Efficient differentiation of vascular smooth muscle cells from Wharton's Jelly mesenchymal stromal cells using human platelet lysate: A potential cell source for small blood vessel engineering

BACKGROUND The development of fully functional small diameter vascular grafts requires both a properly defined vessel conduit and tissue-specific cellular populations.Mesenchymal stromal cells(MSCs) derived from the Wharton's Jelly(WJ) tissue can be used as a source for obtaining vascular smooth muscle cells(VSMCs),while the human umbilical arteries(h UAs) can serve as a scaffold for blood vessel engineering.AIM To develop VSMCs from WJ-MSCs utilizing umbilical cord blood platelet lysate.METHODS WJ-MSCs were isolated and expanded until passage(P) 4. WJ-MSCs were properly defined according to the criteria of the International Society for Cell and Gene Therapy. Then, these cells were differentiated into VSMCs with the use of platelet lysate from umbilical cord blood in combination with ascorbic acid,followed by evaluation at the gene and protein levels. Specifically, gene expression profile analysis of VSMCs for ACTA2, MYH11, TGLN, MYOCD, SOX9,NANOG homeobox, OCT4 and GAPDH, was performed. In addition,immunofluorescence against ACTA2 and MYH11 in combination with DAPI staining was also performed in VSMCs. HUAs were decellularized and served as scaffolds for possible repopulation by VSMCs. Histological and biochemical analyses were performed in repopulated h UAs.RESULTS WJ-MSCs exhibited fibroblastic morphology, successfully differentiating into"osteocytes", "adipocytes" and "chondrocytes", and were characterized by positive expression(> 90%) of CD90, CD73 and CD105. In addition, WJ-MSCs were successfully differentiated into VSMCs with the proposed differentiation protocol. VSMCs successfully expressed ACTA2, MYH11, MYOCD, TGLN and SOX9. Immunofluorescence results indicated the expression of ACTA2 and MYH11 in VSMCs. In order to determine the functionality of VSMCs, h UAs were isolated and decellularized. Based on histological analysis, decellularized h UAs were free of any cellular or nuclear materials, while their extracellular matrix retained intact. Then, repopulation of decellularized h UAs with VSMCs was performed for 3 wk. Decellularized h UAs were repopulated efficiently by the VSMCs. Biochemical analysis revealed the increase of total hydroyproline and s GAG contents in repopulated h UAs with VSMCs. Specifically, total hydroxyproline and s GAG content after the 1 st, 2 nd and 3 rd wk was 71 ± 10, 74 ± 9 and 86 ± 8 μg hydroxyproline/mg of dry tissue weight and 2 ± 1, 3 ± 1 and 3 ± 1μg s GAG/mg of dry tissue weight, respectively. Statistically significant differences were observed between all study groups(P<0.05).CONCLUSION VSMCs were successfully obtained from WJ-MSCs with the proposed differentiation protocol. Furthermore, h UAs were efficiently repopulated by VSMCs. Differentiated VSMCs from WJ-MSCs could provide an alternative source of cells for vascular tissue engineering.

Differentiation of smooth muscle progenitor cells in peripheral blood and its application in tissue engineered blood vessels

Background： A major shortcoming in tissue engineered blood vessels （TEBVs） is the lack of healthy and easily attainable smooth muscle cells （SMCs）. Smooth muscle progenitor cells （SPCs）, especially from peripheral blood, may offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. Methods： SPCs were isolated from 5-ml fresh rat peripheral blood by density-gradient centrifugation and cultured for 3 weeks in endothelial growth medium-2-MV （EGM-2-MV） medium containing platelet-derived growth factoroBB （PDGF BB）. Before seeded on the synthesized scaffold, SPC-derived smooth muscle outgrowth cell （SOC） phenotypes were assessed by immuno-fluorescent staining, Western blot analysis, and reverse transcription polymerase chain reaction （RT-PCR）. The cells were seeded onto the silk fibroin-modified poly（3-hydroxybutyrate-co-3-hydroxyhexanoate） （SF-PHBHHx） scaflblds by 6× 10^4 cells/cm^2 and cultured under the static condition for 3 weeks. The growth and proliferation of the seeded cells on the scaffold were analyzed by 3-（4,5-dimethylthiazol-2-yl）-diphenyltetrazolium bromide （MTT） assay, scanning electron microscope （SEM）, and 4,6-diamidino-2-phenylindole （DAPI） staining. Results： SOCs displayed specific ＂hill and valley＂ morphology, expressed the specific markers of the SMC lineage： smooth muscle （SM） a-actin, calponin and smooth muscle myosin heavy chain （SM MHC） at protein and messenger ribonucleic acid （mRNA） levels. RT-PCR results demonstrate that SOCs also expressed smooth muscle protein 22a （SM22a, a contractile protein, and extracellular matrix components elastin and matrix Gla protein （MGP）, as well as vascular endothelial growth factor （VEGF）. After seeded on the SF-PHBHHx scaffold, the cells showed excellent metabolic activity and proliferation. Conclusion： SPCs isolated from peripheral blood can be differentiated the SMCs in vitro and have an impressive growth potential in the biodegradable synthesized scaffold. Thus, SPCs may be a promising cell sointo urce for constructing TEBVs.

Vascular smooth muscle cell differentiation－2010

Vascular smooth muscle cells have attracted considerable interest as a model for a flexible program of gene expression.This cell type arises throughout the embryo body plan via poorly understood signaling cascades that direct the expression of transcription factors and microRNAs which,in turn,orchestrate the activation of contractile genes collectively defining this cell lineage.The discovery of myocardin and its close association with serum response factor has represented a major break-through for the molecular understanding of vascular smooth muscle cell differentiation.Retinoids have been shown to improve the outcome of vessel wall remodeling following injury and have provided further insights into the molecular circuitry that defines the vascular smooth muscle cell phenotype.This review summarizes the progress to date in each of these areas of vascular smooth muscle cell biology.

The crosstalk between endothelial cells and vascular smooth muscle cells during low shear stress:a proteomic-based approach

Instruction Shear stress,caused by the parallel frictional drag force of blood flow,is a biomechanical force which plays an important role in the control of blood vessels growth and functions [1]. Clinical researches had found out that atherosclerotic le-

Increased serum TREM-1 level is associated with in-stent restenosis,and activation of TREM-1 promotes inflammation,proliferation and migration in vascular smooth muscle cells

Background and Objective In-stent restenosis(ISR)remains a major limitation of percutaneous coronary intervention despite improvements in stent design and pharmacological agents,whereas the mechanism of ISR has not been fully clarified.In the present study,we sought to investigate the potential association of serum soluble TREM-1(sTREM-1)levels with the incidence of ISR.The role of TREM-1 was evaluated in cultured vascular smooth muscle cells(VSMCs).

High glucose decreases the expression of ATP-binding cassette transporter G1 in human vascular smooth muscle cells

Objective：ATP-binding cassette transporters（ABC） A1 and G1 play an important role in mediating cholesterol efflux and preventing macrophage foam cell formation. In this study, we examined the regulation of ABC transporters by high glucose in human vascular smooth muscle cells（VSMCs）, the other precursor of foam cells. Methods：Incubation of human VSMCs with D-glucose（5 to 30 mM） for 1 to 7 days in the presence or absence of antioxidant and nuclear factor（NF）- κ B inhibitors, the expressions of ABCA1 and ABCG1 were analyzed by real time PCR and Western blotting. Results：High glucose decreased ABCG1 mRNA and protein expression in cultured VSMCs, whereas the expression of ABCA1 was not significantly decreased. Down-regulation of ABCG1 mRNA expression by high glucose was abolished by antioxidant N-acetyl-L-cysteine（NAC） and NF- κ B inhibitors, BAY 11-7085 and tosyl-phenylalanine chloromethyl-ketone（TPCK）. Conclusion：High glucose suppresses the expression of ABCG1 in VSMCs, which is the possible mechanism of VSMC derived foam cell transformation.

Influence of Stent Implantation on the Expression of PCNA and Apoptosis in Injured Vascular Smooth Muscle Cells

Objectives To evaluate the impact of stent implantation on proliferation and apop-tosis in injured media vascular smooth muscle cells (VSMC) and to explore the mechanism of restenosis after stent implantation. Methods Fifty male New Zealand rabbits were randomized into two groups, including balloon group and stent group. Control group was set up. The samples were harvested on 3, 7, 14, 28, 56 days after operation and the following investigation was carried out: (1) Assessing the expression of proliferating cell nuclear antigen (PCNA) of media VSMC by the method of immunohistochemistry; (2) Analyzing apoptosis of media VSMC by DNA agarose gel electrophoresis and TUNEL technique. Results The expression of PCNA and apoptosis in stent and balloon groups were markedly increased compared with control groups. (1) Stent group induced significant increased expression of PCNA in the media VSMC compared with balloon group on 3 to 28 days. On day 7, the positive rates of PCNA were 24. 36±0. 55 % vs 18. 74±1. 09 % ( P < 0. 01 ); (2) From 3 to 28 days, stent group appeared obvious DNA ladder, while balloon group only had little trace ; (3) TUNEL method showed that stent group induced much more significant apoptosis than that of balloon group on 3 to 28 days. The highest rate of apoptosis appeared on day 7: 12. 42 ±1.13% vs 5. 54±0.53% (P<0. 01); (4) By calculating the ratio of the positive rate of PCNA to apoptosis, it showed that on 3 to 28 days, the ratio of balloon group was higher than that of stent group. There was obvious difference between two groups. Conclusions Stent group induces augmented proliferation and much more significant apoptosis of media VSMC than that of balloon group. It makes the ratio of proliferation to apotosis reduced and the severity of restenosis relieved after stent implantation.

Advances in the differentiation of pluripotent stem cells into vascular cells

Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood vessels are related to many disorders like stroke,myocardial infarction,aneurysm,and diabetes,which are important causes of death worldwide.Translational research for new appro-aches to disease modeling and effective treatment is needed due to the huge socio-economic burden on healthcare systems.Although mice or rats have been widely used,applying data from animal studies to human-specific vascular physiology and pathology is difficult.The rise of induced pluripotent stem cells(iPSCs)provides a reliable in vitro resource for disease modeling,regenerative medicine,and drug discovery because they carry all human genetic information and have the ability to directionally differentiate into any type of human cells.This review summarizes the latest progress from the establishment of iPSCs,the strategies for differentiating iPSCs into vascular cells,and the in vivo trans-plantation of these vascular derivatives.It also introduces the application of these technologies in disease modeling,drug screening,and regenerative medicine.Additionally,the application of high-tech tools,such as omics analysis and high-throughput sequencing,in this field is reviewed.

A new cerebroside and its anti-proliferation effect on VSMCs from the radix of Cyperus rotundus L.

A new cerebroside,1-O-(β-D-glucopyranosyloxy)-(2S,3R,4E,8Z)-2-[(2′R)-2’-hydroxylignoceranoylamino]-4,8-tetradecene-3- diol was isolated from the 60%EtOH extract of traditional Chinese medical plant Cyperus rotundus L.Its structure was determined on the basis of spectroscopic data.This new compound showed anti-proliferation effect on vascular smooth muscle cells(VSMCs).

橙皮素对PDGF-BB诱导的大鼠原代胸主动脉VSMCs表型转化的影响

目的:探讨橙皮素(HES)对血小板源性生长因子-BB(PDGF-BB)诱导的大鼠胸主动脉血管平滑肌细胞(VSMCs)表型转化的影响。方法:4周龄健康雄性SD大鼠1只,5%水合氯醛麻醉后,采用组织块贴壁法提取大鼠胸主动脉原代VSMCs并培养,取对数生长期细胞,采用细胞免疫荧光化学法鉴定VSMCs平滑肌肌动蛋白-α(α-SMA),采用Cell Counting Kit-8(CCK-8)试剂盒检测2、5、10、25及50μg/L PDGF-BB和10、50、100、150、200、250及300μmol/L的HES对VSMCs增殖活性的影响,筛选PDGF-BB最佳造模条件和HES最佳干预浓度;VSMCs分为对照组(0μg/L PDGF-BB)、造模组(25μg/L PDGF-BB)及加药组(25μg/L PDGF-BB+100μmol/L橙皮素),采用划痕实验检测各组VSMCs的细胞迁移能力,采用Western blot检测各组VSMCs的α-SMA、平滑肌22α(SM22α)、弹性蛋白原(ELN)及增殖细胞核抗原(PCNA)蛋白的表达。结果:原代平滑肌细胞呈长梭形,传代2 d后VSMCs有典型的“峰-谷”状特征,α-SMA鉴定阳性率>95%;PDGF-BB最佳造模浓度是25μg/L,HES最佳干预浓度是100μmol/L;与对照组比较,造模组VSMCs迁移率增加,α-SMA及SM22α蛋白表达下降,但ELN和PCNA蛋白表达上升(P<0.001);与造模组比较,加药组VSMCs迁移率降低,α-SMA及SM22α蛋白表达上升,但ELN、PCNA蛋白表达下降(P<0.001)。结论:PDGF-BB可诱导大鼠胸主动脉VSMCs的增殖及增强其迁移能力,HES可通过抑制PDGF-BB的作用从而影响VSMCs的表型转化。

Synthesis and Anti-VSMC Vegetation Activities of New Aurone Derivatives

A series of new aurone derivatives was prepared by means of a practical route and their anti-vascular smooth muscle cells（VSMC） vegetation activities were evaluated by the 3-（4,5-dimethylthlazol-2-yl）-2,5- diphenyltetrazolium bromide（MTT） method with tetrandrine as a positive contrast drug. The structures of the com- pounds were confirmed by 1H NMR, 13C NMR and electrospray ionization mass spectrometry（ESI-MS）. Several new compounds exhibited promising activity against VSMC proliferation and the preliminary structure-activity relation- ships（SAR） were discussed in order to investigate the essential structures required for their bioactivities.

枸橼酸镁对慢性肾衰竭环境下氧化应激的抑制作用

目的探讨枸橼酸镁(magnesium citrate,MgCit)抑制慢性肾衰竭(chronic renal failure,CRF)环境下氧化应激的作用。方法SD大鼠分为CRF模型组、MgCit组(375、750 mg/kg),另设正常对照组、MgCit对照组(750 mg/kg);透射电镜观察大鼠胸大动脉血管平滑肌细胞(vascular smooth muscle cells,VSMCs)中线粒体形态;利用试剂盒检测大鼠主动脉和血浆中超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malonaldehyde,MDA)的含量。培养VSMCs并分为正常对照组、CRF模型组、MgCit组(1.5、3 mmol/L),流式细胞仪定量检测细胞超氧化物阴离子(dihydroethidium,DHE)和细胞凋亡情况。结果与对照组相比,模型组大鼠胸大动脉VSMCs线粒体肿胀,嵴断裂或消失;MgCit干预能减轻线粒体肿胀,未见嵴断裂;模型组大鼠主动脉和血浆SOD水平降低(P<0.05),MDA水平升高(P<0.05);MgCit干预能增加主动脉和血浆SOD水平,并降低MDA水平(P<0.05)。CRF环境下即CRF模型组VSMCs的DHE含量升高,凋亡增加(P<0.05);MgCit干预能降低DHE含量,并抑制凋亡(P<0.05)。结论MgCit抑制CRF大鼠和VSMCs的氧化应激水平。

Human vascular smooth muscle cells and endothelial cells cocultured on polyglycolic acid (70/30) scaffold in tissue engineered vascular graft

Background Current prosthetic, small diameter vascular grafts showing poor long term patency rates have led to the pursuit of other biological materials. Biomaterials that successfully integrate into surrounding tissue should match not only the mechanical properties of tissues, but also topography. Polyglycolic acid （70130） has been used as synthetic grafts to determine whether human vascular smooth muscle cells and endothelial cells attach, survive and secrete endothelin and 6-keto-prostaglandin F1α （6-keto-PGF1α）. Methods Endothelial cells and smooth muscle cells were isolated from adult human great saphenous vein. They were seeded on polyglycolic acid scaffold in vitro separately to grew vascular patch （Groups A and B respectively） and cocultured in vitro to grow into vascular patch （Group C）. Smooth muscle cells and endothelial cells were identified by immunohistochemical analysis and growth of cells on polyglycolic acid was investigated using scanning electron microscopy. The levels of endothelin and 6-keto-PGF1α in the culturing solutions were examined by radioimmunology to measure endothelial function. Results Seed smooth muscle cells adhered to polyglycolic acid scaffold and over 28 days grew in the interstices to form a uniform cell distribution throughout the scaffold. Then seed endothelial cells formed a complete endothelial layer on the smooth muscle cells. The levels of endothelin and 6-keto-prostaglandin F1 alpha in the culturing solution were （234±29） pg/ml and （428±98） pg/ml respectively in Group C and （196±30） pg/ml and （346±120） pg/ml in Group B; both significantly higher than in Groups A and D （blank control group, all P〈0.05 ）. Conclusions Cells could be grown successfully on polyglycolic acid and retain functions of secretion. Our next step is to use human saphenous vein smooth muscle cells and endothelial cells to grow tubular vascular grafts in vitro.

Effects of rosuvastatin on the production and activation of matrix metalloproteinase-2 and migration of cultured rat vascular smooth muscle cells induced by homocysteine

Objective： To test the influence of homocysteine on the production and activation of matrix metalloproteinase-2 （MMP-2） and tissue inhibitors of matrix metalloproteinase-2 （TIMP-2） and on cell migration of cultured rat vascular smooth muscle cells （VSMCs）. Also, to explore whether rosuvastatin can alter the abnormal secretion and activation of MMP-2 and TIMP-2 and migration of VSMCs induced by homocysteine. Methods： Rat VSMCs were incubated with different concentrations of homocysteine （50-5000 μmol/L）. Western blotting and gelatin zymography were used to investigate the expressions and activities of MMP-2 and TIMP-2 in VSMCs in culture medium when induced with homocysteine for 24, 48, and 72 h. Transwell chambers were employed to test the migratory ability of VSMCs when incubated with homocysteine for 48 h. Different concentrations of rosuvastatin （10^-9-10^-5 mol/L） were added when VSMCs were induced with 1 000 pmol/L homocysteine. The expressions and activities of MMP-2 and TIMP-2 were examined after incubating for 24, 48, and 72 h, and the migration of VSMCs was also examined after incubating for 48 h. Results： Homocysteine （50-1000 μmol/L） increased the production and activation of MMP-2 and expression of TIMP-2 in a dose-dependent manner. However, when incubated with 5000 pmol/L homocysteine, the expression of MMP-2 was up-regulated, but its activity was down-regulated. Increased homocysteine-induced production and ac- tivation of MMP-2 were reduced by rosuvastatin in a dose-dependent manner whereas secretion of TIMP-2 was not significantly altered by rosuvastatin. Homocysteine （50-5000 μmol/L） stimulated the migration of VSMCs in a dose-dependent manner, but this effect was eliminated by rosuvastatin. Conclusions： Homocysteine （50-1000 μmol/L） significantly increased the production and activation of MMP-2, the expression of TIMP-2, and the migration of VSMCs in a dose-dependent manner. Additional extracellular rosuvastatin can decrease the excessive expression and acti- vation of MMP-2 and abnormal migration of VSMCs induced by homocysteine.

Parthenolide inhibits proliferation of vascular smooth muscle cells through induction of G_0/G_1 phase cell cycle arrest

Objective： This study is to determine the effect of the natural product parthenolide, a sesquiterpene lactone isolated from extracts of the herb Tanacetum parthenium, on the proliferation of vascular smooth muscle cells （VSMCs）. Methods： Rat aortic VSMCs were isolated and cultured in vitro, and treated with different concentrations ofparthenolide （l 0, 20 and 30 μmol/L）. [^3H]thymidine incorporation was used as an index of cell proliferation. Cell cycle progression and distribution were determined by flow cytometric analysis. Furthermore, the expression of several regulatory proteins relevant to VSMC proliferation including IκBα, cyclooxygenase-2 （Cox-2）, p21, and p27 was examined to investigate the potential molecular mechanism. Results： Treatment with parthenolide significantly decreased the [^3H]thymidine incorporation into DNA by 30%-56% relative to control values in a dose-dependent manner （P〈0.05）. Addition of parthenolide also increased cell population at G0/G1 phase by 19.2%-65.7% （P〈0.05） and decreased cell population at S phase by 50.7%-84.8% （P〈0.05）, which is consistent with its stimulatory effects on p21 and p27. In addition, parthenolide also increased IκBα expression and reduced Cox-2 expression in a time-dependent manner. Conclusion： Our results show that parthenolide significantly inhibits the VSMC proliferation by inducing G0/G1 cell cycle arrest. IκBα and Cox-2 are likely involved in such inhibitory effect ofparthenolide on VSMC proliferation. These findings warrant further investigation on potential therapeutic implications ofparthenolide on VSMC proliferation in vivo.

心血管纳米医学在血管平滑肌细胞中的应用

血管平滑肌细胞(vascular smooth muscle cell, VSMC)具有调节血管紧张性和维持血管形态的功能,其功能障碍与心血管疾病的发生、发展密切相关。纳米医学(nanomedicine)是指纳米材料在医学中的应用,因其能够克服传统治疗方式存在的药理学局限,如药物选择性差、生物利用度低等问题,被广泛应用于心血管疾病、肿瘤等的诊断和治疗。本文基于VSMC在心血管系统的关键作用和纳米医学在传统给药系统中的优化作用,概述了VSMC表型转换异常、恶性增殖和迁移引发心血管疾病的病理机制,总结了靶向改善VSMC功能来治疗心血管疾病的纳米应用,如靶向药物递送、自身靶向干预和组织工程支架制备等,最后讨论了心血管纳米医学面临的挑战和未来发展的方向,以期为预防和治疗心血管疾病提供潜在方案。

Platelet-derived microvesicles drive vascular smooth muscle cell migration via forming podosomes and promoting matrix metalloproteinase-9 activity

We have shown that platelet-derived microvesicles(PMVs)induce abnormal proliferation,migration,and energy metabolism of vascular smooth muscle cells(VSMCs)after vascular intimal injury.Here,we examined a novel role of podosome in mediating matrix metalloproteinase-9(MMP-9)dependent VSMC migration induced by plateletderived microvesicles(PMVs).VSMCs were isolated from the thoracic aortas of male Sprague Dawley(SD)rats and identified with immunofluorescent staining.Blood samples were collected from SD Rats,the platelets were isolated with density gradient centrifugation from the blood samples and activated by collagen I.Intriguingly,proteins expressed in platelets were found to participate in the positive regulation of podosome assembly using GO analysis by DAVID,and most of the proteins were found in extracellular exosomes.Of note,activated platelets indirectly induced VSMC migration via releasing PMVs which was verified using platelets and VSMCs transwell coculture system.Besides,podosome,an invasive protrusion to mediate extracellular matrix(ECM)remodeling,was formed in VSMCs to induce cell migration.Furthermore,MMP-9 activity detected by gelatin zymography was used to verify the function of the podosome in ECM remodeling.The result indicated that MMP-9 activity was robustly activated in VSMCs to implement the function of the podosome.In addition,gelatin degradation was detected in intact VSMCs using a gelatin degradation assay after co-culture with platelets.Taken together,our data reveal a novel mechanism that PMVs promote VSMC migration via forming podosomes and inducing MMP-9 activity.

Inhibition Mechanism of Emodin on Rabbit Vascular Smooth Muscle Cells Proliferation

The proliferation of vascular smooth muscle cells(VSMCs)contributes to the pathogenesis of atherosclerosis and restenosis after angioplasty and vein graft.In this study,MTT colormetry was used to test the effective scope of emodin to inhibit VSMCs proliferation.Flow cytometry and confocal image were adopted to investigate its inhibitive mechanism.The results show that emodin could inhibit the growth and proliferation of VSMCs and the inhibition rate of emodin on VSMCs is 24.6%-94.58%,which is time-and concentration-dependent.Emodin could reduce S phase entry,increase the apoptosis of VSMCs,and reduce the intensity of[Ca^(2+)]_(i)in hPDGF B/B stimulated VSMCs.This research provides theoretical basis for medical application of emodin.It is concluded that emodin could inhibit the growth and proliferation of VSMCs effectively.Decreasing the DNA synthesis,increasing the cell apoptosis and reducing the intensity of[Ca^(2+)]_(i)in hPDGF B/B stimulated VSMCs may be the inhibitive mechanism of emodin against VSMCs proliferation.