Liver Anti-Fibrosis Therapy with Mesenchymal Stem Cells Secreting Hepatocyte Growth Factor

Abstract

The objective of this study is to investigate the anti-fibrotic effect of combined mesencymal stem cells (MSCs) and gene therapy on liver fibrosis. When transfected by the complex with a plasmid DNA of hepatocyte growth factor (HGF) and the spermine-introduced pullulan of gene carrier, MSCs secreted HGF protein over 1 week. The HGF secreted from transfected MSC had the biological activity to promote the albumin production of hepatocytes. After intravenous injection, the HGF-secreting MSCs (HGF-MSC) accumulated in the liver. The injection of HGF-MSC decreased the fibrosis area in a rat model of liver fibrosis to a significantly great extent compared with that of original MSC. In the in vitro experiment, the higher number of HGF-transfected MSCs was migrated by stromal cell-derived factor (SDF)-1α more strongly than the original MSC. Considering the promotion of SDF-1α secretion in the liver fibrosis, it is possible that, when transplanted, genetically-engineered MSCs are accumulated in the liver due to their higher response to SDF-1α. It is concluded that the intravenous injection of genetically-engineered MSCs is a promising therapy for liver fibrosis.     

Mechanisms by which thrombolytic therapy results in nonuniform lysis and residual thrombus after reperfusion

A transport reaction model describing penetration of plasmin by diffusion and permeation into a dissolving fibrin gel was solved numerically to explore mechanisms that lead to the formation and growth of dissolution fingers through blood clots during thrombolytic therapy. Under conditions of fluid permeation driven by arterial pressures, small random spatial variations in the initial fibrin density within clots (±4 to 25% peak variations) were predicted by the simulation to result in dramatic dissolution fingers that grew in time. Within vitro experiments, video microscopy revealed that the shape of the proximal face of a fibrin gel, when deformed by pressure-driven permeation, led to lytic breakthrough in the center of the clot, consistent with model predictions of increased velocities in this region leading to cannulation. Computer simulation of lysis of fibrin retracted by platelets (where more permeable regions are expected in the middle of the clot due to retraction) predicted cannulation of the clot during thrombolysis. This residual, annular thrombus was predicted to lyse more slowly, because radial pressure gradients to drive inner clot permeation were quite small. In conjunction with kinetic models of systemic pharmacodynamics and plasminogen activation biochemistry, a two-dimensional transport-reaction model can facilitate the prediction of the time and causes of clot cannulation, poor reperfusion, and embolism during thrombolysis.     

在纤维蛋白凝块上诱导脐血间充质干细胞向膀胱尿路上皮细胞的分化◆

背景：在纤维蛋白凝块上诱导脐血间充质干细胞分化为膀胱尿路上皮细胞,尚无明确方案。 目的：探讨脐血来源间充质干细胞在纤维蛋白凝块上分化为膀胱尿路上皮细胞的可能性。 方法：实验组将脐血间充质干细胞接种于人工制备的脐血来源的纤维蛋白凝块上与胎儿膀胱尿路上皮细胞共培养,对照组将脐血间充质干细胞接种于人工制备的脐血来源的纤维蛋白凝胶上仅用DMEM/F1培养基培养。 结果与结论：实验组的脐血间充质干细胞逐渐伸展呈多角形,细胞扁平变大,透射电子显微镜下具有膀胱尿路上皮细胞的超微结构,抗广谱角蛋白(AE1/AE3)免疫组织化学染色阳性。对照组的脐血间充质干细胞呈长梭形,抗广谱角蛋白(CK AE1/AE3)免疫组织化学染色阴性。提示在纤维蛋白凝块上诱导脐血间充质干细胞有分化为膀胱尿路上皮细胞的能力。      

High Sensitivity Micro-Elastometry: Applications in Blood Coagulopathy

Highly sensitive methods for the assessment of clot structure can aid in our understanding of coagulation disorders and their risk factors. Rapid and simple clot diagnostic systems are also needed for directing treatment in a broad spectrum of cardiovascular diseases. Here we demonstrate a method for micro-elastometry, named resonant acoustic spectroscopy with optical vibrometry (RASOV), which measures the clot elastic modulus (CEM) from the intrinsic resonant frequency of a clot inside a microwell. We observed a high correlation between the CEM of human blood measured by RASOV and a commercial thromboelastograph (TEG), (R = 0.966). Unlike TEG, RASOV requires only 150 μL of sample and offers improved repeatability. Since CEM is known to primarily depend upon fibrin content and network structure, we investigated the CEM of purified clots formed with varying amounts of fibrinogen and thrombin. We found that RASOV was sensitive to changes of fibrinogen content (0.5–6 mg/mL), as well as to the amount of fibrinogen converted to fibrin during clot formation. We then simulated plasma hypercoagulability via hyperfibrinogenemia by spiking whole blood to 150 and 200% of normal fibrinogen levels, and subsequently found that RASOV could detect hyperfibrinogenemia-induced changes in CEM and distinguish these conditions from normal blood.     

Thrombin and fibrin-induced growth of fibroblasts: Role in wound repair and thrombus organization

Summary Skin fibroblast cultures were treated with various components of the blood clotting system (thrombin, fibrinogen and fibrin) during the logarithmic growth phase. Fibrin as well as thrombin showed dose-dependent growth promoting activities as revealed by cell counting and³H-thymidine uptake. No effect was seen with fibrinogen. After entrapping in polymerizing fibrin enriched by complete culture medium the cells elongated, multiplied and formed net-like interconnecting cell strands throughout the clots. Nutritional deprivation appeared as a limiting factor for eventual growth cessation. The results demonstrate active growth of fibroblasts in fibrin clots such as present in healing wounds and thrombi. The production of thrombin by the coagulation cascade does not only result in the conversion of fibrinogen to fibrin but has also a long-lasting hormone-like effect on fibroblast proliferation which is of essential importance in wound healing, thrombus organization and progression of chronic atherosclerotic lesions.Supported by Deutsche Forschungsgemeinschaft     

Effect of EGF and bFGF on Fibroblast Proliferation and Angiogenic Cytokine Production from Cultured Dermal Substitutes

Abstract

Growth factors accelerate wound healing but the underlying mechanisms remain poorly understood. The aim of this study was to investigate the effect of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on fibroblast proliferation and production of angiogenic factors from cultured dermal substitutes (CDS). In the first experiment, fibroblasts were seeded into a flask at a density of 1 × 10⁴ cells/cm².Cell proliferation was assessed after culturing in media containing EGF or bFGF at concentrations ranging from 2 to 50 μg. The number of fibroblasts increased significantly in the presence of EGF or bFGF, but fibroblasts detached from the flasks in the presence of 50 μg bFGF. In the second experiment, CDS were prepared by incorporating fibroblasts into collagen gels. To make a wound surface model, the CDS was elevated to the air–liquid interface, on which a spongy sheet of hyaluronic acid (HA) containing EGF or bFGF was placed. The amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released from the CDS after 1 week of cultivation was measured by ELISA. When the CDS was covered with a HA sponge containing EGF (Group 1), fibroblasts released 3.5-times more VEGF compared with a HA-alone sponge (control group). When covered with a HA sponge containing bFGF (Group 2), 8.7-times more VEGF was released compared with the control group. Fibroblasts in Groups 1 and 2 released 9.6- and 9.3-times more HGF, respectively, compared with the control group. Thus, EGF stimulates fibroblasts to produce VEGF and HGF, in addition to its ability to enhance epidermal cell proliferation.     

Improved neovascularization and wound repair by targeting human basic fibroblast growth factor (bFGF) to fibrin

Targeted therapy is a new generation of therapeutics, where two critical factors are involved. One is the particular molecular target, and the other is the specific target-binding drug. In this work, the fibrin, a main component of plasma clot at wound sites, was used as the target for human bFGF, aiming to improve therapeutic neovascularization and wound repair. To endow bFGF with fibrin-targeting ability, a fibrin-binding peptide Kringle1 (K1), derived from human plasminogen, was fused to human bFGF. The recombinant K1bFGF showed high fibrin and plasma-clot-binding ability. When applied to the wound sites with plasma clots, K1bFGF induced robust neovascularization and improved wound healing. To extend the application of K1bFGF to other cases where no plasma clots exist, we developed a fibrin-scaffold/K1bFGF system. This system could induce localized neovascularization by delivery of K1bFGF in a sustained and site-targeting manner, and provide a microenvironment promoting cell growth and tissue regeneration. In summary, we successfully used the pathologic environment fibrin clot as the target for bFGF, and based on which bFGF was designed into a targeting agent by introduction of a fibrin-binding peptide. This provides a potential approach to improve therapeutic neovascularization and wound repair.     

含重组碱性成纤维细胞生长因子培养基中3T3成纤维细胞在血纤维蛋白凝块上生长的可能性（英文）

目的：重组碱性成纤维细胞生长因子对培养条件下３Ｔ３成纤维细胞在血纤维蛋白凝块上生长的可能研究。方法：采用Ｇｉｅｍｓａ染色和ＭＴＴ检测法,经过电镜扫描,分段对比观察,研究３Ｔ３细胞在血纤维蛋白凝块上的生长情况。结果：ｒｈｂＦＧＦ促进细胞生长产维持细胞存活的最佳浓度是１００ｎｇ／ｍｌ,在含有１００ｎｇ／ｍｌ的低血清培养基中细胞可在血纤维蛋白凝块生长。经４８ｈ培养以后仍有大量的细胞存活。结论：３Ｔ３成纤     

Composition of emboli influences the efficacy of thrombolysis with rt-PA in a rat stroke model

Background and purpose: The efficacy of thrombolysis with recombinant tissue plasminogen activator and its dependency upon clot composition was tested in an embolic stroke model.Methods: In 53 rats the carotid territory was embolised with two arterial-like type of clots and one venous-like type of clot. The arterial ones were either fibrin poor (group A) or fibrin rich (group B). Hemispheric cerebral blood flow before and after embolisation was measured by intraarterial 133 Xenon injection method. 15 min after embolisation 18 animals were treated with tissue plasminogen activator 20mg/kg, and 35 animals with saline. Carotid angiography displayed the rate of occlusion of the cerebral arterial supply before and after treatment. Brains were fixed after 2 days, evaluated neuropathologically and infarct volume measured.Results: Cerebral blood flow was reduced by 36 to 62% after embolisation. The comparison of post-treatment angiography of treated animals to controls in group embolised with arterial-like fibrin rich clots showed significant (p=0.0163) reperfusion in thrombolytic treated animals. Thrombolytic therapy significantly reduced the infarct volume from 11.6 to 1.9% of embolised hemisphere volume (p=0.019) in group embolised with fibrin-poor clots and from 18.3 to 0.0% (p=0.0001) in the group embolised with fibrin-rich clots. Among the treated animals embolised with fibrin rich clots, both the completely recanalised animals (p=0.0003) and the partially recanalised animals (p=0.027) developed smaller infarctions than control animals. No hemorrhagic complications were observed. The control animals embolised with venous-like clots recanalised spontaneously and developed almost no infarctions and only temporary clinical damage.Conclusions: Early thrombolytic therapy induced recanalisation and reduced infarct volume after embolic stroke, this effect was particularly apparent in the group embolised with fibrin-rich clots.     

碱性成纤维细胞生长因子培养条件下的三维组织培养

目的:在含重组人碱性成纤维细胞生长因子(rhbFGF)的细胞培养基条件下,研究巨噬细胞和成纤维细胞以血纤维蛋白凝块或脂肪组织为支架,构建三维组织模型的可能性。方法:采用相差显微镜观察、吉姆萨(Giemsa)染色和扫描电子显微镜观察巨噬细胞和成纤维细胞在血纤维蛋白凝块或脂肪组织支架上生长情况。结果:巨噬细胞和成纤维细胞在血纤维蛋白凝块和脂肪组织支架上均生长良好,细胞间无排斥现象,部分细胞间建立了细胞桥连接。结论:血纤维蛋白凝块和脂肪组织支架对两种细胞都表现出无免疫原性和很好的组织相溶性,细胞间不仅能够共存,而且还存在着细胞交流。     

Albumin concentration influences fibrinolytic activity in plasma and purified systems

Delayed clot lysis was observed in 9 patients with nephrotic syndrome. This delay, assessed by fibrin degradation product release, was corrected by supplementing patients' plasma with purified human albumin to reach the normal albumin concentration of 45g/1. The effect of albumin on clot lysis was further assessed in the presence of albumin-depleted plasma and in a purified system containing tissue-type plasminogen activator (t-PA), plasminogen, fibrin monomers and increasing albumin concentrations. An increase in plasminogen uptake was observed when clots were prepared in the presence of such concentrations. We conclude that low albumin levels may alter clot structure and reduce spontaneous thrombolysis, thereby increasing the risk of thrombosis.     

Biochemical characterization of autologous fibrin sealants produced by CryoSeal and Vivostat in comparison to the homologous fibrin sealant product Tissucol/Tisseel

Different principles for production of "autologous fibrin sealant" have been established, and commercial devices employing these methods are nowadays available and used in clinical routine. Users might anticipate for these autologous fibrin sealants features comparable to commercial homologous fibrin sealants, used in surgical routine for many years. However, only little is known about biochemical properties, formation, cross-linking and stability of fibrin sealant clots produced for autologous use with the aid of commercially available devices. We have investigated protein composition, formation and stability of clots obtained from autologuous fibrin sealants produced with commercially available devices (CryoSeal and Vivostat) and compared these parameters to those of the industrially produced homologous fibrin sealant Tissucol/Tisseel. The CryoSeal product is a mixture of many plasma proteins; the Vivostat product and Tissucol/Tisseel appear as comparatively pure plasma derivatives. The products differ in their protein composition and concentrations, including their concentration in fibrin. Significant fibrin alpha and gamma-chain cross-linking by FXIIIa occurs only in Tissucol/Tisseel clots. In test tubes CryoSeal and Vivostat (tranexamic acid-free formulation) fibrin clots liquefy within 1-2 days, but Vivostat (tranexamic acid containing formulation) clots were stable for 4 days and showed partial liquefaction after 5 days. Tissucol/Tisseel clots, containing the protease inhibitor aprotinin, appeared unchanged over the observation period of 5 days. In an in vitro model mimicking in vivo conditions (diffusion of protease inhibitors and proteolytic digestion) clot liquefaction occurs at day 1 for all autologous fibrin sealants clots, with an observable delay for the tranexamic acid containing Vivostat, and day 5 for Tissucol/Tisseel clots. Characterization of the CryoSeal and Vivostat fibrin sealants and Tissucol/Tisseel and their performance show a clear difference in biochemical properties.     

Regulation of extravascular fibrinolysis by factor XIII

The stability of fibrin clots in the extravascular compartment was examined using samples of ¹²⁵I-labelled fibrin implanted subcutaneously into Balb/c mice. These samples of ¹²⁵I-labelled fibrin were prepared in vitro. Crosslinking of fibrin in the presence of a high level of factor XIIIa resulted in complete conversion of γ-chain monomers to γ-γ dimers and extensive covalent polymerisation of the α-chain monomers. This fibrin, categorised as fully crosslinked (FXL), showed a 50% clot lysis time (LT-50) of 9.6±0.8 days. In contrast, fibrin, termed as partially crosslinked (PXL), prepared with a low level of Factor XIIIa, had all of its γ-chain monomers converted γ-γ dimers, but only a portion of its α-chain monomer polymerised. This PXL fibrin showed an LT-50 of 4.8±1.1 days. Non-crosslinked (NXL) fibrin displayed an LT-50 of 5.2±1.2 days. Fibrin produced by the addition of thrombin to plasma was of the PXL type and gave an LT-50 of 4.2±1.0 days. Fibrin produced by the addition of thrombin to whole blood, on the other hand, was found to be FXL and showed an LT-50 of 13.0 ± 2.0 days. Western blot analysis of fibrin samples prepared from plasma and whole blood with the use of antibody to α₂-plasmin inhibitor (α₂-PI) provided evidence that this inhibitor was covalently crosslinked to fibrin from both sources. FXL fibrin proved far more stable than either PXL fibrin or NXL fibrin to fibrinolysis by leukocyte elastase in vitro (p<0.001). Inclusion of the specific elastase inhibitor Elastatinal in the implanted NXL fibrin clot extended the LT-50 from 5 to 11.7 days (p<0.001), whereas a plasmin inhibitor, ϵ-amino caproic acid caused no change in lysis time. These results suggest that extravascular fibrinolysis is mediated chiefly by leukocyte proteases and that factor XIIIa-catalysed α-chain crosslinking is essential for protection of extravascular clots against fibrinolysis by these proteases.     

Secreted klotho protein attenuates osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro via inactivation of the FGFR1/ERK signaling pathway

Abstract

Increasing evidence indicates that the osteogenic differentiation of mesenchymal stem cells (MSCs) is related to bone formation, heterotopic ossification, and even vascular calcification. Therefore, it is essential to understand the microenvironment that regulates these processes. The Klotho gene plays an important role in tissue mineralization, and its secreted protein functions as a hormone. We investigated the effects of secreted Klotho protein on the osteogenesis of human bone marrow MSC (hBMSCs). To this end, the cells received osteogenic medium with or without Klotho protein. The results showed that osteoblast-specific gene expression and mineral deposition were decreased when MSCs were incubated with Klotho. Klotho reduced the expression of fibroblast growth factor receptor 1 (FGFR1) and phosphorylated extracellular signal-regulated kinase 1/2. However, both MEK and FGFR1 inhibitors delayed bone mineral formation more than Klotho. These data suggest that secreted Klotho protein attenuates the osteogenic differentiation of hBMSCs in vitro through FGFR1/ERK signaling.     

Solubility fo fibrin clots in monochloroacetic acid. A reflection of serum pepsinogen levels.

Fibrin clots formed from normal plasma dissolve readily in 1% monochloroacetic acid at 37 C. However, if the clots are washed thoroughly before the acid is added, they are no longer soluble. The agent present in the serum which causes dissolution of the fibrin clot was isolated and identified as pepsinogen. Because of the low pH of monochloroacetic acid the pepsinogen is activated and the clots are digested, simulating the dispersion of a fibrin clot which occurs in the absence of fibrin-stabilizing factor (factor XIII). Because of its higher pH, urea will not activate pepsinogen and is therefore a better agent to screen factor XIII deficiencies.     

Activation of mannan‐binding lectin‐associated serine proteases leads to generation of a fibrin clot

The lectin pathway of complement is activated upon binding of mannan‐binding lectin (MBL) or ficolins (FCNs) to their targets. Upon recognition of targets, the MBL‐and FCN‐associated serine proteases (MASPs) are activated, allowing them to generate the C3 convertase C4b2a. Recent findings indicate that the MASPs also activate components of the coagulation system. We have previously shown that MASP‐1 has thrombin‐like activity whereby it cleaves and activates fibrinogen and factor XIII. MASP‐2 has factor Xa‐like activity and activates prothrombin through cleavage to form thrombin. We now report that purified L‐FCN‐MASPs complexes, bound from serum to N‐acetylcysteine‐Sepharose, or MBL‐MASPs complexes, bound to mannan‐agarose, generate clots when incubated with calcified plasma or purified fibrinogen and factor XIII. Plasmin digestion of the clot and analysis using anti‐D‐dimer antibodies revealed that the clot was made up of fibrin and was similar to that generated by thrombin in normal human plasma. Fibrinopeptides A and B (FPA and FPB, respectively) were released after fibrinogen cleavage by L‐FCN‐MASPs complexes captured on N‐acetylcysteine‐Sepharose. Studies of inhibition of fibrinopeptide release indicated that the dominant pathway for clotting catalysed by the MASPs is via MASP‐2 and prothrombin activation, as hirudin, a thrombin inhibitor that does not inhibit MASP‐1 and MASP‐2, substantially inhibits fibrinopeptide release. In the light of their potent chemoattractant effects on neutrophil and fibroblast recruitment, the MASP‐mediated release of FPA and FPB may play a role in early immune activation. Additionally, MASP‐catalysed deposition and polymerization of fibrin on the surface of micro‐organisms may be protective by limiting the dissemination of infection.     

Osteogenic response of human adipose-derived stem cells to BMP-6, VEGF,and combined VEGF plus BMP-6 in vitro

Exogenous addition of three factors—mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF), and bone morphogenetic proteins (BMPs)—has proven to be more beneficial than delivery of any single factor for fracture repair in animal models. We studied the osteogenic differentiation of human adipose-derived stem cells (hADSCs) in the presence of VEGF, BMP-6, or VEGF plus BMP-6 to better understand their enhancement of osteoblastic differentiation of MSCs. The VEGF plus BMP-6 group demonstrated an additive effect on the enhancement of mineralization and expression of ALP and Msx2 genes. Unlike VEGF or BMP-6 alone, the combination of VEGF and BMP-6 significantly enhanced the expression of COL1A1, osterix, and Dlx5 genes. The data indicate that a cross-talk between VEGF and BMP-6 signaling pathways enhances osteogenic differentiation of hADSCs.     

Soluble fibrin is a useful marker for predicting extracorporeal membrane oxygenation circuit exchange because of circuit clots

A circuit clot is one of the most frequent complications during extracorporeal membrane oxygenation (ECMO) support. We identify coagulation/fibrinolysis markers for predicting ECMO circuit exchange because of circuit clots during ECMO support. Ten patients with acute pulmonary failure who underwent veno-venous ECMO were enrolled between January 2014 and December 2016. ECMO support lasted 106 days. The 6 days on which the ECMO circuits were exchanged were considered as circuit clot (+) group, while the remaining 100 days were considered as circuit clot (?) group. The predictors of ECMO circuit exchange because of circuit clots were identified. The mean duration of ECMO support was 10?±?13 days, and the mean number of ECMO circuit exchange was 0.6?±?1.1 times per patient. Thrombin-antithrombin complex (TAT) and soluble fibrin (SF) were higher in the circuit clot (+) group than in the circuit clot (?) group (both P?<?0.01). According to a multivariate analysis, SF was the only independent predictor of ECMO circuit exchange (P?<?0.01). The odds ratio (confidence intervals) for SF (10 µg/ml) was 1.20 (1.06–1.36). The area under the curve and optimal cut-off value were 0.95 and 101 ng/ml for SF (sensitivity, 100%; specificity, 89%). SF may be useful in predicting ECMO circuit exchange because of circuit clots.     

Potential of a Cryopreserved Cultured Dermal Substitute Composed of Hyaluronic Acid and Collagen to Release Angiogenic Cytokine

Abstract

An allogeneic cultured dermal substitute (CDS) was prepared by culturing fibroblasts on a spongy matrix of hyaluronic acid (HA) and collagen (Col), which was then cryopreserved. This cryopreserved allogeneic CDS (CDS-1; cryopreserved for 1 month, CDS-6; cryopreserved for 6 months) was thawed and re-cultured for a period of 7 days to investigate the potential of the CDS for wound treatment. The cell metabolic activity in the CDS and their cytokine production were measured using an MTT assay and ELISA. Fibroblast metabolic activity in each CDS-1 and CDS-6 immediately after thawing and following 3 and 7 days of re- cultivation was 56, 67 and 93%, and 49, 64 and 86%, respectively, of that before cryopreservation. The amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released from the CDS-1 on days 1, 3 and 7 of re-cultivation after thawing was 8, 44 and 92% (VEGF) and 3, 7 and 28% (HGF), respectively, of that before cryopreservation. The amount of VEGF and HGF released from the CDS-6 on days 1, 3 and 7 of re-cultivation after thawing was 9, 32 and 45% (VEGF) and 6, 10 and 27% (HGF), respectively, of that before cryopreservation. These findings showed that the potential of the CDS was restored to some extent over the first 3 days of re-cultivation after thawing. The potential of the CDS for wound treatment was then evaluated using a wound surface model, in which the each CDS-1 and CDS-6 that was re-cultured for 3 days after thawing was elevated at the air/culture medium interface, and a wound dressing was placed on top, and then cultured for 5 days. Two different types of wound dressing were tested. Fibroblasts in the CDS in Group II (placing a wound dressing with EGF) released increased amount of VEGF and HGF compared with that in Group I (placing a wound dressing without EGF). These findings suggest that re-culture of the CDS for 3 days following thawing results in a CDS with improved wound healing potential and that an EGF-incorporating wound dressing is useful as a top dressing for the CDS.