metalloproteinases in intimal thickening is well established, and we previously showed that adenoviral-delivered overexpression of an endogenous inhibitor, the tissue inhibitor of metalloproteinases-3 (TIMP-3), significantly retarded intimal thickening in short-term autologous porcine arteriovenous interposition grafts (28 days). However, it is essential to determine whether this approach will provide longer-term benefits. We assessed whether a recombinant adenovirus that overexpresses TIMP-3 (RAdTIMP-3) affects vein graft intimal thickening in the longer term (at 3 months). Porcine saphenous veins were subjected to luminal infection with 2.5×10(10) pfu/mL RAdTIMP-3 or RAd60 (control virus) or vehicle control, for 30 minutes before implantation into the carotid artery. Analysis of grafts harvested 3 months after delivery revealed that RAdTIMP-3-infected grafts had significantly reduced intimal areas compared with both controls (3.2 ± 0.4 mm(2) versus 5.6 ± 0.7 mm(2) and 5.9 ± 0.5 mm(2), RAdTIMP-3, RAd60, and vehicle, respectively). Medial areas were also significantly decreased by TIMP-3 (3.8 ± 0.3 mm(2) versus 6.7 ± 1.0 mm(2) and 5.2 ± 0.4 mm(2), RAdTIMP-3, RAd60, and vehicle, respectively). Overexpression of TIMP-3 provides a sustained retardation of vein graft intimal thickening and highlights the translational potential for ex vivo TIMP-3 gene therapy.