Adipose-derived stem cells:Pathophysiologic implications vs therapeutic potential in systemic sclerosis

Adipose-derived stem cells(ADSCs)residing in the stromal vascular fraction(SVF)of white adipose tissue are recently emerging as an alternative tool for stem cell-based therapy in systemic sclerosis(SSc),a complex connective tissue disorder affecting the skin and internal organs with fibrotic and vascular lesions.Several preclinical and clinical studies have reported promising therapeutic effects of fat grafting and autologous SVF/ADSC-based local treatment for facial and hand cutaneous manifestations of SSc patients.However,currently available data indicate that ADSCs may represent a double-edged sword in SSc,as they may exhibit a pro-fibrotic and anti-adipogenic phenotype,possibly behaving as an additional pathogenic source of pro-fibrotic myofibroblasts through the adipocyte-to-myofibroblast transition process.Thus,in the perspective of a larger employ of SSc-ADSCs for further therapeutic applications,it is important to definitely unravel whether these cells present a comparable phenotype and similar immunosuppressive,anti-inflammatory,anti-fibrotic and pro-angiogenic properties in respect to healthy ADSCs.In light of the dual role that ADSCs seem to play in SSc,this review will provide a summary of the most recent insights into the preclinical and clinical studies employing SVF and ADSCs for the treatment of the disease and,at the same time,will focus on the main findings highlighting the possible involvement of these stem cells in SSc-related fibrosis pathogenesis.     

Transplantation of adipose-derived stem cells ameliorates Echinococcus multilocularis-induced liver fibrosis in mice

BackgroundAlveolar echinococcosis (AE) can cause severe liver fibrosis and could be fatal if left untreated. Currently, there are no effective therapeutic options for AE-induced liver fibrosis. In view of the therapeutic potential of adipose-derived stem cells (ADSCs), we investigated whether ADSCs transplantation has the ability to control or reverse fibrosis progression in the liver of Echinococcus multilocularis (E. multilocularis) infected mice.Methodology/Principal findingsC57BL/6 mice infected with E. multilocularis through portal vein inoculation were intravenously injected with ADSCs isolated from inguinal adipose tissues of 6–8 weeks old mice. Histopathological analysis including heamatoxylin & eosin staining as well as Masson’s trichrome staining, and Sirius red staining were performed to access the degree of liver fibrosis. Histopathological examination 30 days after ADSCs transplantation revealed that ADSCs significantly decreased the degree of liver fibrosis in E. multilocularis infected mice by inhibiting the expressions of α-SMA and type 1 collagen deposition. In addition, compared to the non-transplanted group, ADSCs transplantation reduced fibrotic areas in E. multilocularis infected mice. We also found that ADSCs transplantation significantly down-regulated TGF-β1 and TGF-βR expressions, while up-regulating Smad7 expression in the TGF-β/Smad signaling pathway.ConclusionsADSCs can alleviate Echinococcus multilocularis infection-induced liver fibrosis by modulating the activity level of the TGF-β/Smad7 signaling pathway and provide a potential therapeutic approach for E. multilocularis-induced fibrosis.     

Poricoic acid A activates AMPK to attenuate fibroblast activation and abnormal extracellular matrix remodelling in renal fibrosis

BackgroundIn chronic kidney disease, although fibrosis prevention is beneficial, few interventions are available that specifically target fibrogenesis. Poricoic acid A (PAA) isolated from Poria cocos exhibits anti-fibrotic effects in the kidney, however the underlying mechanisms remain obscure.PurposeWe isolated PAA and investigated its effects and the underlying mechanisms in renal fibrosis.Study designUnilateral ureteral obstruction (UUO) and 5/6 nephrectomy (Nx) animal models and TGF-β1-induced renal fibroblasts (NRK-49F) were used to investigate the anti-fibrotic activity of PAA and its underlying mechanisms.MethodsWestern blots, qRT-PCR, immunofluorescence staining, co-immunoprecipitation and molecular docking methods were used. Knock-down and knock-in of adenosine monophosphate-activated protein kinase (AMPK) in the UUO model and cultured NRK-49F cells were employed to verify the mechanisms of action of PAA.ResultsPAA improved renal function and alleviated fibrosis by stimulating AMPK and inhibiting Smad3 specifically in Nx and UUO models. Reduced AMPK activity was associated with Smad3 induction, fibroblast activation, and the accumulation and aberrant remodelling of extracellular matrix (ECM) in human renal puncture samples and cultured NRK-49F cells. PAA stimulated AMPK activity and decreased fibrosis in a dose-dependent manner, thus showing that AMPK was essential for PAA to exert its anti-fibrotic effects. AMPK deficiency reduced the anti-fibrotic effects of PAA, while AMPK overexpression enhanced its effect.ConclusionPAA activated AMPK and further inhibited Smad3 specifically to suppress fibrosis by preventing aberrant ECM accumulation and remodelling and facilitating the deactivation of fibroblasts.     

Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts

Introduction  

Extracellular matrix (ECM) turnover is controlled by the synthetic rate of matrix proteins, including type I collagen, and their enzymatic degradation by matrix metalloproteinases (MMPs). Fibrosis is characterized by an unbalanced accumulation of ECM leading to organ dysfunction as observed in systemic sclerosis. We previously reported that proteasome inhibition (PI) in vitro decreases type I collagen and enhances MMP-1 production by human fibroblasts, thus favoring an antifibrotic fibroblast phenotype. These effects were dominant over the pro-fibrotic phenotype induced by transforming growth factor (TGF)-β. Here we investigate the molecular events responsible for the anti-fibrotic phenotype induced in fibroblasts by the proteasome inhibitor bortezomib.     

Negative regulation of the hepatic fibrogenic response by suppressor of cytokine signaling 1

Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFNγ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFNγ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFNγ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in HSC activation. Liver fibrosis was induced in Socs1^−/−Ifng^−/− mice with dimethylnitrosamine or carbon tetrachloride. Ifng^−/− and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1^−/−Ifng^−/− mice showed elevated serum ALT levels and increased liver fibrosis compared to Ifng^−/− mice. The latter group showed higher ALT levels and fibrosis than C57BL/6 controls. The livers of SOCS1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. SOCS1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from SOCS1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of SOCS1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFNγ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability.     

Reversible Modulation of Myofibroblast Differentiation in Adipose-Derived Mesenchymal Stem Cells

Unregulated activity of myofibroblasts, highly contractile cells that deposit abundant extracellular matrix (ECM), leads to fibrosis. To study the modulation of myofibroblast activity, we used human adipose-derived mesenchymal stem cells (ADSCs), which have much potential in regenerative medicine. We found that ADSCs treated with TGF-β developed a myofibroblastic phenotype with increases in α-smooth muscle actin (α-SMA), a myofibroblast marker, and ECM proteins type I collagen and fibronectin. In contrast, treatment with bFGF had the opposite effect. bFGF-differentiated ADSCs showed marked down-regulation of α-SMA expression, collagen I, and fibronectin, and loss of focal adhesions and stress fibers. Functionally, bFGF-differentiated ADSCs were significantly more migratory, which correlated with up-regulation of tenascin-C, an anti-adhesive ECM protein, and vimentin, a pro-migratory cytoskeletal protein. On the other hand, TGF-β-differentiated ADSCs were significantly more contractile than bFGF-differentiated cells. Interestingly, cells completely reversed their morphologies, marker expression, signaling pathways, and contractility versus migratory profiles when switched from culture with one growth factor to the other, demonstrating that the myofibroblast differentiation process is not terminal. Cell differentiation was associated with activation of Smad2 downstream of TGF-β and of ERK/MAP kinase downstream of bFGF. Reversibility of the TGF-β-induced myofibroblastic phenotype depends, in part, on bFGF-induced ERK/MAP kinase signaling. These findings show that ADSC differentiation into myofibroblasts and re-differentiation into fibroblast-like cells can be manipulated with growth factors, which may have implications in the development of novel therapeutic strategies to reduce the risk of fibrosis.     

Adipose Stromal Cells Amplify Angiogenic Signaling via the VEGF/mTOR/Akt Pathway in a Murine Hindlimb Ischemia Model: A 3D Multimodality Imaging Study

Although adipose-derived stromal cell (ADSC) transplantation has been demonstrated as a promising therapeutic strategy for peripheral arterial disease (PAD), the mechanism of action behind the observed therapeutic efficacy of ADSCs remains unclear. This study was designed to investigate the long-term outcome and therapeutic behavior of engrafted ADSCs in a murine hindlimb ischemia model using multimodality molecular imaging approaches. ADSCs (1.0×10⁷) were isolated from Tg(Fluc-egfp) mice which constitutively express dual-reporter firefly luciferase and enhanced green fluorescent protein (Fluc⁺-eGFP⁺, mADSCs^Fluc+GFP+), then intramuscularly injected into the hindlimb of BALB/c-nu mice after unilateral femoral artery ligation and excision. Abbreviated survival (∼5 weeks) of post-transplant mADSCs within the ischemic hindlimb was longitudinally monitored using noninvasive bioluminescence imaging (BLI), fluorescence imaging (FRI), and bioluminescence tomography with micro-computed tomography (BLT/micro-CT). Use of the BLT/micro-CT system enabled quantitative 3-dimensional (3D) imaging of the cells’ distribution and kinetics in vivo. Engrafted mADSCs improved blood perfusion recovery, ambulatory performance and prognosis of the ischemic hindlimb, probably by inducing angiogenesis and formation of collateral vessels, which could be visualized using laser Doppler perfusion imaging (LDPI), micro-CT angiography, vascular-cast imaging, and immunofluorescence. mADSCs augmented activation of the pro-angiogenic VEGF/mTOR/Akt pathway in vivo, even though the cells failed to incorporate into the host microvasculature as functional components. Downregulation of VEGF/mTOR/Akt signaling using small molecule inhibitors counteracted mADSC-induced angiogenesis and perfusion restoration. This study demonstrates for the first time the spatiotemporal kinetics and functional survival of transplanted mADSCs in a PAD model using in vivo 3D multimodality imaging. Our study indicates that mADSCs potentiate pro-angiogenic signal amplification via a VEGF/mTOR/Akt-dependent pathway, and thereby promote recovery from hindlimb ischemia.     

Salvianolic acid B inhibits myofibroblast transdifferentiation in experimental pulmonary fibrosis via the up-regulation of Nrf2

Salvianolic acid B (SalB) is one of the most bioactive components extracted from Salvia miltiorrhiza, and its antioxidant capacity corresponds with its protective effects against cell injury from oxidative stress. The aim of the present study was to evaluate the effect of SalB on experimental pulmonary fibrosis and its ability to ameliorate the oxidative/antioxidative imbalance during fibrosis pathogenesis. The anti-fibrotic activity of SalB was first confirmed in Transforming growth factor β1(TGF-β1)-stimulated MRC-5 cells. The protection of SalB against oxidative stress during fibrogenesis in vitro was verified by detecting ROS production, the levels of glutathione (GSH) and malondialdehyde (MDA). The Western blot and PCR results indicated that SalB could up-regulate nuclear factor erythroid-derived 2-like 2 (Nrf2) at both the protein and mRNA levels and induce Nrf2 nuclear translocation in vitro, which may be the mechanism underlying the anti-fibrotic capacity of SalB. Furthermore, the anti-fibrotic and antioxidant capacities of SalB in vivo were confirmed in rats with BLM-induced pulmonary fibrosis. The immunohistochemistry results showed that Nrf2 was absent in fibroblastic foci (FF) areas, while the SalB treatment could increase the expression of Nrf2 in lung tissues, especially in FF areas.     

Hepatocyte Growth Factor Gene-Modified Adipose-Derived Mesenchymal Stem Cells Ameliorate Radiation Induced Liver Damage in a Rat Model

Liver damage caused by radiotherapy is associated with a high mortality rate, but no established treatment exists. Adipose-derived mesenchymal stem cells (ADSCs) are capable of migration to injured tissue sites, where they aid in the repair of the damage. Hepatocyte growth factor (HGF) is critical for damage repair due to its anti-apoptotic, anti-fibrotic and cell regeneration-promoting effects. This study was performed to investigate the therapeutic effects of HGF-overexpressing ADSCs on radiation-induced liver damage (RILD). ADSCs were infected with a lentivirus encoding HGF and HGF-shRNA. Sprague-Dawley (SD) rats received 60Gy of irradiation to induce liver injury and were immediately given either saline, ADSCs, ADSCs + HGF or ADSCs + shHGF. Two days after irradiation, a significant reduction in apoptosis was observed in the HGF-overexpressing ADSC group compared with the RILD group, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Scanning electron microscopy showed chromatin condensation after irradiation, which was ameliorated in the group that received ADSCs and was reversed in the group that received HGF-overexpressing ADSCs. HGF-overexpressing ADSCs ameliorated radiation- induced liver fibrosis through down regulation of α-SMA and fibronectin. Hepatocyte regeneration was significantly improved in rats treated with ADSCs compared with rats from the RILD group), as assessed by Ki-67 immunohistochemistry. Rats that received HGF-overexpressing ADSCs showed an even greater level of hepatocyte regeneration. HGF-overexpressing ADSCs completely blocked the radiation-induced increase in the enzymes ALT and AST. The effect of mitigating RILD was compromised in the ADSC + shHGF group compared with the ADSC group. Altogether, these results suggest that HGF-overexpressing ADSCs can significantly improve RILD in a rat model, which may serve as a valuable therapeutic alternative.     

Adipose-Derived Stromal Cells Protect Intervertebral Disc Cells in Compression: Implications for Stem Cell Regenerative Disc Therapy

Introduction: Abnormal biomechanics plays a role in intervertebral disc degeneration. Adipose-derived stromal cells (ADSCs) have been implicated in disc integrity; however, their role in the setting of mechanical stimuli upon the disc''s nucleus pulposus (NP) remains unknown. As such, the present study aimed to evaluate the influence of ADSCs upon NP cells in compressive load culture.Methods: Human NP cells were cultured in compressive load at 3.0MPa for 48 hours with or without ADSCs co-culture (the ratio was 50:50). We used flow cytometry, live/dead staining and scanning electron microscopy (SEM) to evaluate cell death, and determined the expression of specific apoptotic pathways by characterizing the expression of activated caspases-3, -8 and -9. We further used real-time (RT-) PCR and immunostaining to determine the expression of the extracellular matrix (ECM), mediators of matrix degradation (e.g. MMPs, TIMPs and ADAMTSs), pro-inflammatory factors and NP cell phenotype markers.Results: ADSCs inhibited human NP cell apoptosis via suppression of activated caspase-9 and caspase-3. Furthermore, ADSCs protected NP cells from the degradative effects of compressive load by significantly up-regulating the expression of ECM genes (SOX9, COL2A1 and ACAN), tissue inhibitors of metalloproteinases (TIMPs) genes (TIMP-1 and TIMP-2) and cytokeratin 8 (CK8) protein expression. Alternatively, ADSCs showed protective effect by inhibiting compressive load mediated increase of matrix metalloproteinases (MMPs; MMP-3 and MMP-13), disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs; ADAMTS-1 and 5), and pro-inflammatory factors (IL-1beta, IL-6, TGF-beta1 and TNF-alpha).Conclusions: Our study is the first in vitro study assessing the impact of ADSCs on NP cells in an un-physiological mechanical stimulation culture environment. Our study noted that ADSCs protect compressive load induced NP cell death and degradation by inhibition of activated caspase-9 and -3 activity; regulating ECM and modulator genes, suppressing pro-inflammatory factors and preserving CK8. Consequently, the protective impact of ADSCs found in this study provides an essential understanding and expands our knowledge as to the utility of ADSCs therapy for intervertebral disc regeneration.     

Soluble factors derived from human amniotic epithelial cells suppress collagen production in human hepatic stellate cells

BackgroundIntravenous infusion of human amniotic epithelial cells (hAECs) has been shown to ameliorate hepatic fibrosis in murine models. Hepatic stellate cells (HSCs) are the principal collagen-secreting cells in the liver. The aim of this study was to investigate whether factors secreted by hAECs and present in hAEC-conditioned medium (CM) have anti-fibrotic effects on activated human HSCs.MethodsHuman AECs were isolated from the placenta and cultured. Human hepatic stellate cells were exposed to hAEC CM to determine potential anti-fibrotic effects.ResultsHSCs treated for 48 h with hAEC CM displayed a significant reduction in the expression of the myofibroblast markers α-smooth muscle actin and platelet-derived growth factor. Expression of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1) and intracellular collagen were reduced by 45% and 46%, respectively. Human AEC CM induced HSC apoptosis in 11.8% of treated cells and reduced HSC proliferation. Soluble human leukocyte antigen–G1, a hAEC-derived factor, significantly decreased TGF-β1 and collagen production in activated HSCs, although the effect on collagen production was less than that of hAEC CM. The reduction in collagen and TGF-B1 could not be attributed to PGE2, relaxin, IL-10, TGF-B3, FasL or TRAIL.ConclusionsHuman AEC CM treatment suppresses markers of activation, proliferation and fibrosis in human HSCs as well as inducing apoptosis and reducing proliferation. Human AEC CM treatment may be effective in ameliorating liver fibrosis and warrants further study.     

Insulin-like growth factor 2 regulates the proliferation and differentiation of rat adipose-derived stromal cells via IGF-1R and IR

BackgroundInsulin-like growth factor 2 (IGF2), an essential component of the stem cell niche, has been reported to modulate the proliferation and differentiation of stem cells. Previously, a continuous expression of IGF2 in tissues was reported to maintain the self-renewal ability of several types of stem cells. Therefore, in this study, we investigated the expression of IGF2 in adipose tissues and explored the effects of IGF2 on adipose-derived stromal cells (ADSCs) in vitro.MethodsThe expression pattern of IGF2 in rat adipose tissues was determined by gene expression and protein analyses. The effect of IGF2 on proliferation, stemness-related marker expression and adipogenic and osteogenic differentiation was systematically investigated. Furthermore, antagonists of IGF2-specific receptors—namely, BMS-754807 and picropodophyllin—were added to explore the underlying signal transduction mechanisms.ResultsIGF2 levels displayed a tendency to decrease with age in rat adipose tissues. After the addition of IGF2, isolated ADSCs displayed higher proliferation and expression of the stemness-related markers NANOG, OCT4 and SOX2 and greater differentiation potential to adipocytes and osteoblasts. Additionally, both type 1 insulin-like growth factor receptor (IGF-1R) and insulin receptor (IR) participated in the IGF2-mediated promotion of stemness in ADSCs.ConclusionsOur findings indicate that IGF2 could enhance the stemness of rat ADSCs via IGF-1R and IR and may highlight an effective method for the expansion of ADSCs for clinical application.     

Choosing the right type of serum for different applications of human adipose tissue–derived stem cells: influence on proliferation and differentiation abilities

Background aimsAdipose tissue–derived stem cells (ADSCs) are thought to have great potential in regenerative medicine. A xenoprotein-free culture and handling system is desirable. To date, there is only little and contradictory information about the influence of the different types of human serum on ADSC proliferation and differentiation.MethodsFirst, ADSCs were cultured in media containing regular human serum (HS plus) or fetal calf serum (FCS plus) with supplementation of growth factors for three passages. During passage 4, ADSC proliferative activity and adipogenic, osteogenic and chondrogenic differentiation ability was quantified. Second, ADSCs were cultured with three different human sera (regular human serum [HS], human serum from platelet-poor plasma [SPPP] or human serum from platelet-rich plasma [SPRP]) without supplementation of platelet-derived growth factor and assessed accordingly. The growth factor content of the different types of human sera was determined by means of multiplex protein assay and enzyme-linked immunosorbent assay.ResultsThe different sera did not affect ADSC doubling time significantly (P < 0.05). Specific glycerol-3-phosphat-dehydrogenase activity was significantly lower in cultures with SPRP (P < 0.01) compared with the other media compositions. Extracellular calcium deposition was significantly higher in cells differentiated in cultures with HS or SPPP compared with those with SPRP, HS plus or FCS (P < 0.01). Glycosaminoglycan content and collagen 2 were highest in cells cultured with SPRP (P < 0.001).ConclusionsCulturing ADSCs in human serum appears to be a reasonable and efficient alternative compared with FCS. With respect to the outcome of a sighted clinical application, it appears to be feasible to handle the cells in a serum suitable for the intended later use.     

Inhibition of TGF-β pathway reverts extracellular matrix remodeling in T. cruzi-infected cardiac spheroids

Chagasic cardiomyopathy (CC) is the main manifestation of Chagas Disease (CD). CC is a progressive dysfunctional illness, in which transforming growth factor beta (TGF-β) plays a central role in fibrogenesis and hypertrophy. In the present study, we tested in a three-dimensional (3D) model of cardiac cells culture (named cardiac spheroids), capable of mimicking the aspects of fibrosis and hypertrophy observed in CC, the role of TGF-β pathway inhibition in restoring extracellular matrix (ECM) balance disrupted by T. cruzi infection. Treatment of T. cruzi-infected cardiac spheroids with SB 431542, a selective inhibitor of TGF-β type I receptor, resulted in a reduction in the size of spheroids, which was accompanied by a decrease in parasite load and in fibronectin expression. The inhibition of TGF-β pathway also promoted an increase in the activity of matrix metalloproteinase (MMP)-2 and a decrease in tissue inhibitor of matrix metalloproteinase (TIMP)-1 expression, which may be one of the mechanisms regulating extracellular matrix remodeling. Therefore, our study provides new insights into the molecular mechanisms by which inhibition of TGF-β signaling reverts fibrosis and hypertrophy generated by T. cruzi during CC and also highlights the use of cardiac spheroids as a valuable tool for the study of fibrogenesis and anti-fibrotic compounds.     

Rupatadine Protects against Pulmonary Fibrosis by Attenuating PAF-Mediated Senescence in Rodents

A similar immune response is implicated in the pathogenesis of pulmonary fibrosis and allergic disorders. We investigated the potential therapeutic efficacy and mechanism of rupatadine, a dual antagonist of histamine and platelet-activation factor (PAF), in bleomycin- (BLM-) and silica-induced pulmonary fibrosis. The indicated dosages of rupatadine were administered in rodents with bleomycin or silica-induced pulmonary fibrosis. The tissue injury, fibrosis, inflammatory cells and cytokines, and lung function were examined to evaluate the therapeutic efficacy of rupatadine. The anti-fibrosis effect of rupatadine was compared with an H1 or PAF receptor antagonist, and efforts were made to reveal rupatadine’s anti-fibrotic mechanism. Rupatadine promoted the resolution of pulmonary inflammation and fibrosis in a dose-dependent manner, as indicated by the reductions in inflammation score, collagen deposition and epithelial-mesenchymal transformation, and infiltration or expression of inflammatory cells or cytokines in the fibrotic lung tissue. Thus, rupatadine treatment improved the declined lung function and significantly decreased animal death. Moreover, rupatadine was able not only to attenuate silica-induced silicosis but also to produce a superior therapeutic efficacy compared to pirfenidone, histamine H1 antagonist loratadine, or PAF antagonist CV-3988. The anti-fibrotic action of rupatadine might relate to its attenuation of BLM- or PAF-induced premature senescence because rupatadine treatment protected against the in vivo and in vitro activation of the p53/p21-dependent senescence pathway. Our studies indicate that rupatadine promotes the resolution of pulmonary inflammation and fibrosis by attenuating the PAF-mediated senescence response. Rupatadine holds promise as a novel drug to treat the devastating disease of pulmonary fibrosis.     

The significance of CD14+ monocytes in peripheral blood stem cells for the treatment of rat liver cirrhosis

Background aimsCirculating monocytes have been exploited as an important progenitor cell resource for hepatocytes in vitro and are instrumental in the removal of fibrosis. We investigated the significance of monocytes in peripheral blood stem cells (PBSC) for the treatment of liver cirrhosis.MethodsRat CD14⁺ monocytes in PBSC were mobilized with granulocyte-colony-stimulating factor (G-CSF) and harvested by magnetic cell sorting (MACS). Female rats with carbon tetrachloride (CCl₄)-induced liver cirrhosis were injected CM-DiI-labeled monocytes, CD14^? cells (1 × 10⁷ cells/rat) or saline via the portal vein.ResultsRat CD14⁺ and CD11b⁺ monocytes in PBSC were partly positive for CD34, CD45, CD44, Oct3/4 and Sox2, suggesting monocytes with progenitor capacity. Compared with CD14^? cell-infused and saline-injected rats, rats undergoing monocyte transplantation showed a gradually increased serum albumin level and decreased portal vein pressure, resulting in a significantly improved survival rate. Meanwhile, monocyte transplantation apparently attenuated liver fibrosis by analysis for fibronectin, α2-(1)-procollagen, α-smooth muscle aorta (SMA) and transforming growth factor (TGF)-β. Transplanted monocytes mainly clustered in periportal areas of liver, in which 1.8% cells expressed hepatocyte marker albumin and CK18. The expression level of hepatocyte growth factor (HGF), TGF-α, extracellular matrix (EGF) and vascular endothelial growth factor (VEGF) increased, while monocyte transplantation enhanced hepatocyte proliferation. On the other hand, the activities and expression of matrix metalloproteinases (MMP) increased while tissue inhibitor of metalloproteinase (TIMP)-1 expression significantly reduced in monocyte-transplanted livers. Some transplanted monocytes expressed MMP-9 and -13.ConclusionsThe data suggest that CD14⁺ monocytes in PBSC contribute to hepatocyte regeneration and extracellular matrix (ECM) remodeling in rat liver cirrhosis much more than CD14^? cells, and might offer a therapeutic alternative for patients with liver cirrhosis.     

Angiogenic Activity of Breast Cancer Patients’ Monocytes Reverted by Combined Use of Systems Modeling and Experimental Approaches

Angiogenesis plays a key role in tumor growth and cancer progression. TIE-2-expressing monocytes (TEM) have been reported to critically account for tumor vascularization and growth in mouse tumor experimental models, but the molecular basis of their pro-angiogenic activity are largely unknown. Moreover, differences in the pro-angiogenic activity between blood circulating and tumor infiltrated TEM in human patients has not been established to date, hindering the identification of specific targets for therapeutic intervention. In this work, we investigated these differences and the phenotypic reversal of breast tumor pro-angiogenic TEM to a weak pro-angiogenic phenotype by combining Boolean modelling and experimental approaches. Firstly, we show that in breast cancer patients the pro-angiogenic activity of TEM increased drastically from blood to tumor, suggesting that the tumor microenvironment shapes the highly pro-angiogenic phenotype of TEM. Secondly, we predicted in silico all minimal perturbations transitioning the highly pro-angiogenic phenotype of tumor TEM to the weak pro-angiogenic phenotype of blood TEM and vice versa. In silico predicted perturbations were validated experimentally using patient TEM. In addition, gene expression profiling of TEM transitioned to a weak pro-angiogenic phenotype confirmed that TEM are plastic cells and can be reverted to immunological potent monocytes. Finally, the relapse-free survival analysis showed a statistically significant difference between patients with tumors with high and low expression values for genes encoding transitioning proteins detected in silico and validated on patient TEM. In conclusion, the inferred TEM regulatory network accurately captured experimental TEM behavior and highlighted crosstalk between specific angiogenic and inflammatory signaling pathways of outstanding importance to control their pro-angiogenic activity. Results showed the successful in vitro reversion of such an activity by perturbation of in silico predicted target genes in tumor derived TEM, and indicated that targeting tumor TEM plasticity may constitute a novel valid therapeutic strategy in breast cancer.     

Anti-fibrotic effects of nintedanib in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The kinase inhibitor nintedanib specific for vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) significantly reduced the rate of decline of forced vital capacity versus placebo.

Aim

To determine the in vitro effect of nintedanib on primary human lung fibroblasts. Methods: Fibroblasts were isolated from lungs of IPF patients and from non-fibrotic controls. We assessed the effect of VEGF, PDGF-BB and basic FGF (bFGF) ± nintedanib on: (i) expression/activation of VEGFR, PDGFR, and FGFR, (ii) cell proliferation, secretion of (iii) matrix metalloproteinases (MMP), (iv) tissue inhibitor of metalloproteinase (TIMP), and (v) collagen.

Results

IPF fibroblasts expressed higher levels of PDGFR and FGFR than controls. PDGF-BB, bFGF, and VEGF caused a pro-proliferative effect which was prevented by nintedanib. Nintedanib enhanced the expression of pro-MMP-2, and inhibited the expression of TIMP-2. Transforming growth factor-beta-induced secretion of collagens was inhibited by nintedanib.

Conclusion

Our data demonstrate a significant anti-fibrotic effect of nintedanib in IPF fibroblasts. This effect consists of the drug’s anti-proliferative capacity, and on its effect on the extracellular matrix, the degradation of which seems to be enhanced.     

Microencapsulated VEGF gene–modified umbilical cord mesenchymal stromal cells promote the vascularization of tissue-engineered dermis: an experimental study

Background aimsTissue-engineered dermis (TED) is thought to be the best treatment for skin defect wounds; however, lack of vascular structures in these products can cause slow vascularization or even transplant failure. We assessed the therapeutic potential of microencapsulated human umbilical cord mesenchymal stromal cells (hUCMSCs) expressing vascular endothelial growth factor (VEGF) in vascularization of TED.MethodshUCMSCs were isolated by means of enzymatic digestion and identified by means of testing biological characteristics. hUCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. Collagen-chitosan laser drilling acellular dermal matrix (ADM) composite scaffold was prepared by means of the freeze dehydration and dehydrothermal cross-linking method. hUCMSC-derived fibroblasts were implanted on composite scaffolds to construct TED. TED with microencapsulated VEGF gene–modified hUCMSCs was then transplanted into skin defect wounds in pigs. The angiogenesis of TED at 1 week and status of wound healing at 3 weeks were observed.ResultsThe collagen-chitosan laser ADM composite has a uniform microporous structure. This composite has been used to grow hUCMSC-derived fibroblasts in vitro and to successfully construct stem cell–derived TED. Microencapsulated VEGF gene–modified hUCMSCs were prepared with the use of a sodium alginate–barium chloride one-step encapsulation technology. Seven days after the transplantation of the stem cell–derived TED and microencapsulated VEGF gene–modified hUCMSCs into the skin defect wounds on the backs of miniature pigs, the VEGF expression increased and the TED had a higher degree of vascularization. Re-epithelialization of the wound was completed after 3 weeks.ConclusionsMicroencapsulated VEGF gene–modified hUCMSCs can effectively improve the vascularization of TED and consequently the quality of wound healing.