Formation Of Intimal Hyperplasia Research Articles

Nitric Oxide Deficit Drives Intimal Hyperplasia in Mouse Models of Hypertension

To evaluate the impact of different types of hypertension on the development of intimal hyperplasia (IH). Genetic, surgical, and pharmacological models of hypertension were used to compare IH formation in a murine model of carotid artery ligation (CAL). CAL was performed in normotensive WT male mice and in three mouse models of hypertension: (1) L-NAME (Nω-nitro-l-arginine-methyl-ester) treatment for 2 weeks prior to CAL to instate renin-independent hypertension; (2) 2K1C (two kidneys, one clip) surgery 1 week prior to CAL to induce renin-dependent hypertension; (3) Cx40-/- mice, a genetic model of renin-dependent hypertension. Mice were sacrificed prior to CAL or 3, 14, or 28 days post CAL. Data collection included tail blood pressure measurements, and morphometric and histological assessment of the ligated carotids. CAL triggered the formation of a VSMC-rich neointima layer after 14-28 days, which was increased in all hypertensive mice. Despite similarly increased blood pressure, L-NAME treated mice displayed more IH than all other hypertensive groups. In addition, L-NAME induced hypertension triggered more cell proliferation and recruitment of CD45 positive inflammatory cells to the ligated vessel wall compared with Cx40-/- or normotensive WT mice. NO deficiency is a major aspect of vascular inflammation, VSMC proliferation, and IH in hypertensive conditions.

Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

BackgroundTranscription of the myristoylated alanine‐rich C kinase substrate (MARCKS) is upregulated in animal models of intimal hyperplasia. MARCKS knockdown inhibits vascular smooth muscle cell (VSMC) migration in vitro; however, the mechanism is as yet unknown. We sought to elucidate the mechanism of MARCKS‐mediated motility and determine whether MARCKS knockdown reduces intimal hyperplasia formation in vivo.Methods and Results MARCKS knockdown blocked platelet‐derived growth factor (PDGF)‐induced translocation of cortactin to the cell cortex, impaired both lamellipodia and filopodia formation, and attenuated motility of human coronary artery smooth muscle cells (CASMCs). Activation of the small GTPases, Rac1 and Cdc42, was prevented by MARCKS knockdown. Phosphorylation of MARCKS resulted in a transient shift of MARCKS from the plasma membrane to the cytosol. MARCKS knockdown significantly decreased membrane‐associated phosphatidylinositol 4,5‐bisphosphate (PIP 2) levels. Cotransfection with an intact, unphosphorylated MARCKS, which has a high binding affinity for PIP 2, restored membrane‐associated PIP 2 levels and was indispensable for activation of Rac1 and Cdc42 and, ultimately, VSMC migration. Overexpression of MARCKS in differentiated VSMCs increased membrane PIP 2 abundance, Rac1 and Cdc42 activity, and cell motility. MARCKS protein was upregulated early in the development of intimal hyperplasia in the murine carotid ligation model. Decreased MARKCS expression, but not total knockdown, attenuated intimal hyperplasia formation.Conclusions MARCKS upregulation increases VSMC motility by activation of Rac1 and Cdc42. These effects are mediated by MARCKS sequestering PIP 2 at the plasma membrane. This study delineates a novel mechanism for MARCKS‐mediated VSMC migration and supports the rational for MARCKS knockdown to prevent intimal hyperplasia.

Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia.

Venous bypass grafts often fail following arterial implantation due to excessive smooth muscle cells (VSMC) proliferation and consequent intimal hyperplasia (IH). Intercellular communication mediated by Connexins (Cx) regulates differentiation, growth and proliferation in various cell types. Microarray analysis of vein grafts in a model of bilateral rabbit jugular vein graft revealed Cx43 as an early upregulated gene. Additional experiments conducted using an ex-vivo human saphenous veins perfusion system (EVPS) confirmed that Cx43 was rapidly increased in human veins subjected ex-vivo to arterial hemodynamics. Cx43 knock-down by RNA interference, or adenoviral-mediated overexpression, respectively inhibited or stimulated the proliferation of primary human VSMC in vitro. Furthermore, Cx blockade with carbenoxolone or the specific Cx43 inhibitory peptide 43gap26 prevented the burst in myointimal proliferation and IH formation in human saphenous veins. Our data demonstrated that Cx43 controls proliferation and the formation of IH after arterial engraftment.

Circumferentially aligned fibers guided functional neoartery regeneration in vivo

An ideal vascular graft should have the ability to guide the regeneration of neovessels with structure and function similar to those of the native blood vessels. Regeneration of vascular smooth muscle cells (VSMCs) with circumferential orientation within the grafts is crucial for functional vascular reconstruction in vivo. To date, designing and fabricating a vascular graft with well-defined geometric cues to facilitate simultaneously VSMCs infiltration and their circumferential alignment remains a great challenge and scarcely reported in vivo. Thus, we have designed a bi-layered vascular graft, of which the internal layer is composed of circumferentially aligned microfibers prepared by wet-spinning and an external layer composed of random nanofibers prepared by electrospinning. While the internal circumferentially aligned microfibers provide topographic guidance for in vivo regeneration of circumferentially aligned VSMCs, the external random nanofibers can offer enhanced mechanical property and prevent bleeding during and after graft implantation. VSMCs infiltration and alignment within the scaffold was then evaluated in vitro and in vivo. Our results demonstrated that the circumferentially oriented VSMCs and longitudinally aligned ECs were successfully regenerated in vivo after the bi-layered vascular grafts were implanted in rat abdominal aorta. No formation of thrombosis or intimal hyperplasia was observed up to 3 month post implantation. Further, the regenerated neoartery exhibited contraction and relaxation property in response to vasoactive agents. This new strategy may bring cell-free small diameter vascular grafts closer to clinical application.

Glucagon-Like Peptide-1 Receptor Activation Does Not Affect Re-Endothelialization but Reduces Intimal Hyperplasia via Direct Effects on Smooth Muscle Cells in a Nondiabetic Model of Arterial Injury

Diabetic patients have an increased risk of restenosis and late stent thrombosis after angioplasty, i.e. complications that are related to a defective re-endothelialization. Exendin-4, a stable glucagon-like peptide (GLP)-1 receptor agonist, has been suggested to influence the formation of intimal hyperplasia and to increase endothelial cell proliferation in vitro. Thus, the aim of this study was to investigate the mechanisms by which treatment with exendin-4 could influence re-endothelialization and intimal hyperplasia after vascular injury. Methods: Sprague-Dawley rats were subjected to balloon injury of the left common carotid artery and treated for 4 weeks with exendin-4 or vehicle. Intimal hyperplasia and vessel wall elasticity were monitored noninvasively by high-frequency ultrasound, and re-endothelialization was evaluated upon sacrifice using Evans blue dye. Results and Conclusion: Exendin-4 selectively reduced the proliferation of smooth muscle cells (SMCs) and intimal hyperplasia in vivo without affecting the re-endothelialization process, but treatment with exendin-4 improved arterial wall elasticity. Our data also show that exendin-4 significantly decreased the proliferation and increased the apoptosis of SMCs in vitro, effects that appear to be mediated through cAMP signaling and endothelial nitric oxide synthase following GLP-1 receptor activation. Together, these effects of exendin-4 are highly desirable and may lead to an improved outcome for patients undergoing vascular interventions.

Abstract 489: Dual Role of Connexin37 in the Control of Vascular Inflammation and Smooth Muscle Cell Proliferation During Intimal Hyperplasia in Mice

Introduction: The long term patency of vascular interventions is plagued by restenosis due to intimal hyperplasia (IH). Intercellular communication mediated by gap junctions plays a key role in tissue homeostasis and we recently showed that the gap junction protein Connexin43 (Cx43) contributes to smooth muscle cell (SMC) proliferation and IH. We now hypothesized that vascular Cx37 also plays a role in vascular remodeling and IH. Methods and Results: WT and Cx37 deficient mice (Cx37-/-) were subjected to the carotid artery ligation model. In WT mice, the ligation resulted in IH formation, as evidence by the development of a SMC -rich neointima after two to four weeks. This remodeling correlated with a reduced Cx37 expression in the endothelium layer and an increased Cx37 expression in the media layer. Compared to WT mice, Cx37-/- mice displayed a delayed development of neointima due to reduced infiltration of CD45-positive immune cells. However, Cx37-/- mice displayed an accelerated SMC proliferation rate between two and four weeks post surgery, as evidence by the number of PCNA-positive cells. Cx37-/- mouse arteries overexpressed synthetic SMC markers. Finally, in primary human vein SMC, Cx37 overexpression reduced cell proliferation and migration. Conclusions: Cx37 plays a dual role in neointima formation following vascular injury. Cx37 promotes immune cell infiltration and inflammation in the vessel. On the other hand, Cx37 overexpression in SMC counteracts the proliferative role of Cx43. Thus, Cx37 may play both protective and harmful roles in IH.

Abstract 476: Glucagon Like Peptide -1 Receptor Activation Does Not Affect Re-endothelialization But Reduces Intimal Hyperplasia via Direct Effects on Smooth Muscle Cells in a Non-diabetic Model of Arterial Injury

Background/Aims: Diabetic patients have an increased risk of restenosis and late stent thrombosis after angioplasty, complications that are related to a defective re-endothelialization. Exendin-4, a stable GLP-1 receptor agonist has been suggested to influence the formation of intimal hyperplasia and to increase endothelial cell proliferation in vitro. Thus, the aim of the study was to investigate the mechanisms by which treatment with exendin-4 could influence re-endothelialization and intimal hyperplasia after vascular injury. Methods: 40 male Sprague-Dawley rats were subjected to balloon injury of the left common carotid artery (LCCA) and treated for four weeks with exendin-4 (1 nmol/day) or vehicle. Intimal hyperplasia and vessel wall elasticity were monitored non-invasively by high frequency ultrasound and re-endothelialization was evaluated upon sacrifice using Evans blue dye. The LCCA was then sectioned for subsequent morphometric and immunohistochemical analyses. To further investigate if and how smooth muscle cells (SMCs) are directly affected by exendin-4 treatment, we studied proliferation and apoptosis of SMCs in vitro. Results and Conclusion: Exendin-4 selectively reduced proliferation of SMCs and intimal hyperplasia in vivo without affecting the re-endothelialization process, however treatment with exendin-4 improved arterial wall elasticity. Our data also show that exendin-4 significantly decreased the proliferation and increased the apoptosis of SMCs in vitro, effects that appear to be mediated through cAMP signaling and endothelial nitric oxide synthase following GLP-1 receptor activation. Together, these effects of exendin-4 are highly desirable and may lead to improved outcome for patients undergoing vascular interventions.

0377 : Phosphoinositide 3-kinase gamma: a potential clinical target in the prevention of vascular damages inuced by arterial injury

Angioplasty with stent placement, used to treat symptomatic atherosclerosis plaques, is frequently complicated by restenosis. This pathology is characteried by fibroproliferative and immuno-inflammatory mechanisms of the arterial wall, called intimal hyperplasia (IH). Our previous results demonstrated that phosphoinositide 3-kinase (??(PI3K(??) was a key mediator of inflammatory processes of the arterial wall leading to atherosclerosis. We now proposed this enzyme as a potential clinical target to prevent vascular damages occuring after arterial lesion leading to IH. As cellular and molecular aspects of IH are still incompletely described, we intended to offer a better understanding ofimmune responses involved in this pathology. Using a mouse model of arterial mechanical injury, we showed that PI3K(?-deficient mice and mice expressing a catalytically-inactive PI3K(?(PI3K(?KD) showed reduced arterial occlusion and accumulation of monocytes and T cells around site of vascular lesion. The transfer of PI3K(?KD CD4 + T cells into Rag2-deficient mice greatly reduced vascular occlusion compared to WT cells, clearly demonstrating the involvement of PI3K(?in CD4 + T cells during IH formation. In addition, we found that IH is associated with increased levels of Th1 and Th17 cytokines. A specific decrease in the Th1 response was observed in the absence of PI3K(?activity, leading to decreased chemokine production by smooth muscle cells. Finally, we show that a short period of treatment with a PI3K(?inhibitor decreased IH development in arterial mechanical injury in mice but also in a rat carotid artery balloon injury model demonstrating the therapeutic potential of this inhibitor. Our work pintpoint PI3K(?as a good target for IH prevention and brings new insights in the immune biology of the disease.

Preliminary Experience in the Use of an Extracellular Matrix (CorMatrix) as a Tube Graft: Word of Caution

A number of materials have been used for the repair of congenital heart disease. However, an ideal material is yet to be discovered. Decellularized extracellular matrix from porcine small intestinal submucosa (CorMatrix) has been developed and commercialized as a biological tissue substitute. This has been used for valvuloplasty, sepal defect repair, or angioplasty as a patch. In this study, we demonstrate our preliminary experience using CorMatrix as a tube graft. A retrospective review of 13 patients who underwent cardiac surgery using CorMatrix as an interposition graft was performed (10 patients for central pulmonary artery reconstruction in comprehensive stage II surgery for hypoplastic left-sided heart syndrome and 3 patients for aortic arch reconstruction in interrupted aortic arch). At a mean follow-up of 9.7 months, 8 of 10 patients who underwent central pulmonary artery reconstruction using CorMatrix tube showed progressive significant stenosis. One patient underwent replacement of the CorMatrix tube with a homograft because of severe stenosis after the placement of a stent. All 3 patients who had aortic arch reconstruction with the CorMatrix tube demonstrated no stenosis, no dilatation, and no aneurysm formation. Although angioplasty using CorMatrix as an interposition tube vascular graft demonstrated no adverse event in the aortic position in short term, a high rate of intimal hyperplasia formation with significant stenosis was found in the low-pressure small-diameter system. Longer follow-up is required to assess the growth potential of the arterial conduit. CorMatrix may not be the ideal material as conduit in the low-pressure small-diameter system to provide long-term durable outcomes.

Muraglitazar-Eluting Bioabsorbable Vascular Stent Inhibits Neointimal Hyperplasia in Porcine Iliac Arteries

PurposeTo evaluate the biocompatibility of a new muraglitazar-eluting polylactide copolymer stent and investigate its ability to prevent the formation of intimal hyperplasia. Materials and MethodsTen self-expandable muraglitazar-eluting poly-96l/4d-lactic acid (PLA96) stents and 10 self-expandable control PLA96 stents were implanted into porcine common iliac arteries. After 28 days follow-up, all stent-implanted iliac arteries were harvested and prepared for quantitative histomorphometric analysis. ResultsAngiographic analysis revealed that one control PLA96 stent had occluded and one had migrated. Histomorphometric analysis demonstrated that, with the control PLA96 stent, the luminal diameter and area were decreased versus the muraglitazar-eluting PLA96 stents (means ± standard error of the mean, 3.58 mm ± 0.34 vs 4.16 mm ± 0.14 and 9.83 mm2 ± 2.41 vs 13.75 mm2 ± 0.93, respectively). The control PLA96 stent induced more intimal hyperplasia than the bioactive muraglitazar-eluting PLA96 stent (557 µm ± 122 vs 361 µm ± 32). Vascular injury scores demonstrated only mild vascular trauma for both stents (muraglitazar-eluting, 0.68 ± 0.07; control, 0.75 ± 0.08). Inflammation scores also showed mild inflammation for both stents (muraglitazar-eluting, 1.05 ± 0.17; control, 1.23 ± 0.19). ConclusionsThis new muraglitazar-eluting PLA96 stent was shown to be biocompatible with a tendency for better patency and less intimal hyperplasia compared with the control PLA96 stents.

K+ channels expression in hypertension after arterial injury, and effect of selective Kv1.3 blockade with PAP-1 on intimal hyperplasia formation.

K(+) channels are central to vascular pathophysiology. Previous results demonstrated that phenotypic modulation associates with a change in Kv1.3 to Kv1.5 expression, and that Kv1.3 blockade inhibits proliferation of VSMCs cultures. To explore whether the Kv1.3 to Kv1.5 switch could be a marker of the increased risk of intimal hyperplasia in essential hypertension and whether systemic treatment with Kv1.3 blockers can prevent intimal hyperplasia after endoluminal lesion . Morphometric and immunohistochemical analysis were performed in arterial segments following arterial injury and constant infusion of the Kv1.3 blocker PAP-1 during 28 days. Differential expression of K(+) channel genes was studied in VSMC from hypertensive (BPH) and normotensive (BPN) mice, both in control and after endoluminal lesion. Finally, the migration and proliferation rate of BPN and BPH VSMCs was explored in vitro. Changes in mRNA expression led to an increased Kv1.3/Kv1.5 ratio in BPH VSMC. Consistent with this, arterial injury in BPH mice induced a higher degree of luminal stenosis, (84 ± 4% vs. 70 ± 5% in BPN, p < 0.01), although no differences in migration and proliferation rate were observed in cultured VSMCs. The in vivo proliferative lesions were significantly decreased upon PAP-1 systemic infusion (18 ± 6% vs. 58 ± 20% with vehicle, p < 0.05). Hypertension leads to a higher degree of luminal stenosis in our arterial injury model, that correlates with a decreased expression of Kv1.5 channels. Kv1.3 blockers decreased in vitro VSMCs proliferation, migration, and in vivo intimal hyperplasia formation, pointing to Kv1.3 channels as promising therapeutical targets against restenosis.

Inhibitory effects of mesenchymal stem cells in intimal hyperplasia after balloon angioplasty

Intimal hyperplasia is a major cause of restenosis after arterial bypass and balloon angioplasty. Induction of rapid re-endothelialization has been proposed to reduce intimal hyperplasia. The aim of this study was to evaluate the inhibitory effect of mesenchymal stem cells on intimal hyperplasia. Male New Zealand white rabbits were fed 1% cholesterol diets from 1 week before balloon angioplasty to the day of harvest. After dissection of rabbit carotid arteries, balloon angioplasty was performed with a 2F Fogarty embolectomy catheter. The injured carotid artery was coated with a mixture of 7 × 10(6) human umbilical cord mesenchymal stem cells (HUC-MSCs) and fibrin matrix. The carotid arteries were harvested 2, 4, and 8 weeks thereafter, and immunofluorescent staining and quantitative real-time polymerase chain reaction analysis were performed. The intima/media ratio was significantly reduced in the group treated with HUC-MSCs compared with the nontreated group (Student t-tests, *P < .05). The area of re-endothelialization was significantly higher (Student t-tests, *P < .05) in the group treated with HUC-MSCs than in the nontreated group. Expression of angiogenic genes such as vascular endothelial growth factor, platelet-derived growth factor, kinase insert domain receptor 1, angiopoietin 1, and angio-associated migratory cell protein was increased (analysis of variance, P < .05) in the group treated with HUC-MSCs relative to the nontreated group. Our study showed that HUC-MSCs reduce the formation of intimal hyperplasia through rapid re-endothelialization. This result might be applied to development of stem cell-coated stents as well as to development of a stem cell-containing sheet coat for inhibition of intimal hyperplasia after angioplasty or surgery.

Targeting PI3Kγ activity decreases vascular trauma-induced intimal hyperplasia through modulation of the Th1 response.

Interventional strategies to treat atherosclerosis, such as transluminal angioplasty and stent implantation, often cause vascular injury. This leads to intimal hyperplasia (IH) formation that induces inflammatory and fibroproliferative processes and ultimately restenosis. We show that phosphoinositide 3-kinase γ (PI3Kγ) is a key player in IH formation and is a valid therapeutic target in its prevention/treatment. PI3Kγ-deficient mice and mice expressing catalytically inactive PI3Kγ (PI3Kγ KD) showed reduced arterial occlusion and accumulation of monocytes and T cells around sites of vascular lesion. The transfer of PI3Kγ KD CD4(+) T cells into Rag2-deficient mice greatly reduced vascular occlusion compared with WT cells, clearly demonstrating the involvement of PI3Kγ in CD4(+) T cells during IH formation. In addition we found that IH is associated with increased levels of Th1 and Th17 cytokines. A specific decrease in the Th1 response was observed in the absence of PI3Kγ activity, leading to decreased CXCL10 and RANTES production by smooth muscle cells. Finally, we show that treatment with the PI3Kγ inhibitor AS-605240 is sufficient to decrease IH in both mouse and rat models, reinforcing the therapeutic potential of PI3Kγ inhibition. Altogether, these findings demonstrate a new role for PI3Kγ activity in Th1-controlled IH development.

Arteriovenous graft for hemodialysis, graft venous anastomosis closure - current state of knowledge. Minireview.

Backround. The use of artificial vascular grafts (arteriovenous graft, AVG) is indicated in patients in hemodialysis programs if the subcutaneous venous bed is exhausted or unsuitable for arteriovenous fistula (AVF) creation. The native fistula should be the hemodialysis access of first choice: AVF has better results in terms of function and potential complications. However, the use of AVG is necessary in some patients. In these patients, extensive clinical examination, color duplex sonography and angiography should be performed prior to indication. The technique of graft implantation requires respect for geometric relations for the graft anastomoses to minimize the formation of intimal hyperplasia mainly on the venous anastomosis. The main complications of AVG are stenosis on the venous anastomosis (VAG), causing closure of graft and graft infection. The cumulative function of AVG is 59-90% in the first year and 50-82% in the second year. Arteriovenous graft stenosis leading to thrombosis is a major cause of complications in patients undergoing hemodialysis. The purpose of this review is to summarise current knowledge of the diagnostics and treatment of graft thrombosis and discuss the issue in combination with relevant publications via Pubmed database. The most frequent cause of failure of AVG for hemodialysis is stenosis and closure by VAG. AVG closure can be addressed surgically, endovascularly (amenable to thrombectomy by radiological or surgical means) and by hybrid performance.

Abstract 515: Effects of the Glucagon-Like Peptide-1 Analog Exendin-4 on Reendothelialization and Intimal Hyperplasia Formation in an Animal Model of Vascular Injury

Background and aims: Diabetic patients have an increased risk of late stent thrombosis formation and restenosis, after angioplasty. It is hypothesized that delayed re-endothelization is a major underlying problem for late-stent thrombosis and also that rapid re-endothelization is essential for preventing restenosis. Exendin-4 is a stable GLP-1 receptor agonist, which is applied in clinical treatment of diabetes. Beside its insulinotropic action, it may also exert direct beneficial effects on endothelial function. We previously reported that exendin-4 is able to activate the GLP-1 receptor on human coronary artery endothelial cells, leading to increased cell proliferation and decreased apoptosis. Therefore, our aim was to study if exendin-4 can influence endothelialization and decrease neointima formation after vascular injury. Materials and Methods: Balloon injury of the left common carotid artery (LCCA) was performed on Sprague-Dawley rats. Rats were randomized into two groups and treated for four weeks with exendin-4 (1 nmol/day) or saline. Intimal hyperplasia and endothelialization was monitored non-invasively by high frequency ultrasound and upon sacrifice with Evans blue, respectively. The LCCA was then sectioned for subsequent morphometric and immunohistochemical analyses. To further investigate if and how smooth muscle cells (SMCs) are directly affected by exendin-4 treatment, we studied proliferation and apoptosis of SMCs in vitro . Results and conclusions: Our findings show that exendin-4 selectively reduces SMC proliferation and intimal hyperplasia through activation of cAMP signaling and endothelial nitric oxide synthase without influencing re-endothelialization. Treatment with exendin-4 did however improve arterial wall elasticity. Together, these effects of exendin-4 are highly desirable and may lead to improved outcome for patients undergoing vascular interventions

Abstract 347: Chronic Kidney Disease Modulates Vascular Smooth Muscle Cell Phenotype and Increases de Novo Venous Intimal Hyperplasia

Background: Vascular access dysfunction associated with arteriovenous grafts and fistulas contributes significantly to the morbidity and mortality of end-stage renal disease (ESRD) patients receiving hemodialysis. The mechanism involved in the development of peri-anastomotic intimal hyperplasia, a major cause of the arteriovenous access dysfunction, is poorly elucidated. We hypothesized that the uremic condition associated with ESRD promotes a pathophysiological vascular smooth muscle cell (VSMC) phenotype that contributes to accelerated development of intimal hyperplasia in blood vessels, both de novo and following the arteriovenous anastomosis. Methods and Results: We examined the effect of culturing human VSMC with human uremic serum on its phenotype. We utilized quantitative real-time PCR to measure the expression of the VSMC-specific contractile marker genes, and found that these genes were reduced 50-80% in VSMC exposed to serum from hemodialysis patients as compared to serum from patients with normal renal function. Chromatin immunoprecipitation assays demonstrated that there were also significant changes in epigenetic markers associated with contractile gene promoters in VSMC cultured in uremic conditions. Moreover, there was a 60% increase in proliferation rate in cells exposed to uremic conditions, with no change in the levels of apoptosis as compared to cells exposed to non-uremic conditions. Interestingly, uremic serum from ESRD patients inhibited PDGF-induced migration of VSMC. We also examined the histopathology of veins from patients with normal kidney function and chronic kidney disease (CKD). Indeed, we found significant de novo venous intimal hyperplasia exists in CKD and ESRD patients prior to any surgical manipulation as compared to veins from patients with normal kidney function. Conclusion: CKD alters VSMC phenotype in vitro and contributes to intimal hyperplasia formation in vivo resulting in the pathogenesis of vascular access dysfunction in ESRD patients.

Abstract 517: The MicroRNA Cluster 99b/let7-e/125a is Downregulated Following Vein Grafting in a Porcine Model, and Upregulation Alters Vascular Cell Phenotype in vitro

Introduction: Coronary artery bypass surgery is a common surgical procedure. However, 20-50% of patients present vein graft failure within 10 years. The occlusion of the graft is induced by intimal hyperplasia formation promoted by enhanced vascular smooth muscle cells (SMC) proliferation and migration. MicroRNAs are becoming recognized for their role in SMC proliferation and migration associated with vascular pathologies. Here we investigate a cluster of miRNAs (99b/let-7e/125a) in a model of vein graft failure and assessed the effect of modulating its expression in SMCs. Methods: Global miRNA profiles were analyzed in porcine saphenous vein grafts 7 and 28 days post-engraftment (n=6) by a TaqMan low density array. The three miRNAs encoded by the cluster were further validated by qPCR. SMCs were isolated from human saphenous vein and then transduced with a lentiviral vector (Lv-cluster) carrying entire cluster sequence. The effects on proliferation (BrdU incorporation) and migration (scratch wound) were analyzed. Computational analysis identified AKT-1, a known pro-proliferative factor, as a common previously validated target for 99b/let7-e/125a miRNAs. AKT-1 mRNA (qPCR) and protein expression (western blot) were analyzed in SMCs previously infected with Lv-cluster. Results: All three miRNAs encoded by this cluster were downregulated in graft samples 7 and 28 days post-engraftment, compared to ungrafted saphenous vein (Fold change at 7 days: 99b: 0.5±0.07, let-7e: 0.34±0.03, 125a: 0.6±0.07; p&lt;0.05. Fold 28 days: 99b: 0.32±0.05, let-7e: 0.24±0.05, 125a: 0.35+0.07; p&lt;0.001). Infection of SMCs with this Lv-cluster increased the expression of the mature miRNAs by ~ 5 fold compared to controls. This increase was associated with a significant (p&lt;0.01) reduction in SMCs migration (20%±1) and proliferation (90%±4). These results demonstrate that overexpression of this cluster in SMCs significantly (p&lt;0.05) reduced the mRNA and protein levels of AKT-1 (0.6 and 0.4 fold, respectively). Conclusion: MiRNAs levels of the cluster 99b/let-7e/125a are downregulated in a porcine model of saphenous vein grafting. In vitro results suggest that overexpression of this miRNA cluster reduces migration and proliferation in SMCs by targeting AKT/mTOR pathway.

Inhibition of intimal hyperplasia in murine aortic allografts by the oral administration of the transforming growth factor-beta receptor I kinase inhibitor SD-208

Transforming growth factor-beta (TGF-β) plays a significant role in the pathogenesis of the intimal hyperplasia of transplant arteriosclerosis (TA). The aim of this study was to evaluate the efficacy of an oral inhibitor of TGF-β receptor I kinase (SD-208) on the development of TA. BALB/c (H-2(d)) donor aortas were transplanted into C57BL/6 (H-2(b)) recipients, and the mice then received different doses (40 or 60 mg/kg) of SD-208 or control vehicle by daily gavage for 8 weeks. The grafts were analyzed by histology and morphometry at 1, 2, 4, 6 and 8 weeks after transplantation. The effects of TGF-β and SD-208 on neointimal smooth muscle-like cell (SMLC) and vascular smooth muscle cell (VSMC) proliferation and migration were evaluated, and the expression levels of Smad3, P-Smad3, connective tissue growth factor (CTGF) and collagen I were determined by in vitro experiments. The intimal hyperplasia of the SD-208-treated group was significantly reduced compared with the vehicle-treated control group (32% and 48% reduction for 40 mg/kg and 60 mg/kg SD-208 compared with the controls, respectively [n = 5], p < 0.05). SD-208 reduced SMLC proliferation and the production of intimal collagen by 21% and 75%, respectively, in the grafts. SD-208 also abolished the promoting effect of TGF-β on SMLC proliferation and migration but did not affect TGF-β inhibition of VSMCs in vitro. CTGF, a protein downstream of TGF-β, was downregulated with the inhibition of Smad3 phosphorylation by SD-208, both in vitro and in vivo. Moreover, we found that the endogenous Smad3 in SMLCs was upregulated from 2 weeks after transplantation and was 64% higher than in VSMCs at 8 weeks. These results demonstrate that SD-208 can effectively reduce the formation of intimal hyperplasia of TA in the murine aortic allograft model.

Supplemental Oxygen Reverses Hypoxia-induced Smooth Muscle Cell Proliferation by Modulating HIF-alpha and VEGF Levels in a Rabbit Arteriovenous Fistula Model

Numerous mechanisms for the formation of intimal hyperplasia have been proposed but none have been proven or accepted. Our research focuses on the potential role of hypoxia-inducible factors (HIFs), vascular endothelial growth factor (VEGF), and platelet-derived growth factors as well as the extracellular signal-regulated kinase (ERK), phosphatidylinositide 3-kinase /protein Kinase B (PI3-K/AKT) pathway in hypoxia-mediated intimal hyperplasia processes. We hypothesize that HIF and VEGF will be downregulated with supplemental oxygen in our arteriovenous fistula rabbit model. Rabbits were randomized into different experimental groups with varying oxygen exposure (21% O2 or 30% O2) and receipt of surgery (surgery with fistula formation, no surgery, or sham operation with skin incision only). Plasma samples were collected at designated intervals in which cytokines and smooth muscle cell proliferation were measured. In addition, cell specimens were exposed to hyperoxic, normoxic, and hypoxic environments with cytokines measured at various time points. Placement of an arteriovenous fistula resulted in hypoxia-induced HIF stabilization with a concurrent increase in VEGF levels. There was a 4.2-fold induction in HIF-1α levels in animals that were placed in normal air after surgery when compared with animals that were exposed to hyperoxic air. Also, VEGF level significantly increased after surgery in the normoxic group, reaching a maximum of 959 pg/mL. Plasma VEGF levels in the surgery and supplemental oxygen group were significantly lower than the normoxic surgery group with almost a 45% reduction in plasma VEGF levels (524 pg/mL). Activation of VEGF receptors on smooth muscle cells through ERK1 and AKT pathways resulted in significant smooth muscle cell proliferation and migration. These effects are dramatically reduced in animals that are exposed to a hyperoxic environment of 30% oxygen. Our results suggest that short-term administration of supplemental oxygen inhibits HIFs and VEGF signaling to reduce smooth muscle proliferation in the local blood vessel. These results provide strong support for the therapeutic use of supplemental oxygen after arterial surgery to reduce intimal hyperplasia. These findings also provide a nidus for future clinical trials to determine whether this is clinically applicable in humans.

Acute reductions in mechanical wall strain precede the formation of intimal hyperplasia in a murine model of arterial occlusive disease

Intimal hyperplasia (IH) continues to plague the durability of vascular interventions. Employing a validated murine model, ultrasound biomicroscopy, and speckle-tracking algorithms, we tested the hypothesis that reduced cyclic arterial wall strain results in accentuated arterial wall IH. A 9-0 suture was tied around the left mouse (n= 10) common carotid artery and a 35-gauge (outer diameter= 0.14mm) blunt mandrel. We previously showed that mandrel removal results in a ∼78% reduction in diameter and ∼85% reduction in flow, with subsequent delayed induction of IH by day 28. Preoperative, postoperative day-4 (before measurable IH), and postoperative day-27 circumferential wall strains were measured in locations 1, 2, and 3mm proximal to the stenosis and in the same locations on the contralateral (nonstenosed) carotid. At postoperative day 28, arteries were perfusion fixed and arterial wall morphology was assessed microscopically in the same regions. Strains were the same in all locations preoperatively. Wall strain was decreased in all regions proximal to the stenosis by day 4 (0.26± 0.01 to 0.11± 0.02; P< .001), while strains remained unchanged for the contralateral artery (P= .45). No statistical regional differences in mean strain or IH were noted at any time point for the experimental or contralateral artery. Based on the median, regions were divided into those with low strain (≤0.1) and high strain (>0.1). Average preoperative strains in both groups were the same (0.27± 0.09 and 0.27± 0.08). All segments in the low-strain group (n= 13) demonstrated significant IH formation by day 28, while only 31% of the high strain group demonstrated any detectable IH at day 28. (Mean low-strain intimal thickness= 32± 20μm, high strain= 8.0± 16μm; P< .01). Changes in cross-sectional area at diastole drove the reduction in strain in the low-strain group, increasing significantly from preoperatively to day 4 (P= .04), while lumen cross-section at systole remained unchanged (P= .46). Cross-sectional area at diastole and systole in the high-strain group remained unchanged from preoperatively to day 4 (P= .67). Early reduction in arterial wall strain is associated with subsequent development of hemodynamically induced IH.