Engineering calcium deposits on polycaprolactone scaffolds for intravascular applications using primary human osteoblasts

Total atherosclerotic occlusions often include significant calcium deposits. Current animal models do not mimic the pathology of gradual occlusion of arteries and lack cell‐mediated calcium. The primary goal of this project was to establish an animal model incorporating these features into chronic total occlusions, using biodegradable scaffolds. As the first step, this study sought to determine the optimal dosage of TGF‐β1 on polycaprolactone (PCL) scaffolds cultured with primary human osteoblasts (HOBs) to effectively induce in vitro calcification. HOBs were cultured in TGF‐β1 and dexamethsaone (Dex)‐supplemented medium in well plates. Calcium in the cultures was visualized using alizarin red. The highest calcification was observed in groups with both TGF‐β1 (0.02 ng/ml) and Dex (10^?10 M ) in the medium. Next, HOBs were cultured on PCL scaffolds with different loadings of TGF‐β1: 0 (control), 5, 10, 50 and 100 ng. These cultures were performed with or without Dex (10^?10 M ) in the medium. DNA content, ALP activity and the amount and distribution of calcium were examined at 7, 14, 21 and 28 days. TGF‐β1 appeared to have an inhibitory effect on scaffold calcification when grown in Dex‐supplemented medium. When cultured without Dex, the lower amount of TGF‐β1 loading (5 ng) showed the most calcification, high DNA synthesis and high ALP activity on scaffolds. This study demonstrates the potential of implanting a PCL–HOB construct in an animal artery to establish a model of atherosclerotic occlusion with calcification. Copyright © 2010 John Wiley & Sons, Ltd.     

Plasminogen activator inhibitor-1 is an aggregate response factor with pleiotropic effects on cell signaling in vascular disease and the tumor microenvironment

In hemostasis, the serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) functions to stabilize clots via inhibition of tissue plasminogen activator (tPA) with subsequent inhibition of fibrinolysis. In tissues, PAI-1 functions to inhibit extracellular matrix degradation via inhibition of urokinase plasminogen activator (uPA). Elevated levels of PAI-1 in the vasculature and in tissues have long been known to be associated with thrombosis and fibrosis, respectively. However, there is emerging evidence that PAI-1 may participate in the pathophysiology of a number of diseases such as atherosclerosis, restenosis, and cancer. In many of these disease states, the canonical view of PAI-1 as an inhibitor of tPA and uPA cannot fully account for a mechanism whereby PAI-1 contributes to the disease. In these cases, one must consider recent data, which indicates PAI-1 can directly promote pro-proliferative and anti-apoptotic signaling in a variety of cell types. Given the wide variety of inflammatory, hormonal, and metabolic signals that increase PAI-1 expression, it is important to consider mechanisms by which PAI-1 can directly participate in disease etiology.     

Disruption of beta-catenin pathway or genomic instability define two distinct categories of liver cancer in transgenic mice

BACKGROUND & AIMS: Human liver cancer can be divided into 2 categories that are characterized by activation of beta-catenin and genomic instability. Here we investigate whether similar categories exist among 5 transgenic models of liver cancer, including c-myc, transforming growth factor-alpha, E2F-1, c-myc/transforming growth factor-alpha, and c-myc/E2F-1 mice. METHODS: The random amplified polymorphic DNA method was used to assess the overall genomic instability, and chromosomal loci affected by genomic alterations were determined by microsatellite analysis. beta-Catenin mutations and deletions were analyzed by polymerase chain reaction and sequencing screening. Cellular localization of beta-catenin and expression of alpha-fetoprotein, a prognostic marker of hepatocellular carcinoma, were investigated by immunohistochemistry. RESULTS: Liver tumors from the transgenic mice could be divided into 2 broad categories characterized by extensive genomic instability (exemplified by the c-myc/transforming growth factor-alpha mouse) and activation of beta-catenin (exemplified by the c-myc/E2F-1 mouse). The c-myc/transforming growth factor-alpha tumors displayed extensive genomic instability with recurrent loss of heterozygosity at chromosomes 1, 2, 4, 6, 7, 9, 12, 14, and X and a low rate of beta-catenin activation. The genomic instability was evident from the early dysplastic stage and occurred concomitantly with increased expression of alpha-fetoprotein. The c-myc/E2F-1 tumors were characterized by a high frequency of beta-catenin activation in the presence of a relatively stable genome and low alpha-fetoprotein levels. CONCLUSIONS: We have identified 2 prototype experimental models, i.e., c-myc/transforming growth factor-alpha and c-myc/E2F-1 mice, for the 2 categories of human hepatocellular carcinoma characterized by genomic instability and beta-catenin activation, respectively. These mouse models will assist in the elucidation of the molecular basis of human hepatocellular carcinoma.     

SMAD2 Mutations Are Associated with Arterial Aneurysms and Dissections

We report three families with arterial aneurysms and dissections in which variants predicted to be pathogenic were identified in SMAD2. Moreover, one variant occurred de novo in a proband with unaffected parents. SMAD2 is a strong candidate gene for arterial aneurysms and dissections given its role in the TGF‐β signaling pathway. Furthermore, although SMAD2 and SMAD3 probably have functionally distinct roles in cell signaling, they are structurally very similar. Our findings indicate that SMAD2 mutations are associated with arterial aneurysms and dissections and are in accordance with the observation that patients with pathogenic variants in genes encoding proteins involved in the TGF‐β signaling pathway exhibit arterial aneurysms and dissections as key features     

TGF⁃β1 p38MAPK及BMP⁃7蛋白在肝多房棘球蚴病患者肝组织中的表达及意义

目的　检测肝多房棘球蚴病患者肝组织中转化生长因子⁃β1（transforming growth factor⁃β1, TGF⁃β1）、p38MAPK及骨形态发生蛋白⁃7（bone morphogenetic protein⁃7, BMP⁃7）表达水平,探讨其在肝多房棘球蚴病肝纤维化中的潜在作用。方法　以20例肝多房棘球蚴病患者为研究对象,分别采集距肝脏病灶0.5 cm内（A组）、距肝脏病灶0.5～1.5 cm（B组）肝组织及距肝脏病灶2 cm及以上的正常肝组织（C组）。肝组织标本分别行HE和Masson染色观察纤维化病理变化,采用Western blotting检测肝组织中TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平,分析TGF⁃β1、p38MAPK及BMP⁃7蛋白表达与肝纤维化的相关性。结果　HE染色结果显示,A组和B组肝组织中肝细胞结构排列紊乱、肝小叶结构有不同程度破坏,可见不同程度肝细胞变性、萎缩、坏死及纤维组织增生,并有嗜酸性粒细胞浸润;C组肝组织无异常病理改变,肝细胞结构形态正常、大小均匀,未见明显排列紊乱,肝小叶结构清晰,无或轻度细胞变性、坏死及炎性细胞浸润。Masson染色结果显示,A组和B组肝组织可见汇管区较多纤维结缔组织增生,出现不同程度小叶内纤维化;C组肝组织无明显异常病理改变。A、B、C组肝组织中TGF⁃β1（P < 0.001）、p38MAPK（P < 0.01）及BMP⁃7蛋白（P < 0.05）表达水平差异均有统计学意义,A组和B组TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平均显著高于C组（P均< 0.05）,B组TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平亦显著高于C组（P均< 0.05）。TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平均与肝纤维化程度呈正相关（r = 0.866、0.702、0.801,P均< 0.05）,不同纤维化程度肝组织中TGF⁃β1（F = 72.580,P < 0.01）、p38MAPK（[χ2] = 31.705,P < 0.01）及BMP⁃7蛋白（[χ2] = 48.388,P < 0.01）表达水平差异均有统计学意义。TGF⁃β1蛋白与p38MAPK、BMP⁃7蛋白表达水平均呈显著正相关（r = 0.607、0.702,P均 < 0.001）,BMP⁃7与p38MAPK蛋白表达水平亦呈显著正相关（r = 0.456,P < 0.001）。结论　TGF⁃β1、p38MAPK和BMP⁃7蛋白通过相互作用、共同介导了肝多房棘球蚴病肝纤维化发生。     

Cholangiocyte endothelin 1 and transforming growth factor beta1 production in rat experimental hepatopulmonary syndrome

BACKGROUND & AIMS: Hepatic production and release of endothelin 1 plays a central role in experimental hepatopulmonary syndrome after common bile duct ligation by stimulating pulmonary endothelial nitric oxide production. In thioacetamide-induced nonbiliary cirrhosis, hepatic endothelin 1 production and release do not occur, and hepatopulmonary syndrome does not develop. However, the source and regulation of hepatic endothelin 1 after common bile duct ligation are not fully characterized. We evaluated the sources of hepatic endothelin 1 production after common bile duct ligation in relation to thioacetamide cirrhosis and assessed whether transforming growth factor beta1 regulates endothelin 1 production. METHODS: Hepatopulmonary syndrome and hepatic and plasma endothelin 1 levels were evaluated after common bile duct ligation or thioacetamide administration. Cellular sources of endothelin 1 were assessed by immunohistochemistry and laser capture microdissection of cholangiocytes. Transforming growth factor beta1 expression and signaling were assessed by using immunohistochemistry and Western blotting and by evaluating normal rat cholangiocytes. RESULTS: Hepatic and plasma endothelin 1 levels increased and hepatopulmonary syndrome developed only after common bile duct ligation. Hepatic endothelin 1 and transforming growth factor beta1 levels increased over a similar time frame, and cholangiocytes were a major source of each peptide. Transforming growth factor beta1 signaling in cholangiocytes in vivo was evident by increased phosphorylation and nuclear localization of Smad2, and hepatic endothelin 1 levels correlated directly with liver transforming growth factor beta1 and phosphorylated Smad2 levels. Transforming growth factor beta1 also stimulated endothelin 1 promoter activity, expression, and production in normal rat cholangiocytes. CONCLUSIONS: Cholangiocytes are a major source of hepatic endothelin 1 production during the development of hepatopulmonary syndrome after common bile duct ligation, but not in thioacetamide-induced cirrhosis. Transforming growth factor beta1 stimulates cholangiocyte endothelin 1 expression and production. Cholangiocyte-derived endothelin 1 may be an important endocrine mediator of experimental hepatopulmonary syndrome.     

Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb,TGF‐β, and RA treatment

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4⁺ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25⁺Foxp3⁺‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25⁺Foxp3⁺ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.     

Potential involvement of BMP receptor type IB activation in a synergistic effect of chondrogenic promotion between rhTGFβ3 and rhGDF5 or rhBMP7 in human mesenchymal stem cells

Chondrogenic promotion by rhGDF5 with or without rhTGFβ3 was studied in pellet culture of human mesenchymal stem cells (HMSCs). A synergy between rhGDF5 and rhTGFβ3 was observed in promoting chondrogenesis. rhBMP2, rhBMP6, rhBMP7 and rhTGFβ1 were further tested and showed the same effect. To explore the mechanism, the expression of TGFβtype I and II receptors, ALK5, ALK2, ALK3, ALK6, TGFβRII, BMPRII, ActRII was studied. ALK6 showed increase by the rhTGFβ1 or rhTGFβ3 treatment. ALK6 protein expression also showed increase by rhTGFβ3. rhTGFβ1/rhTGFβ3 induced ALK6 up-regulation was inhibited by SD-208, a TGFβ type I receptor inhibitor. Chondrogenesis by rhTGFβ1/rhTGFβ3 or the combination between rhTGFβ1/rhTGFβ3 and rhGDF5 also was diminished by SD-208. SMAD1/5/8 phosphorylation in nascent human mesenchymal stem cells (HMSCs) was stimulated weakly by rhGDF5 but strongly by rhBMP7. The rhGDF5 stimulated SMAD1/5/8 phosphorylation was enhanced by rhTGFβ1/rhTGFβ3 but inhibited by SD-208. The rhBMP7 stimulated SMAD1/5/8 phosphorylation did not show influence by rhTGFβ3 and SD-208. Our results indicated the potential involvement of ALK6 activation by rhTGFβs in the synergy between rhTGFβs and rhBMPs.     

TGF-β1／Smad信号转导通路在子痫前期患者胎盘组织中的表达及其临床意义

目的检测子痫前期患者胎盘组织中TGF—β1／Smad的表达,探求TGF—β1／Smad信号转导通路在子痫前期发病中的作用。方法采用免疫组织化学法检测40例子痫前期（轻、重度）和正常孕妇15例正常妊娠妇女胎盘中TGF-β1以及Smad3、Smad4、Smad7的分布和表达,采用TUNEL法检测三组胎盘滋养细胞凋亡的情况。结果TGF-β以及Smad2、Smad3、Smad4、Smad7在正常妊娠和子痫前期患者胎盘的滋养细胞、内皮细胞、基质细胞中均有表达,以滋养细胞表达为主。TGF-β1以及Smad3、Smad4在正常妊娠组、子痫前期轻度组和重度组的表达强度及范围均呈逐渐升高趋势,Smad7在正常妊娠组、子痫前期轻度和重度组表达强度及范围依次呈逐渐下降趋势。正常妊娠和子痫前期患者胎盘的滋养细胞、内皮细胞、基质细胞均存在凋亡,以滋养细胞凋亡为主。重度子痫前期组胎盘滋养细胞凋亡（1．02）％显著高于子痫前期轻度组（0．73）％,子痫前期轻度组显著高于正常妊娠组（0．38）％,P〈0．05。滋养细胞TGF—β1的表达与滋养细胞凋亡具有相关性（r=0．96,P〈0．05）。结论子痫前期患者胎盘组织中的TGF—β1可能是通过Smad信号转导蛋白抑制滋养细胞浸润,诱导细胞凋亡。