alpha5beta1 integrin expression and luminal edge fibronectin matrix assembly by smooth muscle cells after arterial injury

Fibronectin is secreted from the cell as a soluble protein that must then polymerize to regulate cell function. To elucidate the process of fibronectin matrix assembly in vascular disease, we immunostained sections of balloon-injured rat carotid artery for the fibronectin-binding alpha5beta1 integrin. Whereas alpha5beta1 integrin was not evident in the normal carotid artery, its expression was induced after a vascular injury. By 14 days, the alpha5beta1 integrin was localized exclusively to the less differentiated smooth muscle cells (SMCs) at the luminal surface of the neointima. Platelet-derived growth factor-BB, dominant in neointimal formation, selectively increased the expression of the alpha5beta1 integrin by human SMCs in culture. To track the assembly of fibronectin fibers, fluorescence-labeled soluble fibronectin protomers were added to cultured SMCs and to fresh segments of normal and balloon-injured rat carotid arteries. Fibronectin fiber formation in cultured SMCs could be detected within 10 minutes, and was blocked by an RGD peptide, an anti-beta1 integrin antibody, and an anti-alpha5beta1 integrin antibody, but not by an anti-beta3 integrin antibody. En face confocal microscopy of arterial segments revealed that soluble fibronectin had polymerized on the alpha5beta1 integrin-expressing SMCs of the luminal surface of the injured arterial neointima, but not on the alpha5beta1 integrin-negative neointimal SMCs below this or on the endothelial cells of uninjured arteries. Furthermore, in situ fibronectin assembly by the neointimal SMCs was inhibited by an RGD peptide and by an anti-beta1 integrin antibody. These studies indicate that a subpopulation of SMCs in the repairing artery wall orchestrates integrin-mediated fibronectin assembly.     

Changes in the Organization of the Smooth Muscle Cells in Rat Vein Grafts

Mechanical tensile stress in vein grafts increases suddenly under the influence of arterial blood pressure. In this study, we examined the influence of increased tensile stress on the organization of the smooth muscle cells (SMCs) in the neointima and media of the rat vein grafts. An autogenous jugular vein was grafted into the abdominal aorta of the rat, and changes in the organization of the vein graft SMCs were studied by observing the distribution of SMC actin filaments and nuclei at 3 min and 1, 5, 10, and 30 days after surgery. In a normal jugular vein, the average wall circumferential tensile stress was ~ 3 kPa at an internal pressure of 3 mm Hg. The SMCs, that contained long, slender actin filamentous bundles, were oriented mainly in the circumferential direction of the vessel, and constituted a 2- to 3-cell-thick medial layer underneath the endothelium. In a vein graft, the wall circumferential tensile stress suddenly increased by ~ 140 times compared with the control level. In response to this suddenly increased stress, the SMC layer was stretched into a structure with scattered pores and disrupted SMC actin filamentous bundles within 3 min. This initial change was followed by a rapid reduction in the density of the SMC nuclei and actin filaments within 1 day and progressive SMC proliferation, that was associated with medial thickening and a change in the SMC orientation from 5 to 30 days. Further studies showed that a local inflation of normal jugular veins to 120 mm Hg for 3 min induced a similar change as found in the vein grafts, whereas the organization of the SMCs was not significantly changed in vein-vein grafts, that did not experience a change in tensile stress. These results suggested that increased tensile stress contributed to the initial damage of the SMCs and played a role in the regulation of medial SMC remodeling in vein grafts. © 1998 Biomedical Engineering Society. PAC98: 8722-q, 8745-k     

microRNA-214-3p在周期性张应变诱导内皮祖细胞分化和增殖中的作用

目的探讨microRNA-214-3p(miR-214-3p)在周期性张应变诱导内皮祖细胞(endothelial progenitor cells, EPCs)分化和增殖中的作用。方法采用FX-5000T细胞周期性张应变加载装置对EPCs施加生理水平的周期性张应变(5%幅度、1.25 Hz频率),加载时间24 h。应用miRNAs芯片筛选周期性张应变调控下差异表达的miRNAs,并挑选miR-214-3p进行深入研究。实时荧光定量PCR方法检测EPCs内平滑肌细胞(vascular smooth muscle cells, VSMCs)相标志分子的表达,BrdU结合酶联免疫吸附ELISA法检测EPCs增殖功能。之后,使用miR-214-3p抑制剂抑制miR-214-3p的表达,检测EPC内VSMC相标志分子表达及EPCs增殖。结果周期性张应变显著抑制miR-214-3p表达,并抑制EPCs向VSMC相分化,同时显著促进EPCs增殖。在静态条件下,使用miR-214-3p抑制剂干扰miR-214-3p的表达,miR-214-3p水平下降同样会抑制EPCs向VSMC相分化,并且诱导EPCs增殖能力显著上升。结论生理水平的周期性张应变能够抑制EPCs内miR-214-3p表达,从而抑制EPCs向VSMC相分化,并且促进EPCs增殖。研究结果为血管损伤的治疗提供新的治疗靶点。     

188铼辐射抑制血管平滑肌细胞增殖研究

目的：探讨放射性核素¹⁸⁸铼（¹⁸⁸Re）对培养平滑肌细胞增殖的作用及其机制。方法：应用¹⁸⁸Re β射线进行培养平滑肌细胞的内辐射。通过细胞计数、再增殖试验、[³H]-TdR掺入试验、流式细胞术细胞周期分析、免疫细胞化学及细胞存活率检测等方法,观察辐射抑制平滑肌细胞增殖的有效及最佳剂量、有效抑制时间、细胞增殖率变化、细胞周期分布、细胞存活率及其相关基因表达。结果：5.2 Gy剂量辐射,50%以上平滑肌细胞增殖受抑,细胞增殖率为46%;20.6 Gy剂量辐射,DNA合成抑制率达92%,增殖期细胞占3%;辐射后2周未见细胞再增殖,<20.6 Gy辐射未见细胞存活力下降;辐射后P53表达升高,PCNA表达降低。结论：¹⁸⁸Re β辐射对平滑肌细胞增殖抑制有效且持久半数有效剂量为5 Gy最佳辐射剂量为20 Gy。低剂量辐射抑制平滑肌细胞增殖的主要机制为损伤细胞分裂增殖能力。P53及PCNA调控辐射抑制细胞增殖过程。     

Enhanced proliferation of aortal smooth muscle cells treated by 1,25(OH)2D3 in vitro coincides with impaired formation of elastic fibres

Elastin is the major extracellular matrix component synthesized, secreted and deposited by vascular smooth muscle cells (SMCs) in the arterial media and thus plays an important role in vascular homeostasis. Results of our previous studies showed that 1alpha,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)-calcitriol] accelerates proliferation of SMCs and modulates their growth in vitro. The aim of this study was to find ultrastructural support for the idea that 1,25(OH)(2)D(3)-calcitriol affects elastic fibre formation due to accelerated proliferation of aortal SMCs in vitro. SMCs exposed 10 days to supraphysiological concentration (10 nM) of calcitriol in primary culture were examined by fluorescence and transmission electron microscopy. Morphological studies revealed that calcitriol altered elastin maturation by favouring accumulation of immature rather than fully processed elastic fibres. A substantial decrease in the amorphous elastin deposition and abnormal accumulation of microfibrillar component, in thickened multilayer culture, were observed. These studies suggest that 1,25(OH)(2)D(3) affect formation of elastic fibres due to enhanced proliferation of SMCs in culture.     

Hepatic artery and portal vein remodeling in rat liver: vascular response to selective cholangiocyte proliferation

Three-dimensional reconstruction of the biliary tree, hepatic artery, and portal vein in normal rats and rats fed alpha-naphthylisothiocyanate (ANIT), a compound that causes selective proliferation of epithelial cells (ie, cholangiocytes) that line the bile ducts, was performed. All hepatic structures in ANIT-fed rats branched 1.5 times more often than in normal rats, reflecting an increased number of segments, whereas the length of the biliary tree, hepatic artery, and portal vein remain unchanged. The length of the proximal vessel segments was uniform in both groups of rats whereas the length of distal segments decreased twofold in ANIT-fed rats, suggesting that small vessels preferentially undergo proliferation. In contrast, the length of all bile duct segments decreased twofold, suggesting that ANIT induced proliferation of all compartments of the biliary tree. The total volume of the biliary tree, hepatic artery, and portal vein was increased 18, 4, and 3 times, respectively, after ANIT feeding. The diameters of the bile ducts (range, 20 to 259 microm) and arterial (range, 21 to 276 microm) segments in ANIT-fed rats did not differ from normal rats (range, 21 to 245 microm and 20 to 265 microm, respectively). In contrast, the diameters of proximal venous segments in ANIT-fed rats were significantly less (316 +/- 68 micro m versus 488 +/- 89 micro m, P < 0.001). The data suggest that after experimentally induced cholangiocyte proliferation, the hepatic artery and portal vein also undergo marked proliferation, presumably to support the increased nutritional and functional demands of the proliferated bile ducts. The molecular mechanisms of these vascular changes remain to be determined.     

Effects of venous pressure elevation on myogenic vasoconstrictive responses to static and dynamic arterial pressures

In order to establish the nature of the stretch-evoked dynamic properties of vascular smooth muscle in arterioles, we have examined the static and dynamic effects of both arterial pulse pressure and elevated venous pressure on the resistance vessels (arteries and arterioles) in an intestinal mesenteric preparation derived from dogs. The dynamic myogenic response to stretch stimuli was directly related to both the frequency of arterial pulse pressure (1-20 c/min) and the level of venous pressure (0-45 mmHg). Under elevated venous pressure (20 mmHg), the mean arterial flow decreased with an increase in the frequency of arterial pulse pressure. The arteriolar vascular tone (namely, vascular resistance) was seen to be enhanced. We found that elevated venous pressure promotes active constriction (9-53%) of arteriolar smooth muscle (myogenic mechanism). The elevation of venous pressure also caused a rhythmic constriction (vasomotion) in the site of both vein and artery, which was completely abolished by an alpha-blocker (phentolamine). The results suggest that during venous pressure elevation a very pronounced myogenic constriction in terminal arterioles is caused by either a local neural reflex or a propagated myogenic response in the arteriolar network.     

Mechanisms of arterial graft failure. 1. Role of cellular proliferation in early healing of PTFE prostheses.

Failure of long-term synthetic arterial bypass grafts has been attributed in part to anastomotic stenosis, but the pathologic basis for this has not been determined. Which cells participate in the formation of the stenosis and the relationship between normal healing and the pathologic development of anastomotic narrowing have not been delineated. In this study we have examined early wound healing in 4-mm polytetrafluorethylene arterial bypass grafts placed in baboons. In this primate model, endothelium and smooth muscle cells (SMCs) derived from the cut ends of adjacent artery form the new intima and migrate together along the luminal surface of the graft at approximately 0.2 mm/day. Both cell types proliferate in association with the growing edge. In addition, both endothelium and SMCs located discretely over anastomoses continue to proliferate despite complete endothelial coverage. Intimal cross-sectional area in this region is always greater than over adjacent graft. Fibroblasts are invariably found in graft matrix and adventitia and do not contribute to formation of intima. It is hypothesized that anastomotic narrowing might be due to chronic endothelial injury and turnover associated with continued SMC proliferation and intimal thickening.     

Changes in lectin binding patterns in the developing pulmonary vasculature of the pig lung

Lectin binding patterns to the developing pulmonary vasculature were studied in 10 Large White pigs aged 1 min to 1 week and in three adult animals. Paraffin-embedded tissue sections were exposed to eight lectin peroxidase conjugates: Dolichos biflorus, Triticum vulgaris, Concanavalin A, Ricinus communis type 2, Arachis hypogaea, Ricinus communis type 1, Tetragonolobus purpureas and Ulex europeus. Lectin binding patterns to the pulmonary arterial and venous endothelium, to smooth muscle cells (SMCs) and to the arterial connective tissue were age-related. Changes occurred during the first week of life and between 1 week and adult life. Neither the endothelial binding patterns in the adult nor the SMC patterns in the immature and adult lung conformed to known morphological differences between the different segments of the arterial and venous pathways. Heterogeneity for endothelial binding was seen in the immature lung. These studies indicate biochemical differences in surface structure between the endothelium, SMCs and connective tissue of the immature and mature lung. Ultrastructural localization of the lectins in the vasculature of the developing animal lung ought to help interpret similar data obtained on the vessels of the immature human pulmonary hypertensive lung using lectins which show similar binding patterns in both species.     

Intracellular pH changes in human aortic smooth muscle cells in response to fluid shear stress.

The smooth muscle cell (SMC) layers of human arteries may be exposed to blood flow after endothelium denudation, for example, following balloon angioplasty treatment. These SMCs are also constantly subjected to pressure driven transmural fluid flow. Flow-induced shear stress can alter SMC growth and metabolism. Signal transduction mechanisms involved in these flow effects on SMCs are still poorly understood. In this work, the hypothesis that shear stress alters the intracellular pH (pHi) of SMC is examined. When exposed to venous and arterial levels of shear stress, human aortic smooth muscle cells (hASMC) undergo alkalinization. The alkalinization plateau persisted even after 20 min of cell exposure to flow. Addition of amiloride (10 micromoles) or its 5-(N-ethyl-N-isopropyl) analog (EIPA, 10 micromoles), both Na+/H+ exchanger inhibitors, attenuated intracellular alkalinization, suggesting the involvement of the Na+/H+ exchanger in this response. The same concentrations of these inhibitors did not show an effect on pHi of hASMCs in static culture. 4-Acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM), a Cl-/HCO3- exchange inhibitor, affected the pHi of hASMCs both in static and flow conditions. Our results suggest that flow may perturb the Na+/H+ exchanger leading to an alkalinization of hASMCs, a different response from the flow-induced acidification seen with endothelial cells at the same levels of shear stress. Understanding the flow-induced signal transduction pathways in the vascular cells is of great importance in the tissue engineering of vascular grafts. In the case of SMCs, the involvement of pHi changes in nitric oxide production and proliferation regulation highlights further the significance of such studies.     

Morphology and increased growth rate of atherosclerotic intimal smooth-muscle cells

Atherosclerotic intimal smooth-muscle cells (SMCs) in vitro showed higher growth activity than did medial SMCs obtained from either atherosclerotic or normal aortas. Using an electron microscope, it was proved in primary cultures by an explant method that intimal SMCs had rich organelles and fewer filaments in their cytoplasms. They were regarded as synthetic phenotype. In contrast, most medial SMCs had rich filaments and fewer organelles. They were regarded as contractile phenotype. When atherosclerotic intimal and normal medial SMCs were plated on type I collagen gel, cytoplasmic cyclic adenosine monophosphate concentration increased and DNA synthesis was suppressed. Intimal SMCs cultured on the gel showed contractile phenotype. Dibutyryl cyclic adenosine monophosphate added to culture media decreased DNA synthesis and altered cellular phenotype to a contractile state. Intimal SMCs were more resistant to injury by hyperlipidemic low-density liproprotein and homocysteine. Lysosomal enzyme activity was enhanced in intimal SMCs.     

Mechanical stretching of human saphenous vein grafts induces expression and activation of matrix-degrading enzymes associated with vascular tissue injury and repair.

After coronary artery bypass surgery, saphenous vein graft occlusion occurs through tissue remodeling. Although a likely trigger, the role of preparative mechanical injury incurred by the graft is not yet understood. We studied the early effects of simple mechanical injury on human saphenous vein grafts by exposing them to longitudinal stretch, a deformation which potentially occurs during surgery. We then maintained ex vivo for up to 7 days matched pairs of experimentally stretched and nonstretched (control) vein segments and examined the expression and activation of matrix metalloproteinases (MMPs) and integrin alphav, molecules implicated in vascular remodeling. At peak expression on day 3, stretched vein secreted 177 +/- 16% active MMP-2 (P < 0.01), 161 +/- 36% (P < 0.05) pro-MMP-9, and contained 206 +/- 18% (P < 0.01) alphav, a receptor for active MMP-2, compared to control. In situ gelatinase activity was present in the intima and adventitia of stretched veins, but not of control, and correlated spatially with expression of alphav. Stretch also increased severalfold cell proliferation (1.27 +/- 0.4 vs. 0.23 +/- 0.05% in control, P < 0.05), as assessed by bromodeoxyuridine incorporation. Furthermore, we found that cell proliferation colocalized with gelatinase activity and alphav in the adventitia. Our results show that a single longitudinal stretch of vein grafts produces significant changes in the expression and activation of key molecules in vascular remodeling. We also found support for the notion that the adventitial layer contributes to vein graft remodeling.     

Effect of pH changes on reactivity of rat mesenteric artery segments at different magnitude of stretch

The reaction to noradrenaline (NA) (10 microM) and electrical field stimulation (EFS) was studied in rat mesenteric artery segments at different magnitude of stretch and the solution pH. Alkaline solution (pH 7.8) potentiated and acidic solution (pH 7.0 or 6.6) inhibited the EFS-evoked response of segments stretched to values corresponding to arterial pressure of 5-200 mmHg. These pH changes failed to alter resting tension at any magnitude of stretch. Acedic solution of pH 6.6 caused 2-fold decrease in noradrenaline- and 5-15-fold decrease in the EFS-evoked response of segments stretched to values corresponding to arterial pressure of 50, 125, and 200 mmHg. In segments pre-contracted with noradrenaline (10 microM) acidification caused the decrease of the dilation and appearance of the constriction induced by the EFS. The effect of acidosis on the EFS-evoked response was diminished and the effect on noradrenaline-evoked response was abolished in the presence of nitric oxide synthase blocker, NG-nitro-L-arginine (100 microM). These results suggest that acidosis effectively impairs reactivity of the rat mesenteric artery in a wide range of its stretch, and the inhibition of the response to noradrenaline occurs completely, while to EFS only partially due to nitric oxide (NO) release, presumably by the endothelium. In addition, it was shown that acidosis is able to act not only as the commonly known dilator agent, but also as an agent potentiating constriction in case of the high noradrenaline-induced tone.     

Immunocytochemistry of intermediate filaments in cultured arterial smooth muscle cells: differences in desmin and vimentin expression related to cell of origin and/or plating time

The objective of this study was to determine whether intermediate filament expression, including desmin and vimentin, in cultured smooth muscle cells (SMCs) is related to cyto-differentiation or proliferation. Using antibodies to desmin and vimentin, we studied by immunoperoxidase technique the distribution of these proteins in subcultured SMCs derived from porcine aorta and coronary artery. In addition, the proliferative potentiality of the cells was estimated by the incorporation of [3H]thymidine into DNA. The frequency of desmin-positive cells in coronary arterial SMCs of 3 and 6 population doubling levels was significantly higher as compared to findings with the aortic SMCs and depended on the plating time. No difference was evident at the 12 population doubling level. In contrast, vimentin was present in the majority of both aortic and coronary arterial SMCs. With regard to the localization of vimentin, two cell types were observed, one had reaction products to vimentin in both perinuclear and cell-peripheral areas (type-I cell), the other only in the cell-peripheral region (type-II cell). The relative proportion of the type-I and -II cells varied with the period of culture. Most of the SMCs showed the type-I cell on the first day and the number of type-II cells was increased on the sixth day. Quiescent SMCs in serum-free media had the same percentage of desmin-positive cells and frequency distribution of type-I and -II cells as did the proliferating SMCs incubated in media containing 5% serum. These results suggest that intermediate filament expression, including desmin and vimentin in cultured SMCs, is related to cell origin and/or plating time, but not to the proliferating activity, per se.     

Cyclic stretch affects pulmonary endothelial cell control of pulmonary smooth muscle cell growth

Endothelial cells are subjected to mechanical forces in the form of cyclic stretch resulting from blood pulsatility. Pulmonary artery endothelial cells (PAECs) produce factors that stimulate and inhibit pulmonary artery smooth muscle cell (PASMC) growth. We hypothesized that PAECs exposed to cyclic stretch secrete proteins that inhibit PASMC growth. Media from PAECs exposed to cyclic stretch significantly inhibited PASMC growth in a time-dependent manner. Lyophilized material isolated from stretched PAEC-conditioned media significantly inhibited PASMC growth in a dose-dependent manner. This inhibition was reversed by trypsin inactivation, which is consistent with the relevant factor being a protein(s). To identify proteins that inhibited cell growth in conditioned media from stretched PAECs, we used proteomic techniques and found that thrombospondin (TSP)-1, a natural antiangiogenic factor, was up-regulated by stretch. In vitro, exogenous TSP-1 inhibited PASMC growth. TSP-1-blocking antibodies reversed conditioned media-induced inhibition of PASMC growth. Cyclic stretched PAECs secrete protein(s) that inhibit PASMC proliferation. TSP-1 may be, at least in part, responsible for this inhibition. The complete identification and understanding of the secreted proteome of stretched PAECs may lead to new insights into the pathophysiology of pulmonary vascular remodeling.     

FOXO1参与高血压条件下异常张应变诱导的血管平滑肌细胞增殖

目的 探讨高血压条件下异常升高的周期性张应变刺激对血管平滑肌细胞（vascular smooth muscle cells, VSMCs）增殖的影响,以及Forkhead转录因子1（FOXO1）在其中可能的作用。方法 构建腹主动脉缩窄高血压大鼠模型,并以假手术组为对照,应用FX-4000T体外周期性张应变加载系统,分别对VSMCs施加5%的生理性张应变和15%的高血压病理性张应变。Western blot检测VSMCs的FOXO1及p-FOXO1表达水平,BrdU法检测VSMCs 增殖活性。RNA干扰技术抑制VSMCs的FOXO1表达,检测FOXO1、p-FOXO1表达以及VSMCs增殖活性变化。结果 腹主动脉缩窄术后 2和4周,大鼠血压较假手术大鼠明显增高。与假手术大鼠相比,高血压大鼠血管壁细胞增殖活性明显增高,同时 FOXO1及 p-FOXO1表达水平也显著性升高。细胞实验表明,与5%张应变组相比,15%张应变加载显著上调VSMCs的FOXO1、p-FOXO1表达水平,以及VSMCs增殖活性。静态条件下RNA干扰抑制VSMCs的FOXO1及p-FOXO1表达,VSMCs的增殖活性明显降低。结论 高血压病理条件下,异常增高的周期性张应变可能通过促进 FOXO1表达和磷酸化诱导VSMCs增殖。以动物模型观察现象,在细胞分子水平探讨机制,旨在明确FOXO1在高血压血管重建中的作用及其力学生物学机制,为阐明高血压血管重建的发病机理和药物治疗靶标的研究提供新的实验依据。     

In vitro and in vivo studies of ePTFE vascular grafts treated with P15 peptide

The purpose of this study is to evaluate the effectiveness of P15 cell-binding peptide treated ePTFE vascular grafts in vitro and in vivo. The P15 peptide was covalently immobilized onto ePTFE vascular grafts by an atmospheric plasma coating method. In vitro cell growth properties were studied using primary human umbilical vein endothelial cells (HUVECs) and primary human umbilical artery smooth muscle cells (HUASMCs). X-ray photoelectron spectroscopy and amino-acid analysis were used to analyze the surface characteristics of the peptide treated and untreated grafts. The cell growth study showed that the P15 peptide effectively promoted the adhesion and proliferation of endothelial cells. 700% more endothelial cells were proliferated on the P15-treated ePTFE grafts compared to the untreated ePTFE controls. In contrast, the P15 peptide was significantly less effective for promoting the adhesion and proliferation of smooth muscle cells than endothelial cells; only about 100% more smooth muscle cells proliferated on the P15-treated samples compared to the untreated control samples. The sheep model was used in the in vivo study. The amount of neointimal hyperplasia present at the arterial and venous sides of the anastomosis and the degree of endothelialization on the luminal surface of the grafts were assessed. Four P15-treated grafts and two control grafts were implanted as arteriovenous grafts between the femoral artery and vein or the carotid artery and jugular vein in two sheep (n = 6). The in vivo study showed that the thickness of the neointimal hyperplasia of untreated grafts was 3-times thicker than that of P15-treated grafts (P < 0.05) at the venous side of the anastomosis. P15-treated grafts also had a higher degree of endothelialization on the graft lumen.     

Anti-atherogenic activity of wild grape (Vitis thunbergii) extract antagonizing smooth muscle cell proliferation and migration promoted by neighboring macrophages

The proliferation and migration of vascular smooth muscle cells (SMCs) play critical roles in intimal thickening and neointimal hyperplasia in early-phase atherosclerosis. This study tested whether wild grape extract (WGE) suppressed the proliferation and migration of human aortic SMCs induced by neighboring macrophages. Cellular expression of fibrogenic connective tissue growth factor (CTGF) and secretion of collagen IV and matrix metalloproteinase (MMP)-2 were determined in SMCs exposed to THP-1-differentiated macrophage-conditioned media. Proliferation was enhanced in SMCs exposed to macrophage-conditioned media collected during the early stage of differentiation, which was attenuated by treatment with ≥ 10 μg/ml WGE. Increased secretion of CTGF and collagen IV macrophage-conditioned media was suppressed in WGE-supplemented SMCs. TGF-β1-promoted production of CTGF and collagen IV was suppressed by blocking TGF-β receptors of R1 and R2 in SMCs. WGE repressed macrophage-conditioned media-upregulated MMP-2 secretion, indicating that WGE had an ability to encumber plaque rupture within atherosclerotic lesions. In addition, ≥ 1 μg/ml WGE ameliorated the migration of SMCs promoted by neighboring macrophages. These results demonstrate that WGE retarded neointimal hyperplasia and thickening within atherosclerotic plaques largely comprising of macrophages and SMCs. Therefore, WGE may be developed as an anti-proliferative and anti-migratory agent targeting SMCs in the proximity of newly differentiated and resident macrophages.     

Mechanical properties of tissue-engineered vascular constructs produced using arterial or venous cells

There is a clinical need for better blood vessel substitutes, as current surgical procedures are limited by the availability of suitable autologous vessels and suboptimal behavior of synthetic grafts in small caliber arterial graft (<5 mm) applications. The aim of the present study was to compare the mechanical properties of arterial and venous tissue-engineered vascular constructs produced by the self-assembly approach using cells extracted from either the artery or vein harvested from the same human umbilical cord. The production of a vascular construct comprised of a media and an adventitia (TEVMA) was achieved by rolling a continuous tissue sheet containing both smooth muscle cells and adventitial fibroblasts grown contiguously in the same tissue culture plate. Histology and immunofluorescence staining were used to evaluate the structure and composition of the extracellular matrix of the vascular constructs. The mechanical strength was assessed by uniaxial tensile testing, whereas viscoelastic behavior was evaluated by stepwise stress-relaxation and by cyclic loading hysteresis analysis. Tensile testing showed that the use of arterial cells resulted in stronger and stiffer constructs when compared with those produced using venous cells. Moreover, cyclic loading demonstrated that constructs produced using arterial cells were able to bear higher loads for the same amount of strain when compared with venous constructs. These results indicate that cells isolated from umbilical cord can be used to produce vascular constructs. Arterial constructs possessed superior mechanical properties when compared with venous constructs produced using cells isolated from the same human donor. This study highlights the fact that smooth muscle cells and fibroblasts originating from different cell sources can potentially lead to distinct tissue properties when used in tissue engineering applications.