Pancreatic islet differentiation of human embryonic stem cells by microRNA overexpression

Development of stem cell‐based therapies for the treatment of type 1 diabetes would provide a renewable supply of human β‐cells. Human embryonic stem cells (ESCs) are considered to be one of the stem cell populations with sufficient proliferative capacity to achieve this goal. Currently, differentiation protocols directing ESCs toward a pancreatic fate employ a variety of expensive cytokines and inhibitors. With the known significance of microRNAs in islet development, we present a novel and cost‐effective strategy in which miR‐375 overexpression promotes pancreatic endocrine differentiation in hESCs in the absence of any extrinsic factors. miR‐375 has been shown to be a key regulator of pancreatic development and function in zebrafish, mouse and human. In this study, hESCs were transduced with lentiviral vectors containing human miR‐375 precursor and aggregated to form human embryoid bodies (hEBs) for up to 21 days. Morphological assessment, immunocytochemistry and DTZ staining confirmed that miR‐375‐induced hEBs have similar characteristics to those of mature islets. In addition, the dynamic expression profile of endodermal marker Foxa2 and endocrine‐specific genes, including HNF4α, Pdx1, Pax6, Nkx6.1, Glut2 and insulin, were detected by quantitative real‐time PCR. Finally, insulin release upon glucose stimulation was detected in our differentiated clusters. The data presented here demonstrate the feasibility of using microRNAs to direct differentiation into the pancreatic lineage. Copyright © 2013 John Wiley & Sons, Ltd.     

Dog mastocytoma cells produce transforming growth factor beta 1.

Transforming growth factor-beta (TGF beta) promotes deposition of extracellular matrix and is associated with fibrotic conditions both in experimental animals and in humans. Although a role for mast cells has been suspected in the pathogenesis of fibrosis, no potent mediator capable of stimulating fibroblast growth or extracellular matrix deposition has been identified in mast cell supernatants. We report here the constitutive production of TGF beta 1 by four dog mastocytoma cell lines. TGF beta 1 was identified by characteristic biologic activity, blockade of biologic effect by specific neutralizing antibody, and by recognition of a band with the appropriate migration by western blot. TGF beta 1 mRNA, but not TGF beta 2 or TGF beta 3 mRNA, was also produced constitutively by all four cell lines. Quantitation by bioassay revealed baseline TGF beta secretion of approximately 1 ng/10(6) cells over 48 h. Stimulation of mastocytoma cells with phorbol ester increased the rate of release of TGF beta 1, most markedly in the first 30 min after stimulation, without increasing TGF beta 1 mRNA. Dog mastocytoma cells produced TGF beta 1 primarily in a latent form, inactive until treated with acid. Both pure TGF beta 1 and TGF beta-containing mastocytoma cell-conditioned media inhibited mitogenesis and proliferation in dog mastocytoma cell lines, suggesting that mast cell tumor lines would not grow preferentially based on their ability to produce TGF beta. These studies may make possible further investigation of the mechanism by which mast cells contribute to the induction of fibrosis.     

骨髓间充质干细胞成骨分化中转化生长因子受体的表达

背景:机体受创后尤其是骨折发生后,骨髓间充质干细胞将启动成骨分化,其表面多种生长因子如碱性成纤维细胞生长因子、转化生长因子、骨形态发生蛋白的受体表达将发生变化.目的:观察骨折损伤后不同时相骨髓间充质干细胞表面转化生长因子受体的表达变化.设计、时间及地点:对照观察实验,于2002-02/2003-11在解放军第三军医大学预防医学院复合伤研究所创伤、烧伤与复合伤国家重点实验室完成.材料:选用健康成年兔,建立成兔桡骨骨折模型.方法:在造模当日及造模后1,5,10,15,20d抽取实验兔骨髓液,取骨髓有核细胞,纯化间充质干细胞,成骨分化培养基诱导培养.主要观察指标:通过Western-blot法检测转化生长因子β1,β2受体在间充质干细胞上的表达.结果:造模后1 d转化生长因子β1受体表达与造模当日比较无明显差异(P>0.05):造模后5d,其表达显著高于造模当日(P<0.05);造模后10d与5d比较无明显差异(P>0.05),但显著高于造模当日(P<0.05);造模后15,20d的转化生长因子β1受体表达显著高丁造模当日及造模后5,10d(P<0.01).造模后1,5d转化生长因子β2受体表达与造模当日无明显差异(P>0.05):造模后10,15d,其表达显著高于造模当日(P<0.05);造模后20d转化生长因子β2受体的表达显著高于造模当日及造模1,5,10,15d(P<0.01).结论:创伤后体内骨髓间充质干细胞成骨分化过程中转化生长因子β1,β2受体的表达呈现逐渐增高的趋势.     

Human transforming growth factor-alpha stimulates bone resorption in vitro.

Tumor-derived transforming growth factors (TGF) have been proposed as possible mediators of hypercalcemia in malignancy. We have studied the action of recombinant human TGF-alpha in cultured bone cells and in bone explant cultures. In clonal UMR-106 rat osteosarcoma cells, TGF-alpha and epidermal growth factor (EGF) were equipotent in binding to the EGF receptor. TGF-alpha and EGF both stimulated resorption of neonatal mouse calvaria, and maximal responses were obtained with 10 ng/ml of TGF-alpha after 72 h in culture. The effects of both TGF-alpha and EGF in calvaria, but not those of parathyroid hormone, were inhibited by 5 X 10(-7) M indomethacin. Fetal rat limb bone cultures were less sensitive to TGF-alpha than neonatal mouse calvaria, with a concentration of 30 ng/ml being required to stimulate resorption in this system. The bone-resorbing activity of TGF-alpha in fetal rat bones was inhibited by 10 ng/ml calcitonin but not by 5 X 10(-7) M indomethacin. EGF at concentrations up to 300 ng/ml did not stimulate resorption of the limb bones at time periods up to 66 h. The results indicate that human TGF-alpha is a potent bone-resorbing agent, and support the concept that this growth factor exhibits some effects distinct from those of EGF. TGF-alpha could play an etiologic role in the hypercalcemia of malignancy.     

转化生长因子β1对骨髓干细胞分化类髓核细胞的影响

背景:转化生长因子β1是骨髓间充质干细胞分化为类髓核细胞的首选生长因子,适当浓度能诱导骨髓间充质干细胞的增殖和分化。目的:观察不同质量浓度转化生长因子β1对大鼠骨髓间充质干细胞体外向类髓核细胞分化的影响,优化骨髓间充质干细胞体外培养条件。方法:取成年大鼠股骨骨髓,体外培养、纯化。取第3代成年大鼠骨髓间充质干细胞,实验组用加入不同质量浓度转化生长因子β1(0,1,10,20μg/L)的HG-DMEM无血清培养液诱导3,7,14,21d;对照组普通状态下用含体积分数为10%胎牛血清的HG-DMEM培养液自然分化。结果与结论:10μg/L转化生长因子β1诱导液诱导的骨髓间充质干细胞蛋白聚糖与Ⅱ型胶原蛋白水平明显高于其他实验组和对照组(P<0.01)。各实验组14d时蛋白聚糖的表达均较3,7,21d时高(P<0.01)。对照组各时间点蛋白聚糖Aggrecan及Ⅱ型胶原表达均呈阴性。提示10μg/L的转化生长因子β1能提高大鼠骨髓间充质干细胞体外诱导分化类髓核的数量,从而使骨髓间充质干细胞发挥更高的治疗效率。     

转化生长因子β1对骨髓干细胞分化类髓核细胞的影响

背景：转化生长因子β1是骨髓间充质干细胞分化为类髓核细胞的首选生长因子,适当浓度能诱导骨髓间充质干细胞的增殖和分化。目的：观察不同质量浓度转化生长因子β1对大鼠骨髓间充质干细胞体外向类髓核细胞分化的影响,优化骨髓间充质干细胞体外培养条件。方法：取成年大鼠股骨骨髓,体外培养、纯化。取第3代成年大鼠骨髓间充质干细胞,实验组用加入不同质量浓度转化生长因子β1（0,1,10,20μg/L）的HG-DMEM无血清培养液诱导3,7,14,21d;对照组普通状态下用含体积分数为10%胎牛血清的HG-DMEM培养液自然分化。结果与结论：10μg/L转化生长因子β1诱导液诱导的骨髓间充质干细胞蛋白聚糖与Ⅱ型胶原蛋白水平明显高于其他实验组和对照组（P〈0.01）。各实验组14d时蛋白聚糖的表达均较3,7,21d时高（P〈0.01）。对照组各时间点蛋白聚糖Aggrecan及Ⅱ型胶原表达均呈阴性。提示10μg/L的转化生长因子β1能提高大鼠骨髓间充质干细胞体外诱导分化类髓核的数量,从而使骨髓间充质干细胞发挥更高的治疗效率。     

Mechanism of transforming growth factor beta-induced inhibition of T helper type 1 differentiation

Regulation by transforming growth factor (TGF)-beta plays an important role in immune homeostasis. TGF-beta inhibits T cell functions by blocking both proliferation and differentiation. Here we show that TGF-beta blocks Th1 differentiation by inhibiting the expression of T-bet, the apparent masterregulator of T helper (Th)1 differentiation. Restoration of T-bet expression through retroviral transduction of T-bet into developing Th1 cells abrogated the inhibitory effect of TGF-beta. In addition, we show that, contrary to prior suggestions, downregulation of interleukin 12 receptor beta2 chain is not key to the TGF-beta-mediated effect. Furthermore, we show that the direct inhibitory effect of TGF-beta on T cells is responsible, at least in part, for the inability of BALB/c mice to mount a Leishmania-specific Th1 response and to clear Leishmanial infection.     

Recombinant human transforming growth factor-alpha stimulates the formation of osteoclast-like cells in long-term human marrow cultures.

Transforming growth factor-alpha (TGF-alpha) is synthesized by a variety of tumor cell lines and stimulates osteoclastic bone resorption in vitro. The mechanism by which TGF-alpha increases osteoclast activity is unknown. We used a human marrow culture system that forms osteoclast-like multinucleated cells (MNCs) to determine the effects of recombinant human TGF-alpha on MNC formation. Addition of 0.01 ng/ml TGF-alpha for the 1st week followed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] for the subsequent 2 wk significantly increased MNCs. Treatment of these cultures with TGF-alpha without later addition of 1,25(OH)2D3 did not increase MNC formation. Autoradiographic studies revealed that TGF-alpha stimulated proliferation of precursors for MNCs, and 1,25(OH)2D3 increased their rate of fusion into MNCs. Addition of murine epidermal growth factor (EGF) (0.1 ng/ml) followed by 1,25(OH)2D3 also significantly stimulated MNC formation. These data suggest that TGF-alpha and EGF may stimulate bone resorption by increasing the proliferation of osteoclast precursors, which leads to increased numbers of osteoclasts.     

碱性成纤维细胞生长因子和转化生长因子β1诱导大鼠骨髓间质干细胞分化为心肌细胞的作用比较

目的:对比观察碱性成纤维细胞生长因子和转化生长因子β1促进大鼠骨髓间质干细胞分化为心肌细胞的作用。方法:实验于2005-12/2006-07在中山大学干细胞与组织工程中心实验室进行。实验材料:清洁级大鼠由中山大学实验动物中心提供(SCXR(粤)2004-0011,粤监证字2004A089),三四周,大约50g。实验方法:①分离培养大鼠骨髓间质干细胞,并采用流式细胞仪鉴定。②取第3代骨髓间质干细胞,以1×107L-1接种于24孔培养板和六孔板中,24h后换液并分组:对照组:仅仅更换培养液;5-氮胞苷诱导组:10μmol/L5-氮胞苷孵育24h后更换新鲜培养液;5-氮胞苷 碱性成纤维生长因子诱导组:10μmol/L5-氮胞苷孵育24h后更换含10μg/L碱性成纤维生长因子的新鲜培养液;5-氮胞苷 转化生长因子β1诱导组:10μmol/L5-氮胞苷孵育24h后更换含10μg/L转化生长因子β1的新鲜培养液。以后每3d换液1次,前2组换完全培养基,后2组分别换含10μg/L碱性成纤维生长因子和10μg/L转化生长因子β1的完全培养基。③30d后应用RT-PCR法检测心肌细胞转录因子GATA-4、结蛋白表达,免疫细胞化学染色法检测细胞心肌肌钙蛋白,并在倒置显微镜下观察细胞跳动情况。结果:①骨髓间质干细胞的生长特性及鉴定:原代培养大鼠骨髓质干细胞3d后镜下可见有细胞贴壁,形成克隆,七八天长满,随着换液和传代,细胞逐渐纯化,第3代细胞形成均一的长梭形,流式细胞仪检测发现CD44、CD29表达阳性,CD45、CD11b表达阴性。②骨髓间质干细胞向心肌细胞的分化:大鼠骨髓间质干细胞经5-氮胞苷、5-氮胞苷 碱性成纤维生长因子或5-氮胞苷 转化生长因子β1诱导30d后,有部分细胞心肌肌钙蛋白表达阳性,GATA-4和结蛋白表达明显增强。镜下观察5-氮胞苷 碱性成纤维生长因子诱导组所诱导细胞变细、胞体伸长,呈心肌样细胞跳动,并较其他两组明显,心肌肌钙蛋白阳性细胞的比率较高(P<0.05)。结论:碱性成纤维生长因子在体外有协同5-氮胞苷促进大鼠骨髓间质干细胞分化为心肌细胞的作用,而转化生长因子β1无此作用。     

转化生长因子β1对树突细胞功能的影响

目的研究转化生长因子β1(TGF-β1)对树突细胞(dendritic cells,DC)功能的影响.方法在培养体系中应用不同的细胞因子培养未成熟DC(imDC,GM-CSF)和TGF-β1处理的DC(TGFβ-DC,GM-CSF+TGF-β1),观察其对脂多糖(LPS)刺激的反应.透射电镜观察细胞超微结构,流式细胞仪检测细胞表型,BrdU ELISA法检测DC刺激异基因T细胞增殖的能力,ELISA法检测DC在LPS刺激后分泌IL-12p70的水平,逆转录-聚合酶链反应(RT-PCR)方法检测Toll-like受体4(TLR4)表达.结果与imDC相比,TGFβ-DC在LPS刺激后仍能保持未成熟的细胞形态.TGFβ-DC的CD80,CD86表达明显低于imDC[(4.14±0.95)%和(13.90±7.22)%;(8.60±0.75)%和(20.63±5.03)%,P值均＜0.05].ImDC对LPS有更强的反应性,其中I-Ab、CD80升高的幅度明显高于TGFβ-DC(P值分别＜0.01及＜0.05).TGFβ-DC在96 h的混合淋巴细胞反应中,DC/T细胞为14,11时,TGFβ-DC的异基因刺激能力较imDC弱(P值均＜0.05).LPS刺激TGFβ-DC 24 h后分泌IL-12 p70的能力显著低于imDC(P＜0.01),TGFβ-DC较imDC弱表达TLR4(P＜0.05).结论TGFβ能抑制DC共刺激分子的表达,且能抵抗LPS的促成熟作用,并可能与其TLR4的表达下降有关.     

检测2型糖尿病患者尿转化生长因子-β1的临床意义

目的探讨检测 2型糖尿病患者尿转化生长因子-β1(TGF-β1) 对诊断糖尿病肾病的临床价值.方法检测 61例2型糖尿病患者及 30名正常人的尿TGF-β1、尿视黄醇结合蛋白(RBP)、α1微球蛋白(α1-MG) 并进行比较分析.结果①糖尿病各组尿 TGF-β1明显高于正常对照组(P<0.01), 其中大量白蛋白尿组明显高于微量白蛋白尿组及正常白蛋白尿组(均为 P<0.01); ②尿 TGF-β1与尿 RBP、α1-MG及尿白蛋白排泄率均呈显著正相关(r分别为 0.63, 0.58, 0.52, 均为 P<0.01). 结论尿TGF-β1测定可作为 2型糖尿病肾病早期诊断的一项敏感指标 .     

Mitogenic response of canine fundic epithelial cells in short-term culture to transforming growth factor alpha and insulinlike growth factor I.

We report methods allowing the culture of rapidly dividing gastric epithelial cells to investigate the regulation of mucosal cell replication. Cells from canine fundic mucosa were dispersed by enzyme treatment, enriched by filtration and elutriation, and cultured on collagen gel in DMEM/F12 medium. After 48 h, greater than 95% of the cells displayed immunoreactivity with antibody to cytokeratin, an epithelial marker. The cells formed confluent monolayers by 72 h with a transmembrane resistance of 1,600 ohm.cm2 when mounted in a Ussing chamber indicating retention of epithelial cell characteristics. Calf serum (0.1-2%) produced a dose-dependent mitogenic effect evident by increases in [3H]-thymidine incorporation into acid-precipitated material and in cell number. After an 18-24-h incubation with [3H]-thymidine, approximately 55% of the cells cultured in 2% serum showed evidence of DNA synthesis by autoradiography and all of the replicating cells were cytokeratin positive. Using comparable culture conditions, a similar proportion of cells incubated for 18-24 h with bromodeoxyuridine displayed nuclear anti-bromodeoxyuridine immunoreactivity, thus indicating that over half of the cells in these cultures synthesized DNA during this period. As with serum, epidermal growth factor and transforming growth factor alpha (TGF alpha) (10 pM to 1 nM), insulin (10 nM to 1 microM) and insulinlike growth factor-I (IGF-I, 1-100 nM) increased [3H]-thymidine uptake. The greater potency of IGF-I, compared to insulin, suggests the presence of IGF-I receptors. We conclude that this culture preparation is composed of fundic mucosal epithelial cells and contains a predominance of dividing epithelial cells. EGF/TGF alpha and IGF-I are potential factors directly regulating proliferation of fundic mucosal cells.     

Fibrin stimulates the proliferation of human keratinocytes through the autocrine mechanism of transforming growth factor-alpha and epidermal growth factor receptor

In the field of dermatology and plastic and reconstructive surgery, fibrin gel is regarded as a material that promotes wound healing. To test the hypothesis that fibrin may promote the growth of the epidermis, we examined its effects on the proliferation of cultured keratinocytes. Human keratinocytes were cultivated in fibrin-coated wells, and the cell numbers and transforming growth factor (TGF)-alpha, secreted into the cultured medium, were measured. We also assessed the capacity of epidermal growth factor receptor (EGF-R) that is responsible for all known actions of TGF-alpha and epidermal growth factor. The keratinocytes increased dramatically in their number, and the TGF-alpha secretion and the binding capacity of EGF-R were also increased dramatically in the presence of fibrin. These findings suggest that fibrin supports the proliferation of keratinocytes in an autocrine fashion via EGF-R; namely, fibrin stimulates keratinocytes to secrete TGF-alpha, which in turn increases cell proliferation and EGF-R capacity. We propose that fibrin can support the wound healing process of the epidermis via the TGF-alpha/EGF-R pathway.     

成纤维细胞生长因子对兔纤维环细胞转化生长因子β2的调节

目的:观察成纤维细胞生长因子对培养兔纤维环细胞转化生长因子β2表达的调节作用。方法:实验于2005-03/2006-04在华中科技大学同济医学院附属协和医院骨科实验室完成。培养4月龄雌性日本大白兔的纤维环细胞,在每次换液时加入成纤维细胞生长因子100μg/L,铺满后收集细胞,作纤维环细胞转化生长因子β2常规免疫组织化学研究,细胞呈棕黄色染色为阳性表达信号,同时为进一步明确纤维环细胞转化生长因子β2的定位进行免疫胶体金标记电镜观察。结果:光镜下细胞明显呈棕色,电镜下细胞膜表面有明显的胶金颗粒粘附。结论:成纤维细胞生长因子能促进纤维环细胞转化生长因子β2的表达。     

Tumour necrosis factor alpha stimulates gastrin release from canine and human antral G cells: possible mechanism of the Helicobacter pylori–gastrin link

There is evidence that gastric Helicobacter pylori ( Hp ) infection promotes duodenal ulceration by releasing gastrin. We therefore asked how Hp releases gastrin. Tumour necrosis factor alpha (TNF-α) is up-regulated in Hp gastritis and stimulates hormone release from pituitary cells, so we tested its effect on primary cultures of canine antral G cells and human antral fragments. TNF-α pretreatment (100ngmL^–1) of canine G cells significantly increased both basal (by 89%: P <0.01) and bombesin-stimulated (by 39% P <0.05) gastrin release. A similar pattern of increase was seen following TNF-α (20ngmL⁻¹) pretreatment of human antral fragments: basal gastrin release was increased by 38% ( P  < 0.05) and bombesin-stimulated by 26% ( P  < 0.05). This effect persisted during immunoblockade with anti-somatostatin antibody S6. We propose that TNF-α provides the link between Hp infection and gastrin release and thus contributes to duodenal ulceration.     

Expression of transforming growth factor alpha and epidermal growth factor receptor in human bladder cancer.

We analysed the expression of epidermal growth factor receptor (EGFr) and transforming growth factor alpha (TGF-alpha) in human bladder tumours. Tumour biopsies were obtained from 54 patients with primary bladder cancer (18 stage T1 and 36 stage T2-4). The protein and mRNA expression of EGFr and TGF-alpha were quantified by ELISA and competitive RT-PCR, respectively. The EGFr protein level was significantly increased in T2-4 tumours (0.44 x 10(-11); 0.0-27.5 x 10(-11) mol/g) compared with T1 tumours (0.0; 0.0-2.0 x 10(-11) mol/g) (median; range; 2p<0.01). The EGFr protein and mRNA level correlated (Spearman r=0.45, 2p<0.005, n=40). Co-expression of TGF-alpha protein and EGFr protein was significantly associated with muscle invasive tumours (T2-4) (chi-squared=7.9, df=3, p<0.05) and the TGF-alpha protein level correlated significantly with EGFr protein expression (Spearman r=0.56, 2p<0.0001, n=54). While tumour stage correlated with survival, no correlation was observed between survival and the expression of EGFr and/or TGF-alpha. In conclusion, human bladder tumours express both EGFr and TGF-alpha. The expression of EGFr and TGF-alpha are closely correlated, and the expression of EGFr and co-expression of EGFr and TGF-alpha correlate with tumour stage.     

Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerular mesangial cells.

Angiotensin II (Ang II) has been implicated in the development of progressive glomerulosclerosis, but the precise mechanism of this effect remains unclear. In an experimental model, we have shown previously that TGF-beta plays a key role in glomerulosclerosis by stimulating extracellular matrix protein synthesis, increasing matrix protein receptors, and altering protease/protease-inhibitor balance, thereby inhibiting matrix degradation. We hypothesized that Ang II contributes to glomerulosclerosis through induction of TGF-beta. Ang II treatment of rat mesangial cells in culture increased TGF-beta and matrix components biglycan, fibronectin, and collagen type I at both the mRNA and protein levels in a time- and dose-dependent manner. Saralasin, a competitive inhibitor of Ang II, prevented the stimulation. Ang II also promoted conversion of latent TGF-beta to the biologically active form. Coincubation of mesangial cells with Ang II and neutralizing antibody to TGF-beta blocked the Ang II-induced increases in matrix protein expression. Continuous in vivo administration of Ang II to normal rats for 7 d resulted in 70% increases in glomerular mRNA for both TGF-beta and collagen type I. These results indicate that Ang II induces mesangial cell synthesis of matrix proteins and show that these effects are mediated by Ang II induction of TGF-beta expression. This mechanism may well contribute to glomerulosclerosis in vivo.     

Bacterial cell wall-induced immunosuppression. Role of transforming growth factor beta

Group A streptococcal cell wall (SCW)-injected rats exhibit a profound immunosuppression that persists for months after the initial intraperitoneal injection of SCW. The goal of this study was to determine the mechanisms for the suppressed T lymphocyte proliferative responses in this experimental model of chronic inflammation. When spleen cell preparations were depleted of adherent cells, restoration of T cell proliferative responses to Con A and PHA occurred, implicating adherent macrophages in the regulation of immunosuppression. Furthermore, macrophages from SCW-treated animals, when cocultured with normal spleen cells in the presence of Con A or PHA, effectively inhibited the proliferative response. Supernatants from suppressed spleen cell cultures were found to inhibit normal T cell mitogenesis. Taken together, these results implicated a soluble macrophage-derived suppressor factor in the down regulation of T cell proliferation after exposure to SCW in vivo. Subsequent in vitro studies to identify this suppressor molecule(s) revealed the activity to be indistinguishable from the polypeptide transforming growth factor beta (TGF-beta). Furthermore, TGF-beta was identified by immunolocalization within the spleens of SCW-injected animals. The cells within the spleen that stained positively for TGF-beta were phagocytic cells that had ingested, and were presumably activated by, the SCW. These studies document that TGF-beta, previously shown to be a potent immunosuppressive agent in vitro, also effectively inhibits immune function in chronic inflammatory lesions in vivo.     

Modulation of transforming growth factor beta receptor levels on microvascular endothelial cells during in vitro angiogenesis.

Microvascular endothelial cells (RFCs) cultured in two-dimensional (2D) cultures proliferate rapidly and exhibit an undifferentiated phenotype. Addition of transforming growth factor beta1 (TGFbeta1) increases fibronectin expression and inhibits proliferation. RFCs cultured in three-dimensional (3D) type I collagen gels proliferate slowly and are refractory to the anti-proliferative effects of TGF beta1. TGF beta1 promotes tube formation in 3D cultures. TGF beta1 increases fibronectin expression and urokinase plasminogen activator (uPA) activity and plasminogen activator inhibitor-1 (PAI-1) levels in 3D cultures. Since the TGF beta type I and II receptors have been reported to regulate different activities induced by TGF beta1, we compared the TGF beta receptor profiles on cells in 2D and 3D cultures. RFCs in 3D cultures exhibited a significant loss of cell surface type II receptor compared with cells in 2D cultures. The inhibitory effect of TGF beta1 on proliferation is suppressed in transfected 2D cultures expressing a truncated form of the type II receptor, while its stimulatory effect on fibronectin production is reduced in both 2D and 3D transfected cultures expressing a truncated form of the type I receptor. These data suggest that the type II receptor mediates the antiproliferative effect of TGF beta1 while the type I receptor mediates the matrix response of RFCs to TGF beta1 and demonstrate that changes in the matrix environment can modulate the surface expression of TGF beta receptors, altering the responsiveness of RFCs to TGF beta1.