Generalized gingival enlargement can be caused by a variety of etiological factors. It can be inherited (hereditary gingival fibromatosis [HGF]); associated with other diseases characterizing a syndrome; or induced as a side effect of systemic drugs, such as phenytoin, cyclosporin, or nifedipine. HGF, previously known as elephantiasis gingivae, hereditary gingival hyperplasia, and hypertrophic gingiva, is a genetic disorder characterized by a progressive enlargement of the gingiva. This review will focus on diagnosis, treatment, and control of HGF. The pattern of inheritance, the histopathologic characteristics, and the known biologic and genetic features associated with HGF are also emphasized.

Cell migration is critical for many processes, such as angiogenesis, inflammation, development and wound healing, and is also involved in tumour progression and metastasis. Here we show that CXCL12, complement factor 5a (C5a), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF)-BB, which stimulate cell migration, also activate p38alpha MAPK. Pharmacological inhibition of this protein kinase with SB 203580 or BIRB 0796, or the genetic ablation of p38alpha MAPK, blocked cell migration induced by the aforementioned chemo-attractants. Macrophages from mice lacking one or more of the other p38 MAPK isoforms showed normal cell migration in response to C5a. We also show that the activation of p38alpha MAPK in response to CXCL12 requires the p21-activated protein kinases (PAK)-1 and PAK-2. MAPKAP-K2 is a protein kinase that is activated by p38alpha MAPK. Reducing its expression using RNA interference blocked CXCL12-induced HeLa cell migration, while macrophages from mice that do not express MAPKAP-K2 failed to migrate in response to C5a. Moreover, RNA interference against the small heat shock protein 27 (HSP27), a physiological substrate of MAPKAP-K2, blocked the CXCL12-induced cell migration. These results demonstrate a general and essential role of the PAK-p38alpha MAPK-MAPKAP-K2-HSP27 signalling pathway in mediating the effects of chemotactic stimuli on cell migration.

Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are potent angiogenic factors that have been used clinically to induce angiogenesis. However, concerns have been raised about VEGF because of its proinflammatory actions, which include enhancing the adhesion of leukocytes to endothelial cells. We have examined the possible antiinflammatory effects of HGF on the vasculature. HGF, unlike VEGF, did not alter leukocyte adhesion to endothelial cells. Instead it inhibited VEGF-induced leukocyte-endothelial cell interactions and the endothelial expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). In a skin inflammation model, VEGF-treated mice showed a significant increase of leukocytes infiltrated or adherent to the luminal surface of blood vessels, as compared with vehicle- or HGF-treated mice. The VEGF effect was markedly suppressed by coadministration of HGF. RT-PCR and promoter analysis revealed that HGF downregulated VEGF-mediated expression of ICAM-1 and VCAM-1 at the transcriptional level. Furthermore, these inhibitory effects coincided with suppression of IkappaB kinase activity, and this in turn prevented the activation of the inflammatory transcription factor NF-kappaB. Taken together, our results demonstrate that HGF suppresses VEGF-induced inflammation presumably by inhibiting the endothelial NF-kappaB pathway. This suggests that combined treatment with HGF and VEGF could be superior to treatment with either factor alone for enhancing therapeutic angiogenesis while avoiding inflammation.

Adipose tissue is a reservoir of Mesenchymal Stem Cells (Adipose-derived Mesenchymal Stem Cells, ASCs), endowed with regenerative properties. Fat graft was proposed for breast reconstruction in post-surgery cancer patients achieving good aesthetic results and tissues regeneration. However, recent findings highlight a potential tumorigenic role that ASCs may have in cancer recurrence, raising some concerns about their safety in clinical application. To address this issue, we established a model where autologous ASCs were combined with primary normal or cancer cells from breast of human donors, in order to evaluate potential effects of their interactions, in vitro and in vivo. Surprisingly, we found that ASCs are not tumorigenic per sè, as they are not able to induce a neoplastic transformation of normal mammary cells, however they could exhacerbate tumorigenic behaviour of c-Met-expressing breast cancer cells, creating an inflammatory microenvironment which sustained tumor growth and angiogenesis. Pharmacological c-Met inhibition showed that a HGF/c-Met crosstalk between ASCs and breast cancer cells enhanced tumor cells migration, acquiring a metastatic signature, and sustained tumor self-renewal. The master role of HGF/c-Met pathway in cancer recurrence was further confirmed by c-Met immunostaining in primary breast cancer from human donors, revealing a strong positivity in patients displaying a recurrent pathology after fat grafts and a weak/moderate staining in patients without signs of recurrence. Altogether our findings, for the first time, suggest c-Met expression, as predictive to evaluate risk of cancer recurrence after autologous fat graft in post-surgery breast cancer patients, increasing the safety of fat graft in clinical application.

Mesenchymal stem cells (MSCs) are prototypical adult stem cells with the capacity for self-renewal and differentiation with a broad tissue distribution. MSCs not only differentiate into types of cells of mesodermal lineage but also into endodermal and ectodermal lineages such as bone, fat, cartilage and cardiomyocytes, endothelial cells, lung epithelial cells, hepatocytes, neurons, and pancreatic islets. MSCs have been identified as an adherent, fibroblast-like population and can be isolated from different adult tissues, including bone marrow (BM), umbilical cord, skeletal muscle, and adipose tissue. MSCs secrete factors, including IL-6, M-CSF, IL-10, HGF, and PGE2, that promote tissue repair, stimulate proliferation and differentiation of endogenous tissue progenitors, and decrease inflammatory and immune reactions. In this paper, we focus on the role of BM-derived MSCs in organ repair.

IL-7Rα-mediated signals are essential for naive T lymphocyte survival. Recent studies show that IL-7Rα is internalized and either recycled to cell surface or degraded. However, how the intracellular process of IL-7Rα trafficking is regulated is unclear. In this paper, we show that Vps34, the class III PI3K, plays a critical role in proper IL-7Rα intracellular trafficking. Mice lacking Vps34 in T lymphocytes had a severely reduced T lymphocyte compartment. Vps34-deficient T lymphocytes exhibit increased death and reduced IL-7Rα surface expression, although three major forms of autophagy remain intact. Intracellular IL-7Rα in normal T lymphocytes at steady state is trafficked through either early endosome/multivesicular bodies to the late endosome-Golgi for surface expression or to the lysosome for degradation. However, Vps34-deficient T cells have mislocalized intracellular Eea1, HGF-regulated tyrosine kinase substrate, and Vps36 protein levels, the combined consequence of which is the inability to mobilize internalized IL-7Rα into the retromer pathway for surface display. Our studies reveal that Vps34, though dispensable for autophagy induction, is a critical regulator of naive T cell homeostasis, modulating IL-7Rα trafficking, signaling, and recycling.

MT1-MMP (membrane type-1 matrix metalloproteinase), otherwise known as MMP14 is a proteolytic enzyme known to be involved in degradating extracellular matrix and assist progression of cancer invasion and progression. We investigated the impact of targeting the expression of MT1-MMP in breast cancer and its clinical relevance. Human breast cancer cell line MDA-MB-231 was used. Expression of MT1-MMP in the breast cancer cell line was manipulated by way of retroviral ribozyme transgene. The in vitro invasion, growth and cell migration were determined on cell lines transfected with either the transgene or control plasmid. Protein and message levels of MMP14 was also assessed using immunohistochemistry and real-time quantitative analysis, and correlated with clinical and pathological information of the patients. Retroviral ribozyme transgene to human MT1-MMP successfully knocked down the levels of MT1-MMP mRNA from MDA-MB-231 cells. Reduction of MT1-MMP from the breast cancer cells resulted in significant reduction of in vitro invasiveness and loss of response to an invasion stimulus, HGF, compared with control and wild-type cells. The invasion index for MT1-MMP knockdown cells were 13+/-3.1 (without HGF) and 16.4+/-2.3 (with HGF, p=0.14), and the index for transfection control cells 25.3+/-4.3 (without HGF) and 40.4+/-4.1 (with HGF, p=0.0049). Transfection with the transgenes did not change the rate of cell growth. In clinical breast cancer, MT1-MMP staining was both membranous and cytoplasmic. Tumour cells displayed stronger staining compared with normal mammary epithelial cells. Tumour tissues had a marginally higher levels of the MMP14 transcript (8.6+/-1.9), compared with normal tissues (4.7+/-1.4), p=0.13. No significant difference was observed between node positive and node negative tumours (9.0+/-2.2 vs 8.7+/-3.1, p=0.24). Marginally higher levels of the MMP14 transcript were seen in tumours which developed metastasis and local recurrence. However, tumours from patients who died of breast cancer related causes had significantly higher levels of the transcript, compared with tumours from patients who remained disease-free 10 years after initial surgery (12.2+/-2.5 vs 6.3+/-1.2, p=0.0091). MT1-MMP is a proteolytic enzyme that is pivotal in controlling the invasiveness of breast cancer cells. It is highly expressed in aggressive breast tumours and is associated with clinical outcome. The enzyme is a potential therapeutic target in breast cancer.

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

A gene causing autosomal-recessive, nonsyndromic hearing loss, DFNB39, was previously mapped to an 18 Mb interval on chromosome 7q11.22-q21.12. We mapped an additional 40 consanguineous families segregating nonsyndromic hearing loss to the DFNB39 locus and refined the obligate interval to 1.2 Mb. The coding regions of all genes in this interval were sequenced, and no missense, nonsense, or frameshift mutations were found. We sequenced the noncoding sequences of genes, as well as noncoding genes, and found three mutations clustered in intron 4 and exon 5 in the hepatocyte growth factor gene (HGF). Two intron 4 deletions occur in a highly conserved sequence that is part of the 3' untranslated region of a previously undescribed short isoform of HGF. The third mutation is a silent substitution, and we demonstrate that it affects splicing in vitro. HGF is involved in a wide variety of signaling pathways in many different tissues, yet these putative regulatory mutations cause a surprisingly specific phenotype, which is nonsydromic hearing loss. Two mouse models of Hgf dysregulation, one in which an Hgf transgene is ubiquitously overexpressed and the other a conditional knockout that deletes Hgf from a limited number of tissues, including the cochlea, result in deafness. Overexpression of HGF is associated with progressive degeneration of outer hair cells in the cochlea, whereas cochlear deletion of Hgf is associated with more general dysplasia.

Met tyrosine kinase, the receptor for HGF/SF, is important in various cellular functions, including proliferation, mitogenesis, formation of branching tubules, angiogenesis, and tumor cell invasion and metastasis. However, the role of Met/HGF signaling pathway in nasopharyngeal carcinoma (NPC) has not been evaluated. In this study, we determined the expression profile and clinical correlation of Met/HGF in 66 cases of advanced NPC and the activation mechanisms of Met receptor in five NPC cell lines. Immunofluorescent staining and quantitative image analysis showed that the Met protein was expressed throughout the tumors and normal nasopharyngeal epithelia. Compared with NPC, the Met expression level was higher in columnar nasopharyngeal epithelium but lower in squamous nasopharyngeal epithelium. The normal interstitial stromal tissue expressed the lowest level of Met protein. HGF was detected mainly in the normal interstitial tissue surrounding the tumor. Met protein expression level was inversely correlated with patients' survival time; the correlation coefficient was -0.319 (P = 0.009). The mean survival time was 118 months in low Met expression group versus 52 months in high expression group (P = 0.0004). The cumulative 5-year survival rate was 77.68% in low expression group versus 38.24% in high expression group. The clinical stage was also significantly more advanced in high Met expression group. In the multivariate analysis, both clinical stage and Met protein expression level were independent prognostic indicators for patient survival. All of the five NPC cell lines tested did not express hgf mRNA but expressed met mRNA, and tyrosine phosphorylation of Met protein was mainly induced by exogenous HGF stimulation in these cells. No mutation was found in the tyrosine kinase and the juxtamembrane domains of Met receptor in the five NPC cell lines tested. These results indicate that: (a) high Met protein expression level correlates with poorer survival in late-stage NPC and serves as an independent prognostic indicator; and (b) the Met receptor in NPC is activated by its paracrine ligand HGF from the interstitial tissues rather than by an autocrine loop or its activating mutation.

A distinctive property of Hepatocyte Growth Factor (HGF) is its ability to induce differentiation of tubular structures from epithelial and endothelial cells (branching tubulogenesis). The HGF receptor directly activates PI3 kinase, Ras and STAT signalling pathways and phosphorylates the adaptator GRB2 Associated Binder-1 (Gab1). Gab1 is also phosphorylated in response to Epidermal Growth Factor (EGF) but is unable to induce tubule formation. Comparison of 32P-peptide maps of Gab1 from EGF- versus HGF-treated cells, demonstrates that the same sites are phosphorylated in vivo. However, while both EGF and HGF induce rapid tyrosine phosphorylation of Gab1 with a peak at 15 min, the phosphorylation persists for over 1 h, only in response to HGF. Nine tyrosines are phosphorylated by both receptors. Three of them (Y307, Y373, Y407) bind phospholipase C-gamma (PLC-gamma). Interestingly, the overexpression of a Gab1 mutant unable to bind PLC-gamma (Gab1 Y307/373/407F) did not alter HGF-stimulated cell scattering, only partially reduced the growth stimulation but completely abolished HGF-mediated tubulogenesis. It is concluded that sustained recruitment of PLCgamma to Gab1 plays an important role in branching tubulogenesis.

Previous studies have shown that activity of urokinase-type plasminogen activator (u-PA) increases very rapidly (within 1 minute) after partial hepatectomy. In view of the well-recognized roles of u-PA as one of the major initiators of the matrix proteolysis cascade and as an activator of plasminogen and hepatocyte growth factor (HGF), we studied matrix degradation in liver shortly after partial hepatectomy. The activation of plasminogen to plasmin following partial hepatectomy was examined by Western blot analysis, and a small increase in plasmin at approximately 15 minutes followed by a large elevation at approximately 3 to 6 hours after partial hepatectomy was detected. In addition, we found that fibrinogen, the major substrate for plasmin, begins to be degraded at approximately 15 to 30 minutes following partial hepatectomy. Using immunohistochemical staining, we detected that the distribution of fibrinogen in normal liver is localized to the perisinusoidal space surrounding the periportal region. A decreased distribution of fibrinogen in the periportal region was found by 15 minutes and continued through 24 hours following partial hepatectomy. In addition, the distribution of fibronectin in normal liver was localized to the perisinusoidal space surrounding the periportal and the pericentral regions. A strikingly decreased distribution of fibronectin in the periportal region was found at 5 minutes after partial hepatectomy. Furthermore, we observed that the protein levels of laminin, entactin, and fibronectin in an extracellular matrix (ECM)-enriched preparation decreased shortly after partial hepatectomy, and were restored later. No changes were observed with either vitronectin or the integrin chain alpha(v). In contrast to the protein levels of the ECM components, the messenger RNA (mRNA) levels of fibronectin, integrin chain beta1, and integrin chain alpha(v) gradually increased over 18 hours and then decreased thereafter. Taken together, these results suggest that rapid reorganization of selected ECM components are important for hepatocyte proliferation at the early stages of liver regeneration.

Hepatocyte growth factor (HGF), also known as scatter factor, is believed to play a primary role in liver regeneration. HGF is produced in an inactive single-chain form that can be cleaved in vitro to the active two-chain form by tissue-type and urokinase-type plasminogen (PLG) activators (tPA and uPA). We have now documented the de novo appearance of active uPA in livers from male Fischer F344 rats that underwent 70% partial hepatectomy (PHx) as early as 1 minute after surgery. Western blot analyses of protein extracts from liver remnants that were obtained immediately after surgery and periodically until 24 hours after PHx indicate that the quantity of uPA remains fairly constant in PHx samples. In contrast, the uPA receptor (uPAR) dramatically increases, beginning within 1 minute after PHx. This results in enhanced activity of uPA, as seen by direct zymography on cryostat sections. The uPA present in remnant liver homogenates from rats that underwent PHx is the primary agent that cleaves single-chain HGF to its two-chain form, because cleavage can be prevented when antibody against uPA is included in the liver homogenates. Furthermore, heterodimeric HGF, which is not present in normal liver, increases in the liver remnants from rats that underwent PHx, correlative to uPAR. The presence of active uPA is one of the earliest responses yet documented after PHx. These findings imply that both uPA and uPAR are involved in activating endogenous HGF in the regenerating livers of animals that underwent PHx.

Human amniotic epithelial cells (hAEC) isolated from term placenta have stem cell-like properties, differentiate into tissue specific cells and reduce lung and liver inflammation and fibrosis following transplantation into disease models established in mice. These features together with their low immunogenicity and immunosuppressive properties make hAEC an attractive source of cells for potential therapeutic applications. However, generation of large cell numbers required for therapies through serial expansion in xenobiotic-free media may be a limiting factor. We investigated if hAEC could be expanded in xenobiotic-free media and if expansion altered their differentiation capacity, immunophenotype, immunosuppressive properties and production of immunomodulatory factors. Serial expansion in xenobiotic-free media was limited with cumulative cell numbers and population doubling times significantly lower than controls maintained in fetal calf serum. The epithelial morphology of primary hAEC changed into mesenchymal-stromal like cells by passage 4-5 (P4-P5) with down regulation of epithelial markers CK7, CD49f, EpCAM and E-cadherin and elevation of mesenchymal-stromal markers CD44, CD105, CD146 and vimentin. The P5 hAEC expanded in xenobiotic-free medium differentiated into osteocyte and alveolar epithelium-like cells, but not chondrocyte, hepatocyte, α- and β-pancreatic-like cells. Expression of HLA Class IA, Class II and co-stimulatory molecules CD80, CD86 and CD40 remained unaltered. The P5 hAEC suppressed mitogen stimulated T cell proliferation, but were less suppressive compared with primary hAEC at higher splenocyte ratios. Primary and P5 hAEC did not secrete the immunosuppressive factors IL-10 and HGF, whereas TGF-β1 and HLA-G were reduced and IL-6 elevated in P5 hAEC. These findings suggest that primary and expanded hAEC may be suitable for different cellular therapeutic applications.

OBJECTIVE

The ETS2 transcription factor is an evolutionarily conserved gene that is deregulated in cancer. We analyzed the transcriptome of lung adenocarcinomas and normal lung tissue by expression profiling and found that ETS2 was significantly downregulated in adenocarcinomas. In this study, we probed the yet unknown functional role of ETS2 in lung cancer pathogenesis.

METHODS

Lung adenocarcinomas (n = 80) and normal lung tissues (n = 30) were profiled using the Affymetrix Human Gene 1.0 ST platform. Immunohistochemical (IHC) analysis was conducted to determine ETS2 protein expression in non-small cell lung cancer (NSCLC) histologic tissue specimens (n = 201). Patient clinical outcome, based on ETS2 IHC expression, was statistically assessed using the log-rank and Kaplan-Meier tests. RNA interference and overexpression strategies were used to assess the effects of ETS2 expression on the transcriptome and on various malignant phenotypes.

RESULTS

ETS2 expression was significantly reduced in lung adenocarcinomas compared with normal lung (P < 0.001). Low ETS2 IHC expression was a significant predictor of shorter time to recurrence in NSCLC (P = 0.009, HR = 1.89) and adenocarcinoma (P = 0.03, HR = 1.86). Moreover, ETS2 was found to significantly inhibit lung cancer cell growth, migration, and invasion (P < 0.05), and microarray and pathways analysis revealed significant (P < 0.001) activation of the HGF pathway following ETS2 knockdown. In addition, ETS2 was found to suppress MET phosphorylation and knockdown of MET expression significantly attenuated (P < 0.05) cell invasion mediated by ETS2-specific siRNA. Furthermore, knockdown of ETS2 augmented HGF-induced MET phosphorylation, cell migration, and invasion.

CONCLUSIONS

Our findings point to a tumor suppressor role for ETS2 in human NSCLC pathogenesis through inhibition of the MET proto-oncogene.

Overexpression of genes, through genomic amplification and other mechanisms, can critically affect the behavior of tumor cells. Genomic amplification of the 13q31-32 region is reported in many tumors, including rhabdomyosarcomas that are primarily pediatric sarcomas resembling developing skeletal muscle. The minimum overlapping region of amplification at 13q31-32 in rhabdomyosarcomas was defined as containing two genes: Glypican-5 (GPC5) encoding a cell surface proteoglycan and C13orf25 encompassing the miR-17-92 micro-RNA cluster. Genomic copy number and gene expression analyses of rhabdomyosarcomas indicated that GPC5 was the only gene consistently expressed and up-regulated in all cases with amplification. Constitutive overexpression and knockdown of GPC5 expression in rhabdomyosarcoma cell lines increased and decreased cell proliferation, respectively. A correlation between expression levels of nascent pre-rRNA and GPC5 (P = 0.001), but not a C13orf25 transcript containing miR-17-92, in primary samples supports an association of GPC5 with proliferative capacity in vivo. We show that GPC5 increases proliferation through potentiating the action of the growth factors fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and Wnt1A. GPC5 enhanced the intracellular signaling of FGF2 and HGF and altered the cellular distribution of FGF2. The mesoderm-inducing effect of FGF2 and FGF4 in Xenopus blastocysts was also enhanced. Our data are consistent with a role of GPC5, in the context of sarcomagenesis, in enhancing FGF signaling that leads to mesodermal cell proliferation without induction of myogenic differentiation. Furthermore, the properties of GPC5 make it an attractive target for therapeutic intervention in rhabdomyosarcomas and other tumors that amplify and/or overexpress the gene.

We investigated the in vivo effects of recombinant human hepatocyte growth factor (HGF) on epithelial cell proliferation in normal mouse lung and on the repair process that follows bleomycin-induced lung injury. Intratracheal administration of 100 micrograms of rhHGF to C57BL/6 mice led to proliferation of bronchial and alveolar epithelial cells as indicated by an increased number of cells staining for proliferating cell nuclear antigen (PCNA). The effect of HGF on the lung repair process was examined by administration of 100 micrograms of rhHGF on Day 3 and Day 6 after intratracheal injection of bleomycin to mice. We found that HGF significantly attenuated collagen accumulation induced by bleomycin as determined by quantitation of hydroxyproline content and by scoring of the extent of fibrosis. To explore the potential mechanisms involved in the beneficial effects of HGF, we performed in vitro studies with A549 pulmonary epithelial cells and found that HGF enhanced cell surface plasmin generation, expression of u-PA activity, and cell migration. In summary, HGF has potent in vivo and in vitro effects on epithelial cells, which suggests it may have a role in the therapy of pulmonary fibrosis.

Hepatocyte growth factor (HGF) or scatter factor (SF) has been considered primarily as an endocrine/paracrine factor. We report here that HGF/SF mRNA was expressed by cultured human normal bronchial epithelial cells and many non-small cell lung carcinoma cell lines. Scatter activity was detected in the culture media of these cells, and this activity was inhibited by a neutralizing anti-recombinant human HGF antiserum. Immunostaining confirmed the presence of immunoreactive human HGF-like protein in the cytoplasm of these cultured cells, and in ciliated columnar epithelium of normal human bronchus/bronchioles. The met/HGF/SF receptor of these cultured cells was constitutively phosphorylated on tyrosine residues. A neutralizing anti-recombinant human HGF antiserum decreased the phosphorylation of the receptor, inhibited the proliferation of 45% of the non-small cell lung carcinoma cell lines studied, and stimulated the proliferation of normal bronchial epithelial cells. Altogether, the data demonstrate that HGF/SF and/or HGF-like protein is an autocrine factor for normal and neoplastic human bronchial epithelial cells in culture.

Mesenchymal stem cells (MSCs) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether SDF-1 transfection improve MSC viability and paracrine action in infarcted hearts. We found SDF-1-modified MSCs effectively expressed SDF-1 for at least 21 days after exposure to hypoxia. The apoptosis of Ad-SDF-1-MSCs was 42% of that seen in Ad-EGFP-MSCs and 53% of untreated MSCs. In the infarcted hearts, the number of DAPI-labeling cells in the Ad-SDF-1-MSC group was 5-fold that in the Ad-EGFP-MSC group. Importantly, expression of antifibrotic factor, HGF, was detected in cultured MSCs, and HGF expression levels were higher in Ad-SDF-MSC-treated hearts, compared with Ad-EGFP-MSC or control hearts. Compared with the control group, Ad-SDF-MSC transplantation significantly decreased the expression of collagens I and III and matrix metalloproteinase 2 and 9, but heart function was improved in d-SDF-MSC-treated animals. In conclusion, SDF-1-modified MSCs enhanced the tolerance of engrafted MSCs to hypoxic injury in vitro and improved their viability in infarcted hearts, thus helping preserve the contractile function and attenuate left ventricle (LV) remodeling, and this may be at least partly mediated by enhanced paracrine signaling from MSCs via antifibrotic factors such as HGF.

Chronic renal disease (CRD) is generally thought to be incurable, except through renal transplantation, and the number of patients with CRD is on the increase. Glomerulosclerosis and tubulointerstitial fibrosis represent the morphological equivalent of end-stage CRD. In this study, we demonstrated the preventive effect of hepatocyte growth factor (HGF) on the progression of renal dysfunction and fibrosis, using a spontaneous mouse model for CRD (ICGN strain). The mice progressively developed glomerular sclerotic injury, tubular atrophy, and renal dysfunction until they were 17 wk of age. When recombinant HGF was injected into these mice during a 4-wk-period (from weeks 14-17 after birth), DNA synthesis of tubular epithelial cells was found to be 4.4-fold higher than in mice without HGF injection, thereby suggesting tubular parenchymal expansion promoted by HGF. Notably, HGF suppressed the expression of transforming growth factor-beta and of platelet-derived growth factor as well as myofibroblast formation in the affected kidney. Consequently, the onset of tubulointerstitial fibrosis was almost completely inhibited by HGF, while HGF attenuated the progression of glomerulosclerosis, both leading to preventing manifestation of renal dysfunction. From our results, supplement therapy with HGF may be taken into consideration as a novel option for prevention and treatment of CRD.

Hepatocyte growth factor (HGF) is a potent mitogen for adult rat hepatocytes in primary culture. HGF stimulates growth and DNA synthesis of normal human epidermal melanocytes in culture. The maximal stimulation of DNA synthesis by 4.0-fold occurred with 10 ng/ml HGF. This stimulatory effect was additive with both acidic and basic fibroblast growth factors, while it was inhibited by transforming growth factor-beta 1. Melanocytes expressed a single class of specific, high-affinity receptors for HGF with a Kd of 22 pM and approximately 120 receptors/cell. Thus, HGF is a potent mitogen for normal human epidermal melanocytes.

Metanephric kidney development begins with the formation of the metanephrogenic mesenchyme; this event depends on the prior action in the intermediate mesoderm of transcription factors such as Lim-1, Pax-2, Eya-1, and Foxc-1. Once it has formed, the mesenchyme secretes GDNF; this induces the nearby wolffian duct to produce a ureteric bud which invades the metanephrogenic mesenchyme and begins to arborize. Ureteric bud development and branching depends on the transcription factor Emx-2, the GDNF-cRet and probably the HGF/cMet, signalling systems, and the intracellular regulatory molecules formin IV and timeless. Proteins of the BMP family modulate ureteric bud branching and keep bud development in step with that of other tissue types. Proteins and glycosaminoglycans of the matrix, and their receptors, and also required. The metanephrogenic mesenchyme has a default fate of apoptosis and is dissuaded from suicide by factors secreted from the bud such as TGF-alpha, TIMP-2, EGF, and FGF-2. Other factors such as LIF and TGFbeta2 cooperate with these to induce clumps of mesenchyme cells to differentiate into nephrons, while BMP-7 appears to lead them instead to form stroma. As nephrons form, they express critical transcription factors such as WT-1, Pax-2, and Hoxa11 and d11, condense, and secrete Wnt-4. Wnt-4 acts in an autocrine loop to stimulate its own synthesis and is required for cells to differentiate into epithelia; its action is antagonized by sFRP-1, secreted by stroma, but this antagonism is itself inhibited by sFRP-2 made by the developing nephron. This system probably acts both to limit the spread of Wnt-4's influence and also to commit responding cells to their epithelial fate. As nephrons mature, regions of them differentiate to perform specific physiological functions, a process that requires the proteins WT-1, Lmx-1b, Notch-2, Jagged-1, and Hnf-1.

A human genomic DNA library was screened by using conditions of reduced stringency with a bovine cDNA probe coding for the kringle domains in prothrombin in order to isolate the human prothrombin gene. Twelve positives were identified, three of which coded for prothrombin (Degen & Davie, 1987). Phage L5 was characterized in more detail because of its strong hybridization to the cDNA probe and its unique restriction map compared to the gene coding for human prothrombin. The gene in L5 was sequenced and found to code for a kringle-containing protein. A human liver cDNA library was screened by using a genomic probe from the gene in L5. cDNAs were isolated that contained sequence identical with regions in the gene in L5. Comparison of the cDNA with the gene indicated that the gene in L5 was composed of 18 exons separated by 17 intervening sequences and is 4690 bp in length. Exons ranged in size from 36 to 242 bp in length while intervening sequences ranged from 77 to 697 bp in length. The putative protein encoded by the gene in L5 contains four kringle domains followed by a serine protease-like domain. This domain structure is identical with that found in hepatocyte growth factor (HGF), although the two proteins are only about 50% identical. On the basis of the similarity of the protein encoded by L5 and HGF, we propose that the putative L5 protein be tentatively called HGF-like protein until a function is identified. The DNA sequence of the gene and cDNA and its translated amino acid sequence were compared against GenBank and NBRF databases. Sequences homologous to DNF15S1 and DNF15S2, human DNF15S2 lung mRNA, and rat acyl-peptide hydrolase were identified in exon 17 to the 3' end of the characterized sequence for the gene. From our results, it is apparent that the gene coding for human HGF-like protein is located at the DNF15S2 locus on human chromosome 3 (3p21). The gene for acyl-peptide hydrolase is 444 bp downstream of the gene coding for HGF-like protein, but on the complementary strand. The DNF15S2 locus has been proposed to code for one or more tumor suppressor genes since this locus is deleted in DNA from small cell lung carcinoma, other lung cancers, renal cell carcinoma, and von Hippel-Lindau syndrome.

Tubulointerstitial fibrosis is considered to be common endpoint result of many forms of chronic renal diseases. Except for renal replacement, chronic renal fibrosis is presently incurable. This study demonstrates that the combination of hepatocyte growth factor (HGF) gene therapy with inhibition of the renin-angiotensin system produced synergistic beneficial effects leading to dramatic attenuation of renal tubulointerstitial fibrosis in obstructive nephropathy in mice. The combined treatment with human HGF gene and losartan, an angiotensin II (AngII) type I receptor blocker, preserved renal mass and gross morphology of the obstructed kidneys. Although HGF gene therapy alone inhibited the expression of alpha-smooth muscle actin (alpha SMA) by approximately 54% and 60% at day 7 and day 14 after surgery, respectively, its combination with losartan almost completely abolished alpha SMA induction in the obstructed kidneys. The combined therapy also synergistically inhibited the accumulation of interstitial matrix components, such as fibronectin and collagen I, and suppressed renal expression of transforming growth factor-beta1 (TGF-beta1) and its type I receptor. In vitro studies revealed that AngII by itself did not induce alpha SMA, but it drastically potentiated TGF-beta1-initiated alpha SMA expression in tubular epithelial cells. Furthermore, HGF abrogated de novo alpha SMA expression induced by TGF-beta1 plus AngII. These results suggest that many factors are implicated in the pathogenesis of renal interstitial fibrosis; therefore, a combined therapy aimed at simultaneously targeting multiple pathologic pathways may be necessary for halting the progression of chronic renal diseases. These findings may provide the basis for designing future therapeutic regimens for blocking progressive renal fibrosis in patients.