Balloon Angioplasty Model Research Articles

Targeted NAD+ repletion via biomimetic nanoparticle enables simultaneous management of intimal hyperplasia and accelerated re-endothelialization: A proof-of-concept study toward next-generation of endothelium-protective, anti-restenotic therapy

Endovascular interventions often fail due to restenosis, primarily caused by smooth muscle cell (SMC) proliferation, leading to intimal hyperplasia (IH). Current strategies to prevent restenosis are far from perfect and impose significant collateral damage on the fragile endothelial cell (EC), causing profound thrombotic risks. Nicotinamide adenine dinucleotide (NAD+) is a co-enzyme and signaling substrate implicated in redox and metabolic homeostasis, with a pleiotropic role in protecting against cardiovascular diseases. However, a functional link between NAD+ repletion and the delicate duo of IH and EC regeneration has yet to be established. NAD+ repletion has been historically challenging due to its poor cellular uptake and low bioavailability. We have recently invented the first nanocarrier that enables direct intracellular delivery of NAD+ in vivo. Combining the merits of this prototypic NAD + -loaded calcium phosphate (CaP) nanoparticle (NP) and biomimetic surface functionalization, we created a biomimetic P-NAD + -NP with platelet membrane coating, which enabled an injectable modality that targets IH with excellent biocompatibility. Using human cell primary culture, we demonstrated the benefits of NP-assisted NAD+ repletion in selectively inhibiting the excessive proliferation of aortic SMC, while differentially protecting aortic EC from apoptosis. Moreover, in a rat balloon angioplasty model, a single-dose treatment with intravenously injected P-NAD + -NP immediately post angioplasty not only mitigated IH, but also accelerated the regeneration of EC (re-endothelialization) in vivo in comparison to control groups (i.e., saline, free NAD+ solution, empty CaP-NP). Collectively, our current study provides proof-of-concept evidence supporting the role of targeted NAD+ repletion nanotherapy in managing restenosis and improving reendothelialization.

The role of 6-phosphogluconate dehydrogenase in vascular smooth muscle cell phenotypic switching and angioplasty-induced intimal hyperplasia

BackgroundRestenosis poses a significant challenge for individuals afflicted with peripheral artery diseases, often leading to considerable morbidity and necessitating repeated interventions. The primary culprit behind the pathogenesis of restenosis is intimal hyperplasia (IH), in which the hyperproliferative and migratory vascular smooth muscle cell (VSMC) accumulate excessively in the tunica intima. 6-Phosphogluconate dehydrogenase (6PGD), sometimes referred to as PGD, is one of the critical enzymes in pentose phosphate pathway (PPP). In this study, we sought to probe whether 6PGD is aberrantly regulated in IH and contributes to VSMC phenotypic switching. MethodsWe used clinical specimens of diseased human coronary arteries with IH lesions and observed robust upregulation of 6PGD at protein level in both the medial and intimal layers in comparison with healthy arterial segments. Results6PGD activity and protein expression were profoundly stimulated upon platelet-derived growth factor-induced VSMC phenotypic switching. Using gain-of-function (dCas9-mediated transcriptional activation) and loss-of-function (small interfering RNA-mediated) silencing, we were able to demonstrate the pathogenic role of 6PGD in driving VSMC hyperproliferation, migration, dedifferentiation, and inflammation. Finally, we conducted a rat model of balloon angioplasty in the common carotid artery, with Pluronic hydrogel-assisted perivascular delivery of Physcion, a selective 6PGD inhibitor with poor systemic bioavailability, and observed effective mitigation of IH. ConclusionsWe contend that aberrant 6PGD expression and activity—indicative of a metabolic shift toward pentose phosphate pathway—could serve as a new disease-driving mechanism and, hence, an actionable target for the development of effective new therapies for IH and restenosis after endovascular interventions.

Abstract 12931: Development of Novel Targeted, Membrane-Coated Polymer Hybrid Nanoparticles for Endothelial Repair Following Balloon Angioplasty in Rat Carotid Artery Model

Background: Coronary and peripheral arterial disease remains one of the leading causes of death worldwide. Treatments include balloon angioplasty, stent placement, or bypass grafting, all of which damage the endothelium and increase the risk of thrombosis or intimal hyperplasia. Thus, there is a critical need for rapid reendothelialization following these treatments. Endothelial progenitor cell-derived (EPC) extracellular vesicles (EVs) containing proangiogenic microRNAs, such as miR126, is one of the natural mechanisms of vascular repair. However, the translational application of EVs is limited by low EV yield, functional heterogeneity, and difficult tissue targeting. Hypothesis: We hypothesize that EV-inspired hybrid nanoparticles can improve reendothelialization and modification with a specific collagen binding peptide SILY can target vascular injury. Aims: (1) Engineer and characterize a SILY modified hybrid EPC membrane coated nanoparticles (SILY-ENPs) containing a miRNA-126-loaded poly-lactic-co-glycolic acid core (miR126-PLGA) in vitro . (2) Test the therapeutic efficacy of the SILY-ENPs in an in vivo rat carotid balloon angioplasty model. Methods: EPC plasma membranes were isolated from umbilical cord blood EPCs and conjugated with SILY using copper-free Click chemistry. Membrane fragments were coextruded with miR126-PLGA to form SILY-ENPs. Particles were characterized in vitro for collagen and platelet binding and angiogenic function. In vivo function was tested in a rat carotid balloon angioplasty model. Results: SILY-ENPs were found to have increased collagen binding and decreased platelet activity. Delivery of miR126 cargo slightly improved pro-angiogenic protein expression and increased rates of reendothelialization. However, SILY-ENPs were found to stimulate hyperplasia as an adverse side-effect. Conclusions: SILY-ENPs exhibited target specificity, decreased platelet activity, and increased rates of reendothelialization, but increased intimal hyperplasia. This technology represents a novel EV inspired nanoplatform which can be tailored for treating various diseases by altering the cargo or membrane coating.

Biomimetic, ROS-detonable nanoclusters — A multimodal nanoplatform for anti-restenotic therapy

The long-term success of endovascular intervention has long been overshadowed by vessel re-occlusion, also known as restenosis. Mainstream anti-restenotic devices, such as drug-eluting stent (DES) and drug-coated balloon (DCB), were recently shown with suboptimal performances and life-threatening complications, thereby underpinning the urgent need for alternative strategies with enhanced efficacy and safety profile. In our current study, we engineered a multimodal nanocluster formed by self-assembly of unimolecular nanoparticles and surface coated with platelet membrane, specifically tailored for precision drug delivery in endovascular applications. More specifically, it incorporates the combined merits of platelet membrane coating (lesion targetability and biocompatibility), reactive oxygen species (ROS)-detonable “cluster-bomb” chemistry (to trigger the large-to-small size transition at the target site, thereby achieving longer circulation time and higher tissue penetration), and sustained drug release. Using RVX-208 (an emerging anti-restenotic drug under clinical trials) as the model payload, we demonstrated the superior performances of our nanocluster over conventional poly(lactic-co-glycolic acid) (PLGA) nanoparticle. In cultured vascular smooth muscle cell (VSMC), the drug-loaded nanocluster induced effective inhibition of proliferation and protective gene expression (e.g., APOA-I) with a significantly reduced dosage of RVX-208 (1 μM). In a rat model of balloon angioplasty, intravenous injection of Cy5.5-tagged nanocluster led to greater lesion targetability, improved biodistribution, and deeper penetration into injured vessel walls featuring enriched ROS. Moreover, in contrast to either free drug solution or drug-loaded PLGA nanoparticle formulation, a single injection with the drug-loaded nanocluster (10 mg/kg of RVX-208) was sufficient to substantially mitigate restenosis. Additionally, this nanocluster also demonstrated biocompatibility according to in vitro cytotoxicity assay and in vivo histological and tissue qPCR analysis. Overall, our multimodal nanocluster offers improved targetability, tissue penetration, and ROS-responsive release over conventional nanoparticles, therefore making it a highly promising platform for development of next-generation endovascular therapies.

Preventive Effect and Mechanism of Crossostephium chinense Extract on Balloon Angioplasty-Induced Neointimal Hyperplasia.

Balloon angioplasty-induced neointimal hyperplasia remains a clinical problem that must be resolved. The bioactivities of the Crossostephium chinense extract (CCE) have demonstrated potential in preventing the progression of restenosis. The present study evaluated whether CCE can suppress balloon angioplasty-induced neointima formation and elucidated its possible pharmacological mechanisms. A rat model of carotid arterial balloon angioplasty was established to evaluate the inhibitory effect of CCEs on neointimal hyperplasia. Two cell lines, A10 vascular smooth muscle cells (VSMCs) and RAW264.7 macrophages, were used to investigate the potential regulatory activities and pharmacological mechanisms of CCEs in cell proliferation and migration and in inflammation. Our in vitro results indicated that CCE3, the ethanolic extract of C. chinense, exerted the strongest growth inhibitory and antimigratory effects on VSMCs. CCE3 blocked the activation of focal adhesion kinase, platelet-derived growth factor receptor-β (PDGFRB), and its downstream molecules (AKT and mTOR) and reduced the expression of matrix metalloproteinase-2. In addition, our findings revealed that CCE3 significantly increased the expression of miRNA-132, an inhibitory regulator of inflammation and restenosis, and suppressed the expression of inflammation-related molecules (inducible nitric oxide synthase, cyclooxygenase-2, interleukin- (IL-) 1β, and IL-6). Our in vivo study results indicated that balloon injury-induced neointimal hyperplasia was inhibited by CCE3. CCE3 could reduce neointima formation in balloon-injured arteries, and this effect may be partially attributed to the CCE3-induced suppression of PDGFRB-mediated downstream pathways and inflammation-related molecules.

Cilostazol as a noninferiority pharmacologic option to paclitaxel in early intimal hyperplasia inhibition after venous balloon angioplasty in a rabbit model: a preliminary study

The development of venous intimal hyperplasia (VIH) has not been fully studied. At present, there are no drugs approved for VIH inhibition; to investigate such alternatives, we aimed to compare paclitaxel with cilostazol in VIH early inhibition in a preliminary experimental model of balloon angioplasty. Twenty-eight male New Zealand rabbits were randomly divided into two groups: cilostazol (A) and paclitaxel (B), which underwent femoral vein barotrauma by a 4 mm balloon angioplasty. The VIH model was previously tested in controls obtaining an 80% increase of subintimal area (SIA) compared with veins without injury (from 0.12 mm2 [standard deviation (SD), 0.05] to 0.86 mm2 [SD, 0.08]). Group A received 20 mg/kg twice daily; group B angioplasty was performed with a single-dose paclitaxel-coated balloon. Seven days later rabbits were euthanized, and vein tissue samples were taken for histological analysis. The primary end point was SIA measure expressed in mm2, and the anticipated difference between treatments was 0.21 mm2. Other measurements were immunohistochemistry expression of hypoxia inducible factor-1 alpha, platelet derived growth factor, and smooth muscle actin, as surrogates of cell migration and oxidative stress. SIA of group A was 0.33 mm2 (SD, 0.15; 95% CI, 0.24-0.42 mm2), and that of group B was 0.31 mm2 (SD, 0.14; 95% CI, 0.22-0.40 mm2). Both drugs showed a reduction of 61% and 63%, respectively, in SIA, compared with controls. The difference between both drugs was 0.0193 mm2 (95% CI, −0.1175 to 0.156 mm2); the statistical difference was found in hypoxia inducible factor-1 alpha expression between both groups. Clinical RelevanceAlthough veins have a thinner middle layer compared with arteries, smooth muscle cells appear to play an important role in venous stenosis after angioplasty. The study of smooth muscle cell response after barotrauma may have clinical applications in the endovascular treatment of venous stenosis, because at the moment, there is no medication indicated to prolong patency after venous endovascular procedures, for example in May Thurner syndrome. Paclitaxel and cilostazol seem to have a promising role. Finally, the present study could inspire a research line to reduce stent placement and increase patency after venous angioplasty.

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation.

SummaryVascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.

MiR-140-3p is Involved in In-Stent Restenosis by Targeting C-Myb and BCL-2 in Peripheral Artery Disease.

Aim: In-Stent Restenosis (ISR) is the major reason for recurrent ischemia and amputation after endovascular treatment of Peripheral Artery Disease (PAD). Our previous study demonstrated that miR-140-3p is significantly down-regulated in PAD arteries. However, expression and function of miR-140-3p in ISR of human PAD are currently unclear.The aim of this study is to determine the miR-140-3p expression and its regulative role in ISR of PAD.Methods: The RNA level was determined by quantitative real-time polymerase chain Reaction (qRT-PCR) and in situ hybridization. Primary cultured ASMCs were isolated from human femoral arterial of the healthy donors or ISR patients. Cell proliferation was determined by Edu incorporation and CCK-8 assay. Apoptosis was determined by Annexin-V/PI Double-Staining assay and TUNEL assay. A rat carotid artery balloon angioplasty model was used to investigate the effect of miR-140-3p on restenosis.Results: MiR-140-3p was significantly down-regulated in PAD and ISR arteries than normal arteries. Primary cultured ISR ASMCs exhibited elevated proliferation and down-regulated miR-140-3p than normal ASMCs. Transfection of miR-140-3p mimic attenuated PDGF-BB-induced proliferation in cultured ASMCs and induced apoptosis. Luciferase reporter assay indicated that miR-140-3p transfection significantly down-regulated C-Myb and BCL-2 in ISR ASMCs by targeting to their 3′-UTRs. MiR-140-3p transfection induced anti-proliferation and apoptosis in ASMCs, which were ameliorated by over-expression of C-Myb or BCL-2. Moreover, the animal study showed that miR-140-3p can reduce restenosis following angioplasty via targeting C-Myb and BCL-2.Conclusions: The result suggests that miR-140-3p regulates ASMC function via targeting C-Myb and BCL-2 in the process of ISR in PAD. The novel findings may offer a hopeful therapeutic target for human PAD.

Proresolving Lipid Mediators Resolvin D1 and Protectin D1 Isomer Attenuate Neointimal Hyperplasia in the Rat Carotid Artery Balloon Injury Model

BackgroundSpecialized proresolving mediators from ω-3 polyunsaturated fatty acid may control resolution of inflammation. We evaluated the influence of two specialized proresolving mediators, resolvin D1 (RvD1) and protectin D1 isomer (PD1 iso) on neointimal hyperplasia after balloon injury. Materials and methodsSprague Dawley male rats at 12-14 wk of age were injured as a model of balloon angioplasty. Then, 1 μg/rat of RvD1 or PD1 iso was administered intravenously via the tail vein immediately and 2 d after angioplasty. The proliferation of injured artery and the infiltration of leukocytes, monocytes, and macrophages at 3 d after injury were evaluated by immunostaining. The activity of the inflammatory transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in the injured artery at 3 d after injury was evaluated using an enzyme-linked immuno sorbent assay kit. The proliferation of the neointima was evaluated by calculating the ratio of the neointimal and medial areas using specimens at 14 d after injury. ResultsRvD1 and PD1 iso attenuated proliferation of medial cells (P < 0.05) and infiltration of leukocytes (P < 0.05) and monocytes/macrophages (P < 0.01). Although both RvD1 and PD1 iso mitigated NFκB activity (P < 0.01), RvD1 attenuated this activity more strongly (P < 0.01). RvD1 decreased neointimal hyperplasia by 37.3% (P < 0.01), whereas PD1 iso decreased neointimal hyperplasia by 31.8% (P < 0.05) (RvD1 versus PD1 iso: P = 0.51). ConclusionsRvD1 and PD1 iso reduced the activity of inflammatory transcription factor NFκB within the injured artery and attenuated inflammatory cell infiltration, leading to a reduction in early inflammation and subsequent neointimal hyperplasia.

Targeting Delivery of Rapamycin with Anti-Collagen IV Peptide Conjugated Fe₃O₄@Nanogels System for Vascular Restenosis Therapy.

Coronary arterial disease (CAD) remains the leading cause of death globally. Although percutaneous coronary interventions (PCI) are most of the important clinical procedure for CAD treating, unfortunately, vascular restenosis is proved as the major drawback of PCI. Because of the complex nature of the restenotic process, the efficacy of drug administration is emphasized. Targeting drug delivery systems become a promising experimental approach for restenosis therapy. Hence, we design and fabricate a thermo/pH-responsive nanogel system with the magnetic inner core as the multifunctional nanocarrier for drug delivery and MRI/fluorescence imaging. To this end, NIPAm-based Fe3O4 core-shell structure nanogel is synthesized as the responsive nanosystem for rapamycin (RAPA) delivery, and the surface conjugation with anti-collagen IV peptide makes the nanosystem an ideal candidate for targeting delivery of RAPA. Based on its stimuliresponsive properties, the nanosystem shows desirable intracellular release behavior of RAPA and significantly reduces the adverse effect of RAPA. The in vitro cytotoxicity evaluations present the biosafety profiles and antiproliferation performance of the drug-loaded nanogels. Meanwhile, the magnetic Fe3O4 inner core shows enhanced T2 weight sensitivity, providing a high potential for imaging-guiding therapy. In the balloon angioplasty model, targeting nanogels are demonstrated accumulation at the injured site of artery. Compared with the non-targeting nanogels, treatment with peptide conjugated nanogels attenuates neointimal hyperplasia more effectively. The biochemical assays further reveal that the enhanced restenosis prevention effect is contributed to the selective release of RAPA at the injured sites of artery, which notably potentiate the nanosystem as a systemically targeting delivered treatment.

Abstract 129: Parthenolide Inhibits Inflammatory Dysfunction of Human Aortic Endothelial Cells and Proliferation of Smooth Muscle Cells in vitro and Restenosis in a Rat Model

Background: Cardiovascular diseases remain the leading cause of death in developed countries. Endovascular surgical interventions trigger uncontrolled proliferation of smooth muscle cells (SMCs) in the intima (intimal hyperplasia) leading to lumen re-narrowing (restenosis). Despite extensive efforts devoted to therapeutic methods with a focus on inhibiting SMC proliferation, restenosis persists at a significant rate. In recent years, a growing body of knowledge has underscored a crucial role for endothelium damage in the pathophysiology of restenosis, suggesting that preservation of the endothelium would provide an effective approach for effectively curbing restenosis. Methods and Results: We utilized high-throughput screening methods to search for small molecules that could inhibit the proliferation of human aortic SMCs without damaging human aortic endothelial cells (ECs). We identified such a lead compound in Prestwick Library; i.e. Parthenolide, a sesquiterpene lactone extracted from feverfew. Treatment with a low dose (1 μM) of Parthenolide for 96h inhibited SMC but not EC proliferation. When the cells were stimulated with inflammatory cytokines (TNF-α or IL-1β), Parthenolide mitigated cytokine-induced proliferation and MCP-1 production of SMCs, but rescued cytokine-induced endothelial dysfunction, including apoptosis, proliferation, decrease of eNOS, and increase of inflammatory markers. Mechanistic studies showed that while Parthenolide treatment in both SMCs and ECs induced NRF-2 activation (nuclear translocation), NRF-2 knockdown with siRNA diminished the aforementioned beneficial effects of Parthenolide in both cell types. In contrast, NFkB activation was not significantly affected by Parthenolide. In a rat balloon angioplasty model, perivascular delivery of Parthenolide in Pluronic gel effectively inhibited intimal hyperplasia and restenosis 14 days after surgery. Conclusion: These results indicate that the natural compound Parthenolide differentially attenuates pathological behaviors of both human vascular SMCs and ECs, whereas known agents as such are scarce. Thus Parthenolide may serve as a promising lead compound for future development of next-generation anti-restenotic therapeutics.

The Hemoglobin Homolog Cytoglobin in Smooth Muscle Inhibits Apoptosis and Regulates Vascular Remodeling.

The role of hemoglobin and myoglobin in the cardiovascular system is well established, yet other globins in this context are poorly characterized. Here, we examined the expression and function of cytoglobin (CYGB) during vascular injury. We characterized CYGB content in intact vessels and primary vascular smooth muscle (VSM) cells and used 2 different vascular injury models to examine the functional significance of CYGB in vivo. We found that CYGB was strongly expressed in medial arterial VSM and human veins. In vitro and in vivo studies indicated that CYGB was lost after VSM cell dedifferentiation. In the rat balloon angioplasty model, site-targeted delivery of adenovirus encoding shRNA specific for CYGB prevented its reexpression and decreased neointima formation. Similarly, 4 weeks after complete ligation of the left common carotid, Cygb knockout mice displayed little to no evidence of neointimal hyperplasia in contrast to their wild-type littermates. Mechanistic studies in the rat indicated that this was primarily associated with increased medial cell loss, terminal uridine nick-end labeling staining, and caspase-3 activation, all indicative of prolonged apoptosis. In vitro, CYGB could be reexpressed after VSM stimulation with cytokines and hypoxia and loss of CYGB sensitized human and rat aortic VSM cells to apoptosis. This was reversed after antioxidant treatment or NOS2 (nitric oxide synthase 2) inhibition. These results indicate that CYGB is expressed in vessels primarily in differentiated medial VSM cells where it regulates neointima formation and inhibits apoptosis after injury.

Inhibitory effects of ASP6537, a selective cyclooxygenase-1 inhibitor, on thrombosis and neointima formation in rats

IntroductionPercutaneous coronary interventions (PCIs), such as balloon angioplasty and stent placement, are effective in the treatment of coronary artery disease. PCI has drawbacks, however, including acute thrombosis after the procedure and restenosis of the vascular lumen due to abnormal neointimal hyperplasia. ASP6537 is a selective COX-1 inhibitor that has been investigated as a possible candidate for clinical development as an antiplatelet agent. In the present study, we evaluated the in vivo antithrombotic effect of ASP6537 and its effect on neointima formation after balloon angioplasty. Material and methodsThe antithrombotic effect of ASP6537 was examined using an arteriovenous shunt thrombosis model in rats while the effect of ASP6537 on neointima formation was evaluated in a rat carotid arterial balloon angioplasty model. ResultsIn the thrombosis study, ASP6537 dose-dependently decreased the protein content of the thrombus, while no prolongation of template bleeding time was observed. In the neointima study, ASP6537 reduced neointima formation. ConclusionsASP6537 may be a promising agent for the prevention of acute thrombosis and restenosis after PCI in place of aspirin.

Abstract 329: ST266 Attenuates Neointima Formation After Arterial Balloon Angioplasty in Rats

Objective: Post-angioplasty restenosis due to neointima formation has been attributed to the inflammatory response after acute endoluminal injury. ST266 (Noveome Biotherapeutics, Inc.) a novel secretome derived from proprietary GMP-cultured human Amnion-Derived Multipotent Progenitor (AMP) cells, has been shown to be anti-inflammatory and to promote wound healing. This study examined the therapeutic potential of ST266 in a rat arterial balloon angioplasty model. Methods: Animals were randomly divided in the following groups (N=7): no-treatment (noTx), systemic ST266, systemic AMPs and local AMP implants. Neointima hyperplasia was induced in the iliac artery of Sprague-Dawley male rats using a 2F Fogarty embolectomy catheter. After surgery, animals in ST266 groups received 0.1, 0.5 or 1ml IV ST266 q.d. In the systemic AMP groups, single-dose (SD) of 0.5 million (M) or 1 M AMPs was injected via Inferior Vena Cava after the angioplasty. In AMP implant experiment 1 M, 5 M or 20 M AMPs were implanted in 300 μL Matrigel (MTG) around the iliac artery after balloon angioplasty. 28 days after the surgery, the iliac arteries were removed for histologic analysis. Re-endothelialization index was measured 10 days after balloon angioplasty. Results: 1ml ST266 decreased Neointima/Neointima+Media ratio (N/NM) compared to noTx group (0.34±0.01 vs 0.54±0.04 resp.; p =0.004). ST266 also decreased Luminal Stenosis (LS) compared to noTx group (18.18±1.86 vs 39.23±5.75%; p =0.008). SD 1 M AMPs decreased LS compared to noTx group (39.23±5.75 vs 19.50±5.35%; p =0.033). Significant differences in N/NM were found between 20 M implanted AMPs and noTx group (0.35±0.02 vs 0.54±0.04 resp.; p =0.003) and the MTG-only group (0.53±0.05, p =0.007). 20 M implanted AMPs decreased the LS (16.78±2.47%) compared to both noTx (39.23±5.75%, p =0.001) and MTG-only groups (37.51±8.55%, p =0.016). 1ml ST266 significantly increased the re-endothelialization index 10 days after balloon angioplasty compared to noTx group (0.40±0.04 vs 0.14±0.037% resp., p =0.002). Conclusion: ST266 reduces neointima formation and luminal stenosis and increases the re-endothelialization index after balloon angioplasty. Further research to elucidate the underlying mechanisms is ongoing.

Roscovitine attenuates intimal hyperplasia via inhibiting NF-κB and STAT3 activation induced by TNF-α in vascular smooth muscle cells

Roscovitine is a selective CDK inhibitor originally designed as anti-cancer agent, which has also been shown to inhibit proliferation in vascular smooth muscle cells (VSMCs). However, its effect on vascular remodeling and its mechanism of action remain unknown. In our study, we created a new intimal hyperplasia model in male Sprague-Dawley rats by trypsin digestion method, which cause to vascular injury as well as the model of rat carotid balloon angioplasty. Roscovitine administration led to a significant reduction in neointimal formation and VSMCs proliferation after injury in rats. Western blot analysis revealed that, in response to vascular injury, TNF-α stimulation induced p65 and STAT3 phosphorylation and promoted translocation of these molecules into the nucleus. p65 can physically associate with STAT3 and bind to TNF-α-regulated target promoters, such as MCP-1 and ICAM-1, to initiate gene transcription. Roscovitine can interrupt activation of NF-κB and reduce expression of TNF-α-induced proinflammatory gene, thus inhibiting intimal hyperplasia. These findings provide a novel mechanism to explain the roscovitine-mediated inhibition of intimal hyperplasia induced by proinflammatory pathways.

Intraluminal delivery of thrombospondin-2 small interfering RNA inhibits the vascular response to injury in a rat carotid balloon angioplasty model.

In an effort to inhibit the response to vascular injury that leads to intimal hyperplasia, this study investigated the in vivo efficacy of intraluminal delivery of thrombospondin-2 (TSP-2) small interfering RNA (siRNA). Common carotid artery (CCA) balloon angioplasty injury was performed in rats. Immediately after denudation, CCA was transfected intraluminally (15 min) with one of the following: polyethylenimine (PEI)+TSP-2 siRNA, saline, PEI only, or PEI+control siRNA. CCA was analyzed at 24 h or 21 d by using quantitative real-time PCR and immunohistochemistry. TSP-2 gene and protein expression were significantly up-regulated after endothelial denudation at 24 h and 21 d compared with contralateral untreated, nondenuded CCA. Treatment with PEI+TSP-2 siRNA significantly suppressed TSP-2 gene expression (3.1-fold) at 24 h and TSP-2 protein expression, cell proliferation, and collagen deposition up to 21 d. These changes could be attributed to changes in TGF-β and matrix metalloproteinase-9, the downstream effectors of TSP-2. TSP-2 knockdown induced anti-inflammatory M2 macrophage polarization at 21 d; however, it did not significantly affect intima/media ratios. In summary, these data demonstrate effective siRNA transfection of the injured arterial wall and provide a clinically effective and translationally applicable therapeutic strategy that involves nonviral siRNA delivery to ameliorate the response to vascular injury.-Bodewes, T. C. F., Johnson, J. M., Auster, M., Huynh, C., Muralidharan, S., Contreras, M., LoGerfo, F. W., Pradhan-Nabzdyk, L. Intraluminal delivery of thrombospondin-2 small interfering RNA inhibits the vascular response to injury in a rat carotid balloon angioplasty model.

BET Bromodomain Blockade Mitigates Intimal Hyperplasia in Rat Carotid Arteries

BackgroundIntimal hyperplasia is a common cause of many vasculopathies. There has been a recent surge of interest in the bromo and extra-terminal (BET) epigenetic “readers” including BRD4 since the serendipitous discovery of JQ1(+), an inhibitor specific to the seemingly undruggable BET bromodomains. The role of the BET family in the development of intimal hyperplasia is not known. MethodsWe investigated the effect of BET inhibition on intimal hyperplasia using a rat balloon angioplasty model. ResultsWhile BRD4 was dramatically up-regulated in the rat and human hyperplastic neointima, blocking BET bromodomains with JQ1(+) diminished neointima in rats. Knocking down BRD4 with siRNA, or treatment with JQ1(+) but not the inactive enantiomer JQ1(−), abrogated platelet-derived growth factor (PDGF-BB)-stimulated proliferation and migration of primary rat aortic smooth muscle cells. This inhibitory effect of JQ1(+) was reproducible in primary human aortic smooth muscle cells. In human aortic endothelial cells, JQ1(+) prevented cytokine-induced apoptosis and impairment of cell migration. Furthermore, either BRD4 siRNA or JQ1(+) but not JQ1(−), substantially down-regulated PDGF receptor-α which, in JQ1(+)-treated arteries versus vehicle control, was also reduced. ConclusionsBlocking BET bromodomains mitigates neointima formation, suggesting an epigenetic approach for effective prevention of intimal hyperplasia and associated vascular diseases.

Abstract 18363: ApoA-I Suppresses Neointimal Hyperplasia Following Stent Deployment and Modulates Neointimal Cellular Phenotype

Background: Increases in smooth muscle cell (SMC) cholesterol content reduce the expression of SMC phenotypic markers and increase macrophage related markers. The balance of neointimal SMCs to macrophage-like cells may influence neointimal hyperplasia following stent deployment. Apolipoprotein (apo) A-I, the main protein component of high-density lipoproteins (HDL), mediates the efflux of cholesterol from cells via the cholesterol transporter ABCA1. In a murine model of balloon angioplasty and stenting, we aimed to investigate the effect of apoA-I on neointimal hyperplasia and neointimal cellular phenotype. Methods and Results: Thoracic aortic segments from apoE knockout mice underwent balloon angioplasty alone or balloon angioplasty with stenting then were carotid-interposition grafted into recipient mice. Mice received PBS or apoA-I (40mg/kg) injections on alternate days in the week prior to surgery and then until sacrifice (28 days). There were no changes in total cholesterol, LDL and HDL between groups at sacrifice and thrombosis rates were 16.7% lower in apoA-I injected mice. Histological analysis revealed that stented aortic sections from mice injected with apoA-I had significantly smaller neointimal areas (18%±6.9), compared to PBS control mice p&lt;0.05. The percentage of SMC alpha-actin positive staining was strikingly higher in the neointimas of apoA-I stented arteries (76%±34.3, p&lt;0.05), while RT-PCR analysis of balloon angioplasty injured aortas found mRNA levels of macrophage marker CD68 were lower in apoA-I infused mice (30%±8.9, p&lt;0.01). Furthermore, mRNA levels of ABCA1 and the cholesterol efflux regulator PPAR-gamma were significantly higher in apoA-I balloon-injured aortic segments (30%±13.8 and 48%±20.7, respectively), p&lt;0.05. Conclusion: ApoA-I infusions reduce neointimal area following stent deployment. In apoA-I infused mice, the cells of the neointima had significantly higher SMC alpha-actin expression and lower macrophage CD68 expression. Mechanistically, this may be due to increased SMC cholesterol efflux as apoA-I increased the expression of ABCA1 and PPAR-gamma. These findings have significant implications for therapeutic modulation of neointimal hyperplasia following stent deployment.

Deep sea water prevents balloon angioplasty-induced hyperplasia through MMP-2: an in vitro and in vivo study.

Major facts about the development of restenosis include vascular smooth muscle cells (VSMCs) proliferation and migration. A previous study showed that in vitro treatment with magnesium chloride has the potential to affect the proliferation and migration of VSMCs. Magnesium is the major element in deep sea water (DSW) and is a biologically active mineral. It is unclear whether DSW intake can prevent abnormal proliferation and migration of VSMCs as well as balloon angioplasty-induced neointimal hyperplasia. Thus, we attempted to evaluate the anti-restenotic effects of DSW and its possible molecular mechanisms. Several concentrations of DSW, based on the dietary recommendations (RDA) for magnesium, were applied to a model of balloon angioplasty in SD rats. The results showed that DSW intake markedly increased magnesium content within the vascular wall and reduced the development of neointimal hyperplasia. The immunohistochemical analysis also showed that the expression of proteins associated with cell proliferation and migration were decreased in the balloon angioplasty groups with DSW supplement. Furthermore, in vitro treatment with DSW has a dose-dependent inhibitory effect on serum-stimulated proliferation and migration of VSMCs, whose effects might be mediated by modulation of mitogen-activated protein kinase (MAPK) signaling and of the activity of matrix metalloproteinase-2 (MMP-2). Our study suggested that DSW intake can help prevent neointimal hyperplasia (or restenosis), whose effects may be partially regulated by magnesium and other minerals.

Adventitial Nab-Rapamycin Injection Reduces Porcine Femoral Artery Luminal Stenosis Induced by Balloon Angioplasty via Inhibition of Medial Proliferation and Adventitial Inflammation

Endovascular interventions on peripheral arteries are limited by high rates of restenosis. Our hypothesis was that adventitial injection of rapamycin nanoparticles would be safe and reduce luminal stenosis in a porcine femoral artery balloon angioplasty model. Eighteen juvenile male crossbred swine were included. Single-injury (40%-60% femoral artery balloon overstretch injury; n=2) and double-injury models (endothelial denudation injury 2 weeks before a 20%-30% overstretch injury; n=2) were compared. The double-injury model produced significantly more luminal stenosis at 28 days, P=0.002, and no difference in medial fibrosis or inflammation. Four pigs were randomized to the double-injury model and adventitial injection of saline (n=2) or 500 μg of nanoparticle albumin-bound rapamycin (nab-rapamycin; n=2) with an endovascular microinfusion catheter. There was 100% procedural success and no difference in endothelial regeneration. At 28 days, nab-rapamycin led to significant reductions in luminal stenosis, 17% (interquartile range, 12%-35%) versus 10% (interquartile range, 8.3%-14%), P=0.001, medial cell proliferation, P<0.001, and fibrosis, P<0.001. There were significantly fewer adventitial leukocytes at 3 days, P<0.001, but no difference at 28 days. Pharmacokinetic analysis (single-injury model) found rapamycin concentrations 1500× higher in perivascular tissues than in blood at 1 hour. Perivascular rapamycin persisted ≥8 days and was not detectable at 28 days. Adventitial nab-rapamycin injection was safe and significantly reduced luminal stenosis in a porcine femoral artery balloon angioplasty model. Observed reductions in early adventitial leukocyte infiltration and late medial cell proliferation and fibrosis suggest an immunosuppressive and antiproliferative mechanism. An intraluminal microinfusion catheter for adventitial injection represents an alternative to stent- or balloon-based local drug delivery.