Release Of HGF Research Articles

Altered hypoxia-induced cellular responses and inflammatory profile in lung fibroblasts from COPD patients compared to control subjects

BackgroundChronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by chronic bronchitis, emphysema and vascular remodelling. The disease is associated with hypoxia, inflammation and oxidative stress. Lung fibroblasts are important cells in remodelling processes in COPD, as main producers of extracellular matrix proteins but also in synthesis of growth factors and inflammatory mediators.MethodsIn this study we aimed to investigate if there are differences in how primary distal lung fibroblasts obtained from COPD patients and healthy subjects respond to hypoxia (1% O2) and pro-fibrotic stimuli with TGF-β1 (10 ng/mL). Genes and proteins associated with oxidative stress, endoplasmic reticulum stress, remodelling and inflammation were analysed with RT-qPCR and ELISA.ResultsHypoxia induced differences in expression of genes involved in oxidative stress (SOD3 and HIF-1α), ER stress (IRE1, PARK and ATF6), apoptosis (c-Jun and Bcl2) and remodelling (5HTR2B, Collagen7 and VEGFR2) in lung fibroblasts from COPD subjects compared to control subjects, where COPD fibroblasts were in general less responsive. The release of VEGF-C was increased after hypoxia, whereas TGF-β significantly reduced the VEGF response to hypoxia and the release of HGF. COPD fibroblasts had a higher release of IL-6, IL-8, MCP-1 and PGE2 compared to lung fibroblasts from control subjects. The release of inflammatory mediators was less affected by hypoxia, whereas TGFβ1 induced differences in inflammatory profile between fibroblasts from COPD and control subjects.ConclusionThese results suggest that there is an alteration of gene regulation of various stress responses and remodelling associated mediator release that is related to COPD and hypoxia, where fibroblasts from COPD patients have a deficient response.

Effect of controlled release of HGF on extracellular vesicle secretion by urine-derived stem cells.

The hepatic growth factor (HGF) stimulates DNA synthesis and cell proliferation and plays a role in tissue protection and regeneration. In this study, we have examined the effect of incubation of HGF with urine-derived stem cells (USCs) on the secretion of small extracellular vesicles (sEV) by the cells. HGF in the incubation medium was either a bolus administration or a controlled release of an equivalent amount from microbeads within the size range of 50-200µm made with ultrapurified low-viscosity high-guluronic acid (UP-LVG) alginate. USCs were incubated with or without HGF for 3days or 7days before removal of the incubation media, followed by harvesting sEV by the precipitation method. The protein content of isolated sEV was measured by bicinchoninic acid assay (BCA) for these three groups: control (no HGF beads), bolus HGF, and HGF beads. We also performed nanoparticle tracking analysis (NTA), Western blot assay, and ELISA for the HGF content of samples. We found a significantly higher concentration of proteins in the HGF microbead group (control release group) compared to the bolus group and the control group after 7days (p < 0.0017). The NTA data aligned with the BCA; they showed a significantly higher concentration of particles within the size range of sEV (<200nm) in the group treated with HGF beads compared to the two other groups on day 7 (p < 0.0001). We found that administration of HGF to USCs by controlled release of the growth factor significantly enhances the levels of sEV secretion during 7days of incubation.

Design of Hybrid Polymer Nanofiber/Collagen Patches Releasing IGF and HGF to Promote Cardiac Regeneration.

Cardiovascular diseases are the leading cause of death globally. Myocardial infarction in particular leads to a high rate of mortality, and in the case of survival, to a loss of myocardial functionality due to post-infarction necrosis. This functionality can be restored by cell therapy or biomaterial implantation, and the need for a rapid regeneration has led to the development of bioactive patches, in particular through the incorporation of growth factors (GF). In this work, we designed hybrid patches composed of polymer nanofibers loaded with HGF and IGF and associated with a collagen membrane. Among the different copolymers studied, the polymers and their porogens PLA-Pluronic-PLA + PEG and PCL + Pluronic were selected to encapsulate HGF and IGF. While 89 and 92% of IGF were released in 2 days, HGF was released up to 58% and 50% in 35 days from PLA-Pluronic-PLA + PEG and PCL + Pluronic nanofibers, respectively. We also compared two ways of association for the loaded nanofibers and the collagen membrane, namely a direct deposition of the nanofibers on a moisturized collagen membrane (wet association), or entrapment between collagen layers (sandwich association). The interfacial cohesion and the degradation properties of the patches were evaluated. We also show that the sandwich association decreases the burst release of HGF while increasing the release efficiency. Finally, we show that the patches are cytocompatible and that the presence of collagen and IGF promotes the proliferation of C2C12 myoblast cells for 11 days. Taken together, these results show that these hybrid patches are of interest for cardiac muscle regeneration.

Prospects on the Potential In Vitro Regenerative Features of Mechanically Treated-Adipose Tissue for Osteoarthritis Care

Gathering a better grasp on the adipose stromal vascular fraction (SVF) is demanding among clinicians for osteoarthritis (OA) care because of its promising but multifaceted clinical outcomes. The aim of this preclinical in vitro study was to test whether the mechanical approach with Hy-Tissue SVF system, a class IIa CE marked device of adipose tissue micro-fragmentation, influences the biological features and functions of SVF. We compared mechanical generated-SVF (mSVF) with the enzymatic generated-SVF (eSVF) by testing cell survival, phenotype, differentiation, and paracrine properties using ELISA assays. Both adipose SVF showed 80% viable cells and enrichment for CD-44 marker. The mSVF product preserved the functions of cell populations within the adipose tissue; however, it displayed lowered nucleated cell recovery and CFU-F than eSVF. As for multipotency, mSVF and eSVF showed similar differentiation commitment for osteochondral lineages. Both adipose SVF exhibited an increased release of VEGF, HGF, IGF-1 and PDGF-bb, involved in pathways mediating osteochondral repair and cell migration. Both mSVF and eSVF also displayed high release for the anti-inflammatory cytokine IL-10. After in vitro culture, supernatants from both mSVF and eSVF groups showed a low release of cytokines except for IL-10, thereby giving evidence of functional changes after culture expansion. In this study, mSVF showed active cell populations in the adipose tissue comparable to eSVF with excellent survival, differentiation and paracrine properties under a new mechanical adipose tissue micro-fragmentation system; thereby suggesting its potential use as a minimally invasive technique for OA treatment.Graphical abstract

TLR3 promotes hepatocyte proliferation after partial hepatectomy by stimulating uPA expression and the release of tissue-bound HGF.

TLR3 is implicated in anti-viral immune responses, but may also act as a sensor of tissue damage in the absence of infection. Here, we provide evidence for an essential role of TLR3 in liver regeneration after an acute loss of tissue due to partial hepatectomy. Mice lacking TLR3 had a severe and sustained defect in the restoration of liver tissue with reduced liver-to-body weight ratios even after an extended recovery period of 2weeks. Hepatocyte cell cycle progression into S phase was impaired in TLR3-deficient mice. Mechanistic analyses revealed that TLR3-deficient mice had markedly reduced systemic levels of active HGF, but had increased amounts of inactive tissue-bound HGF. Importantly, expression of uPA, which orchestrates the processing and release of HGF from the hepatic extracellular matrix, was reduced in regenerating livers of TLR3-deficient mice. In addition, expression of the HGF maturation factor HGFAC was transiently diminished in TLR3-deficient mice. In vitro, engagement of TLR3 directly stimulated expression of uPA by hepatic stellate cells. Thus, TLR3 supports liver regeneration through upregulation of uPA, which promotes the release of preformed HGF from extracellular matrix stores.

The endocannabinoid receptors CB1 and CB2 affect the regenerative potential of adipose tissue MSCs

Human adipose tissue includes large quantities of mesenchymal stromal cells (atMSCs), which represent an abundant cell source for therapeutic applications in the field of regenerative medicine. Adipose tissue secrets various soluble factors including endocannabinoids, and atMSCs express the cannabinoid receptors CB1 and CB2. This indicates that adipose tissue possesses an endocannabinoid system (ECS). The ECS is also ascribed great significance for wound repair, e.g. by modulating inflammation. However, the exact effects of CB1/CB2 activation in human atMSCs have not been investigated, yet. In the present study, we stimulated human atMSCs with increasing concentrations (1–30 μM) of the unspecific cannabinoid receptor ligand WIN55,212–2 and the specific CB2 agonist JWH-133, either alone or co-applied with the receptor antagonist Rimonabant (CB1) or AM 630 (CB2). We investigated the effects on metabolic activity, cell number, differentiation and cytokine release, which are important processes during tissue regeneration. WIN decreased metabolic activity and cell number, which was reversed by Rimonabant. This suggests a CB1 dependent mechanism, whereas the number of atMSCs was increased after CB2 ligation. WIN and JWH increased the release of VEGF, TGF-β1 and HGF. Adipogenesis was enhanced by WIN, which could be reversed by blocking CB1. There was no effect on osteogenesis, and only WIN increased chondrogenic differentiation. Our results indicate that definite activation of the cannabinoid receptors exerted different effects in atMSCs, which could be of specific value in cell-based therapy for wound regeneration.

Linking cell function with perfusion: insights from the transcatheter delivery of bone marrow-derived CD133+ cells in ischemic refractory cardiomyopathy trial (RECARDIO)

BackgroundCell therapy with bone marrow (BM)-derived progenitors has emerged as a promising therapeutic for refractory angina (RA) patients. In the present study, we evaluated the safety and preliminary efficacy of transcatheter delivery of autologous BM-derived advanced therapy medicinal product CD133+ cells (ATMP-CD133) in RA patients, correlating perfusion outcome with cell function.MethodsIn the phase I “Endocavitary Injection of Bone Marrow Derived CD133+ Cells in Ischemic Refractory Cardiomyopathy” (RECARDIO) trial, a total of 10 patients with left ventricular (LV) dysfunction (ejection fraction ≤ 45%) and evidence of reversible ischemia, as assessed by single-photon emission computed tomography (SPECT), underwent BM aspiration and fluoroscopy-based percutaneous endomyocardial delivery of ATMP-CD133. Patients were evaluated at 6 and 12 months for safety and preliminary efficacy endpoints. ATMP-CD133 samples were used for in vitro correlations.ResultsPatients were treated safely with a mean number of 6.57 ± 3.45 × 106 ATMP-CD133. At 6-month follow-up, myocardial perfusion at SPECT was significantly ameliorated in terms of changes in summed stress (from 18.2 ± 8.6 to 13.8 ± 7.8, p = 0.05) and difference scores (from 12.0 ± 5.3 to 6.1 ± 4.0, p = 0.02) and number of segments with inducible ischemia (from 7.3 ± 2.2 to 4.0 ± 2.7, p = 0.003). Similarly, Canadian Cardiovascular Society and New York Heart Association classes significantly improved at follow-up vs baseline (p ≤ 0.001 and p = 0.007, respectively). Changes in summed stress score changes positively correlated with ATMP-CD133 release of proangiogenic cytokines HGF and PDGF-bb (r = 0.80, p = 0.009 and r = 0.77, p = 0.01, respectively) and negatively with the proinflammatory cytokines RANTES (r = − 0.79, p = 0.01) and IL-6 (r = − 0.76, p = 0.02).ConclusionResults of the RECARDIO trial suggested safety and efficacy in terms of clinical and perfusion outcomes in patients with RA and LV dysfunction. The observed link between myocardial perfusion improvements and ATMP-CD133 secretome may represent a proof of concept for further mechanistic investigations.Trial registrationClinicalTrials.gov, NCT02059681. Registered 11 February 2014.

Abstract 13.55 A COMPARISON OF THE REGENERATIVE GENE EXPRESSION IN ASCS DERIVED FROM UAL, SAL AND ABDOMINOPLASTY SAMPLES

BACKGROUND: Due to their regenerative potential, Mesenchymal stem cells are already in clinical use in several medical specialties. In plastic surgery, mainly the adipose-derived stromal cells (ASCs) are in the focus of recent research to remedy defects across the whole body and to reverse ischemic tissue states. The ability of the regenerative cytokine release is the key mechanism in the therapeutic use of ASCs. Through the intensive research effort of the last decades in the area of liposuction for aesthetic or reconstructive use, various methods and devices have been developed. However which method derives the most potent cells for regenerative applications is still disputed. Here we assess ASCs from Ultrasound assisted (UAL) and suction assisted (SAL) lipoaspirat as well as ASCs from excised fat for their ability to release key cytokines for tissue regeneration. MATERIAL AND METHODS: UAL and SAL lipoaspirate was collected from 5 patients and compared with excisional fat from abdominoplasty samples from the same patients. ASCs were characterized by their surface marker profile CD45- / CD31- / CD34 + by means of FACS and the expression of differentiation cytokines (HGF, FGF-2, MCP-1, SDF-1, VEGF) was compared. RESULTS: There was no significant difference between the release of HGF, FGF-2, SDF-1 and VEGF between the individual samples. However, we observed an increased expression of MCP-1 in the UAL sample. CONCLUSION: Utilizing an elegant study design with the patients serving as their own controls we were able to show that UAL and SAL both derive ASCs comparable to cells from excised fat. This demonstrates these methods have potential to harvest ASCs for autologous fat transfer and regenerative medicine.

Abstract IA04: Using comprehensive proteomic approaches to map signaling adaptations to BRAF and BRAF/MEK inhibition

Abstract Although small molecule inhibitors of BRAF have shown great promise in advanced melanoma, resistance is commonplace. Many patients with acquired BRAF and BRAF/MEK inhibitor resistance develop disease at new sites, suggesting that drug-induced selection pressure drives metastasis. Here we used mass spectrometry-based phosphoproteomic screening and network modeling to uncover two novel adaptations to BRAF inhibitor therapy. The first involved ligand-independent EphA2 signaling that led to the adoption of an invasive/metastatic phenotype. The EphA2-mediated invasion was epigenetically mediated, AKT-dependent and was readily reversible upon removal of drug as well as through PI3K or HDAC inhibition. In a human melanoma mouse xenograft model, chronic BRAF inhibition led to the development of metastases, which stained positively for EphA2. A second phosphoproteomic screen showed BRAF inhibition to enhance the survival of both melanoma cells and fibroblasts through the induction of fibronectin (FN)/integrin α5β1 signaling. Adhesion to FN amplified the adaptive EGFR, HER2 and c-MET signals required for PI3K/AKT-mediated survival when BRAF was inhibited. At the same time, BRAF inhibition led, directly and indirectly, to the paracrine release of HGF and neuregulin from fibroblasts, with TGF-β release from the melanoma cells increasing both fibroblast differentiation and survival. These effects were partly mediated through paradoxical MAPK activation in the fibroblasts and could be reversed through BRAF/MEK inhibition. Thus, we have used comprehensive phosphoproteomics to identify two key non-genetic adaptations to BRAF inhibitor therapy that offer new insights into the process of therapeutic escape. Citation Format: Inna V. Fedorenko, Kim H.T. Paraiso, Bin Fang, John M. Koomen, Keiran S.M. Smalley. Using comprehensive proteomic approaches to map signaling adaptations to BRAF and BRAF/MEK inhibition. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA04.

Abstract 3597: Malignancy of bladder cancer cells is enhanced by tumor associated fibroblasts through a cytokine-chemokine loop

Abstract The tumor microenvironment plays an important role in cancer progression. In particular, stromal fibroblasts have profound influence on migration and invasiveness of tumor cells. We employ bladder carcinoma cell lines of different de-differentiation grade to pursue the hypothesis that tumor cell malignancy correlates with the cells' potential to recruit support by tumor associated fibroblasts (TAFs). Bladder cancer cell lines RT112 and Cal29 were used as models for tumor cells of low or high de-differentiation grade, respectively. They were grown separately or in co-culture with tumor-associated fibroblast isolated from kidney carcinomas. Migratory and invasive cell properties were quantified by Boyden chamber (Matrigel) and wound healing assays. Cytokine production of cells was measured by intracellular staining followed by cytometry, by semi-quantitative PCR, or by subjecting conditioned medium to ELISA and Western blot analysis. Migration and invasiveness of bladder cancer cell lines Cal29 and RT112 was enhanced by co-cultivation with TAFs. The low grade cell line RT112, however, was much more dependent on TAF-derived signals to increase the malignancy parameters. Conditioned medium from tumor cells induced the secretion of Matrix Metalloproteinases (MMPs) and the release of GM-CSF, HGF, and Monocyte Chemotactic Protein (MCP-1, CCL2) by TAFs. We could identify tumor cell-derived IL-1α and β as well as TGF-β as stimuli responsible for the upregulation of GM-CSF and MCP-1, respectively, in fibroblasts. MCP-1 is implicated in inflammatory disease states characterized by monocytic infiltrates and was shown to promote cell migration of both bladder cancer cell lines under investigation. Employing pathway-specific reporter gene constructs we are currently addressing signal transduction reactions in both tumor cells and fibroblasts underlying this crosstalk. Supported by: IZKF Jena, European Regional Development Fund ERDF. Citation Format: Susanne Grimm, Susanne Jennek, Jens Bratsch, Andreas Wohlmann, Vrinda Mohta, Kerstin Junker, Karlheinz Friedrich. Malignancy of bladder cancer cells is enhanced by tumor associated fibroblasts through a cytokine-chemokine loop. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3597. doi:10.1158/1538-7445.AM2014-3597

Pharmacologic targeting of the PI3K/mTOR pathway controls release of angioregulators from primary human acute myeloid leukemia cells and their neighboring stromal cells

Acute myeloid leukemia (AML) is a heterogeneous and aggressive malignancy with poor overall survival. Constitutive as well as cytokine-initiated activation of PI3K/Akt/mTOR signaling is a common feature of AML patients, and inhibition of this pathway is considered as a possible therapeutic strategy in AML. Human AML cells and different stromal cell populations were cultured under highly standardized in vitro conditions. We investigated the effects of mTOR inhibitors (rapamycin and temsirolimus) and PI3K inhibitors (GDC-0941 and 3-methyladenin (3-MA)) on cell proliferation and the constitutive release of angioregulatory mediators by AML and stromal cells. Primary human AML cells were heterogeneous, though most patients showed high CXCL8 levels and detectable release of CXCL10, Ang-1, HGF and MMP-9. Hierarchical clustering analysis showed that disruption of PI3K/Akt/mTOR pathways decreased AML cell release of CXCL8-11 for a large subset of patients, whereas the effects on other mediators were divergent. Various stromal cells (endothelial cells, fibroblasts, cells with osteoblastic phenotype) also showed constitutive release of angioregulatory mediators, and inhibitors of both the PI3K and mTOR pathway had anti-proliferative effects on stromal cells and resulted in decreased release of these angioregulatory mediators. PI3K and mTOR inhibitors can decrease constitutive cytokine release both by AML and stromal cells, suggesting potential direct and indirect antileukemic effects.

Release kinetics of platelet-derived and plasma-derived growth factors from autologous plasma rich in growth factors

Many studies have evaluated the biological effects of platelet rich plasma reporting the final outcomes on cell and tissues. However, few studies have dealt with the kinetics of growth factor delivery by plasma rich in growth factors. Venous blood was obtained from three healthy volunteers and processed with PRGF-Endoret technology to prepare autologous plasma rich in growth factors. The gel-like fibrin scaffolds were then incubated in triplicate, in a cell culture medium to monitor the release of PDGF-AB, VEGF, HGF and IGF-I during 8 days of incubation. A leukocyte-platelet rich plasma was prepared employing the same technology and the concentrations of growth factors and interleukin-1β were determined after 24h of incubation. After each period, the medium was collected, fibrin clot was destroyed and the supernatants were stored at -80°C until analysis. The growth factor delivery is diffusion controlled with a rapid initial release by 30% of the bioactive content after 1h of incubation and a steady state release when almost 70% of the growth factor content has been delivered. Autologous fibrin matrix retained almost 30% of the amount of the growth factors after 8 days of incubation. The addition of leukocytes to the formula of platelet rich plasma did not increase the concentration of the growth factors, while it drastically increased the presence of pro-inflammatory IL-1β. Further studies employing an in vitro inflammatory model would be interesting to study the difference in growth factors and pro-inflammatory cytokines between leukocyte-free and leukocyte-rich platelet rich plasma.

Dual Delivery of Hepatocyte and Vascular Endothelial Growth Factors via a Protease-Degradable Hydrogel Improves Cardiac Function in Rats

Acute myocardial infarction (MI) caused by ischemia and reperfusion (IR) is the most common cause of cardiac dysfunction due to local cell death and a temporally regulated inflammatory response. Current therapeutics are limited by delivery vehicles that do not address spatial and temporal aspects of healing. The aim of this study was to engineer biotherapeutic delivery materials to harness endogenous cell repair to enhance myocardial repair and function. We have previously engineered poly(ethylene glycol) (PEG)-based hydrogels to present cell adhesive motifs and deliver VEGF to promote vascularization in vivo. In the current study, bioactive hydrogels with a protease-degradable crosslinker were loaded with hepatocyte and vascular endothelial growth factors (HGF and VEGF, respectively) and delivered to the infarcted myocardium of rats. Release of both growth factors was accelerated in the presence of collagenase due to hydrogel degradation. When delivered to the border zones following ischemia-reperfusion injury, there was no acute effect on cardiac function as measured by echocardiography. Over time there was a significant increase in angiogenesis, stem cell recruitment, and a decrease in fibrosis in the dual growth factor delivery group that was significant compared with single growth factor therapy. This led to an improvement in chronic function as measured by both invasive hemodynamics and echocardiography. These data demonstrate that dual growth factor release of HGF and VEGF from a bioactive hydrogel has the capacity to significantly improve cardiac remodeling and function following IR injury.

The use of lipid microtubes as a novel slow‐release delivery system for laryngeal injection

The potential utility of direct injection of bioactive substances in the treatment of vocal fold tissue fibrosis is limited by rapid clearance from the injection site. The objective of this study is to evaluate the potential of a lipid-based microtube delivery system to preserve the biological activity of injected substances and prolong their duration of pharmacological effects in the larynx. Prospective in vitro and case-control in vivo murine study Lipid-based microtubes were loaded with Texas red-dextran (MT-TR) and hepatocyte growth factor (MT-HGF). In vitro and in vivo (using a murine vocal fold injection model) release of MT-TR and MT-HGF were determined to assess duration of microtube-mediated delivery. The biologic effects of MT-HGF on fibroblasts were assessed after treatment in the presence of transforming growth factor (TGF)-β. In vitro release kinetics demonstrated slow release of MT-TR and MT-HGF, correlating with in vivo results demonstrating persistence of MT-HGF at 4 weeks postinjection. Bioefficacy was maintained, as MT-HGF was shown to inhibit TGF-β-mediated induction of procollagen mRNA levels in vitro 24 hours after treatment in fibroblast cells. Sustained release of HGF from microtubes at 6 days exacerbated the effects of TGF-β and increased levels of procollagen mRNA. Microtubes have significant potential utility as an efficacious means of sustained-release delivery of bioactive agents to the larynx. Atthough the role of HGF as an antifibrotic agent is questioned, its sustained bioefficacy after microtube encapsulation distinguishes microtubes from other delivery vehicles.

The antifibrotic effects of plasminogen activation occur via prostaglandin E2 synthesis in humans and mice

Plasminogen activation to plasmin protects from lung fibrosis, but the mechanism underlying this antifibrotic effect remains unclear. We found that mice lacking plasminogen activation inhibitor-1 (PAI-1), which are protected from bleomycin-induced pulmonary fibrosis, exhibit lung overproduction of the antifibrotic lipid mediator prostaglandin E2 (PGE2). Plasminogen activation upregulated PGE2 synthesis in alveolar epithelial cells, lung fibroblasts, and lung fibrocytes from saline- and bleomycin-treated mice, as well as in normal fetal and adult primary human lung fibroblasts. This response was exaggerated in cells from Pai1-/- mice. Although enhanced PGE2 formation required the generation of plasmin, it was independent of proteinase-activated receptor 1 (PAR-1) and instead reflected proteolytic activation and release of HGF with subsequent induction of COX-2. That the HGF/COX-2/PGE2 axis mediates in vivo protection from fibrosis in Pai1-/- mice was demonstrated by experiments showing that a selective inhibitor of the HGF receptor c-Met increased lung collagen to WT levels while reducing COX-2 protein and PGE2 levels. Of clinical interest, fibroblasts from patients with idiopathic pulmonary fibrosis were found to be defective in their ability to induce COX-2 and, therefore, unable to upregulate PGE2 synthesis in response to plasmin or HGF. These studies demonstrate crosstalk between plasminogen activation and PGE2 generation in the lung and provide a mechanism for the well-known antifibrotic actions of the fibrinolytic pathway.

D025 Phagocytosis is pivotal in the beneficial effect of bone marrow mononuclear cellsbased therapy for myocardial infarction

Cell-based therapy is a promising option for treatment of cardiovascular diseases. Based on experimental studies demonstrating that bone marrow-derived mononuclear cells (BMMNCs) improve the functional recovery after ischemia, clinical trials were initiated to address this new therapeutic concept. BMMNCs improve neovascularization of ischemic tissue by a broad repertoire of potential therapeutic actions. Whereas initial studies documented that the cells incorporate and differentiate to cardiovascular cells, other studies suggested that short-time paracrine mechanisms mediate the beneficial effects. Here, we hypothesized that BMMNCs have a phagocytic ability, and switch to a proangiogenic phenotype after engulfment of apoptotic cells. Activation of such angiogenic program may be pivotal in the beneficial effect of BMMNCs-based therapy. In vitro, wildtype (WT) BMMNCs ingestion of apoptotic cells upregulated the release of proangiogenic factors VEGF and HGF by 15- and 5-fold, respectively. In contrast, BMMNCs collected from mice deficient in MFG-E8, a protein that is required for attachment and engulfment of apoptotic cells by phagocytes, displayed lower phagocytic ability, leading to decrease in VEGF and HGF release. The capacity of BMMNCs to differentiate into cells with endothelial phenotype was similar in control and MFG-E8-deficient cells. In an in vivo model of mice myocardial infaction (MI), transplantation of WT BMMNCs increased fractionnal shortening (120 % of untreated control, p < 0.05), 14 days after MI. Size of the infarct scar was reduced by 30 % (p < 0.05 vs untreated control), and capillary density in the infarct border zone was raised by 10 % (p < 0.05 vs untreated control) in the WT BMMNCs group. In contrast, transplantation of MFG-E8 deficient BMMNCs displayed no significant effect on cardiac function, infarct size or angiogenesis in the ischemic myocardium. Four days after MI, VEGF protein levels were raised by 1.4 fold in the myocardium of WT BMMNCs treated animals compared to untreated controls (p < 0.05), while treatment with MFG-E8 deficient BMMNCs failed to raise VEGF levels. Taken together, these results suggest that phagocytosis of apoptotic cells reprograms BMMNCs into a proangiogenic phenotype. Hence, the therapeutic effect of transplanted BMMNCs depends, at least in part, on their phagocytic ability.

Effectiveness of autologous preparation rich in growth factors for the treatment of chronic cutaneous ulcers

Autologous Preparation Rich in Growth Factors (PRGF), a small volume of plasma enriched in platelets, is a novel therapeutic strategy for the acceleration of the wound healing of a wide range of tissues because of the continuous release of multiple growth factors, including PDGF-AB, TGF-beta1, IGF-I, HGF, VEGF-A, and EGF. In this article, we have characterized the PRGF preparation and designed a randomized open-label controlled pilot trial to evaluate the effectiveness of PRGF in the treatment of chronic cutaneous ulcers. Results showed that at 8 weeks, the mean percentage of surface healed in the PRGF group was 72.94% +/- 22.25% whereas it was 21.48% +/- 33.56% in the control group (p < 0.05). These results, with the limitations of a pilot study, suggest that topical application of PRGF is more effective than standard therapy in helping a chronic ulcer to heal.

Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-α abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.

Presence of a mobilizable intracellular pool of hepatocyte growth factor in human polymorphonuclear neutrophils

AbstractHepatocyte growth factor (HGF), a heparin-binding factor, is synthesized as a single-chain inactive precursor (pro-HGF), which is converted by proteolysis to an active heterodimer (mature HGF). HGF has pleiotropic activities and has been implicated in the regulation of mitogenesis, motogenesis, and morphogenesis of epithelial and endothelial cells. As polymorphonuclear neutrophils (PMNs) secrete numerous cytokines involved in the modulation of local inflammation, we investigated their ability to produce HGF. We found that HGF was stored in secretory vesicles and in gelatinase/specific granules. This intracellular stock was rapidly mobilized by degranulation when neutrophils were stimulated with phorbol myristate acetate or N-formylmethionyl-leucyl-phenylalanine. Cycloheximide did not affect the release of HGF. Moreover, HGF messenger RNA and protein expression was found in bone marrow myeloid cells, suggesting that HGF synthesis likely occurs during PMN maturation. In mature circulating PMNs, intracellular HGF was in the pro-HGF form, whereas the HGF secreted by degranulation was the mature form. Furthermore, PMNs pretreated with diisopropyl fluorophosphate only released the pro-HGF form, suggesting that PMN-derived serine protease(s) are involved in the proteolytic process. We also obtained evidence that secreted mature HGF binds PMN-derived glycosaminoglycans (probably heparan sulfate). These findings suggest that PMNs infiltrating damaged tissues may modulate local wound healing and repair through the production of HGF, a major mediator of tissue regeneration.

Mechanical Stretch Induces Activation of Skeletal Muscle Satellite Cells in Vitro

Cultured quiescent satellite cells were subjected to mechanical stretch in a FlexerCell System. In response to stretch, satellite cells entered the cell cycle earlier than if they were under control conditions. Only a brief period of stretch, as short as 2 h, was necessary to stimulate activation. Additionally, conditioned medium from stretched cells could activate unstretched satellite cells. The presence of HGF on c-met-positive myogenic cells was detected by immunofluorescence at 12 h in culture, and immunoblots demonstrated that HGF was released by stretched satellite cells into medium. Also, stretch activation could be abolished by the addition of anti-HGF antibodies to stretched cultures, and activity in conditioned medium from stretched cells could be neutralized by anti-HGF antibodies. In addition, stretch appeared to cause release of preexisting HGF from the extracellular matrix. These experiments suggest that HGF may be involved in linking mechanical perturbation of muscle to satellite cell activation.