OBJECTIVE

The purpose of this study was to determine whether messenger RNA coding for hepatocyte growth factor (HGF), HGF receptor (MET), keratinocyte growth factor (KGF), KGF receptor, and fibroblast growth factor (FGF) receptor-2 were produced in primary cultures of human corneal epithelial, stromal fibroblast, and endothelial cells, as well as ex vivo corneal epithelium, endothelial cells transfected with the SV40 large T antigen, and control embryonic lung fibroblasts. The effects of exogenous HGF and KGF, compared to epidermal growth factor, on the proliferation of first passage corneal cells were also examined.

METHODS

Polymerase chain reaction was used to amplify complementary DNA for each modulator from each cell type. Hot blotting was used to demonstrate the specificity of amplification products. Proliferation of first passage corneal epithelial, stromal fibroblast, and endothelial cells in response to varying concentrations of HGF, KGF, and epidermal growth factor was measured.

RESULTS

Specific amplification products for messenger RNA coding for each modulator were detected in each corneal cell type, although very low levels of HGF and KGF messenger RNA appeared to be present in corneal epithelial cells relative to stromal fibroblasts and corneal endothelial cells. Amplification products that may have been derived from alternative transcripts were detected for several of the modulators. HGF and KGF stimulated proliferation in a dose-response manner in first passage corneal epithelial and endothelial cells, but not stromal fibroblast cells.

CONCLUSIONS

Human corneal epithelial, stromal fibroblasts, and endothelial cells produce messenger RNA coding for HGF and KGF, although low levels appear to be present in the epithelial cells. All three major cell types of the cornea produce messenger RNA coding for HGF receptor, KGF receptor, and FGF receptor-2. The proliferation of human corneal epithelial and endothelial cells, but not stromal fibroblasts, was stimulated by exogenous HGF and KGF. HGF and KGF likely have intracrine, autocrine, and/or paracrine functions in the cornea. Exogenous HGF and KGF may be useful in corneal preservation and for regulating corneal wound healing.

The use of autologous fibrin matrices has been proposed as a therapeutic strategy for the local and physiological delivery of growth factors in the treatment of several clinical conditions requiring tendon healing or tendon graft remodelling. In the present work, we investigated the proliferation, synthesis of type-I collagen and angiogenic factors by tendon cells seeded on platelet-rich (PR) and platelet-poor (PP) matrices. Furthermore, in vivo cellular and vascular effects of each treatment were examined after infiltration in Achilles tendon in sheep. Results showed that the presence of platelets within the fibrin matrices increased significantly the proliferation of tendon cells. Additionally, cultured tendon cells synthesised type I collagen and angiogenic factors such as VEGF and HGF. The synthesis of VEGF, but not of HGF, was significantly higher when platelets were present within the matrix. In the sheep model, the injection of pre-clotted plasma within tendons increased cellular density and promoted neovascularization. These results indicate that administration of fibrin matrices is a safe and easy strategy that may open new avenues for enhancing tissue healing and remodelling and influences the process of regeneration in clinical situations characterised by a poor healing outcome.

Hepatocyte growth factor (HGF), initially identified and molecularly cloned as a potent mitogen of primary cultured hepatocytes, has multiple activities in a variety of tissues during the course of development and also in various disease states. HGF plays key roles in the attenuation of disease progression as an intrinsic repair factor. It is also evident that HGF levels are regulated under different conditions, for example, during the course of pregnancy, aging, and disease. This review focuses on the levels of HGF in normal and pathophysiological situations and examines the relationships between HGF levels and disease, disease stage, and disease prognosis. The clinical potential of HGF as a treatment for subjects with various diseases is also given attention.

Muscle injuries represent a major part of sports injuries and are a challenging problem in traumatology. Strain injuries are the most common muscle injuries after contusions. These injuries can lead to significant pain and disability causing time to be lost to training and competition. Despite the frequency of strain injuries the treatment available is limited and is generally not sufficient to enhance muscle regeneration efficiently when fast resumption of sport activity is a primary target. A number of growth factors play a specific role in regeneration and it has been proven that a previously described method of physically and chemically stimulating whole blood (to produce autologous conditioned serum) induces concentration increases in FGF-2, HGF, and TGF-beta1. A preliminary study was conducted on muscle strain injuries in professional sportsmen receiving either: 1. autologous conditioned serum (ACS) or 2. Actovegin/Traumeel treatment as control. Assessment of recovery from injury was done by: 1. sport professional's ability to participate to 100 % under competition conditions in their respective sport and 2. MRI analysis. A significant difference in the recovery time from injury was demonstrated: 16.6 +/- 0.9 in the ACS treated instead of 22.3 +/- 1.2 (mean +/- SEM) days in the Actovegin/Traumeel control group (p = 0.001). MRI analysis supported the observed acceleration of the lesion recovery time. We conclude that ACS injection is a promising approach to reduce the time to recovery from muscle injury.

OBJECTIVE

Bone marrow-derived mononuclear cells (BMCs) improve the functional recovery after ischemia. However, BMCs comprise a heterogeneous mixture of cells, and it is not known which cell types are responsible for the induction of neovascularization after cell therapy. Because cell recruitment is critically dependent on the expression of the SDF-1-receptor CXCR4, we examined whether the expression of CXCR4 may identify a therapeutically active population of BMCs.

RESULTS

Human CXCR4(+) and CXCR4(-) BMCs were sorted by magnetic beads. CXCR4(+) BMCs showed a significantly higher invasion capacity under basal conditions and after SDF-1 stimulation. Hematopoietic or mesenchymal colony-forming capacity did not differ between CXCR4(+) and CXCR4(-) BMCs. Injection of CXCR4(+) BMCs in mice after induction of hindlimb ischemia significantly improved the recovery of perfusion compared to injection of CXCR4(-) BMCs. Likewise, capillary density was significantly increased in CXCR4(+) BMC-treated mice. Because part of the beneficial effects of cell therapy were attributed to the release of paracrine effectors, we analyzed BMC supernatants for secreted factors. Importantly, supernatants of CXCR4(+) BMCs were enriched in the proangiogenic cytokines HGF and PDGF-BB.

CONCLUSIONS

CXCR4(+) BMCs exhibit an increased therapeutic potential for blood flow recovery after acute ischemia. Mechanistically, their higher migratory capacity and their increased release of paracrine factors may contribute to enhanced tissue repair.

Here we address the molecular mechanism of serum-independent survival and growth of human bladder carcinoma cell line 5637. Serum starvation promoted tyrosine phosphorylation of a 145-kDa protein and activation of the tyrosine kinase Src and the receptor for epidermal growth factor (EGFR) over a slow time course (>8 hours). The phosphorylated 145-kDa protein was identified as the beta-subunit of c-Met/hepatocyte growth factor (HGF) receptor, p145(met), in which tyrosine residues 1003, 1234, and 1235 were phosphorylated. Inhibitors of Src (PP2, SU6656) or EGFR (AG99), but not p145(met) (K252a), effectively blocked tyrosine phosphorylation of p145(met) and promoted cell death accompanied by activation of caspase-like proteases. Conditioned medium from the serum-starved 5637 cells or purified EGF readily promoted the activation of Src and EGFR, and tyrosine phosphorylation of p145(met) in normally grown 5637 cells, suggesting that autocrine signaling of EGFR ligands is responsible for signal transduction events in serum-starved cells. Consistent with this idea, a monoclonal antibody against EGFR that would interfere with the ligand binding to EGFR blocked tyrosine phosphorylation events and promoted the caspase activation and cell death in serum-free conditions. Such apoptotic cell death was also induced by pretreatment of cells with a high concentration of HGF that downregulated endogenous p145(met). Nevertheless, Cu2+ ions, competitive inhibitors for HGF-binding to p145(met), did not show any effect on cellular functions in serum-free conditions. These results suggest that the serum-independent growth of 5637 cells involves the transmembrane signaling cascade via EGFR ligand(s) (but not HGF), EGFR, Src and p145(met).

Gastric cancer (GC) is the third leading cause of cancer mortality worldwide. Although surgical resection is a potentially curative approach for localized cases of GC, most cases of GC are diagnosed in an advanced, non-curable stage and the response to traditional chemotherapy is limited. Fortunately, recent advances in our understanding of the molecular mechanisms that mediate GC hold great promise for the development of more effective treatment strategies. In this review, an overview of the morphological classification, current treatment approaches, and molecular alterations that have been characterized for GC are provided. In particular, the most recent molecular classification of GC and alterations identified in relevant signaling pathways, including ErbB, VEGF, PI3K/AKT/mTOR, and HGF/ MET signaling pathways, are described, as well as inhibitors of these pathways. An overview of the completed and active clinical trials related to these signaling pathways are also summarized. Finally, insights regarding emerging stem cell pathways are described, and may provide additional novel markers for the development of therapeutic agents against GC. The development of more effective agents and the identification of biomarkers that can be used for the diagnosis, prognosis, and individualized therapy for GC patients, have the potential to improve the efficacy, safety, and cost-effectiveness for GC treatments.

Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteolytic enzymes are implicated in a variety of physiological processes, such as collagen maturation, organogenesis, angiogenesis, reproduction and inflammation. Moreover, deficiency or overexpression of certain ADAMTS proteins is directly involved in serious human diseases, including cancer. However, the functional roles of other family members, such as ADAMTS12, remain unknown. Here, by using different in vitro and in vivo approaches, we have evaluated the possible role of ADAMTS12 in the development and progression of cancer. First, we show that expression of ADAMTS12 in Madin-Darby canine kidney (MDCK) cells prevents the tumorigenic effects of hepatocyte growth factor (HGF) by blocking the activation of the Ras-MAPK signalling pathway and that this regulation involves the thrombospondin domains of the metalloproteinase. We also show that addition of recombinant human ADAMTS12 to bovine aortic endothelial cells (BAE-1 cells) abolishes their ability to form tubules upon stimulation with vascular endothelial growth factor (VEGF). Additionally, tumours induced in immunodeficient SCID mice injected with A549 cells overexpressing ADAMTS12 show a remarkable growth deficiency in comparison with tumours formed in animals injected with parental A549 cells. Overall, our data suggest that ADAMTS12 confers tumour-protective functions upon cells that produce this proteolytic enzyme.

OBJECTIVE

Mesenchymal stromal cells (MSC) isolated from bone marrow (BM), adipose tissue and umbilical cord blood can be induced to differentiate into hepatocyte-like cells. MSC can also be isolated from umbilical cord Wharton's jelly (UC MSC), which can be easily obtained. UC MSC are more primitive MSC than those isolated from other tissue sources and do not express the major histocompatibility complex (MHC) class II (HLA-DR) antigens. Previous studies have shown that UC MSC are still viable and not rejected 4 months after transplantation as xenografts, without the need for immune suppression, suggesting that they are a favorable cell source for transplantation.

METHODS

UC MSC were induced to differentiate into hepatocyte-like cells by a simple one-step protocol with hepatotic growth factor (HGF) and fibroblast growth factor-4 (FGF-4). Differentiated cells were examined for the expression of hepatocyte-specific markers and hepatocyte functions.

RESULTS

UC MSC were isolated. Flow cytometry analysis showed that they expressed the MSC-specific markers. They differentiated into osteoblast-, adipocyte- and chondrocyte-like cells, showing their multipotent differentiation potential. Immunocytochemistry, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis demonstrated that UC MSC expressed the hepatocyte-specific markers albumin (ALB), human alpha-fetoprotein (AFP) and cytokeratin 18 (CK-18) following hepatocyte induction. Periodic acid-Schiff staining showed that differentiated UC MSC could store glycogen, and an low-density lipoprotein (LDL)-uptake assay showed that they could uptake LDL.

CONCLUSIONS

This study demonstrates that UC MSC can differentiate into functional hepatocyte-like cells following the induction of HGF and FGF-4. UC MSC can serve as a favorable cell source for tissue engineering in the treatment of liver disease.

BACKGROUND

Interleukin-17 (IL-17) is exclusively produced by activated T cells, and this cytokine can induce inflammatory responses, support immune responses (Th1), and stimulate osteoclastic bone resorption in combination with receptor activator of NF-kappaB (RANK) and RANK ligand (RANKL). These biological functions are relevant to the aetiopathogenesis of periodontitis, and thus we sought to investigate whether IL-17 is produced in periodontal lesions and to assess the relationship of gene expression between IL-17 and other cytokines, and to determine the effect of IL-17 on IL-6 production in human gingival fibroblasts (HGF).

METHODS

IL-17 was detected and measured in periodontal tissues obtained as biopsy samples during periodontal surgery and in the cell-free culture supernatants cultured ex vivo, by using Western immunoblotting and enzyme-linked immunosorbent assay, respectively. IL-17 and other cytokine gene expression were investigated by the reverse transcription-polymerase chain reaction (RT-PCR) method. The contribution of IL-17 to IL-6 production by HGF was studied.

RESULTS

IL-17 protein was moderately detected in periodontal tissues. In contrast, IL-17 mRNA was expressed only in nine of 23 periodontitis tissue samples by RT-PCR. The IL-17 mRNA-positive samples simultaneously expressed mRNAs encoding interferon (IFN)-gamma, IL-2, RANK, and RANKL, but not IL-4. IL-10 (Th2 cytokine) was detected more frequently in the samples than IFN-gamma and IL-2 (Th1 cytokine). Recombinant human IL-17 induced IL-6 production from HGF in a dose- and time-dependent fashion.

CONCLUSIONS

These results indicate that IL-17 is produced in periodontal lesions, which may be involved in Th1 modulation and enhance inflammatory reactions via gingival fibroblast-derived mediators in periodontal disease. Thus, IL-17, together with other cytokines, has a potential role in the aetiopathogenesis of periodontal disease.

We examined a panel of 25 small cell lung cancer (SCLC) cell lines and nude mouse xenografts for expression of the proto-oncogenes c-met and c-kit, and for expression of the corresponding ligands, hepatocyte growth factor (HGF) (also known as scatter factor (SF)), and stem cell factor (SCF), respectively. Expression of mRNA was detected by Northern blotting, and c-met and c-kit protein expression was detected by Western blotting and immunocytochemistry. c-met and c-kit mRNA was expressed in 22 of the examined cell lines or xenografts, and coexpression of the two proto-oncogenes was observed in 20 tumours. Expression of c-met and c-kit protein paralleled in the mRNA expression. HGF/SF mRNA was expressed in two of the examined tumours, and only one of these also expressed the c-met proto-oncogene. SCF mRNA was expressed in 19 of the examined tumours, and in 18 of these coexpression of c-kit and SCF was present. The high percentage of SCLC tumours expressing c-met and c-kit indicates that these proto-oncogenes may have an important function in this disease. The rare coexpression of c-met and HGF/SF is evidence that an autocrine regulatory pathway is not present for this receptor/ligand system in SCLC, while the frequent coexpression of c-kit and SCF indicates that this receptor/ligand system may have an autocrine function in SCLC.

Glycogen storage disease type Ia (GSD-Ia) profoundly impairs glucose release by the liver due to glucose-6-phosphatase (G6Pase) deficiency. An adeno-associated virus (AAV) containing a small human G6Pase transgene was pseudotyped with AAV8 (AAV2/8) to optimize liver tropism. Survival was prolonged in 2-week-old G6Pase (-/-) mice by 600-fold fewer AAV2/8 vector particles (vp), in comparison to previous experiments involving this model (2 x 10(9) vp; 3 x 10(11) vp/kg). When the vector was pseudotyped with AAV1, survival was prolonged only at a higher dose (3 x 10(13) vp/kg). The AAV2/8 vector uniquely prevented hypoglycemia during fasting and fully corrected liver G6Pase deficiency in GSD-Ia mice and dogs. The AAV2/8 vector has prolonged survival in three GSD-Ia dogs to >11 months, which validated this strategy in the large animal model for GSD-Ia. Urinary biomarkers, including lactate and 3-hydroxybutyrate, were corrected by G6Pase expression solely in the liver. Glycogen accumulation in the liver was reduced almost to the normal level in vector-treated GSD-Ia mice and dogs, as was the hepatocyte growth factor (HGF) in GSD-Ia mice. These preclinical data demonstrated the efficacy of correcting hepatic G6Pase deficiency, and support the further preclinical development of AAV vector-mediated gene therapy for GSD-Ia.

Primary hepatocytes are commonly used as liver surrogates in toxicology and tissue engineering fields, therefore, maintenance of functional hepatocytes in vitro is an important topic of investigation. This paper sought to characterize heparin-based hydrogel as a three-dimensional scaffold for hepatocyte culture. The primary rat hepatocytes were mixed with a prepolymer solution comprised of thiolated heparin and acrylated poly(ethylene glycol) (PEG). Raising the temperature from 25 degrees to 37 degrees C initiated Michael addition reaction between the thiol and acrylated moieties and resulted in formation of hydrogel with entrapped cells. Analysis of liver-specific products, albumin and urea, revealed that the heparin hydrogel was non-cytotoxic to cells and, in fact, promoted hepatic function. Hepatocytes entrapped in the heparin-based hydrogel maintained high levels of albumin and urea synthesis after three weeks in culture. Because heparin is known to bind growth factors, we incorporated hepatocyte growth factor (HGF)-an important liver signaling molecule - into the hydrogel. HGF release from heparin hydrogel matrix was analyzed using enzyme linked immunoassay (ELISA) and was shown to occur in a controlled manner with only 40% of GF molecules released after 30 days in culture. Importantly, hepatocytes cultured within HGF-containing hydrogels exhibited significantly higher levels of albumin and urea synthesis compared to cells cultured in the hydrogel alone. Overall, heparin-based hydrogel showed to be a promising matrix for encapsulation and maintenance of difficult-to-culture primary hepatocytes. In the future, we envision employing heparin-based hyrogels as matrices for in vitro differentiation of hepatocytes or stem cells and as vehicles for transplantation of these cells.

Diabetic nephropathy is characterized by progressive loss of renal function, persistent proteinuria, and relentless accumulation of extracellular matrix leading to glomerulosclerosis and interstitial fibrosis. This study investigated the potential effects of long-term expression of exogenous hepatocyte growth factor (HGF) on normal and diabetic kidneys. Intravenous injection of human HGF gene via naked plasmid vector resulted in abundant HGF protein specifically localized in renal glomeruli, despite an extremely low level of transgene mRNA in the kidney. In uninephrectomized mice made diabetic with streptozotocin, delivery of exogenous HGF gene ameliorated the progression of diabetic nephropathy. HGF attenuated urine albumin and total protein excretion in diabetic mice. Exogenous HGF also mitigated glomerular mesangial expansion, reduced fibronectin and type I collagen deposition, and prevented interstitial myofibroblast activation. In addition, HGF prevented kidney cells from apoptotic death in the glomeruli and tubulointerstitium. Moreover, expression of HGF inhibited renal expression of TGF-beta1 and reduced urine level of TGF-beta1 protein. Therefore, despite the effects of HGF on diabetic nephropathy being controversial, these observations suggest that supplementation of HGF is beneficial in ameliorating diabetic renal insufficiency in mice.

Transplantation of human islets is an attractive alternative to daily insulin injections for patients with type 1 diabetes. However, the majority of islet recipients lose graft function within five years. Inflammation is a primary contributor to graft loss, and inhibiting pro-inflammatory cytokine activity can reverse inflammation mediated dysfunction of islet grafts. As mesenchymal stem cells (MSCs) possess numerous immunoregulatory properties, we hypothesized that MSCs could protect human islets from pro-inflammatory cytokines. Five hundred human islets were co-cultured with 0.5 or 1.0 × 10(6) human MSCs derived from bone marrow or pancreas for 24 hours followed by 48 hour exposure to interferon-γ, tumor necrosis factor-α and interleukin 1β. Controls include islets cultured alone (± cytokines) and with human dermal fibroblasts (± cytokines). For all conditions, glucose stimulated insulin secretion (GSIS), total islet cellular insulin content, islet β cell apoptosis, and potential cytoprotective factors secreted in the culture media were determined. Cytokine exposure disrupted human islet GSIS based on stimulation index and percentage insulin secretion. Conversely, culture with 1.0 × 10(6) bMSCs preserved GSIS from cytokine treated islets. Protective effects were not observed with fibroblasts, indicating that preservation of human islet GSIS after exposure to pro-inflammatory cytokines is MSC dependent. Islet β cell apoptosis was observed in the presence of cytokines; however, culture of bMSCs with islets prevented β cell apoptosis after cytokine treatment. Hepatocyte growth factor (HGF) as well as matrix metalloproteinases 2 and 9 were also identified as putative secreted cytoprotective factors; however, other secreted factors likely play a role in protection. This study, therefore, demonstrates that MSCs may be beneficial for islet engraftment by promoting cell survival and reduced inflammation.

Hepatocyte Growth Factor (HGF) receptor, encoded by the protooncogene c-met, is overexpressed in many human tumours, including those of thyroid epithelium. The absence in most cases of any primary structural abnormality of the met gene suggests that overexpression is secondary to mutation of other gene(s). To test this hypothesis we investigated the effect on met expression of two activated oncogenes known to play a major role in thyroid oncogenesis, ras and ret. To minimize the possibility of unknown co-operating events, we introduced these genes directly into normal human thyrocytes in primary culture using amphotropic retroviral vectors and assessed met expression as early as possible in the resulting epithelial colonies. Double immunofluorescence revealed expression of met protein, strictly localized to cells expressing the mutant ras and ret vectors, expression in background normal cells being barely detectable. In contrast, colonies induced to proliferate at a comparable rate by a vector expressing SV40 T showed no increase in met expression. To permit quantitation by Western blotting we also extended these findings to a thyroid cell line (R18) containing a zinc-inducible mutant ras gene. Induction of the oncogene led to a fourfold increase in met protein expression. We conclude that overexpression of met is induced by activation of the ras or ret signalling pathway and not simply by deregulation of the cell cycle per se. The data suggest that the proliferative advantage conferred by these oncogenes may be in part due to the resulting sensitization of tumour epithelium to paracrine HGF secreted by stromal cells.

Hepatocyte growth factor (HGF) is involved in malignant behavior of cancers as a mediator in tumor-stromal interactions through enhancing tumor invasion and metastasis. We found recently that NK4, a four-kringle fragment of HGF, functions as both an HGF-antagonist and an angiogenesis inhibitor. We have now determined whether blockade of the HGF-c-Met/HGF receptor pathway and tumor angiogenesis by administration of recombinant NK4 would inhibit growth, invasion, and metastasis of human pancreatic carcinoma implanted into the pancreas of nude mice. When treatment with NK4 or anti-HGF neutralizing antibody was initiated from the third day after orthotopic injection of SUIT-2 human pancreatic cancer cells, both NK4 and anti-HGF antibody suppressed the conversion of orthotopic pancreatic tumors from carcinoma in situ to aberrantly invading cancers during days 3-14. On the other hand, when the treatment was begun on day 10, a time when cancer cells were already invading surrounding tissues, NK4 but not anti-HGF antibody inhibited tumor growth, peritoneal dissemination, and ascites accumulation at 4 weeks after the inoculation. Antitumor effects of NK4 correlated with decreased microvessel density in pancreatic tumors thereby indicating that the antiangiogenic activity of NK4 may have mainly contributed to its antitumor effects. Moreover, although NK4-treatment was initiated from the end stage (day 24 after tumor inoculation), NK4 prolonged survival time of mice, and the suppression of peritoneal dissemination, ascites accumulation, and invasion of metastasized cancer cells into the peritoneal wall were remarkable. We propose that simultaneous targeting of both tumor angiogenesis and the HGF-mediated invasion-metastasis may prove to be a new approach to treating patients with pancreatic cancer.

During development, cranial motor neurons extend their axons along distinct pathways into the periphery. For example, branchiomotor axons extend dorsally to leave the hindbrain via large dorsal exit points. They then grow in association with sensory ganglia, to their targets, the muscles of the branchial arches. We have investigated the possibility that pathway tissues might secrete diffusible chemorepellents or chemoattractants that guide cranial motor axons, using co-cultures in collagen gels. We found that explants of dorsal neural tube or hindbrain roof plate chemorepelled cranial motor axons, while explants of cranial sensory ganglia were weakly chemoattractive. Explants of branchial arch mesenchyme were strongly growth-promoting and chemoattractive for cranial motor axons. Enhanced and oriented axon outgrowth was also elicited by beads loaded with Hepatocyte Growth Factor (HGF); antibodies to this protein largely blocked the outgrowth and orientation effects of the branchial arch on motor axons. HGF was expressed in the branchial arches, whilst Met, which encodes an HGF receptor, was expressed by subpopulations of cranial motor neurons. Mice with targetted disruptions of HGF or Met showed defects in the navigation of hypoglossal motor axons into the branchial region. Branchial arch tissue may thus act as a target-derived factor that guides motor axons during development. This influence is likely to be mediated partly by Hepatocyte Growth Factor, although a component of branchial arch-mediated growth promotion and chemoattraction was not blocked by anti-HGF antibodies.

BACKGROUND

A transcription factor, ets-1, regulates the transcription of metalloproteinase genes, the activity of which is necessary for matrix degradation and the migration of endothelial cells. However, no study has demonstrated that ets-1 itself has an angiogenic action in vivo. Thus, we examined (1) the effects of overexpression of the ets-1 gene on angiogenesis in a rat hindlimb ischemia model, and (2) how ets-1 induced angiogenesis.

RESULTS

In this study, we used the HVJ-liposome method, which is highly effective for transfection, to transfect the human ets-1 gene. At 4 weeks after transfection, the capillary density and blood flow were significantly increased in a hindlimb transfected with the human ets-1 gene compared with control. These data clearly demonstrated that ets-1 has the ability to stimulate angiogenesis in vivo. To elucidate the molecular mechanisms by which ets-1 induced angiogenesis, we focused especially on the expression of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), potent angiogenic growth factors, because the promoter regions of both genes contain ets binding sites. Interestingly, overexpression of ets-1 upregulated both tissue HGF and VEGF concentrations in rat hindlimb. More importantly, administration of neutralizing antibody against HGF and VEGF attenuated the increase in blood flow and BrdU-positive cells induced by ets-1. Upregulation of HGF and VEGF by ets-1 was also confirmed by in vitro experiments using human vascular smooth muscle cells.

CONCLUSIONS

The present study demonstrated that ets-1 regulated angiogenesis through the induction of angiogenic growth factors (VEGF and HGF). Overexpression of ets may provide a new therapeutic strategy to treat peripheral arterial disease.

OBJECTIVE

Mammalian extraocular muscles (EOMs) are both physiologically and biochemically unique when compared with nonocular skeletal muscles. Recent studies have demonstrated a process of continuous myonuclear addition in normal uninjured myofibers in adult EOMs of rabbits and mice. The current study was conducted to determine whether this process of myonuclear addition is a universal phenomenon in mammalian EOMs.

METHODS

The EOMs from adult uninjured monkeys and humans were examined immunohistochemically for the expression of specific markers of activated satellite cells: hepatocyte growth factor (HGF); the myogenic regulatory factors MyoD, myogenin, and Pax7; and a marker for nuclei in all proliferative phases of the cell cycle, Ki-67. The satellite cell identity of the cells positive for Ki-67, HGF, and Pax7 was determined by colabeling sets of serial sections with either laminin or dystrophin.

RESULTS

In cross sections of monkey and human EOMs, approximately 7% to 8% of the myofiber profiles were associated with Pax7-positive satellite cells and between 2% and 4% were associated with MyoD-positive satellite cells or HGF-positive satellite cells. Similar percentages of satellite cells were positive for myogenin in the orbital layer, but the global layer had few satellite cells that were myogenin positive. An average of 0.72% of the myofibers had Ki-67-positive cells associated with them in the satellite cell position.

CONCLUSIONS

Activated satellite cells were present on myofibers in normal uninjured adult monkey and human EOMs, as visualized with these five distinct markers. The data support the hypothesis that the process of continuous myonuclear addition is most likely active in primate and human EOMs. The presence of continuous myofiber remodeling in EOM suggests new mechanisms that may be responsible for EOM sparing or involvement in skeletal muscle diseases.

Ozone has been proposed as an alternative antiseptic agent in dentistry based on reports of its antimicrobial effects in both gaseous and aqueous forms. This study investigated whether gaseous ozone (4 x 10(6) microg m(-3)) and aqueous ozone (1.25-20 microg ml(-1)) exert any cytotoxic effects on human oral epithelial (BHY) cells and gingival fibroblast (HGF-1) cells compared with established antiseptics [chlorhexidine digluconate (CHX) 2%, 0.2%; sodium hypochlorite (NaOCl) 5.25%, 2.25%; hydrogen peroxide (H(2)O(2)) 3%], over a time of 1 min, and compared with the antibiotic, metronidazole, over 24 h. Cell counts, metabolic activity, Sp-1 binding, actin levels, and apoptosis were evaluated. Ozone gas was found to have toxic effects on both cell types. Essentially no cytotoxic signs were observed for aqueous ozone. CHX (2%, 0.2%) was highly toxic to BHY cells, and slightly (2%) and non-toxic (0.2%) to HGF-1 cells. NaOCl and H(2)O(2) resulted in markedly reduced cell viability (BHY, HGF-1), whereas metronidazole displayed mild toxicity only to BHY cells. Taken together, aqueous ozone revealed the highest level of biocompatibility of the tested antiseptics.

BACKGROUND

Low-level laser irradiation has been reported to enhance wound healing. Activation of gingival fibroblasts (GF) has a potential for early wound healing in periodontal treatment. The present study aimed to investigate the direct effect of low-level Er:YAG laser irradiation on gingival fibroblasts proliferation in order to clarify the laser effect on healing.

METHODS

Cultured human gingival fibroblasts (hGF) were exposed to low-power, pulsed Er:YAG laser irradiation with different energy densities ranging from 1.68 to 5.0 J/cm(2). The cultures were analyzed by means of trypan blue staining and counted under a light microscope. The effect of Er:YAG laser on hGF was also evaluated using a transmission electron microscope (TEM).

RESULTS

Cultures irradiated with Er:YAG laser presented faster cell growth when compared with untreated controls. This difference was statistically significant. Transmission electron microscopy revealed rough endoplasmic reticulum, prominent Golgi complexes, and mitochondria after laser irradiation.

CONCLUSIONS

Our results showed that the low-level Er:YAG laser irradiation stimulates the proliferation of cultured gingival fibroblasts. The optimal stimulative energy density was found to be 3.37 J/cm(2). This result suggests that Er:YAG laser irradiation may be of therapeutic benefit for wound healing.

Bile acid synthesis in the liver is regulated by the rate-limiting enzyme cholesterol 7alpha-hydroxylase (CYP7A1). Transcription of the CYP7A1 gene is inhibited by bile acids and cytokines. The rate of bile acid synthesis is reduced immediately after partial hepatectomy and during the early stage of liver regeneration. Hepatocyte growth factor (HGF) released from stellate cells activates a receptor tyrosine kinase c-Met, in hepatocytes and stimulates signaling pathways that regulate cell growth, proliferation, and apoptosis. This study demonstrated that HGF strongly and rapidly repressed CYP7A1 mRNA expression and the rate of bile acid synthesis in primary human hepatocytes. HGF rapidly induced c-Jun and small heterodimer partner mRNA and protein expression and increased phosphorylation of ERK1/2, JNK, and c-Jun. Specific inhibitors of protein kinase C, extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun N-terminal kinase (JNK) blocked HGF inhibition of CYP7A1 expression. Knockdown of c-Met by small interfering RNA resulted in a significant increase in CYP7A1 and blocked HGF inhibition of CYP7A1 mRNA expression. Chromatin immunoprecipitation assays showed that HGF induced recruitment of c-Jun and small heterodimer partner (SHP) but reduced recruitment of the coactivators peroxisome proliferators activated receptor rho coactivator 1alpha (PGC-1alpha) and cAMP response element binding protein (CREB)-binding protein (CBP) to chromatin.

CONCLUSIONS

This study demonstrated that HGF is a novel regulator of CYP7A1 and bile acid synthesis in human hepatocytes and may protect hepatocytes from accumulating toxic bile acids and developing intrahepatic cholestasis during the early stage of liver regeneration.

In tumour-induced angiogenesis of gliomas, vascular endothelial growth factor (VEGF) and its receptors fms-like tyrosine kinase (Flt-1) and kinase-insert-domain-containing receptor (KDR) play a major role and are promising targets for tumour therapy. Nevertheless, preliminary results of such therapies could not prove clinical efficacy and thus make a profound knowledge of VEGF regulation essential. Based on earlier results, which demonstrated an inhibitory influence of VEGF on Flt-1-expressing glioblastoma cells, in the present study we focused on the extent of VEGF and VEGF receptor coexpression and possible therapeutical consequences. Protein expression of VEGF, Flt-1 and KDR was analysed by immunohistochemistry in native tumour tissues of 63 glioblastomas. VEGF could be detected in all glioblastomas. Additionally and independently to the expected Flt-1 and KDR expression in tumour endothelia, we found a coexpression of VEGF with Flt-1 in tumour cells of 46 and with KDR in 45 glioblastomas. After exposure of glioblastoma cells to X-ray radiation we observed a strong dose-dependent increase of VEGF secretion in two glioblastoma cell cultures by up to 46% and 96%, respectively that originated from an increased VEGF mRNA expression. In contrast, under the same conditions secretion of HGF/SF was only slightly elevated and bFGF despite being strongly increased remained at very low overall amounts compared to VEGF. Based on previous data on an autocrine function of VEGF in Flt-1-expressing glioblastoma cells we hypothesise that the X-ray radiation induced upregulation of VEGF might result in a downregulation of tumour cell proliferation and thus lead to a reduced sensitivity to radiation therapy. Therefore our results support the idea that a combination of anti-VEGF and radiation therapy might prove a promising new option in fighting against one of the most fatal tumour types.