Artery Balloon Injury Research Articles

Synthetically Engineered Targeted Gold Nanoparticles for the Prevention of Restenosis after Vascular Interventions

Wasserman, Molly A. MD; Jiang, Qun MD; Rink, Jonathan PhD; Kibbe, Melina R. MD, FACS, FAHA; Thaxton, Colby S. MD, PhD Author Information

Endothelial cell-targeted pVEGF165 polyplex plays a pivotal role in inhibiting intimal thickening after vascular injury

Upregulation of vascular endothelial growth factor (VEGF) expression can inhibit intimal thickening after vascular injury. However, the lack of efficient gene delivery systems leads to insufficient VEGF expression, which prevents its application in gene therapy. In the present study, to improve the delivery of the plasmid vector with the VEGF gene (pVEGF165) to the injured vessel wall, we explored the potentially important difference between endothelial cell-targeted and nontargeted polymeric carriers. The αvβ3 integrin is overexpressed on activated endothelial cells but not on normal quiescent vessels. In this study, CDG2-cRGD, synthesized by conjugating an αvβ3 integrin-binding cyclic arginylglycylaspartic acid (cRGD) peptide with the Generation 2 polycation polyamidoamine (PAMAMG2)-g-cyclodextrin (termed as CDG2), was developed as a targetable carrier. It was observed that the specific integrin–ligand interactions greatly enhanced cellular internalization of CDG2-cRGD in human umbilical vein endothelial cells (HUVECs), which are notoriously difficult to transfect. Consequently, HUVECs were found to show remarkably high levels of VEGF165 expression induced by the CDG2-cRGD polyplex. Interestingly, VEGF165 overexpression in vivo was more complex than that in vitro, and in vivo assays demonstrated that the stimulus response to balloon injury in arteries could obviously upregulate VEGF165 expression in the saline-treated group, although it was not enough to prevent intimal thickening. In gene-transfected groups, intravascular delivery of pVEGF165 with the CDG2-cRGD polyplex into rabbits after vascular injury resulted in a significant inhibition of intimal thickening at 4 weeks, whereas the low therapeutic efficacy in the nontargeted CDG2-treated group was only comparable to that in the saline-treated group. It is becoming clear that the conflicting results of VEGF165 gene therapy in two gene-transfected groups are reflective of the pivotal role of the cRGD-conjugated carriers in achieving the beneficial therapeutic effects of vascular gene therapy.

Carbon Monoxide Releasing Molecule Accelerates Reendothelialization after Carotid Artery Balloon Injury in Rat

ObjectiveThis study was aimed to investigate the effects of carbon monoxide releasing molecule (CORM-2), a novel carbon monoxide carrier, on the reendothelialization of carotid artery in rat endothelial denudation model. MethodsMale rats subjected to carotid artery balloon injury were treated with CORM-2, inactive CORM-2 (iCORM-2) or dimethyl sulfoxide (DMSO). The reendothelialization capacity was evaluated by Evans Blue dye and the immunostaining with anti-CD31 antibody. The number of circulating endothelial progenitor cells (EPCs) was detected by flow cytometry. The proliferation, migration, and adhesion of human umbilical vein endothelial cells (HUVECs) were assessed by using [3H]thymidine, Boyden chamber and human fibronectin respectively. The expressions of protein were detected by using western blot analysis. ResultsCORM-2 remarkably accelerated the re-endothelialization 5 d later and inhibited neointima formation 28 d later. In addition, the number of peripheral EPCs significantly increased in CORM-2-treated rats than that in iCORM-2 or DMSO-treated rats after 5 d later. In vitro experiments, CORM-2 significantly enhanced the proliferation, migration and adhesion of HUVECs. The levels of Akt, eNOS phosphorylation, and NO generation in HUVECs were also much higher in CORM-2 treated group. Blocking of PI3K/Akt/eNOS signaling pathway markedly suppressed the enhanced migration and adhesion of HUVECs induced by CORM-2. ConclusionsCORM-2 could promote endothelial repair, and inhibit neointima formation after carotid artery balloon injury, which might be associated with the function changes of HUVECs regulated by PI3K/Akt/eNOS pathway.

Dynamic expressions of monocyte chemo attractant protein-1 and CC chamomile receptor 2 after balloon injury and their effects in intimal proliferation.

ObjectiveThe dynamic expressions of monocyte chemo attractant protein-1 (MCP-1) and CC chamomile receptor 2 (CCR2) after balloon injury and their effects in intimal proliferation were discussed. In this study, the expression of MCP-1 and its receptor during the intimal proliferation in rat artery after balloon injury were studied.MethodsUsing the model of balloon injury of rats’ arteries, the changes of intimal proliferation were observed with optical microscopy and the expressions of MCP-1 and CCR2 at different times were examined with the methods of RT-PCR and immunohistochemistry. The expressions of MCP-1 and CCR2 in the arterial tissues were detected using reverse transcription polymerase chain reaction (RT-PCR) and analyzed by semi-quantitative method.ResultsThe expressions of MCP-1 and CCR2 mRNA began to gradually increase after balloon injury. The MCP-1 reached to the peak on the first day, but decreased gradually later on. Expressions of CCR2 mRNA began to increase on the first day and reached to the peak on the 7th day, but then started to decrease gradually until 28th day when we can still detect it. The expressions of MCP-1 proteins began to increase gradually after balloon injury and were obviously detected in the VSMC on the 4th and 7th day, until 14th day when we can still detect it clearly in the proliferating intima.ConclusionThe dynamic expressions of MCP-1, MCP-1 proteins and CCR2 mRNA after balloon injury were shown to play an important role in intimal proliferation.

Down-regulation of miR-23b induces phenotypic switching of vascular smooth muscle cells in vitro and in vivo.

Phenotypic switch of vascular smooth muscle cells (VSMCs) plays a key role in the pathogenesis of different vascular diseases, such as atherosclerosis and restenosis after coronary intervention. MicroRNAs have been identified as key regulators of VSMC biology. The miR-23b is highly expressed in VSMCs and it is involved in differentiation, proliferation, and migration of several non-vascular cell types. However, the role of miR-23b in vascular disease is currently unknown. Thus, the aim of the present study was to evaluate the role of miR-23b on VSMC phenotypic switch in vitro and after vascular injury in vivo. To determine the changes of miR-23b expression in the injured arterial wall, we used the standard rat carotid artery balloon injury model. In vivo studies demonstrated that miR-23b is down-regulated after vascular injury. Gain-of-function studies showed that overexpression of miR-23b inhibited VSMC proliferation and migration, whereas the opposite effect was obtained with the in vitro inhibition of miR-23b. We further demonstrated that miR-23b can significantly promote the expression of VSMC marker genes such as smooth muscle α-actin (ACTA2) and smooth muscle myosin heavy chain (MYH11). Overexpression of miR-23b in balloon-injured arteries by Ad-miR-23b markedly decreased neointimal hyperplasia. Finally, miR-23b specifically suppresses urokinase-type plasminogen activator, SMAD family member 3, and transcription factor forkhead box O4 (FoxO4) expression in phenotypically modulated VSMCs. By luciferase reporter assay, we validated the transcription factor FoxO4 as a direct target of miR-23b in VSMCs. We identify miR-23b as a novel regulator of VSMC phenotypic switch in vitro and following vascular injury in vivo.

Abstract 367: Pcsk6 Is a Key Protease Modulating Smooth Muscle Cell Activation in Vascular Remodeling and Plaque Vulnerability

Proprotein convertases (PCSKs) are conserved among species and involved in processing of MMPs and growth factors, but poorly characterised in atherosclerosis. Previously we demonstrated upregulation of PCSK6 in a large cohort of plaques from symptomatic vs. asymptomatic patients. This protease localized to smooth muscle cells (SMCs) and showed a positive correlation to markers of inflammation, extracellular matrix remodeling and cytokines in plaques. Here we aimed at elucidating its role in vascular development and disease. In zebrafish embryos PCSK6 localized to heart and vasculature and its ablation caused defective peripheral vascular patterning with cerebral and myocardial hemorrhage. Increased expression of PCSK6 in vascular pathologies was validated by microarrays from carotid plaques vs. undiseased arteries (n=32 patients, p<0.0001), abdominal (AAA, n=14, p<0.0001) and thoracic aortic aneurysms (TAA, n=244, p=0.012). By immunohistochemistry, PCSK6 localized mainly to SMCs in the fibrous cap and neo-vessels in plaques, AAA and TAA tissues. Investigation of mouse carotid ligation, rat artery balloon injury and human restenosis tissues with pronounced intimal hyperplasia confirmed induction of PCSK6 in proliferating SMCs. By microarrays following progression of rat carotid injury, PCSK6 was downregulated in early phases defined by inflammatory pathways, while upregulated in later phases with SMCs activation and localized in SMCs. Expression of PCSK6 in this model was positively correlated to PDGFB and IGF1 (Spearman r>0.7, p<0.0001). PCSK6 overexpression in SMCs in vitro increased their migration in a wound-healing assay, especially upon PDGFBB stimulation. By eQTL analyses several polymorphisms in the PCSK6 gene were found to influence its expression in plaques and aneurysm tissue. Among these, rs6598465 showed association with maximum progression of arterial intima-media thickness in high-risk coronary artery disease subjects (n=3400, p=0.037). We identified a functional link between elevated expression of PCSK6 and vascular remodeling characterized by SMC activation. The present study establishes PCSK6 as one of the key modulators of pathological processes in relation to plaque vulnerability.

P55γ functional mimetic peptide N24 blocks vascular proliferative disorders.

Proliferation and migration disorders of vascular smooth muscle cells (VSMCs) contribute to the pathogenesis of proliferative cardiovascular diseases. Although, over the past two decades, a large panel of drugs has been developed for targeting VSMC proliferation, cardiovascular disease remains the leading cause of death worldwide. Thus, there is a compelling need to identify novel signaling pathways and molecules controlling VSMC proliferation and migration, to provide not only mechanistic insights but also safe and effective therapies for the treatment of cardiovascular diseases. Our recent studies have demonstrated that p55γ, a regulatory subunit of phosphoinositide 3-kinase, functions as an endogenous brake on VSMC proliferation. Here, we demonstrate that the small peptide N24, the first 24 amino acids of the NH2 terminus of p55γ, is a functional mimetic which negatively regulates VSMC proliferation and migration. Specifically, luminal delivery of adenovirus expressing N24 or local administration of Tat transactivator protein (TAT)-tagged N24 by pluronic gel alleviates neointimal formation following balloon injury in rat carotid arteries. Enforced expression of N24 suppresses the proliferation and migration of VSMCs induced by serum- or platelet-derived growth factor-BB. Mechanistically, N24 induces cell cycle arrest via activating the p53-p21 signal pathway, without triggering cell death. N24 interacts with and stabilizes p53 by blocking its ubiquitin-dependent degradation, subsequently promotes p21 transcription, and arrests cell cycle progression. Indeed, knockdown of p21 or p53 abrogates the N24-mediated cell growth arrest. Thus, N24 is a p55γ mimetic inhibiting VSMC proliferation as well as migration, thereby conferring important therapeutic implications for anti-proliferative treatment. • N24 attenuates balloon injury-induced neointimal formation. • Overexpression of N24 inhibits cultured VSMC proliferation and migration. • Overexpression of N24 arrests the cell cycle at S phase. • N24 interacts with and stabilizes p53 resulting in growth suppression.

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.

Targeted inhibitory effect of Lenti-SM22alpha-p27-EGFP recombinant lentiviral vectors on proliferation of vascular smooth muscle cells without compromising re-endothelialization in a rat carotid artery balloon injury model.

AimsIn-stent restenosis remains a serious problem after the implantation of drug-eluting stents, which is attributable to neointima formation and re-endothelialization. Here, we tried to find a new method which aims at selectively inhibiting proliferation of vascular smooth muscle cells (VSMC) proliferation without inhibition of re-endothelialization.Methods and ResultsWe used the smooth muscle-specific SM22alpha promoter in a recombinant lentiviral vector to drive overexpression of cell-cycle inhibitor, p27, in VSMCs. p27 effectively inhibited VSMC proliferation mediated by cell cycle arrest at the G0/G1 checkpoint. The SM22alpha-p27 lentiviral vector inhibited VSMC proliferation more effectively than paclitaxel. Rats infected with Lenti-SM22alpha-p27 had a significantly lower intima/media (I/M) ratio and also showed inhibition of restenosis on day 28 after balloon injury. Moreover, the repair of injured endothelium, and re-endothelialization of the carotid artery wall, was not affected by the smooth muscle cell-specific expression of p27.ConclusionA recombinant lentiviral vector carrying the SM22alpha promoter was used to effectively infect and selectively overexpress p27 protein in VSMCs, leading to inhibition of intimal hyperplasia without compromising endothelial repair.

Nitric oxide affects UbcH10 levels differently in type 1 and type 2 diabetic rats

BackgroundNitric oxide (NO) more effectively inhibits neointimal hyperplasia in type 2 diabetic versus nondiabetic and type 1 diabetic rodents. NO also decreases the ubiquitin-conjugating enzyme UbcH10, which is critical to cell-cycle regulation. This study seeks to determine whether UbcH10 levels in the vasculature of diabetic animal models account for the differential efficacy of NO at inhibiting neointimal hyperplasia. Materials and methodsVascular smooth muscle cells (VSMCs) harvested from nondiabetic lean Zucker (LZ) and type 2 diabetic Zucker diabetic fatty (ZDF) rats were exposed to high glucose (25 mM) and high insulin (24 nM) conditions to mimic the diabetic environment in vitro. LZ, streptozotocin-injected LZ (STZ, type 1 diabetic), and ZDF rats underwent carotid artery balloon injury (±10 mg PROLI/NO), and vessels were harvested at 3 and 14 d. UbcH10 was assessed by Western blotting and immunofluorescent staining. ResultsNO more effectively reduced UbcH10 levels in LZ versus ZDF VSMCs; however, addition of insulin and glucose dramatically potentiated the inhibitory effect of NO on UbcH10 in ZDF VSMCs. Three days after balloon injury, Western blotting showed NO decreased free UbcH10 and increased polyubiquitinated UbcH10 levels by 35% in both STZ and ZDF animals. Fourteen days after injury, immunofluorescent staining showed increased UbcH10 levels throughout the arterial wall in all animal models. NO decreased UbcH10 levels in LZ and STZ rats but not in ZDF. ConclusionsThese data suggest a disconnect between UbcH10 levels and neointimal hyperplasia formation in type 2 diabetic models and contribute valuable insight regarding differential efficacy of NO in these models.

Regulation of mitochondrial morphology by positive feedback interaction between PKCδ and Drp1 in vascular smooth muscle cell.

Dynamin-related protein-1 (Drp1) plays a critical role in mitochondrial fission which allows cell proliferation and Mdivi-1, a specific small molecule Drp1 inhibitor, is revealed to attenuate proliferation. However, few molecular mechanisms-related to Drp1 under stimulus for restenosis or atherosclerosis have been investigated in vascular smooth muscle cells (vSMCs). Therefore, we hypothesized that Drp1 inhibition can prevent vascular restenosis and investigated its regulatory mechanism. Angiotensin II (Ang II) or hydrogen peroxide (H2 O2 )-induced proliferation and migration in SMCs were attenuated by down-regulation of Drp1 Ser 616 phosphorylation, which was demonstrated by in vitro assays for migration and proliferation. Excessive amounts of ROS production and changes in mitochondrial membrane potential were prevented by Drp1 inhibition under Ang II and H2 O2 . Under the Ang II stimulation, activated Drp1 interacted with PKCδ and then activated MEK1/2-ERK1/2 signaling cascade and MMP2, but not MMP9. Furthermore, in ex vivo aortic ring assay, inhibition of the Drp1 had significant anti-proliferative and -migration effects for vSMCs. A formation of vascular neointima in response to a rat carotid artery balloon injury was prevented by Drp1 inhibition, which shows a beneficial effect of Drp1 regulation in the pathologic vascular condition. Drp1-mediated SMC proliferation and migration can be prevented by mitochondrial division inhibitor (Mdivi-1) in in vitro, ex vivo and in vivo, and these results suggest the possibility that Drp1 can be a new therapeutic target for restenosis or atherosclerosis.

Vascular wall hypoxia promotes arterial thrombus formation via augmentation of vascular thrombogenicity.

Atherosclerotic lesions represent a hypoxic milieu. However, the significance of this milieu in atherothrombosis has not been established. We aimed to assess the hypothesis that vascular wall hypoxia promotes arterial thrombus formation. We examined the relation between vascular wall hypoxia and arterial thrombus formation using a rabbit model in which arterial thrombosis was induced by 0.5 %-cholesterol diet and repeated balloon injury of femoral arteries. Vascular wall hypoxia was immunohistochemically detected by pimonidazole hydrochloride, a hypoxia marker. Rabbit neointima and THP-1 macrophages were cultured to analyse prothrombotic factor expression under hypoxic conditions (1 % O2). Prothrombotic factor expression and nuclear localisation of hypoxia-inducible factor (HIF)-1α and nuclear factor-kappa B (NF-κB) p65 were immunohistochemically assessed using human coronary atherectomy plaques. Hypoxic areas were localised in the macrophage-rich deep portion of rabbit neointima and positively correlated with the number of nuclei immunopositive for HIF-1α and NF-κB p65, and tissue factor (TF) expression. Immunopositive areas for glycoprotein IIb/IIIa and fibrin in thrombi were significantly correlated with hypoxic areas in arteries. TF and plasminogen activator inhibitor-1 (PAI-1) expression was increased in neointimal tissues and/or macrophages cultured under hypoxia, and both were suppressed by inhibitors of either HIF-1 or NF-κB. In human coronary plaques, the number of HIF-1α-immunopositive nuclei was positively correlated with that of NF-κB-immunopositive nuclei and TF-immunopositive and PAI-1-immunopositive area, and it was significantly higher in thrombotic plaques. Vascular wall hypoxia augments the thrombogenic potential of atherosclerotic plaque and thrombus formation on plaques via prothrombotic factor upregulation.

Histone Demethylase JMJD2A Inhibition Attenuates Neointimal Hyperplasia in the Carotid Arteries of Balloon-Injured Diabetic Rats via Transcriptional Silencing: Inflammatory Gene Expression in Vascular Smooth Muscle Cells

Background/Aims: Diabetic patients suffer from severe neointimal hyperplasia following angioplasty. The epigenetic abnormalities are increasingly considered to be relevant to the pathogenesis of diabetic cardiovascular complications. But the epigenetic mechanisms linking diabetes and coronary restenosis have not been fully elucidated. In this study, we explored the protective effect and underlying mechanisms of demethylases JMJD2A inhibition in balloon-injury induced neointimal formation in diabetic rats. Methods: JMJD2A inhibition was achieved by the chemical inhibitor 2,4-pyridinedicarboxylic acid (2,4-PDCA) and small interfering RNA (siRNA). In vitro, we investigated the proliferation, migration and inflammation of rat vascular smooth muscle cells (VSMCs) in response to high glucose (HG). In vivo, diabetic rats induced using high-fat diet and low-dose streptozotocin (35mg/kg) underwent carotid artery balloon injury. Morphometric analysis was performed using hematein eosin and immumohistochemical staining. Chromatin Immunoprecipitation (ChIP) was conducted to detect modification of H3K9me3 at inflammatory genes promoters. Results: The global JMJD2A was increased in HG-stimulated VSMCs and balloon-injured arteries of diabetic rats, accompanied by decreased H3K9me3. The inhibition of JMJD2A suppressed VSMCs proliferation, migration and inflammation induced by high glucose (HG) in vitro. And JMJDA2A inhibition attenuated neointimal formation in balloon-injured diabetic rats. The underlying mechanisms were relevant to the restoration of H3K9me3 levels at the promoters of MCP-1 and IL-6, and then the suppressed expression of MCP-1 and IL-6. Conclusion: The JMJD2A inhibition significantly attenuated neointimal formation in balloon injured diabetic rats via the suppression of VSMCs proliferation, migration, and inflammation by restoring H3K9me3.

Shuangshen Ningxin Capsule, a Traditional Chinese Medicinal Preparation, Alleviates Myocardial Ischemia through Autophagy Regulation.

Shuangshen Ningxin capsule (SSNX), a modern Chinese formula, has been used to treat cardiovascular diseases in Eastern Asia. Our study focuses on the autophagy regulation of SSNX against coronary artery injuries. Myocardial infarction model was established in Chinese miniswines (CMS) by coronary artery balloon injury. SSNX was administered to the CMS for 8 weeks with 4 mg/kg or 16 mg/kg. Myocardial cells were incubated with 20% SSNX medicated serum for 2 hours. Assays were performed to detect the effects of SSNX on (i) coronary artery diameter by angiography, (ii) hemodynamics by noninvasive hemodynamic monitoring system, (iii) plaque burden and plaque volume by intravenous ultrasound (iv) coronary artery histology by H&E staining, (v) autophagosome by transmission electron microscopy, (vi) lactate dehydrogenase (LDH) leakage, and (vii) Beclin-1 and LC3-I/II expressions by Western blot. The results showed that CMS treated with SSNX exhibited the correction for the disturbed cardiac hemodynamics, increase of coronary artery diameter, reduction of high plaque burden and plaque volume, and decrease of LDH. The inhibitory effect of SSNX on CMS autophagy was demonstrated by the reduction of autophagosome and the downregulation of beclin-1 and LC3-I/II. SSNX may protect coronary artery and increase the stability of plaque through the suppression of myocardial cellular autophagy, which suggests the potentially therapeutic effect of SSNX on ischemic cardiovascular disease.

Vascular balloon injury and intraluminal administration in rat carotid artery.

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

Abstract 17269: Extracelllular Protein Disulfide Isomerase Reshapes Vascular Architecture Against Constrictive Remodeling

INTRODUCTION: Vascular remodeling orchestrates a complex network of signaling pathways responsible for pathological changes in many vascular diseases such as atherosclerosis. We investigated the role of endoplasmic reticulum chaperone Protein Disulfide Isomerase (PDI) and the extracellular PDI (ecPDI) pool in vascular caliber and architecture during vascular repair and remodeling after injury (AI). METHODS AND RESULTS: After rabbit iliac artery balloon injury, PDI is markedly increased at mRNA and protein levels (25-fold vs. basal 14 days AI), with increase in both intracellular and ecPDI. Silencing PDI by siRNA in vitro induced ER stress markers upregulation and apoptosis (assessed by TUNEL assay). PDI knockdown also upregulated proliferation marker PCNA and decreased differentiation marker calponin-C. Furthermore, ecPDI inhibition prevents injury-increased hydrogen peroxide generation and decreases arterial nitrate (NO3-) level. EcPDI neutralization in vivo with PDIAb-containing perivascular gel from days 12-14AI promoted 25% decrease in vascular caliber at arteriography and similar decreases in total vessel circumference at optical coherence tomography, without changing neointima, indicating increased constrictive remodeling. EcPDI neutralization promoted striking changes in collagen, with switch from circumferential to radial fiber orientation towards a more rigid fiber type. Collagen type I and III were decreased after ecPDI inhibition in arteries 14 days AI. Cytoskeleton architecture was also disrupted, with loss of stress fiber coherent organization and switch from thin to medium-thickness actin fibers. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling. There was decreased PDI expression in media and intima from plaques exhibiting constrictive remodeling and, conversely, enhanced PDI expression in media of plaques depicting outward remodeling. CONCLUSIONS: Thus, PDI is highly upregulated after injury and reshapes matrix and cytoskeleton architecture to support an anticonstrictive remodeling effect. Such findings suggest an important role for PDI in lumen maintenance during vascular remodeling.

Vessel Ultrasound Sonographic Assessment of Soluble Receptor for Advanced Glycation End Products Efficacy in a Rat Balloon Injury Model

ObjectiveWe aimed to assess the therapeutic efficacy of differentially modified soluble receptor for advanced glycation end products (sRAGE) in vivo using vessel ultrasound sonography and to compare the sonography data with those from postmortem histomorphologic analyses to have a practical reference for future clinical applications. MethodsVessel ultrasound sonography was performed in a sRAGE-treated rat carotid artery balloon injury model at different time points after the surgery, and therapeutic efficacy of different doses of sRAGE produced in Chinese hamster ovary cells and with different N-glycoform modifications were assessed. ResultsVessel ultrasound sonography found that sRAGE produced in Chinese hamster ovary cells with complex N-glycoform modifications is highly effective, and is consistent with our recent findings in the same model assessed with histology. We also found that sonography is less sensitive than histology when a higher dose of sRAGE is administered. ConclusionsSonograph results are consistent with those obtained from histology; that is, sRAGE produced in Chinese hamster ovary cells has significantly higher efficacy than insect cell-originated sRAGE cells.

Nitric oxide inhibits neointimal hyperplasia following vascular injury via differential, cell-specific modulation of SOD-1 in the arterial wall

Superoxide (O2•−) promotes neointimal hyperplasia following arterial injury. Conversely, nitric oxide (•NO) inhibits neointimal hyperplasia through various cell-specific mechanisms, including redox regulation. What remains unclear is whether •NO exerts cell-specific regulation of the vascular redox environment following arterial injury to inhibit neointimal hyperplasia. Therefore, the aim of the present study was to assess whether •NO exerts cell-specific, differential modulation of O2•− levels throughout the arterial wall, establish the mechanism of such modulation, and determine if it regulates •NO-dependent inhibition of neointimal hyperplasia. In vivo, •NO increased superoxide dismutase-1 (SOD-1) levels following carotid artery balloon injury in a rat model. In vitro, •NO increased SOD-1 levels in vascular smooth muscle cells (VSMC), but had no effect on SOD-1 in endothelial cells or adventitial fibroblasts. This SOD-1 increase was associated with an increase in sod1 gene expression, increase in SOD-1 activity, and decrease in O2•− levels. Lastly, to determine the role of SOD-1 in •NO-mediated inhibition of neointimal hyperplasia, we performed the femoral artery wire injury model in wild type and SOD-1 knockout (KO) mice, with and without •NO. Interestingly, •NO inhibited neointimal hyperplasia only in wild type mice, with no effect in SOD-1 KO mice. In conclusion, these data show the cell-specific modulation of O2•− by •NO through regulation of SOD-1 in the vasculature, highlighting its importance on the inhibition of neointimal hyperplasia. These results also shed light into the mechanism of •NO-dependent redox balance, and suggest a novel VSMC redox target to prevent neointimal hyperplasia.

Periadventitial atRA citrate-based polyester membranes reduce neointimal hyperplasia and restenosis after carotid injury in rats.

Oral all-trans retinoic acid (atRA) has been shown to reduce the formation of neointimal hyperplasia; however, the dose required was 30 times the chemotherapeutic dose, which already has reported side effects. As neointimal formation is a localized process, new approaches to localized delivery are required. This study assessed whether atRA within a citrate-based polyester, poly(1,8 octanediolcitrate) (POC), perivascular membrane would prevent neointimal hyperplasia following arterial injury. atRA-POC membranes were prepared and characterized for atRA release via high-performance liquid chromatography with mass spectrometry detection. Rat adventitial fibroblasts (AF) and vascular smooth muscle cells (VSMC) were exposed to various concentrations of atRA; proliferation, apoptosis, and necrosis were assessed in vitro. The rat carotid artery balloon injury model was used to evaluate the impact of the atRA-POC membranes on neointimal formation, cell proliferation, apoptosis, macrophage infiltration, and vascular cell adhesion molecule 1 (VCAM-1) expression in vivo. atRA-POC membranes released 12 μg of atRA over 2 wk, with 92% of the release occurring in the first week. At 24 h, atRA (200 μmol/l) inhibited [(3)H]-thymidine incorporation into AF and VSMC by 78% and 72%, respectively (*P = 0.001), with negligible apoptosis or necrosis. Histomorphometry analysis showed that atRA-POC membranes inhibited neointimal formation after balloon injury, with a 56%, 57%, and 50% decrease in the intimal area, intima-to-media area ratio, and percent stenosis, respectively (P = 0.001). atRA-POC membranes had no appreciable effect on apoptosis or proliferation at 2 wk. Regarding biocompatibility, we found a 76% decrease in macrophage infiltration in the intima layer (P < 0.003) in animals treated with atRA-POC membranes, with a coinciding 53% reduction in VCAM-1 staining (P < 0.001). In conclusion, perivascular delivery of atRA inhibited neointimal formation and restenosis. These data suggest that atRA-POC membranes may be suitable as localized therapy to inhibit neointimal hyperplasia following open cardiovascular procedures.

A selective microRNA-based strategy inhibits restenosis while preserving endothelial function.

Drugs currently approved to coat stents used in percutaneous coronary interventions do not discriminate between proliferating vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). This lack of discrimination delays reendothelialization and vascular healing, increasing the risk of late thrombosis following angioplasty. We developed a microRNA-based (miRNA-based) approach to inhibit proliferative VSMCs, thus preventing restenosis, while selectively promoting reendothelialization and preserving EC function. We used an adenoviral (Ad) vector that encodes cyclin-dependent kinase inhibitor p27(Kip1) (p27) with target sequences for EC-specific miR-126-3p at the 3' end (Ad-p27-126TS). Exogenous p27 overexpression was evaluated in vitro and in a rat arterial balloon injury model following transduction with Ad-p27-126TS, Ad-p27 (without miR-126 target sequences), or Ad-GFP (control). In vitro, Ad-p27-126TS protected the ability of ECs to proliferate, migrate, and form networks. At 2 and 4 weeks after injury, Ad-p27-126TS-treated animals exhibited reduced restenosis, complete reendothelialization, reduced hypercoagulability, and restoration of the vasodilatory response to acetylcholine to levels comparable to those in uninjured vessels. By incorporating miR-126-3p target sequences to leverage endogenous EC-specific miR-126, we overexpressed exogenous p27 in VSMCs, while selectively inhibiting p27 overexpression in ECs. Our proof-of-principle study demonstrates the potential of using a miRNA-based strategy as a therapeutic approach to specifically inhibit vascular restenosis while preserving EC function.