Trimethylamine-N-oxide (TMAO) promotes balloon injury-induced neointimal hyperplasia via upregulating Beclin1 and impairing autophagic flux.

BACKGROUND AND AIMS: TMAO is a microbiota-dependent metabolite associated with increased risk of various cardiovascular diseases. However, the relationship between TMAO and vascular injury-related neointimal hyperplasia is unclear. This study aimed to explore whether TMAO promotes neointimal hyperplasia after balloon injury and elucidate the underlying mechanism. METHODS AND RESULTS: Through hematoxylin and eosin staining and immunohistochemistry staining, we found that supplementary TMAO promoted balloon injury-induced neointimal hyperplasia, while reducing TMAO by antibiotic administration produced the opposite result. TMAO showed limited effect on rat aortic vascular smooth muscle cells (RAOSMCs) proliferation and migration. However, TMAO notably induced dysfunction of rat aortic vascular endothelial cells (RAOECs) in vitro and attenuated reendothelialization of carotid arteries after balloon injury in vivo. Autophagic flux was measured by fluorescent mRFP-GFP-LC3, transmission electron microscopy, and western blot. TMAO impaired autophagic flux, as evidenced by the accumulation of p62 and LC3II and high autophagosome to autolysosome ratios. Furthermore, we confirmed that Beclin1 level increased in TMAO-treated RAOECs and carotid arteries. Knocking down Beclin1 alleviated TMAO-induced autophagic flux impairment and neointimal hyperplasia. CONCLUSIONS: TMAO promoted neointimal hyperplasia through Beclin1-induced autophagic flux blockage, suggesting that TMAO is a potential target for improvement of vascular remodeling after injury.

α-Iso-cubebene attenuates neointima formation by inhibiting HMGB1-induced monocyte to macrophage differentiation via suppressing ROS production.

α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis, a medicinal herb used to improve cardiovascular symptoms. To investigate the mechanisms involved, the effects of ICB on cellular production of reactive oxygen species (ROS) was determined using cultured human THP-1 cells. When THP-1 cells were stimulated with HMGB1, cellular concentration of ROS was increased in dose- and time-dependent manners. These increases were significantly attenuated in cells pretreated with NADPH oxidase inhibitors, diphenyleneiodonium chloride and apocynin, but not by other inhibitors related to ROS generation in monocytes. The expression of constitutively expressed NADPH oxidase (NOX) subunits including NOX1, NOX2, NOX4 and NOX5 was not affected by HMGB1, but HMGB1-induced ROS production was exclusively attenuated in NOX2-deficient cells using siRNA, suggesting an enhanced NOX2 complex assembly. When cells were stimulated with HMGB1, p47phox phosphorylation at ser345, ser359 and ser370 was increased in dose- and time-dependent manners, which were significantly attenuated in ICB (3-10 µg/mL)-pretreated cells. In addition, HMGB1-induced monocyte-macrophage differentiation (MMD) in bone marrow-derived cells isolated from mice were significantly attenuated in cells treated with apocynin and ICB. Also, macrophage infiltration and intimal hyperplasia in the wire-injured femoral artery were significantly attenuated in ICB-treated mice compared to wild-type control mice. The results of this study show that ICB inhibits HMGB1-induced MMD by suppressing ROS production in monocytes, thus suggest that ICB has therapeutic potential for vascular inflammation with subsequent intimal hyperplasia related to vascular injury.

Plantamajoside Attenuates Neointima Formation via Upregulation of Tissue Inhibitor of Metalloproteinases in Balloon-Injured Rats.

The abnormal change of vascular smooth muscle cell (VSMC) behavior is an important cellular event leading to neointimal hyperplasia in atherosclerosis and restenosis. Plantamajoside (PMS), a phenylethanoid glycoside compound of the Plantago asiatica, has been reported to have anti-inflammatory, antioxidative, and anticancer activities. In this study, the protective effects of PMS against intimal hyperplasia and the mechanisms underlying the regulation of VSMC behavior were investigated. MTT and BrdU assays were performed to evaluate the cytotoxicity and cell proliferative activity of PMS, respectively. Rat aortic VSMC migrations after treatment with the determined concentration of PMS (50 and 150 µM) were evaluated using wound healing and Boyden chamber assays. The inhibitory effects of PMS on intimal hyperplasia were evaluated in balloon-injured (BI) rat carotid artery. PMS suppressed the proliferation in platelet-derived growth factor-BB-induced VSMC, as confirmed from the decrease in cyclin-dependent kinase (CDK)-2, CDK-4, cyclin D1, and proliferating cell nuclear antigen levels. PMS also inhibited VSMC migration, consistent with the downregulated expression and zymolytic activities of matrix metalloproteinase (MMP)2, MMP9, and MMP13. PMS specifically regulated MMP expression through p38 mitogen-activated protein kinase and focal adhesion kinase pathways. Tissue inhibitor of metalloproteinase (TIMP)1 and TIMP2 levels were upregulated via Smad1. TIMPs inhibited the conversion of pro-MMPs to active MMPs. PMS significantly inhibited neointimal formation in BI rat carotid arteries. In conclusion, PMS inhibits VSMC proliferation and migration by upregulating TIMP1 and TIMP2 expression. Therefore, PMS could be a potential therapeutic agent for vascular atherosclerosis and restenosis treatment.

Immunomodulation of IL-33 and IL-37 with Vitamin D in the Neointima of Coronary Artery: A Comparative Study between Balloon Angioplasty and Stent in Hyperlipidemic Microswine.

Inflammation is a major contributor to the development and progression of atherosclerosis. Interleukin (IL)-33 and IL-37, members of the IL-1 family, modulate inflammation, with IL-33 having a pro-inflammatory effect and IL-37 having anti-inflammatory properties. IL-37 is constitutively expressed at low levels but upregulated in inflammatory contexts. The aim of this study was to evaluate the effect of vitamin D on the expression of IL-33, IL-37, macrophages, and caspase-1 in the neointimal tissue of coronary artery in Yucatan microswine with vitamin D deficient, sufficient, and supplemented status. The intimal injury was induced by balloon angioplasty and stenting in the coronary artery, and tissues were harvested after 6 months. The expression of various proteins of interest was evaluated by immunostaining. Increased expression of IL-33 and IL-37 in the neointimal tissue of the vitamin D deficient, as compared to the sufficient and supplemented microswine, as revealed by histological evaluation and semi-quantitative analysis, suggested the immunomodulatory effect of vitamin D on the expression of IL-33 and IL-37. The minimal expression or absence of IL-33 and IL-37 expression in stented arteries is suggestive of an attenuated inflammatory response in stented arteries, compared to balloon angioplasty. The decreased IL-33 expression in the sufficient and supplemented microswine could be a potential mechanism for controlling the inflammatory process and neointima formation leading to attenuated luminal narrowing of the coronary artery. Overall, these results support supplementation of vitamin D to attenuate inflammation, neointima formation, and restenosis.

Vitamin D attenuates HMGB1-mediated neointimal hyperplasia after percutaneous coronary intervention in swine.

Intracoronary stenting is a common procedure in patients with coronary artery disease (CAD). Stent deployment stretches and denudes the endothelial layer, promoting a local inflammatory response, resulting in neointimal hyperplasia. Vitamin D deficiency associates with CAD. In this study, we examined the association of vitamin D status with high mobility group box 1 (HMGB1)-mediated pathways (HMGB1, receptor for advanced glycation end products [RAGE], and Toll-like receptor-2 and -4 [TLR2 and TLR4]) in neointimal hyperplasia in atherosclerotic swine following bare metal stenting. Yucatan microswine fed with a high-cholesterol diet were stratified to receive vitamin D-deficient (VD-DEF), vitamin D-sufficient (VD-SUF), and vitamin D-supplemented (VD-SUP) diet. After 6 months, PTCA (percutaneous transluminal balloon angioplasty) followed by bare metal stent implantation was performed in the left anterior descending (LAD) artery of each swine. Four months following coronary intervention, angiogram and optical coherence tomography (OCT) were performed and swine euthanized. Histology and immunohistochemistry were performed in excised LAD to evaluate the expression of HMGB1, RAGE, TLR2, and TLR4. OCT analysis revealed the greatest in-stent restenosis area in the LAD of VD-DEF compared to VD-SUF or VD-SUP swine. The protein expression of HMGB1, RAGE, TLR2, and TLR4 was significantly higher in the LAD of VD-DEF compared to VD-SUF or VD-SUP swine. Vitamin D deficiency was associated with both increased in-stent restenosis and increased HMGB1-mediated inflammation noted in coronary arteries following intravascular stenting. Inversely, vitamin D supplementation was associated with both a decrease in this inflammatory profile and in neointimal hyperplasia, warranting further investigation for vitamin D as a potential adjunct therapy following coronary intervention.

l-Theanine attenuates neointimal hyperplasia via suppression of vascular smooth muscle cell phenotypic modulation.

Neointimal hyperplasia is a prominent pathological phenomenon in the process of stent restenosis. Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) play major pathological processes involved in the development of restenosis. l-Theanine, one of the major amino acid components in green tea, has been reported to improve vascular function. Here we display the effects of l-theanine on neointima formation and the underlying mechanism. In the rat carotid-artery balloon-injury model, l-theanine greatly inhibited neointima formation and prevented VSMCs from a contractile phenotype switching to a synthetic phenotype. In vitro study showed that l-theanine significantly inhibited PDGF-BB-induced VSMC proliferation and migration, which was comparable with the effect of l-theanine on AngII-induced VSMC proliferation and migration. Western blot analysis demonstrated that l-theanine suppressed PDGF-BB and AngII-induced reduction of SMA and SM22α and increment of OPN, suggesting that l-theanine inhibited the transformation of VSMCs from contractile to the synthetic phenotype. Further experiments showed that l-theanine exhibits potential preventive effects on neointimal hyperplasia and related vascular remodeling via inhibition of phosphorylation of Elk-1 and activation of MAPK1. The present study provides the new experimental evidence that l-theanine has potential clinical application as an anti-restenosis agent for the prevention of restenosis.

Activation of heme oxygenase-1 by Ginkgo biloba extract differentially modulates endothelial and smooth muscle-like progenitor cells for vascular repair.

Vascular progenitors such as endothelial progenitor cells (EPCs) and smooth muscle-like progenitor cells (SMPCs) may play different roles in vascular repair. Ginkgo biloba extract (GBE) is an exogenous activator of heme oxygenase (HO)-1, which has been suggested to improve vascular repair; however, the detailed mechanisms have yet to be elucidated. This study aimed to investigate whether GBE can modulate different vascular progenitor cells by activating HO-1 for vascular repair. A bone marrow transplantation mouse model was used to evaluate the in vivo effects of GBE treatment on wire-injury induced neointimal hyperplasia, which is representative of impaired vascular repair. On day 14 of GBE treatment, the mice were subjected to wire injury of the femoral artery to identify vascular reendothelialization. Compared to the mice without treatment, neointimal hyperplasia was reduced in the mice that received GBE treatment for 28 days in a dose-dependent manner. Furthermore, GBE treatment increased bone marrow-derived EPCs, accelerated endothelial recovery, and reduced the number of SMPCs attached to vascular injury sites. The effects of GBE treatment on neointimal hyperplasia could be abolished by co-treatment with zinc protoporphyrin IX, an HO-1 inhibitor, suggesting the in vivo role of HO-1. In this in vitro study, treatment with GBE activated human early and late EPCs and suppressed SMPC migration. These effects were abolished by HO-1 siRNA and an HO-1 inhibitor. Furthermore, GBE induced the expression of HO-1 by activating PI3K/Akt/eNOS signaling in human late EPCs and via p38 pathways in SMPCs, suggesting that GBE can induce HO-1 in vitro through different molecular mechanisms in different vascular progenitor cells. Accordingly, GBE could activate early and late EPCs, suppress the migration of SMPCs, and improve in vivo vascular repair after mechanical injury by activating HO-1, suggesting the potential role of pharmacological HO-1 activators, such as GBE, for vascular protection in atherosclerotic diseases.

Role of polypyrimidine tract-binding protein 1/yin yang 2 signaling in regulating vascular smooth muscle cell proliferation and neointima hyperplasia.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular restenosis. We investigated whether polypyrimidine tract-binding protein 1 (PTBP1), a novel regulator of cell proliferation and differentiation, is implicated in VSMC proliferation and neointima hyperplasia responding to injury. C57BL/6 J mice of 10-12 weeks old were randomly divided into sham and carotid artery injury group. Primary VSMCs obtained from thoracic aortas of 10- to 12-week-old mice were treated with physiological saline and platelet derived growth factor-BB (PDGF-BB). Adenovirus expressing shCon, shPTBP1 or shYY2 were transfected into the injured common carotid artery or VSMCs. qRT-PCR and immunoblotting were used to determine the mRNA and protein expression levels, respectively. Immunohistochemical staining of H&E and Ki-67 were used to evaluate restenosis of vessels. Cell counting kit-8 assay and Ki-67 immunofluorescent staining were utilized to evaluate the rate of VSMC proliferation. The expression of PTBP1 were upregulated both in injured arteries and in PDGF-BB-treated VSMCs. PTBP1 inhibition significantly attenuated neointima hyperplasia and Ki-67 positive area induced by injury. Knockdown of PTBP1 in vitro also suppressed VSMC proliferation after PDGF-BB treatment. The effects of PTBP1 inhibition mentioned above were all abolished by knockdown of YY2. Finally, we identified four cell cycle regulators (p53, p21, Cdkn1c, Cdkn2b) that were regulated by PTBP1/YY2 axis both in vitro and in vivo. These findings demonstrated that PTBP1 is a critical regulator of VSMC proliferation and neointima hyperplasia via modulating the expression of YY2.

Salvianolic acid B inhibits Ang II-induced VSMC proliferation in vitro and intimal hyperplasia in vivo by downregulating miR-146a expression.

BACKGROUND: Salvianolic acid B (Sal B), a water-soluble compound extracted from Salvia miltiorrhiza that has been widely used to treat cardiovascular diseases for hundreds of years in China, exerts cardiovascular protection by multiple mechanisms. miR-146a is involved in vascular smooth muscle cell (VSMC) phenotypic modulation and proliferation. However, it has yet to be investigated whether the cardiovascular protective effect of Sal B is mediated by miR-146a. PURPOSE: To determine the relationship among the cardiovascular protective effect of Sal B, miR-146a expression, and VSMC proliferation. METHODS: MTS assay and cell counting were performed to evaluate the effect of Ang II, Sal B and miR-146a on VSMC proliferation. The neointima hyperplasia was assessed by hematoxylin/eosin staining. qRT-PCR was used to detect the expression of miR-146a, KLF5, cyclin D1 and PCNA. Western blot analysis was used to detect the expressions of KLF5, cyclin D1 and PCNA after miR-20b-5p was knocked down or overexpressed in VSMC. RESULTS: Sal B suppressed intimal hyperplasia induced by carotid artery ligation and decreased Ang II-induced VSMC proliferation by down-regulating the positive cell-cycle regulators KLF5 and cyclin D1. Further experiments showed that VSMC proliferation and upregulation of KLF5 and cyclin D1 induced by Ang II were accompanied by elevated miR-146a level. Furthermore, overexpression of miR-146a promoted and knockdown of miR-146a reduced Ang II-induced VSMC proliferation and ameliorated intimal hyperplasia induced by carotid artery ligation. Sal B inhibited Ang II-induced VSMC proliferation by suppressing miR-146a expression. CONCLUSION: Sal B inhibited Ang II-induced VSMC proliferation in vitro and intimal hyperplasia in vivo by downregulating miR-146a expression.

Catalpol Ameliorates Neointimal Hyperplasia in Diabetic Rats.

Catalpol, an iridoid glycoside, is an isolated natural product of Rehmannia glutinosa, which has been reported to have antidiabetic properties. This study investigated the vascular protective effects of catalpol in hyperglycemic rats with balloon-injured carotid arteries. Balloon injury stress led to the upregulation of monocyte chemoattractant protein-1 expression in rats with streptozotocin-induced diabetes. Western blotting and real-time PCR were performed. In situ hybridization, immunohistochemistry, and confocal analyses were employed. Monocyte chemoattractant protein-1 levels were increased through streptozotocin induction or balloon injury. After treatment with catalpol, the neointimal hyperplasia area was reduced 2 weeks after balloon injury in hyperglycemic rats. Real-time PCR and immunohistochemical analysis demonstrated reduced levels of monocyte chemoattractant protein-1 2 weeks after the balloon injury. Monocyte chemoattractant protein-1 expression was significantly increased in balloon-injured rats compared with the control groups. Thus, treatment with catalpol affected monocyte chemoattractant protein-1 expression. This study demonstrated that catalpol downregulated monocyte chemoattractant protein-1 expression in carotid arteries and ameliorated neointimal hyperplasia in hyperglycemic rats. The suppressive effect of monocyte chemoattractant protein-1 suggests that it plays a key role in neointimal hyperplasia. The results imply that catalpol is potentially effective for preventing hyperglycemia-related ischemic cardiac diseases.

Dihydromyricetin from ampelopsis grossedentata protects against vascular neointimal formation via induction of TR3.

Vine tea has been used as a medicinal herb in traditional Chinese medicine for hundreds of years. As the most abundant ingredient in vine tea, Dihydromyricetin (DHM) has been reported to exert anti-inflammatory, antioxidant, and anti-cardiovascular disease. However, the role of DHM in injury-induced neointimal formation remains poorly characterized. We determined the effects of DHM on ligation-induced carotid artery neointimal formation. We found that ligation-induced carotid artery neointimal formation could be significantly attenuated by DHM treatment. We provide evidence that DHM increases orphan nuclear receptor TR3 expression in smooth muscle cell (SMC) and carotid artery. Moreover, overexpression and loss-of-function strategies of TR3 were done to overexpression and knockdown of TR3, and demonstrate that DHM promotes SMC differentiation, however, inhibits SMC proliferation and migration, via regulating expression of TR3. Collectively, we reveal that DHM may be a therapeutic agent for the treatment of injury-induced vascular diseases.

Suppressive activities and mechanisms of ugonin J on vascular smooth muscle cells and balloon angioplasty-induced neointimal hyperplasia.

Neointimal hyperplasia (or restenosis) is primarily attributed to excessive proliferation and migration of vascular smooth muscle cells (VSMCs). In this study, we investigated the inhibitory effects and mechanisms of ugonin J on VSMC proliferation and migration as well as neointimal formation. Cell viability and the cell-cycle distribution were, respectively, analyzed using an MTT assay and flow cytometry. Cell migration was examined using a wound-healing analysis and a transwell assay. Protein expressions and gelatinase activities were, respectively, measured using Western blot and gelatin zymography. Balloon angioplasty-induced neointimal formation was induced in a rat carotid artery model and then examined using immunohistochemical staining. Ugonin J induced cell-cycle arrest at the G0 /G1 phase and apoptosis to inhibit VSMC growth. Ugonin J also exhibited marked suppressive activity on VSMC migration. Ugonin J significantly reduced activations of focal adhesion kinase, phosphoinositide 3-kinase, v-akt murine thymoma viral oncogene homolog 1, and extracellular signal-regulated kinase 1/2 proteins. Moreover, ugonin J obviously reduced expressions and activity levels of matrix metalloproteinase-2 and matrix metalloproteinase-9. In vivo data indicated that ugonin J prevented balloon angioplasty-induced neointimal hyperplasia. Our study suggested that ugonin J has the potential for application in the prevention of balloon injury-induced neointimal formation.

Desalted Salicornia europaea extract attenuated vascular neointima formation by inhibiting the MAPK pathway-mediated migration and proliferation in vascular smooth muscle cells.

Salicornia europaea L. (SE) has been used as folk medicine for the treatment of various diseases such as obesity, diabetes, and cancer. However, its effects on atherosclerotic events in vascular smooth muscle cells (VSMCs) remain unknown. The present study explored the effects of the ethyl acetate fraction of desalted SE hot water extract (SEWEAF) on atherosclerotic responses (especially migration and proliferation) in VSMCs and vascular neointima formation. Treatment with the SEWEAF significantly suppressed the platelet-derived growth factor (PDGF)-BB-induced VSMC migration and proliferation as well the phosphorylation of mitogen-activated protein kinases (MAPKs) such as the p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2. Moreover, oral administration of the SEWEAF resulted in the attenuation of neointima formation in balloon-injured rat carotid arteries. Additionally, HPLC analysis showed that the major components in the two subfractions of the SEWEAF were five phenolic acids and four flavonols. In the SEWEAF components, for which atherosclerosis-linked responses in VSMCs have not been known, p-coumaric acid, quercetin-3-ß-d-glucoside, and isorhamnetin-3-ß-d-glucoside inhibited both PDGF-BB-induced migration and proliferation and isorhamnetin attenuated only PDGF-BB-stimulated VSMC proliferation. These results suggest that the SEWEAF may suppress PDGF-BB-induced VSMC migration by downregulating the phosphorylation of p38 MAPK and ERK1/2, thus leading to the reduction of neointimal hyperplasia during vascular remodeling. Therefore, the desalted SE extract, SEWEAF may be a potential ingredient for dietary supplements or nutraceuticals to ameliorate and/or prevent vascular remodeling-related disorders.

Hyperbaric oxygen inhibits venous neointimal hyperplasia following arteriovenous fistulization.

Hypoxia following arteriovenous fistulization results in venous neointimal hyperplasia (VNH), potentially causing early arteriovenous fistula (AVF) dysfunction. In this study, we used hyperbaric oxygen (HBO) in a rabbit model of AVF to determine whether it could ameliorate early AVF failure. Chronic renal failure was induced by adenine in 96 adult rabbits randomly divided into 3 groups (n=32 in each group). The sham + HBO group underwent sham operation and received HBO. The AVF alone group underwent fistulization, but did not receive HBO. The AVF + HBO group underwent fistulization and received HBO. Each group was further divided into 4 subgroups of 8 rabbits each that were euthanized at 1, 7, 14 or 28 days post-operatively. At each time point, blood flow changes in the AVF venous segment were detected using a high-frequency duplex ultrasonography system. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and hematoxylin and eosin staining were performed to evaluate VNH. Western blot analysis was performed to confirm the expression of hypoxia-inducible factor (HIF)-1α. At 14 and 28 days following HBO treatment, blood flow in the AVF + HBO group was greater than that at day 0. The AVF + HBO group had a smaller ratio of intima to media area, a lower HIF-1α protein expression, and a smaller percentage of PCNA-positive cells in the proximal vein than did the AVF alone group. Our results thus suggest that continuous HBO treatment following AVF significantly inhibits VNH and promotes blood flow. Therefore, early AVF failure may be prevented by the use of HBO therapy.

Alkaloid rich fraction from Nelumbo nucifera targets VSMC proliferation and migration to suppress restenosis in balloon-injured rat carotid artery.

AIMS: Restenosis- an adverse consequence following angioplasty, and atherosclerosis are characterized by abnormal vascular smooth muscle cell (VSMC) proliferation and migration leading to neo-intima formation. In the present study, we investigated the inhibitory effects of alkaloid rich fraction (ARF) from Nelumbo nucifera and isolated compound neferine on platelet-derived growth factor (PDGF-BB) induced VSMC proliferation and migration in vitro and neo-intima formation in a rat carotid artery injury model. METHODS: PDGF-BB induced VSMC proliferation and migration was assessed using colorimetric assay and modified Boyden chamber method respectively. Gene expression of cell cycle associated molecules was determined by reverse transcription-polymerase chain reaction (RT-PCR). The signaling molecules such as PDGF-Rß, extracellular regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), P38, metalloproteinase (MMP)-9 and nuclear factor-kappa B (NF-κB) were determined by western blot analysis. Stress fiber formation was evaluated using immunofluorescence microscopy. The rat carotid artery balloon injury model was performed to assess the effect of ARF on neo-intima formation. RESULTS: ARF possessed the strongest anti-oxidant activities. The anti-proliferative activity of both ARF and neferine was due to suppression of cyclin D1, cyclin E and cyclin-dependent kinase (Cdk) gene expression. Moreover, ARF and neferine inhibited PDGF-Rß, ERK1/2, JNK and P38 activations and NF-κB translocation. Also, ARF and neferine inhibited VSMC migration by inhibiting MMP-9 activity without affecting cytoskeleton remodeling. In a rat carotid artery injury model, ARF inhibited neo-intima formation. CONCLUSION: Our results indicate that ARF targets VSMC proliferation and migration to attenuate neo-intima formation by inhibition of PDGF-Rß mediated signaling.

Long-term effect of stent coating with zedoary essential components on neointimal formation in the porcine coronary artery.

OBJECTIVE: To examine the effect of the zedoary essential component-eluting stent (ZES) on a porcine coronary neointimal formation. METHODS: ZES, sirolimus-eluting stents (SES), and bare metal stents (BMS) were randomly implanted in three different major epicardial vessels in 36 balloon-injured pigs. Coronary angiography, optical coherence tomography, and histomorphological analysis were used to determine antihyperplasia effects. RESULTS: ZES and SES had a significantly larger lumen diameter and area, and reduced diameter and area of stenosis in arteries at 30 and 90 days compared with arteries implanted with BMS (P<0.01). Histomorphometric analysis showed moderate inflammatory responses, such as infiltration of mononuclear cells, lymphocytes, and multinucleated giant cells in some arteries with SES compared with ZES (P<0.05). Injury scores were not different among the three groups at 30 and 90 days. The endothelialization score in the SES group was 2.69 ± 0.42 at 30 days and 2.83 ± 0.39 at 90 days compared with the ZES and BMS groups (both were 3.00 ± 0.00 at either 30 or 90 days, P<0.05). Well developed endothelium was observed in the ZES group, while incomplete endothelium and inflammatory cells were observed with stent struts partly naked at the vessel lumen in the SES group. CONCLUSION: The ZES inhibits neointimal hyperplasia with good endothelia coverage in the porcine balloon injury coronary model.

Cinaciguat prevents neointima formation after arterial injury by decreasing vascular smooth muscle cell migration and proliferation.

AIMS: Vascular smooth muscle cell (VSMC) migration, proliferation and remodeling of the extracellular matrix contribute to lumen loss after arterial injury leading to restenosis. Several studies indicated the role of the cyclic guanosine monophosphate signaling in neointimal formation. Cinaciguat, the novel soluble guanylate cyclase activator, currently being in phase IIb clinical trial, has been shown to exert antiplatelet and anti-remodeling effects in animal models of vascular pathology. In this study we investigated the effects of cinaciguat on post-injury arterial stenosis. METHODS AND RESULTS: Male Sprague-Dawley rats (n=100) underwent endothelial denudation by wire injury of the right common carotid artery. Cinaciguat (10mg/kg/day orally) were administered to 50 rats (1-, 2-, 3-day and 1-, 3-week treatment time), while 50 rats received placebo. A 3-week treatment resulted in a significantly reduced vascular stenosis (17.53 ± 10.84% in the treatment group vs. 43.25 ± 30.83% in the control wire injury group) and neointima/media area ratio (0.45 ± 0.32 in the treatment group vs. 1.09 ± 0.69 in the control wire injury group). By using quantitative real-time PCR, Western blot and immunohistochemistry, matrix-metallopreoteinase-9 (MMP-9) was found to be upregulated in the control-injured carotids over the whole follow-up, and cinaciguat significantly decreased MMP-9 expression by 3 weeks. As assessed by protein immunoblot, injury-induced local decrease of soluble guanylate cyclase ß1 subunit could be recovered by cinaciguat. In vitro wound healing assay with VSMCs revealed dose-dependent antimigratory and antiproliferative effects of cinaciguat. Plasma level of cyclic guanosine monophosphate was significantly elevated after 3 weeks of treatment. CONCLUSION: Our results show that cinaciguat prevents injury-induced neointimal hyperplasia by decreasing VSMC migration and proliferation through the regulation of MMP-9.

Nelumbo nucifera leaf extract inhibits neointimal hyperplasia through modulation of smooth muscle cell proliferation and migration.

OBJECTIVE: Endovascular injury induced by balloon withdrawal leads to the increased activation of matrix metalloproteinases (MMPs) in the vascular wall allowing the proliferated smooth muscle cells (SMCs) to digest the surrounding extracellular matrix and migrate from the media into the intima leading to the intimal thickening. The objective of this study was to examine the effect of Nelumbo nucifera leaf extract (NL) on intimal thickening of rat carotid artery injured by balloon catheter and on the proliferation and migration of cultured vascular smooth muscle cells (VSMCs) induced by tumor necrosis factor-α. METHODS: NL was administered orally using gastric sonde at three different doses, 100 mg kg(-1) (NL100), 400 mg kg(-1) (NL400), and 800 mg kg(-1) (NL800) for 4 wk from the day of balloon injury in the rats. VSMC proliferation and migration were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Boyden chamber methods, whereas enzymatic action of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) was carried out by gelatin zymography, and MMP-9 protein expression, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase phosphorylations were assessed by Western blot analyses. RESULTS: NL reduced the intimal thickening by suppressing VSMC's proliferation through inhibition of extracellular signal-regulated kinase 1/2 phosphorylation and their migration by reducing the expression of MMP-2 and -9 through inhibition of JNK1/2 phosphorylation. CONCLUSION: Thus, the results suggest that NL can be considered of therapeutic value in the prevention of atherosclerosis because restenosis after percutaneous transluminal coronary angioplasty can be considered a model of "accelerated atherosclerosis."

Laser-induced carotid artery injury model in the rat for therapeutic agent screening.

The aims of this study were to establish a rat model of carotid artery injury and to evaluate its suitability for evaluating therapeutic agents active against endothelial proliferation. Wistar-Kyoto rats were injected intravenously with the photochemically reactive dyes rose bengal or Evans blue, and the carotid artery was then focally irradiated with laser light of the appropriate wavelength. Histological sections of the carotid artery were analyzed to determine the appropriate parameters for this model. Ferulic acid was used to assess the suitability of this model for drug screening. No animal died as a result of the photochemical treatment. Endothelial proliferation in the carotid artery was observed in rats injected with rose Bengal and exposed to green laser light. Ferulic acid (400 mg/kg per day) significantly (p<0.05) reduced endothelial proliferation in the carotid artery 28 days after injury in dye-treated animals compared with vehicle-treated animals. This simple experimental rat model is suitable for studying factors inhibiting endothelial thickening after vessel damage and for developing therapeutic strategies active against endothelial proliferation.

NFAT regulates the expression of AIF-1 and IRT-1: yin and yang splice variants of neointima formation and atherosclerosis.

AIMS: Alternative transcription and splicing of the allograft inflammatory factor-1 (AIF-1) gene results in the expression of two different proteins: AIF-1 and interferon responsive transcript-1 (IRT-1).  Here, we explore the impact of AIF-1 and IRT-1 on vascular smooth muscle cell (VSMC) activation and neointima formation, the mechanisms underlying their alternative splicing, and associations of AIF-1 and IRT-1 mRNA with parameters defining human atherosclerotic plaque phenotype. METHODS AND RESULTS: Translation of AIF-1 and IRT-1 results in different products with contrasting cellular distribution and functions. Overexpression of AIF-1 stimulates migration and proliferation of human VSMCs, whereas IRT-1 exerts opposite effects. Adenoviral infection of angioplasty-injured rat carotid arteries with AdAIF-1 exacerbates intima hyperplasia, whereas infection with AdIRT-1 reduces neointima. Expression of these variants is modulated by changes in nuclear factor of activated T-cells (NFAT) activity.  Pharmacological inhibition of NFAT or targeting of NFATc3 with small interfering RNA (siRNA) lowers the AIF-1/IRT-1 ratio and favours an anti-proliferative outcome.  NFAT acts as a repressor on the IRT-1 transcriptional start site, which is also sensitive to interferon-γ stimulation. Expression of AIF-1 mRNA in human carotid plaques associates with less extracellular matrix and a more pro-inflammatory plaque and plasma profile, features that may predispose to plaque rupture. In contrast, expression of IRT-1 mRNA associates with a less aggressive phenotype and less VSMCs at the most stenotic region of the plaque. CONCLUSION: Inhibition of NFAT signalling, by shifting the AIF-1/IRT-1 ratio, may be an attractive target to regulate the VSMC response to injury and manipulate plaque stability in atherosclerosis.