Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

Background

Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy.

Methods and Results

HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS.

Conclusion

VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease.     

Liver damage and systemic inflammatory responses are exacerbated by the genetic deletion of CD39 in total hepatic ischemia

Liver ischemia reperfusion injury is associated with both local damage to the hepatic vasculature and systemic inflammatory responses. CD39 is the dominant vascular endothelial cell ectonucleotidase and rapidly hydrolyses both adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate. These biochemical properties, in tandem with 5′-nucleotidases, generate adenosine and potentially illicit inflammatory vascular responses and thrombosis. We have evaluated the role of CD39 in total hepatic ischemia reperfusion injury (IRI). Wildtype mice, Cd39-hemizygous mice (+/−) and matched Cd39-null mice (−/−); (n = 25 per group) underwent 45 min of total warm ischemia with full inflow occlusion necessitating partial hepatectomy. Soluble nucleoside triphosphate diphosphohydrolase (NTPDases) or adenosine/amrinone were administered to wildtype (n = 6) and Cd39-null mice (n = 6) in order to study protective effects in vivo. Parameters of liver injury, systemic inflammation, hepatic ATP determinations by P³¹-NMR and parameters of lung injury were obtained. All wildtype mice survived up to 7 days with minimal biochemical disturbances and minor evidence for injury. In contrast, 64% of Cd39+/− and 84% of Cd39-null mice required euthanasia or died within 4 h post-reperfusion with liver damage and systemic inflammation associated with hypercytokinemia. Hepatic ATP depletion was pronounced in Cd39-null mice posthepatic IRI. Soluble NTPDase or adenosine administration protected Cd39-deficient mice from acute reperfusion injury. We conclude that CD39 is protective in hepatic IRI preventing local injury and systemic inflammation in an adenosine dependent manner. Our data indicate that vascular CD39 expression has an essential protective role in hepatic IRI.     

Exosomes from mesenchymal stem cells expressing miR‐125b inhibit neointimal hyperplasia via myosin IE

Intercellular communication between mesenchymal stem cells (MSCs) and their target cells in the perivascular environment is modulated by exosomes derived from MSCs. However, the potential role of exosome‐mediated microRNA transfer in neointimal hyperplasia remains to be investigated. To evaluate the effects of MSC‐derived exosomes (MSC‐Exo) on neointimal hyperplasia, their effects upon vascular smooth muscle cell (VSMC) growth in vitro and neointimal hyperplasia in vivo were assessed in a model of balloon‐induced vascular injury. Our results showed that MSC‐Exo were internalised by VSMCs and inhibited proliferation and migration in vitro. Further analysis revealed that miR‐125b was enriched in MSC‐Exo, and repressed the expression of myosin 1E (Myo1e) by targeting its 3? untranslated region. Additionally, MSC‐Exo and exosomally transferred miR‐125b repressed Myo1e expression and suppressed VSMC proliferation and migration and neointimal hyperplasia in vivo. In summary, our findings revealed that MSC‐Exo can transfer miR‐125b to VSMCs and inhibit VSMC proliferation and migration in vitro and neointimal hyperplasia in vivo by repressing Myo1e, indicating that miR‐125b may be a therapeutic target in the treatment of vascular diseases.     

Nitric Oxide Inhibits Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia by Increasing the Ubiquitination and Degradation of UbcH10

Nitric oxide (NO) limits formation of neointimal hyperplasia in animal models of arterial injury in large part by inhibiting vascular smooth muscle cell (VSMC) proliferation through cell cycle arrest. The ubiquitin-conjugating enzyme UbcH10 is responsible for ubiquitinating cell cycle proteins for proper exit from mitosis. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing levels of UbcH10. Western blotting and immunofluorescent staining showed that NO reduced UbcH10 levels in a concentration-dependent manner in VSMC harvested from the abdominal aortas of Sprague-Dawley rats. Treatment with NO or siRNA to UbcH10 decreased both UbcH10 levels and VSMC proliferation (P<0.001), while increasing UbcH10 levels by plasmid transfection or angiotensin II stimulation increased VSMC proliferation to 150% (P=0.008) and 212% (P=0.002) of control, respectively. Immunofluorescent staining of balloon-injured rat carotid arteries showed a ~4-fold increase in UbcH10 levels, which was profoundly decreased following treatment with NO. Western blotting of carotid artery lysates showed no UbcH10 in uninjured vessels, a substantial increase in the injury alone group, and a significant decrease in the injury+NO group (~3-fold reduction versus injury alone). Importantly, in vitro and in vivo, a marked increase in polyubiquitinated UbcH10 was observed in the NO-treated VSMC and carotid arteries, respectively, indicating that NO may be decreasing unmodified UbcH10 levels by increasing its ubiquitination. Central to our hypothesis, we report that NO decreases UbcH10 levels in VSMC in vitro and following arterial injury in vivo in association with increasing polyubiquitinated-UbcH10 levels. These changes in UbcH10 levels correlate with VSMC proliferation and neointimal hyperplasia, making UbcH10 a promising therapeutic target for inhibiting this proliferative disease.     

血管损伤的新靶点：干扰素调节因子

内膜增生是血管损伤后动脉重塑过程中普遍存在的现象。血管平滑肌细胞（vascular smooth muscle cells,VSMCs）的增殖、迁移、表型转换是血管损伤性疾病高血压、动脉粥样硬化、血管成形术后再狭窄等的共同病理生理学过程。干扰素调节因子（interferon regulatory factors,IRFs）是一类能对干扰素基因表达起到免疫调节作用的转录因子。近来研究发现,其在血管损伤病理过程具有调节作用,其中IRF1与细胞生长、分化和损伤密切相关,IRF3与IRF7可以抑制新生内膜的形成,而IRF8和IRF9则促进VSMCs增殖、迁移及血管内膜增生。本文重点介绍了IRFs的结构特征、信号途径及在血管重塑过程中作为新型调控因子的功能。     

Effect of sinomenine on vascular smooth muscle cell dedifferentiation and neointima formation after vascular injury in mice

Sinomenine, a pure alkaloid extract from Sinomenium acutum, has anti-inflammatory and immunoregulatory functions. This study investigated the efficiency and the signalling pathways involved in the effect of sinomenine on vascular smooth muscle cell (VSMC) dedifferentiation in response to platelet-derived growth factor (PDGF)-BB stimulation and vascular injury. VSMCs were isolated from rat aorta and preincubated with sinomenine before being stimulated with PDGF-BB. WST and BrdU incorporation assays were used to evaluate VSMC proliferation. Flow cytometric analysis was performed for testing the cell cycle progression. The cell migration of VSMCs were analysed using a Transwell system. The expression of VSMC specific genes and signalling proteins were tested by Western blot. For the animal study, C57/BL6 mice were fed either normal rodent chow diets or sinomenine chow diets that supplemented with 0.09 % sinomenine (w/w) in the normal chows for 14 days before carotid artery wire injury. PDGF-BB activated the dedifferentiation of VSMCs characterised by decreased expression of SMA, Smoothelin and SM22α. However, sinomenine treatment preserved the dedifferentiation in response to PDGF-BB. The activations of mitogen-activated protein kinase extracellular signal-regulated kinases, Akt, GSK3β and STAT3 induced by PDGF-BB were also inhibited in sinomenine-treated VSMCs. In vivo evidence with wire-injured mice exhibited a reduction in neointimal area and an increase in smooth muscle-specific gene expression in the sinomenine-treated group. In this study, we found that sinomenine-suppressed VSMC phenotype switching induced by PDGF-BB in vitro and neointimal formation in vivo. Therefore, sinomenine is a potential candidate to be used in the treatment of vascular proliferative disease.     

Endothelial stress induces the release of vitamin D-binding protein, a novel growth factor

Endothelial cells (EC) under stress release paracrine mediators that facilitate accumulation of vascular smooth muscle cells (VSCM) at sites of vascular injury. We found that medium conditioned by serum-starved EC increase proliferation and migration of VSCM in vitro. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as vitamin D-binding protein (DBP). DBP induced both proliferation and migration of VSMC in vitro in association with increased phosphorylation of ERK 1/2. PD 98059, a biochemical inhibitor of ERK 1/2, abrogated these proliferative and migratory responses in VSMC. DBP is an important carrier for the vitamin-D sterols, 25-hydroxyvitamin-D, and 1alpha,25-dihydroxyvitamin-D. Both sterols inhibited the activity of DBP on VSMC, suggesting that vitamin D binding sites are important for initiating the activities of DBP on VSMC. Release of DBP at sites of endothelial injury represents a novel pathway favoring accumulation of VSMC at sites of vascular injury.     

Effects of TH-142177 on angiotensin II-induced proliferation, migration and intracellular signaling in vascular smooth muscle cells and on neointimal thickening after balloon injury.

We investigated the effects of TH-142177 (N-n-butyl-N-[2'-(1-H-tetrazole-5-yl) biphenyl-4-yl]-methyl-(N-carboxy methyl-benzylamino)-acetamide), a novel selective antagonist of angiotensin II type 1-receptor (AT1-R) on angiotensin II (AII)-induced proliferation and migration of vascular smooth muscle cells (VSMC), and on neointimal formation in the rat carotid artery after balloon injury, and on the intracellular signaling by the stimulation of AT1-R. High affinity AII receptor sites were detected in rat VSMC by the use of [125I]Sar1,Ile8-AII. TH-142177 and losartan competed with [125I]Sar1,Ile8-AII for the binding sites in VSMC in a monophasic manner, although PD123177, a selective antagonist of angiotensin II type 2-receptor (AT2-R), had little inhibitory effect, demonstrating the predominant existence of AT1-R in rat VSMC. TH-142177 prevented AII-induced DNA synthesis and migration, with a significant inhibition of 74 and 55%, respectively, at the concentration of 100 nM. AII-induced activation of p21ras, mitogen-activated protein kinase (p42MAPK and p44MAPK), and protein kinase C was significantly (50-78%) inhibited by TH-142177 (100 nM), suggesting that the activation of these enzymes is mediated through the stimulation of AT1-R. Balloon-injured left carotid arteries in rats showed extensive neointimal thickening, and TH-142177 (3 mg/kg) brought out a marked decrease in the neointimal thickening after balloon injury. In conclusion, TH-142177 inhibited AII-induced proliferation and migration of rat VSMC and neointimal formation in the carotid artery after balloon injury, and these effects may be related, in part, to the suppression of ras, p42MAPK and p44MAPK, and protein kinase C activities through the blockade of AT1-R. Thus, TH-142177 may have therapeutic potential for the treatment of vascular diseases such as atherosclerosis and restenosis.     

Role of Pin1 in neointima formation: Down-regulation of Nrf2-dependent heme oxygenase-1 expression by Pin1

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intima formation after stenting and balloon angioplasty. Pin1, a peptidyl prolyl isomerase recognizing phosphorylated Ser/Thr–Pro, isomerizes the peptide bond. Because Pin1 overexpression is associated with transformation and the uncontrolled cell growth of tumors, we hypothesized that Pin1 functions as a chronic stimulator of VSMC proliferation. Pin1-positive smooth muscle cells were seen in the neointimal region of the femoral artery after guidewire injury. Exposure of VSMCS to platelet-derived growth factor (PDGF) increased Pin1 expression in a concentration-dependent manner. Basal cell growth rate and cyclin D1 expression were enhanced in Pin1-overexpressing VSMCs (Pin1-VSMCs). Moreover, PDGF-induced production of reactive oxygen species (ROS) in Pin1-VSMCs was higher than in control VSMCs. In Pin1-VSMCs, heme oxygenase-1 (HO-1) induction in response to nitric oxide donor was suppressed compared to control VSMCs. Nuclear translocation of nuclear factor E2-related factor-2 (Nrf2) was also diminished in Pin1-VSMCs. In contrast, the activity of the inducible minimal antioxidant response element (ARE) was potentiated in Pin1-null mouse embryonic fibroblasts (MEFs), compared to Pin1-wild-type MEFs. Moreover, Nrf2 ubiquitination was stimulated by Pin1 overexpression. Intraperitoneal injection of juglone (a Pin1 inhibitor) for 3 weeks (1 mg/kg, two times a week) significantly suppressed neointimal formation induced by wire injury. In conclusion, Pin1 induction during neointimal formation may be associated with ROS-mediated VSMC proliferation via down-regulation of Nrf2/ARE-dependent HO-1 expression. Pin1 may be a novel therapeutic target for several vascular diseases including atherosclerosis and stenosis.     

Effect of nitric oxide on neointimal hyperplasia based on sex and hormone status

Nitric oxide (NO)-based therapies decrease neointimal hyperplasia; however, studies have been performed only in male animal models. Thus, we sought to evaluate the effect of NO on vascular smooth muscle cells (VSMC) in vitro and neointimal hyperplasia in vivo based on sex and hormone status. In hormone-replete medium, male VSMC proliferated at greater rates than female VSMC. In hormone-depleted medium, female VSMC proliferated at greater rates than male VSMC. However, in both hormone environments, NO inhibited proliferation and migration to a greater extent in male compared to female VSMC. These findings correlated with greater G₀/G₁ cell cycle arrest and changes in cell cycle protein expression in male compared to female VSMC after exposure to NO. Next, the rat carotid artery injury model was used to assess the effect of NO on neointimal hyperplasia in vivo. Consistent with the in vitro data, NO was significantly more effective at inhibiting neointimal hyperplasia in hormonally intact males compared to females using weight-based dosing. An increased weight-based dose of NO in females was able to achieve efficacy equal to that in males. Surprisingly, NO was less effective at inhibiting neointimal hyperplasia in castrated animals of both sexes. In conclusion, these data suggest that NO inhibits neointimal hyperplasia more effectively in males compared to females and in hormonally intact compared to castrated rats, indicating that the effects of NO in the vasculature may be sex- and hormone-dependent.     

Manganese superoxide dismutase inhibits neointima formation through attenuation of migration and proliferation of vascular smooth muscle cells

Superoxide anion is elevated during neointima development and is essential for neointimal vascular smooth muscle cell (VSMC) proliferation. However, little is known about the role of manganese superoxide dismutase (MnSOD, SOD2) in the neointima formation following vascular injury. SOD2 in the mitochondria plays an important role in cellular defense against oxidative damage. Because of its subcellular localization, SOD2 is considered the first line of defense against oxidative stress and plays a central role in metabolizing superoxide. Because mitochondria are the most important sources of superoxide anion, we speculated that SOD2 may have therapeutic benefits in preventing vascular remodeling. In this study, we used a rat carotid artery balloon-injury model and an adenoviral gene delivery approach to test the hypothesis that SOD2 suppresses vascular lesion formation. SOD2 was activated along with the progression of neointima formation in balloon-injured rat carotid arteries. Depletion of SOD2 by RNA interference markedly promoted the lesion formation, whereas SOD2 overexpression suppressed the injury-induced neointima formation via attenuation of migration and proliferation of VSMCs. SOD2 exerts its inhibitory effect on VSMC migration induced by angiotensin II by scavenging superoxide anion and suppressing the phosphorylation of Akt. Our data indicate that SOD2 is a negative modulator of vascular lesion formation after injury. Therefore, SOD2 augmentation may be a promising therapeutic strategy for the prevention of lesion formation in proliferative vascular diseases such as restenosis.     

Carotid artery ligation induced intimal thickening and proliferation is unaffected by ageing

Following interventions to treat atherosclerosis, such as coronary artery bypass graft surgery, restenosis occurs in approximately 40% of patients. Identification of proteins regulating intimal thickening could represent targets to prevent restenosis. Our group previously demonstrated that in a murine model of vascular occlusion, Wnt4 protein expression and β-catenin signalling was upregulated which promoted vascular smooth muscle cell (VSMC) proliferation and intimal thickening. In this study, the effect of age on VSMC proliferation, intimal hyperplasia and Wnt4 expression was investigated. In vitro proliferation of VSMCs isolated from young (2 month) or old (18–20 month) C57BL6/J mice was assessed by immunocytochemistry for EdU incorporation. As previously reported, 400 ng/mL recombinant Wnt4 protein increased proliferation of VSMCs from young mice. However, this response was absent in VSMCs from old mice. As our group previously reported reduced intimal hyperplasia in Wnt4^+/? mice compared to wildtype controls, we hypothesised that impaired Wnt4 signalling with age may result in reduced neointimal formation. To investigate this, carotid artery ligation was performed in young and old mice and neointimal area was assessed 21 days later. Surprisingly, neointimal area and percentage lumen occlusion were not significantly affected by age. Furthermore, neointimal cell density and proliferation were also unchanged. These data suggest that although Wnt4-mediated proliferation was impaired with age in primary VSMCs, carotid artery ligation induced neointimal formation and proliferation were unchanged in old mice. These results imply that Wnt4-mediated proliferation is unaffected by age in vivo, suggesting that therapeutic Wnt4 inhibition could inhibit restenosis in patients of all ages.     

Protective roles of endogenous carbon monoxide in neointimal development elicited by arterial injury

We reported that carbon monoxide (CO) generated through heme oxygenase (HO) inhibits mitogen-induced proliferation of vascular smooth muscle cells (VSMCs). We report that balloon injury induces HO-1, the stress-inducible isozyme of HO, in VSMCs and inhibits neointimal formation through the action of endogenous CO. Northern blot analysis and immunohistochemistry revealed that HO-1 is markedly induced in the media as early as 1 day after injury, whereas only a little expression was detected in the intact carotid artery. The neointimal proliferative changes were augmented or inhibited by the HO inhibitors or inducer, respectively, and effects of these interventions were not altered by suppression of endogenous nitric oxide (NO), if any. To elucidate the mechanisms by which HO controls the proliferative changes, effects of alterations in the HO reaction were examined by determining angiotensin II-elicited VSMC proliferation in vitro: the HO inducer attenuated and its inhibitor restored the proliferative response to angiotensin II (1 nM and 100 nM). Hemoglobin, a reagent trapping both NO and CO, but not met-hemoglobin, which can capture NO but not CO, augmented the proliferative response. These data suggest that endogenous CO serves as a protective factor that limits the excessive VSMC proliferation associated with vascular diseases.     

Apatinib attenuates phenotypic switching of arterial smooth muscle cells in vascular remodelling by targeting the PDGF Receptor‐β

Apatinib (YN968D1) is a small‐molecule tyrosine kinase inhibitor（TKI）which can inhibit the activity of vascular endothelial growth factor receptor‐2 (VEGFR‐2). It has been reported that apatinib has anti‐tumour effect of inhibiting proliferation and inducing apoptosis of a variety of solid tumour cells, whereas its effect on vascular smooth muscle cells (VSMC) remains unclear. This study investigated the effect of apatinib on phenotypic switching of arterial smooth muscle cells in vascular remodelling. Compared to the vehicle groups, mice that were performed carotid artery ligation injury and treated with apatinib produced a reduction in abnormal neointimal area. For in vitro experiment, apatinib administration inhibited VSMC proliferation, migration and reversed VSMC dedifferentiation with the stimulation of platelet‐derived growth factor type BB (PDGF‐BB).In terms of mechanism, with the preincubation of apatinib, the activations of PDGF receptor‐β (PDGFR‐β) and phosphoinositide‐specific phospholipase C‐γ1 (PLC‐γ1) induced by PDGF‐BB were inhibited in VSMCs. With the preincubation of apatinib, the phosphorylation of PDGFR‐β, extracellular signal‐related kinases (ERK1/2) and Jun amino‐terminal kinases (JNK) induced by PDGF‐BB were also inhibited in rat vascular smooth muscle cell line A7r5. Herein, we found that apatinib attenuates phenotypic switching of arterial smooth muscle cells induced by PDGF‐BB in vitro and vascular remodelling in vivo. Therefore, apatinib is a potential candidate to treat vascular proliferative diseases.     

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.     

ERK1/2 mediates PDGF-BB stimulated vascular smooth muscle cell proliferation and migration on laminin-5

During restenosis following arterial injury, vascular smooth muscle cells (VSMCs) form a neointimal layer in arteries by changing from a differentiated, contractile phenotype to a dedifferentiated, migratory, and proliferative phenotype. Several growth factors, cytokines, and extracellular matrix components released following injury have been implicated in these phenotypic changes. We have recently detected the expression of laminin-5, an ECM protein found predominantly in epithelial tissues, in the arterial vasculature. Here we report that ln-5 expression by VSMC is upregulated by platelet-derived growth factor (PDGF-BB), epidermal growth factor, basic fibroblast growth factor, and transforming growth factor-beta1. Adhesion to ln-5 specifically enhances PDGF-BB-stimulated VSMC proliferation and migration. PD98059, a specific inhibitor of the ERK1/2 members of the Mitogen Activated Protein kinase family, increases both VSMC adhesion to ln-5 and blocks PDGF-BB-stimulated VSMC migration on ln-5. These results suggest that adhesion to ln-5 mediates a PDGF-BB-stimulated VSMC response to vascular injury via an ERK1/2 signaling pathway.     

Thymoquinone suppresses platelet‐derived growth factor‐BB–induced vascular smooth muscle cell proliferation,migration and neointimal formation

The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are mainly responsible for vascular occlusion diseases, such as pulmonary arterial hypertension and restenosis. Our previous study demonstrated thymoquinone (TQ) attenuated monocrotaline‐induced pulmonary arterial hypertension. The aim of the present study is to systematically examine inhibitory effects of TQ on platelet‐derived growth factor‐BB (PDGF‐BB)–induced proliferation and migration of VSMCs in vitro and neointimal formation in vivo and elucidate the potential mechanisms. Vascular smooth muscle cells were isolated from the aorta in rats. Cell viability and proliferation were measured in VSMCs using the MTT assay. Cell migration was detected by wound healing assay and Transwell assay. Alpha‐smooth muscle actin (α‐SMA) and Ki‐67‐positive cells were examined by immunofluorescence staining. Reactive oxygen species (ROS) generation and apoptosis were measured by flow cytometry and terminal deoxyribonucleotide transferase–mediated dUTP nick end labelling (TUNEL) staining, respectively. Molecules including the mitochondria‐dependent apoptosis factors, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), PTEN/AKT and mitogen‐activated protein kinases (MAPKs) were determined by Western blot. Neointimal formation was induced by ligation in male Sprague Dawley rats and evaluated by HE staining. Thymoquinone inhibited PDGF‐BB–induced VSMC proliferation and the increase in α‐SMA and Ki‐67‐positive cells. Thymoquinone also induced apoptosis via mitochondria‐dependent apoptosis pathway and p38MAPK. Thymoquinone blocked VSMC migration by inhibiting MMP2. Finally, TQ reversed neointimal formation induced by ligation in rats. Thus, TQ is a potential candidate for the prevention and treatment of occlusive vascular diseases.     

Attenuation of neointimal vascular smooth muscle cellularity in atheroma by plasminogen activator inhibitor type 1 (PAI-1).

Rupture of vulnerable atheroma often underlies acute coronary syndromes. Vulnerable plaques exhibit a paucity of vascular smooth muscle cells (VSMCs) in the cap. Therefore, decreased VSMC migration into the neointima may predispose to vulnerability. The balance between cell surface plasminogen activator activity and its inhibition [mediated primarily by plasminogen activator inhibitor type 1 (PAI-1)] modulates migration of diverse types of cells. We sought to determine whether increased expression of PAI-1 would decrease migration of VSMCs in vitro and neointimal cellularity in vivo in apolipoprotein E knockout (ApoE(-/-)) mice fed a high-fat diet. Increased vessel wall expression of PAI-1 in transgenic mice was induced with the SM22alpha promoter. VSMC migration through Matrigel in vitro was quantified with laser scanning cytometry. Expression of PAI-1 was increased threefold in the aortic wall of SM22-PAI transgene-positive mice. Neointimal cellularity of vascular lesions was decreased by 26% (p=0.01; n=5 each) in ApoE(-/-) mice with the SM22-PAI transgene compared with ApoE(-/-) mice. VSMCs explanted from transgene-positive mice exhibited twofold greater expression of PAI-1 and their migration was attenuated by 27% (p=0.03). Accordingly, increased expression of PAI-1 protein by VSMCs reduces their migration in vitro and their contribution to neointimal cellularity in vivo.