Including Hepatocyte Growth Factor Research Articles

Differentiation of bone marrow‐derived mesenchymal stem cells into hepatocyte‐like cells in an alginate scaffold

Alginate scaffolds are the most frequently investigated biomaterials in tissue engineering. Tissue engineering techniques that generate liver tissue have become important for treatment of a number of liver diseases and recent studies indicate that bone marrow-derived stem cells (BMSCs) can differentiate into hepatocyte-like cells. The goal of the study described here, was to examine in vitro hepatic differentiation potential of BMSCs cultured in an alginate scaffold. To investigate the potential of BMSCs to differentiate into hepatocyte-like cells, we cultured BMSCs in alginate scaffolds in the presence of specific growth factors including hepatocyte growth factor, epidermal growth factor and fibroblast growth factor-4. We can demonstrate that alginate scaffolds are compatible for growth of BMSCs and when cultured in alginate scaffolds for several days they display several liver-specific markers and functions. Specifically, they expressed genes encoding alpha-foetoprotein, albumin (ALB), connexin 32 and CYP7A1. In addition, these BMSCs produced both ALB and urea, expressed cytokeratin-18 (CK-18) and were capable of glycogen storage. Percentage of CK-18 positive cells, a marker of hepatocytes, was 56.7%. Our three-dimensional alginate scaffolds were highly biocompatible with BMSCs. Furthermore, culturing induced their differentiation into hepatocyte-like cells. Therefore, BMSCs cultured in alginate scaffolds may be applicable for hepatic tissue engineering.

Phosphorylation of focal adhesion kinase on tyrosine 194 by Met leads to its activation through relief of autoinhibition

Focal adhesion kinase (FAK) has a crucial role in integration of signals from integrins and growth factor receptors. In this study, we demonstrate that growth factor receptors including hepatocyte growth factor receptor Met, epidermal growth factor receptor, and platelet-derived growth factor receptor directly phosphorylate FAK on Tyr194 in the FERM domain (band 4.1 and ezrin/radixin/moesin homology domain). Upon binding to Met or phosphoinositides, FAK may undergo conformational changes, which renders Tyr194 accessible for phosphorylation. Substitution of Tyr194 with Phe significantly suppresses the activation of FAK by Met. In contrast, substitution of Tyr194 with Glu (Y194E substitution) leads to constitutive activation of FAK. The phosphorylation of FAK on Tyr194 may cause conformational changes in the FERM domain, which disrupts the intramolecular inhibitory interaction between the FERM and kinase domains of FAK. Moreover, substitution of the basic residues in the (216)KAKTLRK(222) patch in the FERM domain with Ala antagonizes the effect of the Y194E substitution on FAK activation, thus suggesting that the interactions between the phosphorylated Tyr194 and the basic resides in the (216)KAKTLRK(222) patch may allow FAK to be activated through relief of its autoinhibition. Collectively, this study provides the first example to explain how FAK is activated by receptor tyrosine kinases.

Association of serum cytokines with pancreatic function in patients after acute pancreatitis

Objective To investigate the effect of cytokines on pancreatic function in patients after acute pancreatitis(AP) and its mechanisms. Methods Fifty-nine patients (mild in 25 and severe in 34) after AP and 20 healthy controls were enrolled in the study. Serum levels of cytokines including hepatocyte growth factor (HGF), epidermal growth factor(EGF), basic fibroblast growth factor (bFGF), regeneration protein(Reg)-1 and Reg-4 were determined using enzyme-linked immunosorbent assay (ELISA). Fasting blood-glucose, insulin, C-peptide and fecal elastase 1 (FE1) were detected for evluation of endocrine and exocrine pancreatic function. The association of pancreatic function with clinical parameters and serum cytokines was analyzed. Results The expression of FE1 was lower in patients [(205.9±18.3) μg/g] after AP in comparison with the controls [(333.9±19.7) μg/g, P<0. 01], but levels of fasting blood-glucose, C-peptide and insulin were higher in patients group (P<0.01). Serum level of HGF was higher in patients with insufficient pancreatic exoerine [(983.76±372.65) pg/ml] than those with normal exocrine function [(263.44±110. 35) pg/ml]. Meanwhile,EGF level was higher in patients with DM after AP [(704.41±190. 37) pg/ml] than those without DM [(360. 03±48.39) pg/mh P<0.05]. There was a negatively correlation between FE1 and HGF (P ＜0. 01). The abnormal fasting blood glucose was correlated with CT grading (P<0. 05).Conclusions The patients after AP develope insufficient exocrine and endocrine function. Serum EGF and HGF may be associated with restoration of pancreatic endocrine and exocrine function. Key words: Pancreatitis; Pancreatic function tests; Cytokines

Matriptase Activation, an Early Cellular Response to Acidosis

Extracellular acidosis often rapidly causes intracellular acidification, alters ion channel activities, and activates G protein-coupled receptors. In this report, we demonstrated a novel cellular response to acidosis: induction of the zymogen activation of matriptase. Acid-induced matriptase activation is ubiquitous among epithelial and carcinoma cells and is characterized by rapid onset, fast kinetics, and the magnitude of activation seen. Trace amounts of activated matriptase can be detected 1 min after cells are exposed to pH 6.0 buffer, and the vast majority of latent matriptase within the cells is converted to activated matriptase within 20 min. Matriptase activation may be a direct response to proton exposure because acid-induced matriptase activation also occurs in an in vitro, cell-free setting in which intracellular signaling molecules and ion channel activities are largely absent. Acid-induced matriptase activation takes place both on the cell surface and inside the cells, likely due to the parallel intracellular acidification that activates intracellular matriptase. Following matriptase activation, the active enzyme is immediately inhibited by binding to hepatocyte growth factor activator inhibitor 1, resulting in stable matriptase-hepatocyte growth factor activator inhibitor 1 complexes that are rapidly secreted. As an early response to acidosis, matriptase activation can also be induced by perturbation of intracellular pH homeostasis by 5-(N-methyl-N-isobutyl)-amiloride and 5-(N-ethyl-N-isopropyl)-amiloride, both of which inhibit Na(+)/H(+) exchangers, and diisothiocyanostilbene-2,2'-disulfonic acid, which can inhibit other acid-base ion channels. This study uncovers a novel mechanism regulating proteolysis in epithelial and carcinoma cells, and also demonstrates that a likely function of matriptase is as an early response to acidosis.

Hepatocyte growth factor protects against Fas‐mediated liver apoptosis in transgenic mice

Apoptosis via the Fas/Fas ligand signalling system plays an important role in the development of various liver diseases. The administration of an agonistic anti-Fas antibody to mice causes massive hepatic apoptosis and fulminant hepatic failure. Several growth factors including hepatocyte growth factor (HGF) have been found to prevent apoptosis. In this study, we demonstrated the overexpression of HGF to have a protective effect on Fas-mediated hepatic apoptosis using a transgenic mice (Tg mice) model. In HGF Tg mice, the elevation of alanine aminotransferase was dramatically inhibited at 12 and 24 h after the administration of 0.15 mg/kg anti-Fas antibody. HGF Tg mice showed a significantly lower number of apoptotic hepatocytes at 12 h compared with wild-type (WT) mice. Furthermore, 85% (six of seven) HGF Tg mice were able to survive after the administration of 0.3 mg/kg anti-Fas antibody, while none of the WT mice survived. The Bcl-xL expression was increased in HGF Tg mice, while there was no difference in the expression of Bax, Bid, Mcl-1 and bcl-2 between WT mice and HGF Tg mice. In addition, the HGF Tg mice showed more Akt phosphorylation than the WT mice both before and after the anti-Fas antibody injection. Taken together, our findings suggest that HGF protects against Fas-mediated liver apoptosis in vivo, and the upregulation of Bcl-xL via Akt activation may also play a role in the protective effects of HGF.

Mesenchymal Stem Cell Transition to Tumor-Associated Fibroblasts Contributes to Fibrovascular Network Expansion and Tumor Progression

BackgroundTumor associated fibroblasts (TAF), are essential for tumor progression providing both a functional and structural supportive environment. TAF, known as activated fibroblasts, have an established biological impact on tumorigenesis as matrix synthesizing or matrix degrading cells, contractile cells, and even blood vessel associated cells. The production of growth factors, cytokines, chemokines, matrix-degrading enzymes, and immunomodulatory mechanisms by these cells augment tumor progression by providing a suitable environment. There are several suggested origins of the TAF including tissue-resident, circulating, and epithelial-to-mesenchymal-transitioned cells.Methodology/Principal FindingsWe provide evidence that TAF are derived from mesenchymal stem cells (MSC) that acquire a TAF phenotype following exposure to or systemic recruitment into adenocarcinoma xenograft models including breast, pancreatic, and ovarian. We define the MSC derived TAF in a xenograft ovarian carcinoma model by the immunohistochemical presence of 1) fibroblast specific protein and fibroblast activated protein; 2) markers phenotypically associated with aggressiveness, including tenascin-c, thrombospondin-1, and stromelysin-1; 3) production of pro-tumorigenic growth factors including hepatocyte growth factor, epidermal growth factor, and interleukin-6; and 4) factors indicative of vascularization, including alpha-smooth muscle actin, desmin, and vascular endothelial growth factor. We demonstrate that under long-term tumor conditioning in vitro, MSC express TAF–like proteins. Additionally, human MSC but not murine MSC stimulated tumor growth primarily through the paracrine production of secreted IL6.Conclusions/SignificanceOur results suggest the dependence of in vitro Skov-3 tumor cell proliferation is due to the presence of tumor-stimulated MSC secreted IL6. The subsequent TAF phenotype arises from the MSC which ultimately promotes tumor growth through the contribution of microvascularization, stromal networks, and the production of tumor-stimulating paracrine factors.

Mechanisms Underlying the Effects of Leukocyte Apheresis with a Fiber Filter in a Rat Model of Dextran Sulfate Sodium-Induced Colitis

While several clinical trials have suggested that leukocytapheresis (LCAP) by filtration can benefit patients with active ulcerative colitis, the mechanisms underlying these benefits are largely unknown. The aim of this study was to address the mechanisms that may underlie the therapeutic effects of LCAP using a dextran sulfate sodium-induced colitis model in rats. Treatment with the active column, but not the sham column, improved disease severity by down-regulating pro-inflammatory events, including the cell-proliferative responses and inflammatory cytokine and reactive oxygen production, as well as by up-regulating protective events, including hepatocyte growth factor production, bone marrow-derived endothelial progenitor cell induction, and colonic blood flow levels, which were mediated predominantly by calcitonin gene-related peptide. The improvement was also associated with the increase of Ki-67 labeling in the colonic epithelium. In conclusion, the LCAP procedure was used in a dextran sulfate sodium-induced colitis model in rats under extracorporeal circulation conditions. This approach down-regulated pro-inflammatory events and up-regulated protective events in association with disease improvement. These data suggest that LCAP is feasible in animals and should shed light on the mechanisms of LCAP in clinical settings.

Differentiation of human umbilical cord mesenchymal stromal cells into low immunogenic hepatocyte-like cells

Mesenchymal stromal cells (MSC) isolated from several human tissues have been known to differentiate into the hepatic lineage in vitro, but the immunogenicity of the differentiated hepatocyte-like cells (DHC) has not been reported. Umbilical cord (UC) MSC are thought to be an attractive cell source for cell therapy because of their young age and low infection rate compared with adult tissue MSC. Hepatic differentiation of UC-MSC was induced with a 2-step protocol. The expressions of hepatic markers were detected by RT-PCR and immunofluorescence staining. Albumin production and urea secretion were measured by ELISA and colorimetric assay respectively. The immunosuppressive properties of DHC was detected by mixed lymphocyte culture. After incubation with specific growth factors, including hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), UC MSC exhibited a high hepatic differentiation ability in an adherent culture condition. The differentiated UC MSC showed hepatocyte-like morphology and expressed several liver-specific markers at gene and protein levels. Furthermore, the DHC exhibited hepatocyte-specific functions, including albumin secretion, low-density lipoprotein uptake and urea production. More importantly, DHC did not express major histocompatibility complex (MHC) II antigen and were not able to induce lymphocyte proliferation in mixed lymphocyte culture, as undifferentiated UC MSC did. Our results indicate that UC MSC are able to differentiate into functional hepatocyte-like cells that still retain their low immunogenicity in vitro. More importantly, DHC incorporated into the parenchyma of liver when transplanted into mice with CCl(4)-induced liver injury. Therefore, DHC may be an ideal source for cell therapy of liver diseases.

Defect of Hepatocyte Growth Factor Activator Inhibitor Type 1/Serine Protease Inhibitor, Kunitz Type 1 (Hai-1/Spint1) Leads to Ichthyosis-Like Condition and Abnormal Hair Development in Mice

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1(+/+) blastocysts with Hai-1/Spint1(-/-) embryonic stem cells successfully generated high-chimeric Hai-1/Spint1(-/-) embryos (B6Hai-1(-/-High)) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1(-/-) mice was caused by placental dysfunction. However, newborn B6Hai-1(-/-High) mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development.

Functional Clustering of Metastasis Proteins Describes Plastic Adaptation Resources of Breast-Cancer Cells to New Microenvironments

To examine the molecular mechanisms underlying breast cancer metastasis in liver and search for potential markers of metastatic progression in soft-tissue, we analyzed metastatic variants developed from the highly metastatic MDA-MB 435 cell line through in vivo stepwise selection in the athymic mice. Comparative proteomic analysis using two-dimensional electrophoresis (2DE-DIGE) revealed that 74 protein spots were reproducibly more than doubled in liver metastatic cells compared to parental counterpart. From 22 proteins identified by MALDI-TOF, belonging to intermediate filaments, intracellular transport and ATP synthesis, we generated a protein-protein interaction network containing 496 nodes, 12 of which interacted. GRP 75 was connected with four other proteins: prohibitin, HSP 27, elongin B and macropain delta chain. After functional classification, we found that pathways including hepatocyte growth factor receptor (p = 0.014), platelet-derived growth factor (p = 0.018), vascular endothelial growth factor (p = 0.021) and epidermal growth factor (p = 0.050) were predominant in liver metastatic cells, but not in lung metastatic cells. In conclusion, we suggest that GRP 75 is involved in cell proliferation, tumorigenesis and stress response in metastatic cells by recruiting signals in which the transmembrane receptor protein tyrosine kinase signaling pathway (p-value FDR = 1.71 x 10(-2)) and protein amino acid phosphorylation (p-value FDR = 3.28 x 10(-2)) might be the most significant biological process differentially increased in liver metastasis.

Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells

Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human alpha-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of growth factors including hepatocyte growth factor (HGF), bone morphogenetic protein 4, fibroblast growth factor 4, all-trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and 20 ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.

Mechanisms of hepatocyte growth factor–mediated and epidermal growth factor–mediated signaling in transdifferentiation of rat hepatocytes to biliary epithelium

Previous studies from our laboratory have demonstrated that hepatocytes can transdifferentiate into biliary epithelium (BE) both in vivo and in vitro; however, the mechanisms are unclear. The current study was designed to investigate the mechanisms of hepatocyte transdifferentiation in vitro. Rat hepatocytes were cultured in roller bottles to obtain hepatocyte organoid cultures, which were stimulated with various growth factors (GFs) including hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), stem cell factor (SCF), macrophage-stimulating protein (MSP), fibroblast growth factor-a (FGF-a), fibroblast growth factor-b (FGF-b), and fibroblast growth factor-8b (FGF-8b). Only the cultures treated with HGF, EGF, and their combination exhibited formation of hepatocyte-derived biliary epithelium (BE) despite the presence and activation of all the pertinent cognate membrane receptors of the rest of the GFs. Microarray analysis of the organoid cultures identified specific up-regulation of approximately 500 target genes induced by HGF and EGF, including members of the extracellular matrix (ECM) protein family, Wnt/beta-catenin pathway, transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) pathway, and CXC (cysteine-any amino acid-cysteine) chemokines. To investigate the downstream signaling involved in hepatocyte to biliary epithelial cell (BEC) transdifferentiation, we investigated expression and activities of mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)] as well as serine/threonine kinase AKT. The analysis indicated that AKT phosphorylation was particularly increased in cultures treated with HGF, EGF, and their combination. Whereas phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely inhibited biliary epithelium formation, AKT inhibitor could only moderately reduce formation of BE in the organoid cultures treated with HGF+EGF. Most of the HGF+EGF target genes were altered by LY294002. Taken together, these data indicate that hepatocyte to BE transdifferentiation is regulated by HGF and EGF receptors and that PI3 kinase-mediated signaling independent of AKT is a crucial component of the transdifferentiation process.

Zinc Supplementation Enhances Hepatic Regeneration by Preserving Hepatocyte Nuclear Factor-4α in Mice Subjected to Long-Term Ethanol Administration

Alcoholic liver disease is associated with sustained liver damage and impaired regeneration, as well as significant zinc deficiency. This study was undertaken to examine whether dietary zinc supplementation could improve liver regeneration by increasing the expression of genes involved in hepatic cellular proliferation in a mouse model of alcoholic liver disease. Adult 129S6 mice fed an ethanol-containing liquid diet for 6 months developed alcoholic liver disease as measured by serum alanine transferase activity and histopathological changes. Zinc supplementation to ethanol-exposed mice enhanced liver regeneration as indicated by increased numbers of proliferation cell nuclear antigen (PCNA)-positive and bromodeoxyuridine (BrdU)-labeled hepatocytes. Zinc-enhanced liver regeneration was associated with an increase in hepatocyte nuclear factor-4alpha (HNF-4alpha), a liver-enriched, zinc-finger transcription factor. Studies using cultured HepG2 cells showed that zinc deficiency suppressed cell proliferation and cell proliferation-related proteins, including hepatocyte growth factor (HGF), insulin-like growth factor I (IGF-I), insulin-like growth factor binding protein 1 (IGFBP1), metallothionein (MT), and cyclin D1, as well as HNF-4alpha. HNF-4alpha gene silencing inhibited cell proliferation in association with decreased protein levels of IGF-I, IGFBP1, MT, and cyclin D1. The present study provides evidence that zinc supplementation enhances liver regeneration at least in part by HNF-4alpha through the up-regulation of cell proliferation-related proteins, suggesting that dietary zinc supplementation may have beneficial effects in alcoholic liver disease.

Mechanism of impaired hepatic regeneration in cholestatic liver

The regenerative capacity of the liver is an important factor following liver surgery. The dramatic change in portal venous flow, due to either portal vein embolization or partial hepatectomy, induces a rapid change in liver volume. In response to these stresses, hepatocytes are primed, through the release of inflammatory cytokines, to increase the expression of immediate early genes and increase the activation of transcriptional factors. The primed hepatocytes then respond to growth factors, including hepatocyte growth factor, epidermal growth factor, and transforming growth factor-alpha. Several pathologic conditions have been shown to inhibit hepatic regeneration. These include diabetes mellitus, malnutrition, aging, infection, chronic ethanol consumption, and biliary obstruction. Impaired hepatic regeneration in the setting of biliary obstruction is an especially serious problem because it can be a major determinant in not considering surgical treatment. The mechanism responsible for impaired hepatic regeneration in patients with biliary obstruction includes decreased portal venous flow, attenuated production of liver proliferation-associated factors, an increased rate of apoptosis, and lack of enterohepatic circulation. Restoring these factors may lead to an improvement in regeneration in a cholestatic liver following portal vein embolization or partial hepatectomy. This review article summarizes the current understanding of the mechanism of hepatic regeneration, with particular emphasis on that in the cholestatic liver.

Sulodexide induces hepatocyte growth factor release in humans

Heparin influences numerous pleiotropic growth factors, including hepatocyte growth factor (HGF), partially by their release from endothelial and extracellular matrix stores. The effects of sulodexide, a heparin-like glycosaminoglycan medication of growing importance in medicine, on HGF liberation are not known. We performed a 2-week open-label sulodexide trial in healthy male volunteers. The drug was initially administered intravenously (i.v.) in a single dose of 1200 Lipoprotein Lipase Releasing Units (LRU), then – orally for 12 days (500 LRU twice a day), and – again by i.v. route (1200 LRU) on day 14. Intravenous sulodexide injections were repeatedly found to induce marked and reproducible increases in immunoreactive plasma HGF levels (more than 3500% vs baseline after 10 min, and more than 1200% after 120 min), and remained unchanged when measured 120 min following oral sulodexide administration. The percentage increments in plasma HGF evoked by i.v. sulodexide at both time points and on both days inversely correlated with baseline levels of the growth factor. On day 14, the HGF levels after 120 min and their percentage increase vs baseline were strongly and directly dependent on i.v. sulodexide dose per kg of body weight. This study shows that sulodexide has a novel, remarkable and plausibly biologically important stimulating effect on the release of pleiotropic hepatocyte growth factor in humans.

Inhibitors targeting hepatocyte growth factor receptor and vascular endothelial growth factor receptor tyrosine kinases

Amgen disclosed a series of 4-heteroaryloxy quinoline/quinazoline compounds as multiple kinase inhibitors, including hepatocyte growth factor (HGF) receptor tyrosine kinase c-Met and vascular endothelial growth factor (VEGF) receptor tyrosine kinase. These compounds are stated to have wide therapeutic applications for the treatment of a variety of cancers, hypertension, arteriosclerosis, myocardial infarction and rheumatoid arthritis.

Tumor lymphangiogenesis and metastatic spread—New players begin to emerge

The metastatic spread of tumor cells is the most lethal aspect of cancer and can occur via various routes, including the lymphatic vasculature. Studies of tumor models in animals and clinicopathological data have indicated that growth of lymphatic vessels (lymphangiogenesis) in the vicinity of solid tumors may contribute to lymphatic metastasis. Research over the past 5 years has identified a range of lymphangiogenic growth factors that could conceivably play a role in promoting tumor lymphangiogenesis and lymphatic metastasis. The most extensively studied signaling system that promotes lymphangiogenesis in tumors involves the secreted lymphangiogenic proteins vascular endothelial growth factor-C (VEGF-C) and VEGF-D, and their cognate receptor on lymphatic endothelium VEGF receptor-3 (VEGFR-3). More recent studies have identified other signaling molecules that can also promote lymphangiogenesis in vivo, including hepatocyte growth factor and members of the fibroblast growth factor, angiopoietin, platelet-derived growth factor and insulin-like growth factor families of secreted proteins. This article provides an overview of the molecular mechanisms that control lymphangiogenic signaling, emphasizing the more recently identified lymphangiogenic growth factors and the roles they may play in cancer biology. Molecular approaches for inhibiting lymphangiogenic signaling in cancer, designed to restrict tumor metastasis, are also examined.

Vitreous and aqueous concentrations of proangiogenic, antiangiogenic factors and other cytokines in diabetic retinopathy patients with macular edema: Implications for structural differences in macular profiles

The aim of the study was to determine anatomical and growth factor profiles in patients with clinically significant macular oedema (CSMO) undergoing pars plana vitrectomy (PPV). Twenty patients with moderate nonproliferative diabetic retinopathy (NPDR) with persistent CSMO underwent PPV. Patients had baseline and postoperative clinical assessment including Ocular Coherence Tomography (OCT). Baseline vitreous and aqueous and serial postoperative aqueous samples were analysed for vascular endothelial growth factor-A (VEGF-A), pigment epithelium derived Factor (PEDF) and other factors (pg/ml) including hepatocyte growth factor, MMP 9, soluble flt-1 Receptor, and TGF β1 by ELISA. Vitreous from patients with full thickness macular holes (8) and proliferative diabetic retinopathy (22) were collected for comparison as controls. Vitreous VEGF-A concentration in the NPDR group was 957 pg/ml compared to 239 pg/ml in the macula hole (FTMH) control ( p<0.0001) and 596 pg/ml compared to PDR ( p=0.006). The median diabetic vitreous PEDF concentration was 1.36 μg/ml (FTMH 2.6 μg/ml p=0.05). In NPDR, it was higher (1.59 μg/ml) than PDR (1.27 μg/ml) p=0.02. There were changes to the HGF, soluble flt-1 Receptor and TGF b1 concentrations in the NPDR compared to either PDR or the normal state. In CSMO, two OCT profiles were identified: dome-shaped macular elevation (Group 1) ( n=4) and diffuse-low elevation profile (Group 2) ( n=16) which also showed differences in the postoperative median aqueous VEGF concentrations despite macular volume decreasing for both. The results suggest that there is an up-regulation of VEGF in the vitreous of the diabetic eye with a reciprocal decrease in PEDF. The structural and molecular differences between the two OCT macular profiles may explain the varying response to PPV in patients with diffuse CSMO.

Proteins Associated with Cisplatin Resistance in Ovarian Cancer Cells Identified by Quantitative Proteomic Technology and Integrated with mRNA Expression Levels

Nearly all women diagnosed with ovarian cancer receive combination chemotherapy including cis- or carboplatin. Despite high initial response rates, resistance to cisplatin develops in roughly one-third of women during primary treatment and in all women treated for recurrent disease. ICAT coupled with tandem MS is a quantitative proteomic technique for high throughput protein expression profiling of complex protein mixtures. Using ICAT/MS/MS we profiled the nuclear, cytosolic, and microsomal fractions obtained from IGROV-1 [corrected] (cisplatin-sensitive) and IGROV-1/CP [corrected] (cisplatin-resistant) ovarian cancer cell lines. The proteomes of cisplatin-sensitive and -resistant ovarian cancer cells were compared, and protein expression was correlated with mRNA expression profiles. A total of 1117 proteins were identified and quantified. The relative expression of 121 of these varied between the two cell lines. Sixty-three proteins were overexpressed in cisplatin-sensitive, and 58 were over expressed in cisplatin-resistant cells. Examples of proteins at least 5-fold overexpressed in resistant cells and with biological relevance to cancer include cell recognition molecule CASPR3 (13.3-fold), S100 protein family members (8.7-fold), junction adhesion molecule Claudin 4 (7.2-fold), and CDC42-binding protein kinase beta (5.4-fold). Examples of cancer-related proteins at least 5-fold overexpressed in sensitive cells include hepatocyte growth factor inhibitor 1B (13.3-fold) and programmed cell death 6-interacting protein (12.7-fold). The direction of changes in expression levels between proteins and mRNAs were not always in the same direction, possibly reflecting posttranscriptional control of protein expression. We identified proteins whose expression profiles correlate with cisplatin resistance in ovarian cancer cells. Several proteins may be involved in modulating response to cisplatin and have potential as markers of treatment response or treatment targets.

Mucosal repair and growth factors: recombinant human hepatocyte growth factor as an innovative therapy for inflammatory bowel disease

The repair of intestinal mucosal injuries is a tightly regulated process involving epithelial restitution, cell proliferation and maturation, and the dedifferentiation of epithelial cells. Deeper injuries also require additional repair mechanisms, including inflammatory processes, angiogenesis, and extracellular-matrix deposition. Once intestinal mucosal injury occurs, numerous growth factors and cytokines, including hepatocyte growth factor (HGF), keratinocyte growth factor, endothelial growth factor, epidermal growth factor, transforming growth factor-beta1, intestinal trefoil factor, interleukin (IL)-1, and IL-2, are induced in both the intestinal lumen and submucosa, and these factors cooperatively stimulate epithelial mucosal repair. HGF, a major agent promoting hepatocyte proliferation, also modulates intestinal epithelial cell proliferation and migration, leading to the acceleration of intestinal mucosal repair. Additionally, the proteolytic activation of HGF, which is mediated by HGF activator, is essential for the regeneration of injured intestinal mucosa. Recently, several studies have shown that the administration of recombinant human HGF or HGF gene therapy abrogates disease severity in several animal models of inflammatory bowel disease (IBD). Recombinant human HGF will soon be available for administration to patients with fulminant hepatic failure. Although additional preclinical biological studies are required, HGF has the potential to be an important new treatment modality promoting intestinal mucosal repair in patients with IBD.