Mechanical properties of the extracellular matrix alter expression of smooth muscle protein LPP and its partner palladin; relationship to early atherosclerosis and vascular injury

Lipoma preferred partner (LPP) localizes to focal adhesions/dense bodies, is selectively expressed in smooth muscle cells (SMC) and enhances cell migration. SMCs cultured on denatured collagen or on a rigid substrate, up regulated expression of LPP, its partner palladin, tenascin C (TN-C), phosphorylated focal adhesion kinase (pFAK) and exhibited robust stress fibers. In an endothelial (EC)/SMC hemodynamic flow system, shear stress waveforms mimicking atheroprone flow, applied to the EC layer, significantly decreased expression of SMC LPP and palladin. They were also down regulated with TN-C, in an ApoE murine model of atherosclerosis and with oxidative stress but up regulated in an arterial injury model in response to upstream sequential changes in pFAK, Prx1 and TN-C. In conclusion, expression of LPP and palladin are modulated by a mix of mechanical cues, oxidative stress and substrate composition which translate into their up or down regulation in vessel wall injury and early atherogenesis.     

Development of an Endothelial–Smooth Muscle Cell Coculture Model Using Phenotype-Controlled Smooth Muscle Cells

A coculture of endothelial cells (ECs) and smooth muscle cells (SMCs), which mimics cellular interactions appearing in vivo, has been performed in studies on the relationship between atherogenesis and fluid shear stress conditions. Although healthy arteries in vivo consist of contractile phenotype SMCs, cultured cells used in many studies normally exhibit a synthetic phenotype. Here, we developed an EC–SMC coculture model to investigate the interactions between ECs and contractile SMCs, and examined the effect of shear stress applied to ECs on SMC phenotypes. Cultured human umbilical artery SMCs were differentiated into contractile states by arresting cell growth using a serum-free medium. Western blotting confirmed that SMC expression of contractile protein markers, α-smooth muscle actin (SMA) and calponin, increased to levels similar to those observed in arterial cells. After coculturing contractile SMCs with ECs separated by a collagen gel layer, the expression of α-SMA decreased under static conditions, indicating that the SMC phenotype tended to be synthetic by coculturing with ECs, but shear stress applied to cocultured ECs maintained the level of α-SMA expression in SMCs. The coculture model constructed in the present study will be a useful tool to investigate interactions between ECs and contractile SMCs under shear conditions.     

Hepatocyte growth factor increases expression of vascular endothelial growth factor and plasminogen activator inhibitor-1 in human keratinocytes and the vascular endothelial growth factor receptor flk-1 in human endothelial cells

The pleiotropic growth factor hepatocyte growth factor/scatter factor (HGF/SF) has been implicated by clinical and experimental studies in repair mechanisms in different organs and tissues. However, no data on the impact of HGF/SF in wound healing in the skin are yet available. Proliferating and migrating keratinocytes play a major role in repair processes in the skin by closing the wound. Recent evidence gathered from studies that used gene-deficient mice has implicated the plasminogen activator (PA)/plasmin system in wound healing, which depends on controlled matrix degradation and deposition during cell migration and proliferation. Furthermore, keratinocytes are an important source of vascular endothelial growth factor (VEGF), which is a potent inducer of angiogenesis. In this study, we show that in human keratinocytes HGF/SF but not the related cytokine macrophage stimulating protein (MSP) significantly increases expression of VEGF and plasminogen activator inhibitor-1 (PAI-1) on the level of protein and mRNA. Furthermore, we demonstrate that HGF/SF increases the expression of the VEGF receptor flk-1 in human endothelial cells and that, in an angiogenesis co-culture assay of endothelial cells and keratinocytes, HGF/SF increases endothelial cell tube formation significantly. Therefore, we propose a role for HGF/SF in wound repair in the skin: HGF/SF--produced by activated fibroblasts--increases in keratinocytes the expression of PAI-1, which leads to increased matrix stability during the repair process and which could also limit activation of HGF/SF by proteases such as urokinase-type PA (u-PA) or tissue-type PA (t-PA). Furthermore HGF/SF also increases the expression of VEGF in these cells, thereby initiating angiogenesis in a paracrine manner. This effect would be enhanced by an increased responsiveness of endothelial cells toward VEGF, resulting from the HGF/SF-induced up-regulation of flk-1 on these cells.     

Normal and malignant prostate epithelial cells differ in their response to hepatocyte growth factor/scatter factor

Hepatocyte growth factor/scatter factor (HGF/SF) promotes the proliferation, differentiation, motility, and invasion of epithelial cells by binding to its cell surface receptor, the Met tyrosine kinase. In the prostate, Met is expressed predominantly by prostate epithelial cells (PrEC), whereas HGF/SF is synthesized by prostate stromal cells (PrSC). Met is also expressed in localized and metastatic prostate cancers. Our results show that PrECs in in vitro culture maintain expression of Met at a level comparable to DU145 cancer cell expression. HGF/SF secreted by PrSC stimulates tyrosine phosphorylation of the Met receptor. In normal PrEC, HGF/SF causes growth inhibition, sustained phosphorylation of mitogen-activated protein kinase, and increased CK18 expression consistent with cell differentiation. In contrast, HGF/SF significantly stimulates the proliferation of DU145 prostate cancer cells. HGF/SF in the conditioned medium of PrSC specifically induces migration of both normal and malignant prostate epithelial cells through MatriGel-coated Transwell filters. HGF/SF depletion reduces cell migration by approximately 50%. The response of PrEC is specific for HGF/SF since the other growth factors tested do not significantly affect growth or migration of PrECs. These results support the in vivo importance of the prostate stroma and specifically of HGF/SF as a unique stromal derived factor in the development and progression of prostate cancer.     

Immunohistochemical analysis of hepatocyte growth factor in human coronary atherectomy specimens: comparison with transforming growth factor beta isoforms

The expression and localization of hepatocyte growth factor/scatter factor (HGF/SF) were examined immunohistochemically in 59 human coronary artery lesions retrieved by directional coronary atherectomy and compared with the localization of transforming growth factor beta isoforms (TGF-β1, -β2, and -β3). In 21 of the 59 specimens (35.6%) HGF-like immunoreactivity (HGF-IR) was revealed. The HGF immunopositivity rate of 45% (14/31) in thrombotic tissue was significantly (P < 0.05) higher than the rates of 7.3% (4/55), 7.1% (3/42), and 0% (0/14) in fibrous tissue, neointimal hyperplasia and atheromatous gruel, respectively. Immunoreactivity for HGF was much weaker than that for TGF-β isoforms in these components except in thrombotic tissue. These cells exhibiting strong HGF-IR were inflammatory cells such as monocytes/macrophages in thrombotic tissue, in tissue lesions adjacent to a thrombus, and outside the capillary walls in a portion of the neovascularized lesions. Smooth muscle cells (SMCs) hardly demonstrated HGF-IR. In contrast, in control coronary arteries obtained at autopsy, the HGF-IR was strongly expressed in SMCs. These findings suggest that HGF produced by macrophages play a part in the process of coronary plaque formation attributable to thrombus in man.     

In vitro model of atherosclerosis using coculture of arterial wall cells and macrophage

In order to determine the precise mechanism of the interactions between different types of cells, which are common phenomena in tissues and organs, the importance of coculture techniques are becoming increasingly important. In the area of cardiology, artificial arteries have been developed, based on the understanding of physiological communication of the arterial smooth muscle cells (SMC), endothelial cells (EC), and the extracellular matrix (ECM). In the study of atherosclerosis, the modification of low-density lipoprotein (LDL), which result in the recruitment and accumulation of white blood cells, especially, monocytes/macrophages, and foam cell formation, are hypothesized. Although there are well known animal models, an in vitro model of atherogenesis with a precisely known atherogenesis mechanism has not yet been developed. In this paper, an arterial wall reconstruction model using rabbit primary cultivated aortic SMCs and ECs, was shown. In addition, human peripheral monocytes were used and the transmigration of monocytes was observed by scanning electron and laser confocal microscopy. Monocyte differentiation into macrophages was shown by immunohistochemistry and comprehensive gene expression analysis. With the modified form of LDL, the macrophages were observed to accumulate lipids with a foamy appearance and differentiate into the foam cells in the ECM between the ECs and SMCs in the area of our coculture model.     

Expression of c-Met receptor and hepatocyte growth factor/scatter factor in synovial sarcoma and epithelioid sarcoma

 Overexpression of c-Met receptor/hepatocyte growth factor (scatter factor) system (c-Met/HGF/SF) as a physiologically paracrine cellular signaling system is thought to be involved in the progression of malignant tumours. In 26 synovial sarcomas and epithelioid sarcomas, c-Met and HGF/SF expression was analysed immunohistochemically. There were 10 biphasic synovial sarcomas, 7 of which showed moderate to strong c-Met expression in epithelial areas compared with the fibrous component, with corresponding expression of HGF/SF. Six of 9 monophasic fibrous synovial sarcomas showed only very faint c-Met and corresponding HGF/SF expression. In 7 epithelioid sarcomas strong expression of c-Met and HGF/SF was observed within epithelioid tumour cells. Non-radioactive in situ hybridization demonstrated the synthesis of c-Met receptor in tumor cells by detecting c-met-mRNA. This analysis shows that in synovial sarcomas and epithelioid sarcomas, tumour entities with epithelial and mesenchymal structures, c-Met and HGF/SF overexpression can be detected, indicating a role of this signaling system in these subtypes of sarcoma, and especially in the more epithelioid tumour phenotype. An autocrine interaction between overexpressed c-Met receptor and HGF/SF may be hypothesized. Received: 21 July 1997 / Accepted: 19 November 1997     

Expression of Hepatocyte Growth Factor (HGF) and its Receptor (MET) in Medullary Carcinoma of the Thyroid

The tyrosine kinase receptor encoded by the MET oncogene has the unusual property of mediating the invasive growth of epithelial cells upon binding with the hepatocyte growth factor (HGF). The MET/HGF receptor is known to be overexpressed in thyroid carcinomas originated from follicular cells, but has not been reported in C cell tumors. To investigate the role of HGF and of its receptor (encoded by MET oncogene) in medullary carcinoma of the thyroid (MCT), we studied the expression of HGF and MET in 20 cases by means of different techniques. By RT-PCR, HGF mRNA was found in 10/20 cases, while MET mRNA presence was demonstrated in 8/20, of which 7 also expressed HGF. Northern blot analysis and in situ hybridization were performed in selected cases and confirmed RT-PCR data in some cases, although the lower sensitivity of these procedures did not allow the identification of all RT-PCR positive cases. By immunohistochemistry (using specific monoclonal antibodies) HGF was demonstrated in 8/9 RT-PCR positive cases ald a monoclonal to MET immunostained 5/6 RT-PCR positive cases. All receptor positive cases also expressed the ligand in the same tumor cell population. These findings demonstrate MET and HGF co-expression in a subset of MCT, in which autocrine/paracrine circuits may be active. No correlation was found between HGF/MET expression and clinico-pathological parameters, except for the more common multifocality of HGF/MET positive MCT. Whether the potential activation of MET in MCT is responsible for local invasion and malignant evolution is to be further investigated, especially in multifocal and aggressive tumors.     

Regulation and function of an activation-dependent epitope of the beta 1 integrins in vascular cells after balloon injury in baboon arteries and in vitro.

Migration and proliferation of endothelial cells (ECs) and smooth muscle cells (SMCs) contribute to the response to injury in damaged and atherosclerotic vessels. These events might be regulated by cellular interactions with extracellular matrix through the expression and activation of integrins. To study the functions of beta 1 integrins in the vessel wall, we used monoclonal antibody (MAb) 15/7, which recognizes an activation epitope of beta 1 integrin subunits, and MAb 8A2, which induces a high affinity form of beta 1 integrins recognized by MAb 15/7. Immunohistochemical analyses were done on samples of normal baboon saphenous arteries and from arteries subjected to balloon injury. EC and SMC expressed the activation epitope of beta 1 integrin in uninjured arteries. By contrast, in balloon-injured arteries 6 weeks after injury, regenerating EC did not express the activation epitope, and there was no decrease in the expression of total beta 1 integrin, whereas SMC migrating into the intima exhibited decreased expression of the total and activated beta 1 integrin. Flow cytometer analysis of cultured cells indicated that baboon EC and SMC weakly express the activation epitope of beta 1 integrin. Next, we determined by utilizing MAb 8A2 the effects of increased expression of activation epitope of beta 1 integrin on the functions of SMC and EC. The activation of beta 1 integrins on SMC induced by MAb 8A2 enhanced SMC adhesion and suppressed SMC migration in a Boyden chamber assay. SMC proliferation was inhibited by MAb 8A2 dose-dependently. Similarly, MAb 8A2-induced activation of beta 1 integrins on EC suppressed EC migration into a wound. However, MAb 8A2 did not affect the basic fibroblast growth factor-induced proliferation of EC, although it blocked the decrease in EC number caused by the removal of basic fibroblast growth factor. These results suggest that activation of beta 1 integrins in vascular cells is regulated in a cell-type dependent manner and plays an important role in modulating vascular cell functions.     

Hepatocyte growth factor/scatter factor induces VEGF in human external auditory canal cholesteatoma cell culture

Human external auditory canal cholesteatoma (EACC) is not often seen in otolaryngology. Some authors have noted circulatory disorders of the local blood vessels as the etiologic factor for establishing EACC. Diminished oxygen supply results in the attempt to establish angiogenesis. Hepatocyte growth factor/scatter factor (HGF/SF) and vascular endothelial growth factor (VEGF) are the most important angiogenic factors in this process. In a recent study we described strong expression of VEGF and HGF in EACC. All EACC and normal AMS cell cultures were obtained from 5 patients undergoing surgery and used at passage 3. After 16 to 72 h of incubation with 20 ng/ml HGF/SF, the expression of the VEGF protein in the supernants of the HGF/SF-treated and untreated culture was analyzed. EACC-culture cells showed a stronger baseline expression of VEGF. After 72 h of incubation with 20 ng/ml HGF/SF of HGF/SF, the expression of VEGF in normal keratinocytes was 173.4 pg/ml. The expression level of VEGF in the EACC culture was 275.73 pg/ml. We observed a 2.5-fold induction of VEGF in EACC after 72 h, which started with 1.5-fold baseline VEGF concentrations of normal keratinocytes. Our analysis showed that, in the EACC culture, VEGF was elevated after treatment with HGF/SF. HGF/SF appears to activate cellular pathways inducing release of VEGF. After purification, no fibroblasts were present in our EACC culture so as to exclude possible paracrine effects by fibroblasts.     

Hepatocyte growth factor/scatter factor-induced intracellular signalling

Hepatocyte growth factor (HGF) identical to scatter factor (SF) is a glycoprotein involved in the development of a number of cellular phenotypes, including proliferation, mitogenesis, formation of branching tubules and, in the case of tumour cells, invasion and metastasis. This fascinating cytokine transduces its activities via its receptor encoded by the c-met oncogene, coupled to a number of transducers integrating the HGF/SF signal to the cytosol and the nucleus. The downstream transducers coupled to HGF/MET, most of which participate in overlapping pathways, determine the development of the cell's phenotype, which in most cell types is dual.     

The MET receptor tyrosine kinase contributes to invasive tumour growth in rhabdomyosarcomas

The receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF), have been implicated in the genesis of the paediatric tumour rhabdomyosarcoma (RMS). Addition of exogenous HGF to RH30 RMS cells enhanced non-chemotactic migration. Stable transfection of dominant negative MET into RH30 cells attenuated Matrigel invasion and in vivo tumour growth. To assess the role of a putative HGF–MET pathway in human RMS, we measured their expression in a panel of 68 human primary tumours. All tumours expressed MET but with a three orders of magnitude variation of expression and 62% of tumours co-expressed HGF. In contrast with other tumour types, neither high-MET expression nor HGF/MET coexpression correlated with metastatic disease. In a microarray screen, we identified CCN1 as being 7.8-fold up regulated following addition of HGF to RH30 cells and in RMS tumours, CCN1 expression correlated with HGF expression. Surprisingly, we identified MET as a consistent feature of embryonal and not alveolar RMS.     

A 90-kd surface antigen from a subpopulation of smooth muscle cells from human atherosclerotic lesions.

A monoclonal antibody, designated 10F3, that reacts with an antigen with a molecular weight of 90,000 daltons has been obtained after immunization of BALB/c mice with long-term cultured smooth muscle cells (SMC) originally isolated from fetal human aorta (fSMC). In adults the antigen is present on venous, arterial and capillary endothelial cells of heart, kidney, liver, spleen, intestine, skin, uterus, placenta, and arteries only, as shown by immunohistochemical investigation using the PAP technique. The antigen 10F3 is also present on the mesenchymal cells of human fetal tissues (7 and 18-week-old fetuses) and on SMC of 7-week-old fetal aorta, and a subpopulation of cells reacting with 10F3 antibody also has been found in atherosclerotic intima. Double staining using 10F3 antibody and muscle actin-specific monoclonal antibody HHF-35 showed that the antigen-positive cells are smooth muscle cells. In primary culture of adult SMC, antigen-positive cells were detected 2 days after seeding (about 90% positive in medial and intimal cultures). It is suggested that 10F3 is a mesenchymal antigen that, lost during differentiation by cells other than endothelium, but expressed again by the SMC involved in atherogenesis.     

HGF/MET signaling promotes glioma growth via up-regulation of Cox-2 expression and PGE2 production

Cyclooxygenase2 (Cox-2) is well known for glioma growth through up-regulation of prostaglandin E2 (PGE2) levels. MET, a hepatocyte growth factor (HGF) receptor, is also frequently high expressed in glioma, which promotes glioma growth and invasion. Here, we demonstrate that HGF/MET signaling can promote PGE2 production in glioma cells via Cox-2 up-regulation. RNA inhibition of MET suggested that MET signaling is essential for Cox-2 up-regulation. Moreover, HGF could enhance Cox-2 expression and PGE2 release. Knockdown of Cox-2 inhibited growth-promoting effects of HGF, suggesting that HGF/MET functioned via Cox-2/PGE2 pathway. Therefore, our work reveals a critical relationship of Cox-2/PGE2 and HGF/MET signaling in promoting glioma cells proliferation. Further, targeting MET and Cox-2 may represent an attractive target therapy for glioma.     

Regulation of uveal melanoma interconverted phenotype by hepatocyte growth factor/scatter factor (HGF/SF).

Human uveal melanoma disseminates initially and preferentially to the liver. This study describes the relationship between the expression of the c-met proto-oncogene (receptor for hepatocyte growth factor/scatter factor (HGF/SF)) in interconverted uveal melanoma cells (co-expressing vimentin and keratin intermediate filaments) and the regulation of their motogenic response to HGF/SF, a key step in local invasion and targeted dissemination to the liver. Expression of c-met in uveal melanoma cell lines correlates with both the appearance of an interconverted phenotype and invasive ability (measured in vitro). Using chemotactic checkerboard analysis, the greatest motogenic response to HGF/SF was achieved by invasive, interconverted, c-met-positive uveal melanoma cells. C-met was observed histologically in a uveal melanoma containing interconverted cells but was absent in a tumor composed of non-interconverted cells (vimentin positive/keratin negative). The c-met ligand, HGF/SF, although not expressed by uveal melanoma cell lines, was localized in tissue sections of primary uveal melanomas and metastatic melanoma to the liver. In the primary tumor, staining for HGF/SF was most intense at the level of the choriocapillaris, a finding that is significant because 1) highly remodeled neovascular loops and networks, which appear in tumors likely to disseminate, can be traced to the choriocapillaris and the draining vortex veins and 2) HGF/SF plays a role in tumor angiogenesis. Foci of metastatic melanoma to the liver stain diffusely for HGF/SF. Regulation of the uveal melanoma interconverted phenotype by HGF/SF may play an important role in the dissemination of this tumor.     

Macrophage colony-stimulating factor gene expression in vascular cells and in experimental and human atherosclerosis.

The infiltration of monocytes into the vascular wall and their transformation into lipid-laden foam cells characterizes early atherogenesis. Macrophages are also present in more advanced human atherosclerotic plaques and can produce many mediators that may contribute to lesion formation and progression. Macrophage colony-stimulating factor (MCSF) enhances the proliferation and differentiation of monocyte progenitors and is required for the survival and activation of mature monocytes and macrophages. The authors therefore examined the expression of the MCSF gene in cultured human vascular endothelial (EC) and smooth muscle cells (SMC) as well as in atheromatous lesions from rabbits and humans. Growth arrested EC and SMC contain a low level of MCSF mRNA. Bacterial lipopolysaccharide (LPS), recombinant human interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) induced MCSF mRNA accumulation in a concentration-dependent manner in both EC and SMC. These stimuli induced large increases in MCSF mRNA with peak induction between 4-8 hours after treatment. LPS, IL-1 alpha, and TNF alpha stimulated EC and SMC also showed increased fluorescent antibody staining for MCSF protein and released immunoreactive MCSF in a time-dependent manner. In contrast, phorbol 12-myristate 13-acetate (PMA) was a less potent inducer of MCSF gene expression and iron-oxidized low-density lipoproteins (ox-LDL) did not increase consistently MCSF mRNA or the synthesis and secretion of immunoreactive protein. Northern analysis of mRNA isolated from the atheromatous aorta of rabbits fed a 1% cholesterol diet for 10 weeks showed elevated MCSF mRNA compared with controls. Immunostaining of atheromatous arterial lesions of rabbits demonstrated MCSF protein in association with intimal SMC as well as macrophages. Furthermore, polymerase chain reaction (PCR) analysis of MCSF mRNA in human atheromata showed higher levels than found in nonatherosclerotic arteries and veins. Since the authors found no mRNA for the MCSF receptor, c-fms, in cultured EC or SMC macrophages are likely the primary target for MCSF within atheromatous vessels. The authors therefore investigated the effects of MCSF on monocyte functions related to foam cell development. Treatment of cultured human monocytes with recombinant human MCSF (10(3) U/ml, 72 hr) led to the accumulation of mRNA for the acetyl-LDL (scavenger) receptor and apolipoprotein E (apo E). These studies establish that vascular EC and SMC produce substantial MCSF in response to a variety of stimuli. The local production of MCSF during atherogenesis may contribute to macrophage survival and proliferation or activate specific macrophage functions such as expression of the scavenger receptor and secretion of apo E.     

Expression of hepatocyte growth factor/scatter factor and its receptor c-Met in brain tumors: evidence for a role in progression of astrocytic tumors (Review)

Hepatocyte growth factor (HGF) is a multifunctional cytokine which is believed to have important roles in tissue development and regeneration, and tumor progression. It is indistinguishable from scatter factor (SF), a motility factor. HGF/SF is believed to be a mesenchymal cell-derived cytokine acting for epithelial cells bearing its receptor tyrosine kinase, c-Met. Recently, we found that glioblastoma multiforme (GBM), a highly malignant brain tumor of astrocytic origin, concomitantly express HGF/SF and c-Met. This finding indicates a presence of autocrine loop of HGF/SF signaling pathway in GBM. Moreover, GBM cells also co-express HGF activator, a recently identified serine proteinase having efficient HGF/SF activating activity. The expression of HGF/SF and c-Met was low or hardly detectable in low-grade astrocytoma, and c-Met immunoreactivity was correlated with the histological grade of the tumor suggesting that the creation of HGF/SF autocrine loop occurs along with the progression of astrocytic brain tumors. Experimental evidence indicated that HGF/SF exhibits potent migration/invasion-inducing activity for GBM cells bearing c-Met receptor. It is also a significant angiogenesis factor in GBM, and may serve as a cellular growth factor for certain GBM cells. These lines of evidence suggest that HGF/SF signaling pathway may serve as a promising new target of therapeutic intervention of GBM.     

Absence of mutations in the kinase domain of the Met gene and frequent expression of Met and HGF/SF protein in primary gastric carcinomas

Hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, are known to play important roles in tumor cell migration, invasion, and metastasis. We analyzed the expression of these genes and the mutations in the kinase domain of the Met gene in 43 gastric carcinomas. Of the 43 cases, the Met and HGF/SF protein were expressed in 29 (67%) and 22 (51%), respectively. All of the cases with HGF/SF immunopositivity also expressed Met. Of 22 cases with HGF/SF immunopositivity, 13 (59%) expressed HGF/SF in tumor cells as well as fibroblasts. We detected no aberrant single-strand conformational polymorphism patterns, suggesting that there are no genetic alterations in the kinase domain of the Met gene. These results indicate that HGF/SF-mediated autocrine and/or paracrine stimulation and overexpression rather than structural alteration of its receptor may contribute to the development and progression of gastric carcinoma, and that expression of Met and HGF/SF may confer a growth advantage to neoplastic cells.