Baicalin inhibits PDGF-BB-stimulated vascular smooth muscle cell proliferation through suppressing PDGFRβ-ERK signaling and increase in p27 accumulation and prevents injury-induced neointimal hyperplasia

The increased proliferation and migration of vascular smooth muscle cells （VSMCs） are key events in the development of atherosclerotic lesions. Baicalin, an herb-derived flavonoid compound, has been previously shown to induce apoptosis and growth inhibition in cancer cells through multiple pathways. However, the potential role of baicalin in regulation of VSMC proliferation and prevention of cardiovascular diseases remains unexplored. In this study, we show that pretreatment with baicalin has a dose-dependent inhibitory effect on PDGF-BB-stimulated VSMC pro- liferation, accompanied with the reduction of proliferating cell nuclear antigen （PCNA） expression. We also show that baicalin-induced growth inhibition is associated with a decrease in cyclin E-CDK2 activation and increase in p27 level in PDGF-stimulated VSMCs, which appears to be at least partly mediated by blockade of PDGF recep- tor [~ （PDGFR~）-extracellular signal-regulated kinase 1/2 （ERK1/2） signaling. In addition, baicalin was also found to inhibit adhesion molecule expression and cell migration induced by PDGF-BB in VSMCs. Furthermore, using an animal carotid arterial balloon-injury model, we found that baicalin significantly inhibited neointimal hyperplasia. Taken together, our results reveal a novel function of baicalin in inducing growth arrest of PDGF-stimulated VSMCs and suppressing neointimal hyperplasia after balloon injury, and suggest that the underlying mechanism involves the inhibition of cyclin E-CDK2 activation and the increase in p27 accumulation via blockade of the PDGFR^-ERK1/2 signaling cascade.

Andrographolide inhibits NF-KB activation and attenuates neointimal hyperplasia in arterial restenosis

The NF-kB transcription factors modulate the expression of tissue factor （TF）, E-selectin （CD62E） and vascular cell adhesion molecule-1 （VCAM-1）, which are essential for thrombosis and inflammation. We have previously shown that andrographolide （Andro） covalently modifies the reduced cysteine^62 of p50-a major subunit of NF-kB transcription factors, thus blocking the binding of NF-kB transcription factors to the promoters of their target genes, preventing NF-kB activation and inhibiting inflammation in vitro and in vivo. Here we report that Andro, but not its inactive structural analog 4H-Andro, significantly suppressed the proliferation of arterial neointima （-60% reduction） in a murine model of arterial restenosis. Consistently, p50^-/- mice manifested attenuated neointimal hyperplasia upon arterial ligation. Notably, the same dosage of Andro did not further reduce neointimal formation in p50^-/- mice, which implicates the specificity of Andro on p50 for treating experimental arterial restenosis. The upregulation of NF-kB target genes, including TF, E-selectin and VCAM-1, and the increased deposition of leukocytes （mainly CD68^＋ macrophages） were clearly detected within the injured arterial walls, all of which were significantly abolished by treatment with Andro or genetic deletion of p50. The expression ofTF, E-selectin and VCAM-I was also markedly upregulated in the patient sample of thrombotic vasculitis, indicating the clinical relevance of NF-kB activation in the pathogeneses of occlusive arterial diseases. Our data thus indicate that, by the downregulation of the NF-kB target genes that are critical in thrombosis and inflammation, specific inhibitors of p50, such as Andro, may be therapeutically valuable for preventing and treating thrombotic arterial diseases, including neointimal hyperplasia in arterial restenosis.

Antrodia Cinnamomea ameliorates neointimal formation by inhibiting infl ammatory cell infi ltration through downregulation of adhesion molecule expression in vitro and in vivo

The increased vascular infl ammation is a key event in the development of atherosclerotic lesions.Antrodia cinnamomea has been shown to promote anticancerogenic activity through decreasing infl ammation.However,the potential role of A.cinnamomea in cardiovascular diseases remains unexplored.Herein,using carotid arterial ligation models,we found that ethanol extract from A.cinnamomea(EEAC)signifi cantly inhibited neointimal hyperplasia in a dose-dependent manner,accompanied with the reduced expression of activated p65 and infl ammatory cytokines.We also show that EEAC ameliorated TNF-α-induced phosphorylation of p65 and pro-infl ammatory cytokine expression in both vascular smooth muscle cells(VSMCs)and macrophages in vitro.Mechanistically,EEAC suppressed expression levels of intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule(VCAM-1)in VSMCs,which attenuates the ability of monocytes/macrophages adhesion to VSMCs.Furthermore,the expression level of these adhesion molecules and infi ltration of monocytes/macrophages were also decreased in neointimal VSMCs of arteries pretreated with EEAC.Altogether,our results reveal a novel function of A.cinnamomea in suppressing vascular infl ammation upon ligation injury during neointimal formation,likely through inhibition of infl ammatory cell infi ltration via downregulating the adhesion molecules in VSMCs.Thus,A.cinnamomea may offer a pharmacological therapy to slow down disease progression in patients with vascular injury.

INHIBITORY EFFECT OF ANGIOTENSIN II TYPE 1 RECEPTOR-ASSOCIATED PROTEIN ON VASCULAR SMOOTH MUSCLE CELL GROWTH AND NEOINTIMAL FORMATION

Objective To investigate the mechanism of a novel angiotensin Ⅱ type 1 receptor-associated protein （ATRAP） interfering with angiotensin Ⅱ type 1 （AT1） receptor-mediated vascular smooth muscle cell （VSMC） growth and neointimal formation. Methods VSMCs isolated from thoracic aorta of adult Sprague-Dawley （SD） rats were used in this study. ATRAP cDNA was subcloned into pcDNA3 vector and then transfected into VSMCs. DNA synthesis and extracellular signal-regulated kinase （ERK） and phospho-ERK expressions in VSMCs were assayed by measurement of ^3H thymidine incorporation and Western blotting, respectively. Morphological changes were observed in the balloon injured artery with or without transfection of ATRAP cDNA using 12-week-old male SD rats. Results ATRAP overexpression in VSMCs inhibited angiotensin Ⅱ （Ang Ⅱ）-induced ^3H thymidine incorporation 48 hours after Ang Ⅱ stimulation （ P 〈 0. 05 ）. In VSMC, Ang Ⅱ stimulation increased the phosphorylation of ERK, which reached the peak around 60 minutes. The activation of phospho-ERK was significantly decreased by ATRAP （ P 〈 0. 05 ）. Neointimal formation was markedly inhibited by ATRAP overexpression in injuried arteries.Conclusions The AT1 receptor-derived activation of ERK plays an essential role in Ang Ⅱ-induced VSMC growth. The growth inhibitory effects of ATRAP might be due to interfering with AT1 receptor-mediated activation of ERK in VSMC growth and neointimal formation.

The Origin of Neointimal Smooth Muscle Cells in Transplant Arteriosclerosis from Recipient Bone-marrow Cells in Rat Aortic Allograft

In order to investigate the origin of neointimal smooth muscle cells in transplant arterio- sclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD-female Wistar aortic allografts, male SD-male Wis- tar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohisto- chemistry. The results indicated that excessive accumulation of α-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts. Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were sig- nificantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P<0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a dis- tinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic al- lograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.

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 antihyperpiasia 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 endotheiium 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.

Pattern of instent neointimal formation compared to native atherosclerosis in the coronary bifurcation lesions： volumetric intravascular ultrasound analysis

Background No clinical study has systematically analyzed and compared circumferential neointimal and plaque distribution of stent neointimal proliferation and in native atherosclerotic plaques. This study aimed to investigate and compare the pattern of instent neointimal formation and native atherosclerosis in the coronary bifurcation lesions by volumetric analysis using systematic intravascular ultrasound （IVUS）. Methods We examined bifurcation lesions in native coronary artery （plaque group, n=102） and stented bifurcations at 9-month follow-up （neointima group, n=51） using volumetric IVUS analysis of both the main vessel （MV） and side branch （SB）. Three 5-mm segments were analyzed; the proximal MV （MVp）, distal MV （MVd） and SB ostium （SBo）. For each segment, volumetric analysis was performed in each of four quadrants （divided according to the branch takeoff and the geometric center of the lumen）; carinal, epicardial, abcarinal, and myocardial. The eccentricity index was defined as the ratio of the abcarinal plaque （or neointimal） volume to the carinal plaque （or neointimal） volume. Results The plaque distribution differed significantly between the four quadrants, with the largest in the abcarinal quadrant, followed by the myocardial, epicardial, and carinal quadrants. The distribution of neointima was similar in the MV, but the four quadrants in the SB did not differ significantly. The eccentricity indices of both the MVd （P 〈0.001） and SBo （P=-0.001） were significantly higher for the plaque group than the neointima group. Conclusions The distribution of neointimal proliferation seems to have a similar pattern to that of atherosclerotic plaque in native coronary arteries. Darticularlv in the main vessel, but the trend is less prominent.

Evaluation of neointimal coverage in patients with coronary artery aneurysm formation after drug-eluting stent implantation by optical coherence tomography

Background The vessel healing in patients with coronary artery aneurysms （CAA） that form after drug-eluting stent （DES） implantation is not clear. This study aims to assess the vessel healing in patients with CAA formation after DES implanation. Methods From June 2008 to August 2011, follow-up coronary angiography was conducted on 1160 patients who underwent percutaneous coronary intervention （PCI）. The average period of follow-up was about （18.95±13.05） months. A total of 175 patients who underwent DES implantation into de novo lesions and who underwent coronary angiography and optical coherence tomography （OCT） examination during follow-up were identified. Patients were divided into the CAA group （n=31） and non-CAA group （n=144） based on the results of the coronary angiography. The cardiac events including angina and acute myocardial infarction were noted; in addition, the neointimal thickness and the frequency of strut malapposition and strut uncoverage were also noted. Results A greater proportion of incomplete neointimal coverage （17.17% vs. 1.90%, P 〈0.001） and strut malapposition （18.20% vs. 1.38%, P 〈0.001） were observed in the CAA group. The neointimal thickness in the CAA group was significantly thinner than that in the non-CAA group （（146.6±94.8） μm vs. （192.5＋97.1）μm, P 〈0.001）, as detected via OCT. Patients with CAA formation had a higher frequency of cardiac events including angina pectoris （25.81% vs. 6.25%, P=0.001） and acute myocardial infarction （9.68% vs. 0.13%, P=0.002） and thrombosis （16.13% vs. 0.69%, P 〈0.001）. The longitudinal length of the CAA in the cardiac event group was significantly longer than in the no cardiac event group （（20.0±9.07） mm vs. （12.05±5.38） ram, P=0.005）. Conclusion CAA formation after DES implantation is frequently associated with cardiac events as a result of stent malapposition and incomplete neointimal coverage.

Endovascular Irradiation Prevents Sm ooth Muscle CellPro-liferation and Neointim alHyperplasia in Rabbits

The present study examined the temporal responses and the efficacy of 192Ir HDR endovascular irradiation for preventing smooth muscle cell proliferation of rabbit iliac arteries after PTA with a cutting balloon catheter. Endovascular irradiation with 12 Gy was randomly performed on the one side of iliac arterial segment with the unirradiated side serving as a control. Animals were euthanatized 1, 2, 3, 4, 8 and 12 week(s) after angioplasty. Histopathological and immunohistochemical studies were carried out. Histopathology showed repair of the dissection by cellular accumulation and a striking reduction in the amount of neointimal hyperplasia in the irradiated arteries as compared with control vessels. A peak of PCNA positive ratio was in neointima of the control arterial segments at a week. 2 - 4 weeks after irradiation, the neointimal PCNA positive ratio was still significantly increased in the control arterial segments compared with the irradiated arterial segments. After 8 weeks, PCNA positive ratio was below 1 % in both irradiated arterial segments and the control. Our results showed that the 192Ir HDR afterloading irradiation with a dose of 12 Gy can be considered sufficient for inhibiting neointimal hyperplasia in angioplastized rabbit iliac arteries with cutting balloon catheter.

Angiotensin-converting-enzyme Inhibitors in Treatment of Atherosclerotic Peripheral Arterial Disease

Objective To evaluate the treatment efficacy of perindopril in correcting endothelial dysfunction in patients with atherosclerotic peripheral arterial disease(PAD).Methods 85 patients with atherosclerotic PAD were divided into 3 groups and undergone endotheliotropic treatment with perindopril.The functional state of endothelium(FSE)was evaluated in all patients by checking the following biochemical parameters:nitric oxide(Ⅱ)(NO),endothelin-1(E-1),C-reactive protein(CRP),superoxide dismutase(SOD),integral assessment of lipid peroxidation(LPO),glutathione peroxidase(GPx).Results Basal level of NO secretion in operated patients was the lowest.SOD level in operated patients increased by 102.7%,143.24%,164.86%and 175.67%directly after the operation as well as 1,3 and 6 months following the surgery respectively,but E-1 level decreased by 82%,70%,78%,and 90%.The results of GPx level and LPO analysis confirmed favorable effects of perindopril on biochemical status in patients with atherosclerotic PAD.Conclusion Perindopril is effective in stimulating NO(Ⅱ)secretion and correcting FSE.Perindopril may be used for prophylaxis of restenosis of reconstruction area following operative treatment in patients with atherosclerotic PAD.

The Role of Progenitor Cells in the Pathogenesis of Arteriosclerosis

The increasing incidence of arteriosclerosis has become a significant global health burden.Arteriosclerosis is characterized by the thickening and hardening of arterial walls,which can lead to the narrowing or complete blockage of blood vessels.However,the pathogenesis of the disease remains incompletely understood.Recent research has shown that stem and progenitor cells found in the bone marrow and local vessel walls play a role in the development of arteriosclerosis by differentiating into various types of vascular cells,including endothelial cells,smooth muscle cells,fibroblasts,and inflammatory cells.This review aims to provide a comprehensive understanding of the role of stem and progenitor cells in the pathogenesis of arteriosclerosis,shedding light on the underlying mechanisms and potential therapeutic approaches for this disease.

Clinical characteristics of early and late drug-eluting stent in-stent restenosis and mid-term prognosis after repeated percutaneous coronary intervention

Background:The mechanism and characteristics of early and late drug-eluting stent in-stent restenosis(DES-ISR)have not been fully clarified.Whether there are different outcomes among those patients being irrespective of their repeated treatments remain a knowledge gap.Methods:A total of 250 patients who underwent initial stent implantation in our hospital,and then were readmitted to receive treatment for the reason of recurrent significant DES-ISR in 2016 were involved.The patients were categorized as early ISR(<12 months;E-ISR;n=32)and late ISR(≥12 months;L-ISR;n=218).Associations between patient characteristics and clinical performance,as well as clinical outcomes after a repeated percutaneous coronary intervention(PCI)were evaluated.Primary composite endpoint of major adverse cardiac events(MACEs)included cardiac death,non-fatal myocardial infarction(MI),or target lesion revascularization(TLR).Results:Most baseline characteristics are similar in both groups,except for the period of ISR,initial pre-procedure thrombolysis in myocardial infarction,and some serum biochemical indicators.The incidence of MACE(37.5%vs.5.5%;P<0.001)and TLR(37.5%vs.5.0%;P<0.001)is higher in the E-ISR group.After multivariate analysis,E-ISR(odds ratio[OR],13.267;[95%CI 4.984-35.311];P<0.001)and left ventricular systolic dysfunction(odds ratio[OR],6.317;[95%CI 1.145-34.843];P=0.034)are the independent predictors for MACE among DES-ISR patients in the mid-term follow-up of 12 months.Conclusions:Early ISR and left ventricular systolic dysfunction are associated with MACE during the mid-term follow-up period for DES-ISR patients.The results may benefit the risk stratification and secondary prevention for DES-ISR patients in clinical practice.

Transplantation of human umbilical cord-derived endothelial progenitor cells promotes re-endothelialization of the injured carotid artery after balloon injury in New Zealand white rabbits

Background Cell transplantation has great potential for promoting endothelial repair and reducing the complications of percutaneous coronary intervention （PCI）. The aim of this study was to investigate the effect of transplantation of human umbilical cord blood endothelial progenitor ceils （EPCs） on injured arteries. Methods Umbilical cord blood mononuclear cells were obtained from post-partum lying-in women, and EPCs were isolated, cultured, expanded and identified by immunofiuorescence. The carotid arterial endothelium of New Zealand white rabbits was injured by dilatation with a 3F balloon, and the EPCs were injected into the lumen of the injured artery in the transplanted group （n=16）, while an equal volume of phosphated buffered saline （PBS） was injected into the control group after balloon injury （n=16）. The animals were sacrificed after either 2 or 4 weeks, and the grafted cells were identified by double immunofluorescence staining with human nuclear antigen （HNA） and CD31 antibodies. Arterial cross sections were analyzed by pathology, immunohistochemisty and morphometry to evaluate the reparative effects of EPCs. Proliferating cell nuclear antigen （PCNA） and transforming growth factor （TGF）-131 mRNA expression were detected by reverse transcription-polymerase chain reaction （RT-PCR）. Results Fluorescence-labeled EPCs were found in the neointima. The neointimal area and the neointimal/medial area ratio were significantly lower in the transplanted group than in the control group （P 〈0.05）. von Willebrand factor （vWF） immunohistostaining showed more VWF-positive cells in the transplanted animals than in the controls （8.75±2.92 vs. 4.50±1.77, P 〈0.05）. Compared with the control group, the transplanted group had lower expression of PCNA mRNA （0.67±0.11 vs. 1.25±0.40, P 〈0.01 ）and higher expression of TGF-β1 mRNA （1.10±0.21 vs. 0.82±0.07, P 〈0.05）. Conclusions EPCs derived from human umbilical cord blood were successfully transplanted into injured vessels. The transplanted EPCs inhibited neointimal hyperplasia and promoted vascular re-endothelialization.

SMYD3-PARP16 axis accelerates unfolded protein response and mediates neointima formation

Neointimal hyperplasia after vascular injury is a representative complication of restenosis.Endoplasmic reticulum(ER)stress-induced unfolded protein response(UPR)is involved in the pathogenesis of vascular intimal hyperplasia.PARP16,a member of the poly(ADP-ribose)polymerases family,is correlated with the nuclear envelope and the ER.Here,we found that PERK and IRE1 a are ADPribosylated by PARP16,and this might promote proliferation and migration of smooth muscle cells(SMCs)during the platelet-derived growth factor(PDGF)-BB stimulating.Using chromatin immunoprecipitation coupled with deep sequencing(ChIP-seq)analysis,PARP16 was identified as a novel target gene for histone H3 lysine 4(H3 K4)methyltransferase SMYD3,and SMYD3 could bind to the promoter of Parp16 and increased H3 K4 me3 level to activate its host gene’s transcription,which causes UPR activation and SMC proliferation.Moreover,knockdown either of PARP16 or SMYD3 impeded the ER stress and SMC proliferation.On the contrary,overexpression of PARP16 induced ER stress and SMC proliferation and migration.In vivo depletion of PARP16 attenuated injury-induced neointimal hyperplasia by mediating UPR activation and neointimal SMC proliferation.This study identified SMYD3-PARP16 is a novel signal axis in regulating UPR and neointimal hyperplasia,and targeting this axis has implications in preventing neointimal hyperplasia related diseases.

Very late thrombosis 12 years after bare metal stent deployment

Percutaneous coronary intervention(PCI)is currentlythe most commonly used revascularization techniquein the management of symptomatic coronary disease.However,late stent thrombosis(LST)and in-stent

Efficacy and safety of a novel nano-porous polymer-free sirolimus- eluting stent in pigs

Background Drug-eluting stents represent a major advance in interventional cardiology. However, the current drug- eluting stents have significant limitations. One of the major problems is very late stent thrombosis, which is likely caused by inflammation and a hypersensitivity reaction related to a polymer on the stent. A polymer-free sirolimus-eluting stent with a unique nano-porous surface has been developed. This study aimed to evaluate this novel polymer-free sirolimus- eluting stent for its efficacy and safety in a pig model. Methods Stents were directly coated with sirolimus （a drug concentration of 2.2 μg/mm2 on the stent surface）. The polymer-free sirolimus-eluting stents （PFSES） were compared to standard polymer-coated sirolimus-eluting stents （PCSES） and bare-metal stents （BMS） in 18 pigs. Results At one month the degree of neointimal hyperplasia was similar between the two sirolimus-eluting stent groups and was significantly less compared to BMS （（1.93±0.51） mm2, （1.57±0.69） mm2 vs. （4.45±1.05） mm2, P 〈0.05）At three months, PFSES maintained the low level of neointima （（2.41±0.99） mm2 vs. （4.32±1.16） mm2, P 〈0.05）, whereas PCSES had developed significant neointimal proliferation similar to BMS. The inflammation level was significantly higher in PCSES when compared with BMS three months post-implantation （2.50±0.55 vs. 0.83±0.75, P 〈0.05） whereas PFSES showed a low level of inflammation comparable to PCSES （1.33±0.52 vs. 2.50±0.55, P 〈0.05）. Conclusion The PFSES is effective and safe. and appears to be suoerior to standard PCSEs.

Mechanism of arterial remodeling in chronic allograft vasculopathy

Chronic allograft vasculopathy(CAV)remains a major obstacle for long-term survival of grafts even though therapeutic strategies have improved considerably in recent years.CAV is characterized by concentric and diffuse neointimal formation,medial apoptosis,infiltration of lymphocyte or inflammatory cells,and deposition of extracellular matrix both in arteries and veins.Recent studies have shown that stem cells derived from the recipient contribute to neointimal formation under the regulation of chemokines and cytokines.Arterial remodeling in allografts eventually causes ischemic graft failure.The pathogenesis is multi-factorial with both immunologic and non-immunological factors being involved.The immunological factors have been discussed extensively in other articles.This review focuses mainly on the arterial remodeling that occurs in 3 layers of vessel walls including intimal injury,accumulation of smooth muscle-like cells in the neointimal,medial smooth muscle cell apoptosis,adventitial fibrosis,and deposition of extracellular matrix.