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.

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.

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.

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.

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.

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.