骨髓基质干细胞种植体外修复内皮及对血管平滑肌细胞增生的影响

目的探讨骨髓基质干细胞(MSCs)种植体外修复内皮的可行性及对血管平滑肌细胞增生的影响。方法培养兔血管内皮、平滑肌和人MSCs,通过细胞共培养模拟血管内皮修复过程,用流式细胞仪分析MSCs分子表型特征,免疫荧光细胞化学法观察与内皮共培养的MSCsFlk1和vWF蛋白表达,根据下室内皮生长状态及是否接种MSCs将其分为对照组、单纯MSCs组、融合内皮组、对数内皮组和MSCs种植组。氚胸腺嘧啶脱氧核苷(3HTdR)掺入检测平滑肌细胞DNA合成,Westernblot检测平滑肌细胞中增殖细胞核抗原蛋白表达。结果分离的MSCs表达基质细胞标志CD105和CD166,不表达造血干祖细胞和内皮细胞标志CD34、Flk1、vWF;与内皮共培养5天时,vWF染色仍为阴性,但约25.71%MSCs开始表达Flk1;MSCs种植组平滑肌细胞3HTdR掺入虽高于融合内皮组,但与对数内皮组比较显著降低;MSCs种植组平滑肌细胞PCNA蛋白吸光度相对值虽高于融合内皮组,但与对数内皮组比较明显减少。结论MSCs种植能抑制平滑肌细胞增生,种植在成熟内皮中的MSCs具有微环境依赖向内皮分化的能力。     

紫杉醇联合骨髓基质干细胞种植体外修复内皮及对血管平滑肌增生的影响

目的探讨紫杉醇联合骨髓基质干细胞种植体外修复内皮的可行性及对血管平滑肌细胞增生的影响。方法培养兔主动脉内皮、平滑肌和人骨髓基质干细胞,通过细胞共培养将内皮/骨髓基质干细胞接种于下室、平滑肌细胞接种于上室模拟血管内皮修复过程,分别用3H TdR掺入和Westernblot检测紫杉醇(1,10,100nmol/L)干预20min后第10天平滑肌DNA合成和PCNA蛋白表达,用免疫荧光细胞化学法观察与紫杉醇干预内皮共培养的骨髓基质干细胞vWF和Flk1蛋白表达。结果骨髓基质干细胞种植组平滑肌细胞3H TdR掺入和PCNA蛋白光密度相对值均高于融合内皮组(n=6,P<0.05),低于对数内皮组(n=6,P<0.05)。共培养前骨髓基质干细胞不表达vWF和Flk1蛋白,与紫杉醇干预内皮共培养10天时vWF染色阴性,但部分骨髓基质干细胞开始表达Flk1蛋白。结论骨髓基质干细胞种植能部分抑制紫杉醇引起的平滑肌细胞延迟增生,与紫杉醇干预内皮共培养的骨髓基质干细胞有向内皮分化的能力。     

人骨髓基质干细胞的分子表型特征及微环境依赖向内皮分化的能力

目的 :探讨人骨髓基质干细胞的分子表型特征及与成熟内皮共培养时向内皮分化的能力。方法 :采用密度梯度离心法分离培养人骨髓基质干细胞 ,用荧光激活细胞分选法分析其CD34、CD10 5和CD16 6表达率 ,用免疫荧光细胞化学法观察与成熟兔主动脉内皮共培养的人骨髓基质干细胞Flk -1和vWF蛋白表达 ,并分析anti VEGF抗体对骨髓基质干细胞Flk -1表达的影响。结果 :分离培养的骨髓基质干细胞CD34表达率为 (4 .16± 0 . 16 ) % ,与阴性对照 (4. 0 6± 0 . 2 3) %相比无统计学差异 ,CD10 5表达率为(90 .2 0± 2 . 35 ) % ,CD16 6表达率为 (82. 30± 3. 2 2 ) % ,均明显高于阴性对照 (n =6 ,P <0 .0 5 ) ;与成熟内皮细胞共培养 5d时 ,vWF染色仍为阴性 ,但部分骨髓基质干细胞开始表达Flk -1;anti VEGF抗体呈浓度依赖地抑制Flk- 1阳性骨髓基质干细胞计数 (n =6 ,P <0 .0 5 )。结论 :与成熟内皮共培养的骨髓基质细胞具有微环境依赖向内皮细胞分化的能力。     

小鼠内皮祖细胞的分离、培养与定向诱导分化

目的 建立一种稳定、高效,从小鼠骨髓中分离培养与定向诱导分化内皮祖细胞(EPCs)的方法。方法 从小鼠骨髓中密度梯度离心法分离单个核细胞,经差速贴壁结合特殊培养基扩增并向内皮细胞定向诱导分化EPCs。应用免疫荧光和流式细胞技术鉴定内皮细胞系列标志:CD34、CD31、Flk-1和祖细胞标志CD133。并通过检测其对FITC标记的UEA-1的吸附和内吞DiI-ac-LDL来进行细胞功能学的鉴定。对分化细胞行vWF、CD31 免疫组化染色鉴定,并与血管内皮细胞合成前列腺素能力进行比较。结果 经过梯度密度离心和贴壁法选择的细胞表达内皮细胞特异性抗原CD34、CD31、Flk-1,部分表达CD133。分离所得细胞经EBM-2专用培养基培养后,第4天可见集落形成,培养第9天流式细胞仪检测其CD34、CD133、CD31、Flk-1阳性率分别为(44±4)%、(18±3)%、(49±4)%和(79±6)%,细胞能特异性吸附FITC标记的荆豆凝集素并内吞DiI-ac-LDL,约3周左右可融合近80%,形成铺路石样内皮细胞特有形态。传代后vWF、CD31免疫组化染色阳性率分别为(66±5)%和(56±5)%。诱导后的内皮祖细胞的合成前列腺素能力与血管内皮细胞之间无显著差异。结论 从小鼠骨髓中分离培养与定向诱导分化EPCs的方法,效率高,稳定性和重复性好。     

骨髓源性内皮祖细胞与内皮细胞共培养促进其向成熟内皮细胞分化的研究

目的：探讨共培养的内皮细胞对内皮祖细胞向成熟内皮细胞分化的影响。方法：分离1～2个月龄,SD大鼠股骨髓,个核细胞（MNCs）,MNCs,Dil-ac-LDL与FITC-UEA-1荧光双染鉴定内皮细胞的特性。应用贴块法培养大鼠腹主动脉的内皮细胞,vWF免疫组化染色进行鉴定。采用Transwell培养板,上、下室分别加入内皮祖细胞和内皮细胞,15%胎牛血清的NO.2 DMEM/F12培养液培养14 d,倒置相差显微镜观察培养内皮祖细胞的形态。RT-PCR检测eNOS、vWF mRNA,流式细胞术检测CD31及KDR的表达。结果：荧光双染显示培养的单个核细胞具有内皮祖细胞特性;共培养的内皮祖细胞呈短梭型、铺路石状,培养至4 w时有复杂的网状结构形成。RT-PCR检测显示,eNOS及vWF mRNA表达均较对照组显著增加（P〈0.05）。流式细胞术分析表明,共培养组的内皮祖细胞CD31及KDR的表达,也显著高于对照组（分别为P〈0.05及P〈0.01）。结论：与内皮细胞共培养可促进内皮祖细胞向成熟内皮细胞分化。     

Endothelial cells lining transjugular intrahepatic portasystemic shunts originate in hepatic sinusoids: implications for pseudointimal hyperplasia

The phenotype of the endothelial cells (ECs) in the pseudointima of transjugular intrahepatic portasystemic shunts (TIPS) and the mechanisms of pseudointima formation after TIPS were unknown. We hypothesized that TIPS were lined by hepatic sinusoidal ECs, which stimulated the migration of smooth muscle cells (SMCs) into the pseudointima and their proliferation. Studies were done with the following specific aims: (1) isolation of ECs from TIPS pseudointima and comparison of their phenotype with human cirrhotic sinusoidal and vascular ECs derived from hepatic and portal veins as well as aorta, and (2) testing of the effects of TIPS ECs on TIPS-derived SMC migration and proliferation. ECs were isolated from eight TIPS retrieved from liver explants by immunomagnetic separation using monodispersed magnetizable polystyrene beads (Dynabeads M-450) coated with Ulex Europeus 1. EC phenotypes were examined by transmission electron microscopy, factor VIII-related antigen, CD31, CD14, and CD34 expression, uptake of acetylated LDL and secretion of type IV collagen. The effects of EC-conditioned media on SMC migration and proliferation were tested in multiwell chemotaxis chambers and by cell counting, respectively. ECs were obtained from TIPS pseudointima with >95% purity. The phenotype of TIPS-derived ECs matched that of cirrhotic sinusoidal endothelium (both expressed CD14) and differed from that of vascular endothelium (CD14 negative, Weibel-Palade positive). Conditioned media from both stenosed (n = 3) and nonstenosed (n = 3) TIPS-derived endothelial cells produced a marked (>100%) P <.001 increase in migration as well as (up to 88%) P <.01 proliferation of SMCs from both stenosed (n = 3) as well as nonstenosed TIPS (n = 3). These data indicate that TIPS pseudointima are lined by hepatic sinusoidal endothelial cells, which stimulate pseudointima formation by increasing SMC migration and proliferation.     

Cultured bovine aortic endothelial cells secrete factor(s) chemotactic for aortic smooth muscle cells

Interactions of vascular endothelial cells (ECs) and smooth muscle cells (SMCs) were studied by testing the ability of cultured bovine aortic ECs to secrete factors influencing the migration of cultured aortic SMCs from the same species. Migration of SMCs was examined in blind-well chambers using gelatin-coated polycarbonate filters. Conditioned culture medium obtained by incubating confluent monolayers of ECs in serum-free RPMI-1640 medium for 48 hours caused a 2.4-fold increase in the migration of SMCs as compared with nonconditioned medium (p less than 0.001). The effect was dependent on the length of conditioning with the ECs and was chemotactic in nature as judged on the basis of checkerboard analysis. Preliminary characterization of the migration stimulating activity indicates that it is sensitive to trypsin, nondialyzable, and stable at 56 degrees C for 30 min. The activity was abolished by heating to 100 degrees C for 20 min but was not significantly inhibited by protamine sulphate, which suggests that most of the activity was not due to platelet-derived growth factor (PDGF)-like proteins. Our results thus show that ECs secrete polypeptide(s) chemotactic for vascular SMCs. Such interactions between ECs and SMCs in vivo might contribute to the migration of medial SMCs into the intima during atherogenesis.     

细胞间接触诱导大鼠骨髓间质干细胞分化为平滑肌细胞

目的体外诱导骨髓间质干细胞分化为平滑肌细胞,探讨已分化成熟的平滑肌细胞及细胞因子对骨髓间质干细胞分化的影响。方法分别用骨髓间质干细胞加平滑肌细胞条件培养液;骨髓间质干细胞与平滑肌细胞分层培养;骨髓间质干细胞EGFP标记后,与平滑肌细胞混合培养。用平滑肌细胞抗体SM-a-Actin、Calponin免疫荧光染色,检测骨髓间质干细胞的分化情况。结果骨髓间质干细胞与平滑肌细胞混合培养7d后免疫荧光染色,可见EGFP(绿色)和抗SM-a-Actin、Calponin(红色)的双标细胞存在。而加平滑肌细胞条件培养液与分层培养组,骨髓间质干细胞均不表达Calponin。结论细胞间直接接触对诱导骨髓间质干细胞定向分化为平滑肌细胞起决定作用。     

The interactions between brain microvascular endothelial cells and mesenchymal stem cells under hypoxic conditions

The purpose of the present study was to investigate the interactions between brain microvascular endothelial cells (BMEC) and mesenchymal stem cells (MSC) under hypoxic conditions. Primary cultured human bone marrow MSC and rat BMEC were isolated, cultured and identified. Vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were detected in the conditioned media of BMEC and MSC under normal and hypoxic conditions using ELISA. MSC differentiation was analyzed using flow cytometry and fluorescence immunocytochemistry. Transendothelial electrical resistance (TEER) techniques were employed to measure changes in permeability across the BMEC monolayer. Under hypoxic conditions, the concentration of VEGF and MMP-9 in the conditioned media increased significantly, with greater levels in the MSC than the BMEC media. Primary MSC did not express vWF and Flk-1. MSC were co-cultured with BMEC under hypoxic conditions 5 days later. MSC expressing Flk-1 accounted for 23.64+/-2.50% (n=6, P<0.001) of the total number of cells. Interestingly, some Flk-1 positive cells began to coexpress vWF simultaneously. Under hypoxic conditions, MSC conditioned media significantly enhanced the proliferation and migration of BMEC. In addition, MSC decreased the TEER of the BMEC monolayer (lowest values: 50.5+/-2.6% of the original), which could partially be inhibited by both anti-VEGF antibody and MMP-9 inhibitor. These data indicate that under hypoxic conditions BMEC induce MSC to differentiate into endothelial cells, and MSC enhance the proliferation and migration of BMEC through paracrine functions, while simultaneously increasing the permeability of the BMEC monolayer.     

Direct and indirect effects of pulsatile shear stress on the smooth muscle cell.

BACKGROUND: Anastomotic intimal hyperplasia is still an unsolved problem after small caliber prosthetic bypass grafting. Oscillatory turbulent flow occurs at the end to side anastomosis, and produces various effects on smooth muscle cells (SMCs) and endothelial cells (ECs), which compose intimal hyperplasia. We examined the influences of pulsatile oscillating shear stress on smooth muscle cells mitogenic activity induced by sheared endothelial cells. METHODS:1) Smooth muscle cells were cultured under three different pulsatile shear conditions (mean: 0, 6, and 60 dyne/cm2). 2) Endothelial cells were cultured under both static and sheared condition (mean: 60 dyne/cm2). Using the conditioned media from each well, SMCs were cultured under static and sheared conditions (60 dyne/cm2). Four groups of SMCs were devised by combining the two types of media and the two culture conditions. SMC colony spreading distances were measured as an index of combined migration and proliferation activity. An MTT assay and a cell counting assay were used to determine the proliferation activities of SMCs. RESULTS: 1) SMC spreading activity was suppressed by shear stress. SMC proliferative activity was stimulated by pulsatile turbulent shear stress. 2) SMC spreading activity was stimulated by mitogens derived from ECs under shear stress. However, this augmented SMC spreading activity was attenuated under sheared conditions. The mitogens derived from ECs under pulsatile shear stress had no effects on SMC proliferation activity. CONCLUSIONS: Pulsatile oscillating shear stress attenuates SMC migration activity induced by EC-denve mitogens and stimulates SMC proliferative activity.     

Jun N-terminal kinase inhibitor blocks angiogenesis by blocking VEGF secretion and an MMP pathway

AIM: The excessive proliferation and migration of vascular smooth muscle cells (SMCs) and angiogenesis of endothelial cells (ECs) participate in the growth and instability of atherosclerotic plaques. It is unclear whether Jun N-terminal kinase (JNK) is pro-or anti-atherogenic. METHODS: We examined the direct effect of JNK inhibitor (JNK-I) on the proliferation and formation of tubes by human coronary SMCs and human coronary ECs. RESULTS: Culture medium from JNK-I-treated SMCs prevented ECs from forming tubes in an in vitro model of angiogenesis indirectly by reducing the amount of vascular endothelial growth factor (VEGF) released from SMCs. In addition, JNK-I attenuated the expression of pro-matrix metalloproteinase-2 in ECs. When added back to the medium of SMCs treated with JNK-I, VEGF blocked the inhibitory effect on the formation of tubes. CONCLUSION: Our results indicate JNK-I to have a direct anti-atherogenic effect in SMCs and ECs.     

Effects of C-reactive protein on human pulmonary vascular cells in chronic thromboembolic pulmonary hypertension

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterised by proximal pulmonary vascular obstruction by thrombo-fibrotic material, the origin of which has not been elucidated. Enhanced inflammation could contribute to persistent obstruction by impairing pulmonary vascular cell function in CTEPH. We investigated C-reactive protein (CRP) effects on pulmonary vascular cell function in vitro. Primary cultures of proximal pulmonary endothelial cells (ECs) and smooth muscle cells (SMCs) from CTEPH and nonthromboembolic pulmonary hypertension (PH) patients were established. Recombinant CRP effects on mitogenic activity, adhesion capacity, endothelin-1 and von Willebrand factor (vWF) secretion and intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule-1 expression were investigated in ECs and/or SMCs. Expression of the CRP receptor, lectin-like oxidised low-density lipoprotein receptor (LOX)-1, was evaluated in proximal pulmonary arterial tissue and cells by Western blotting and immunofluorescence. CRP increased CTEPH-SMC proliferation by 250%. CRP increased adhesion capacity, endothelin-1 and vWF secretion by CTEPH-ECs by 37%, 129% and 694%, respectively. CRP-induced adhesion of CTEPH-ECs to monocytes was mediated by ICAM-1. CRP had no effect on cells from nonthromboembolic PH patients, probably because of overexpression of LOX-1 in CTEPH. Local expression of CRP was detected in ECs and SMCs within pulmonary arterial tissue. CRP may contribute to persistent obstruction of proximal pulmonary arteries in CTEPH by promoting vascular remodelling, endothelial dysfunction and in situ thrombosis.     

Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels

A prerequisite for successful tissue engineering is the existence of a functional microvascular network. We hypothesized that such networks can be created and quantified in an in vitro setting by co-culturing endothelial cells (ECs) with tissue-specific ‘bystander cells’ in 3-D gel matrices. To test this hypothesis we adapted a previously described in vitro microcarrier-based angiogenesis assay (V. Nehls and D. Drenckhahn, 1995, Microvasc Res 50: 311–322). On optimizing this assay, we noted that the initial EC-microcarrier coverage depended on EC type and seeding technique employed to coat the microcarrier beads with the ECs. A confluent EC monolayer on the microcarrier surfaces formed only when bovine aortic endothelial cells (BAECs) were admixed to the beads under gentle agitation on an orbital shaker. After embedding BAEC-covered microcarrier beads into a sandwich-like arrangement of collagen or fibrin gels, we assessed cellular outgrowth at different serum concentrations in terms of migration distance and sprout formation. Quantifiable sprout formation was highest at 1% fetal bovine serum (FBS) in collagen matrices and at 0.1% FBS in fibrin matrices. At higher serum concentration, excess cell migration and formation of clusters prevented quantitative analysis of sprouting. Following the fine-tuning of this angiogenesis assay, we co-cultured BAECs with adipose tissue-derived fibroblasts (FBs) and vascular smooth muscle cells (SMCs). While FBs were able to increase the average migration distance of BAECs in both matrices, SMCs enhanced BAEC migration in fibrin, but not in collagen gels. By contrast, the number of newly formed sprouts in fibrin gels was increased by both cell types. We conclude that in this model bystander cells enhance EC network formation in a matrix-dependent manner. Additionally, these results stress the importance of carefully selecting␣the experimental parameters of a given in vitro angiogenesis model.     

骨髓间质干细胞对系统性红斑狼疮患者血管内皮细胞损伤模型修复作用的观察

目的 观察骨髓间质干细胞(MSCs)对系统性红斑狼疮(SLE)患者血清刺激后血管内皮细胞中血管假性血友病因子(vWF)和可溶性血栓调理蛋白(sTM)表达的影响,探讨MSCs对SLE血管内皮损伤后修复的可能作用.方法 人脐静脉内皮细胞株ECV-304和活动期SLE患者血清体外共培养12 h,诱导血管内皮细胞损伤,加入MSCs继续共培养3 d.采用酶联免疫吸附试验(ELISA)法检测上清液中vWF和sTM的表达.结果 SLE血清刺激ECV-304细胞后,上清液中vWF和sTM的表达显著高于未刺激组(P＜0.05)[(30.1±6.1)ng/ml vs(10.2±3.1)ng/ml;(2.41±0.16)vs(1.19±0.14)];加入MSCs后,能显著降低vWF的表达(P＜0.05)[(2.41±0.16)vs(2.19±0.14)],但是对sTM值无明显影响(P＞0.05)[(30.1±6.1)ng/ml vs(26.2±6.0)ng/ml].结论 活动期SLE患者血清可损伤血管内皮细胞,MSCs能降低血管内皮细胞中vWF表达,提示可能对SLE血管内皮损伤有一定的修复作用.     

Irradiation enhances the support of haemopoietic cell transmigration, proliferation and differentiation by endothelial cells

Endothelial cells (ECs) are a critical component of the bone marrow stroma in the regulation of haemopoiesis. Recovery of bone marrow aplasia after radiation exposure depends, in part, on the repair of radiation-induced endothelial damage. Therefore, we assessed the ability of an irradiated human bone marrow EC line (TrHBMEC) to support transmigration, proliferation and differentiation of CD34+ bone marrow cells either irradiated or not in transendothelial migration or co-culture models. Radiation-induced EC damage was reflected by an increased release of soluble intercellular adhesion molecule (sICAM)-1 and platelet endothelial cell adhesion molecule (PECAM)-1. Irradiation of TrHBMECs with a 10 Gy dose strongly enhanced the transmigration of CD34+ cells, granulo-monocytic progenitors (CFU-GM) and erythroid progenitors (BFU-E). While ICAM-1 and PECAM-1 expression on irradiated TrHBMECs was increased, only antibodies against PECAM-1 inhibited the radiation-induced enhanced transmigration of haemopoietic cells. Irradiation of TrHBMECs (5-15 Gy) also increased proliferation and differentiation towards the granulo-monocytic lineage of co-cultured CD34+ cells, as well as colony formation by those cells and the production of interleukin 6 (IL-6), IL-8, granulocyte colony-stimulating factor (CSF) and granulocyte-macrophage CSF. Irradiated TrHBMECs were more capable of stimulating irradiated (1,2 Gy) CD34+ cells and haemopoietic progenitors than non-irradiated TrHBMECs. Together, these results suggest that, despite the radiation-induced damage, irradiated ECs may favour haemopoietic reconstitution after radiation exposure.     

Platelet derived bFGF mediates vascular integrative mechanisms of mesenchymal stem cells in vitro

Patients with myocardial infarction reveal an altered number of circulating mesenchymal stem cells (MSCs). Recently, it was shown that MSCs are able to regenerate myocardial tissue and to differentiate into endothelial cells. The homing mechanisms of MSCs from the circulation into the target tissue, however, are not understood so far. In this study, we evaluated the impact of platelets on MSC recruitment, proliferation, migration and integration into the endothelium. MSCs expressing α_vβ₃ integrin were recruited to human arterial endothelial cells exposed to isolated platelets or IL-1β under high shear conditions. Furthermore, induction of vascular injury in vivo resulted in increased recruitment of injected MSCs as assessed by intravital microscopy and depletion of platelets significantly reduced this adhesion. The interaction of platelets and MSCs was inhibited by pre-incubation with the mAb 7E3 or an RGD protein both blocking β₃ integrin mediated adhesion. Platelets had a chemotactic effect on MSCs, promoted a migratory MSC phenotype and dose- and activation-dependently enhanced migration of MSCs, a process, which was mediated by basic fibroblast growth factor (bFGF). Similarly, platelet derived bFGF increased proliferation of MSCs. Coincubation of MSCs with platelets facilitated integration into an endothelial monolayer, which was significantly reduced by pre-incubation with a blocking mAb to bFGF. We conclude that platelets may play a critical part in the recruitment of MSCs to the endothelium, influence MSC function and promote integration of MSCs into the endothelium.     

JTT-705 blocks cell proliferation and angiogenesis through p38 kinase/p27(kip1) and Ras/p21(waf1) pathways

The excessive proliferation and migration of vascular smooth muscle cells (SMCs) participate in the growth and instability of atherosclerotic plaque. We examined the direct role of a newly developed chemical inhibitor of cholesteryl ester transfer protein, JTT-705, on SMC proliferation and angiogenesis in endothelial cells (ECs). JTT-705 inhibited human coronary artery SMC proliferation. JTT-705 induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular-signal-regulated kinases (ERK) in SMCs. In addition, the anti-proliferative effects of JTT-705 in SMCs were blocked by p38 MAPK inhibitor. JTT-705 induced the upregulation of p-p21(waf1), and this effect was blocked by dominant-negative Ras (N17), but not by inhibitors of p38 MAPK or ERK. In addition, JTT-705 also induced the upregulation of p27(kip1), and this effect was blocked by p38 MAPK inhibitor. Interestingly, culture medium from JTT-705-treated SMCs blocked human coronary artery EC tube formation in an in vitro model of angiogenesis indirectly via a decrease in vascular endothelial growth factor (VEGF) from SMCs and directly via an anti-proliferative effect in ECs. JTT-705 blocked the proliferation of SMCs through the activation of p38 kinase/p27(kip1) and Ras/p21(waf1) pathways, and simultaneously blocked EC tube formation associated with a decrease in VEGF production from SMCs and an anti-proliferative effect in ECs. Our results indicate that JTT-705 may induce a direct anti-atherogenic effect in addition to its inhibitory effect of CETP activity.     

Migration of mononuclear cells expressing β-actin through the adventitia into media and intima in coronary arteriogenesis and venogenesis in ischemic myocardium

It was previously thought that arteriogenesis and venogenesis are induced not only by proliferation of vessel-resident smooth muscle cells (SMCs) and endothelial cells (ECs) but also by migration of their precursors. However, it is not well understood through what route(s) the precursors migrate into the existing vessels.We examined through what route or routes circulating mononuclear cells expressing β-actin (β-MNCs), which we identified in canine coronary vessels, migrate into coronary vessel walls and cause arteriogenesis and venogenesis at 1, 2, 4 and 8 weeks after induction of myocardial infarction.The following changes were observed: (1) The β-MNCs migrated via coronary microvessels to the interstitial space at one week; (2) β-MNCs traversed the adventitia into the media and settled in parallel with pre-existing smooth muscle cells (SMCs) in arterioles and arteries and lost β-actin and acquired α-smooth muscle actin (α-SMA) to become mature SMCs at 2-4 weeks; (3) at the same time, other β-MNCs migrated across the adventitia and media into the intima and settled in parallel with pre-existing endothelial cells (ECs) and lost β-actin, while acquiring CD(31), to become mature ECs, resulting in arteriogenesis; (4) Similarly, β-MNCs migrated into venular and venous walls and became SMCs or ECs, resulting in venogenesis.β-MNCs in the interstitial space expressed CD(34) but not other major vascular cell markers.β-MNCs, possibly a vascular progenitor, migrate not from the lumen but across the adventitia into the media or intima of coronary vessels and transit to SMCs or ECs, and participate in arteriogenesis and venogenesis in ischemic myocardium.     

Overexpression of endothelial NO synthase induces angiogenesis in a co-culture model

OBJECTIVE: Angiogenesis is a complex multistep process that involves endothelial cell (EC) migration, proliferation and differentiation into vascular tubes. NO has been reported to be a downstream mediator in the angiogenic response to a variety of growth factors, but the mechanisms by which NO promotes neovessel formation is not clear. We hypothesized that NO directly contributes to EC migration and capillary tube formation. METHODS: Since previous studies have noted important biological differences between NO produced pharmacologically by NO-donor compounds compared to that from NO synthase (NOS), we used a cell-based gene transfer approach to increase NO production in a co-culture model of in vitro angiogenesis. Rat smooth muscle cells (SMCs) were transfected with plasmids containing VEGF(121), VEGF(165) (SMC(VEGF)), endothelial NOS (SMC(eNOS)) or the empty vector (SMC(Cont)). Expression of the eNOS in SMC(eNOS) was confirmed by Northern analysis, NADPH-diaphorase activity, and nitrite/nitrate levels, whereas VEGF production was confirmed using ELISA. Calf pulmonary artery ECs (CPAECs) were cultured on the fibrin matrix with (co-culture) or without underlying SMCs (monoculture). RESULTS: Co-culture of ECs with SMC(Cont) had no effect on EC differentiation compared with EC in monoculture (differentiation index, DI=2.8+/-3.4 vs. 2.1+/-2.8, respectively, NS). In contrast, co-culture with SMC(eNOS) resulted in the formation of extensive capillary-like structures within 48 h (DI=17.2+/-5.9, P<0.001 versus SMC(Cont)), which was significantly inhibited using a NOS inhibitor, L-NAME (3 mM) (DI=4.5+/-3.04, P<0.001 versus SMC(eNOS)). Similarly, SMC(VEGF121) induced an angiogenic response (DI=14.2+/-3.8), which was also significantly inhibited by L-NAME (DI=5.9+/-1.8, P<0.05). In using the Boyden chamber model, SMC(eNOS), but not SMC(Cont) increased EC migration to a similar extent as SMC(VEGF121), and both were significantly inhibited with L-NAME. CONCLUSIONS: These data support an important paracrine role for endogenously produced NO in EC migration and differentiation in vitro, and suggest that the cell-based eNOS gene transfer may be a useful approach to increase new blood vessel formation in vivo.