In Vivo Modulation of Angiogenesis and Immune Response on a Collagen Matrix via Extracorporeal Shockwaves.

The effective management of tissue integration and immunological responses to transplants decisively co-determines the success of soft and hard tissue reconstruction. The aim of this in vivo study was to evaluate the eligibility of extracorporeal shock wave therapy (ESWT) with respect to its ability to modulate angiogenesis and immune response to a collagen matrix (CM) for tissue engineering in the chorioallantoic membrane (CAM) assay, which is performed with fertilized chicken eggs. CM were placed on the CAM on embryonic development day (EDD) 7; at EDD-10, ESWT was conducted at 0.12 mJ/mm2 with 500 impulses each. One and four days later, angiogenesis represented by vascularized area, vessel density, and vessel junctions as well as HIF-1α and VEGF gene expression were evaluated. Furthermore, immune response (iNOS2, MMP-9, and MMP-13 via qPCR) was assessed and compared between ESWT- and non-ESWT-groups. At EDD-14, the vascularized area (+115% vs. +26%) and the increase in vessel junctions (+751% vs. +363%) were significantly higher in the ESWT-group. ESWT significantly increased MMP-9 gene expression at EDD-11 and significantly decreased MMP-13 gene expression at EDD-14 as compared to the controls. Using the CAM assay, an enhanced angiogenesis and neovascularization in CM after ESWT were observed. Furthermore, ESWT could reduce the inflammatory activity after a latency of four days.

Transcriptional regulation of the VEGF gene in dependence of individual genomic variations.

Overexpression of the vascular endothelial growth factor (VEGF) gene has been associated with advanced stage and poor survival in several cancers. The majority of disease-associated VEGF-single nucleotide polymorphisms (SNPs) locate within regulatory regions. Therefore, an influence of SNPs located in the promoter/5'-untranslated region (5'UTR) on transcription factor binding (TFB) and gene expression seems feasible. We reviewed the literature investigating a potential connection of VEGF-SNPs and transcriptional regulation of the VEGF gene. In addition, we employed transcription factor databases to search for VEGF-SNPs which have already been associated with diseases. The objective of this review is to gain an overview about an association of VEGF-SNPs and the transcription factor dependent regulation of the VEGF gene. A decreasing binding specificity of the transcription factor MZF1 in presence of the VEGF-SNP +405 C-allele has been reported. TF databases indicated a potential HIF binding site for the -2578 C-allele representing an important potential inducer of VEGF expression. Additionally, linkage disequilibrium of the -2578 A-allele and an 18 bp insertion increases the number of potential TFB sites. For the VEGF promoter SNP -1154 A/G an interaction with the HRE under participation of the SNP +405 C/G was supposed. The comprehension of the association of specific SNPs and TFB could be an essential part in our understanding of individual differences of VEGF regulation and course of diseases.

Reactive oxygen species activation of MAPK pathway results in VEGF upregulation as an undesired irradiation response.

BACKGROUND: Radioresistance limits the effectiveness of radiotherapy in head and neck squamous cell carcinoma. We previously demonstrated post-radiogenic mitogen-activated protein kinase (MAPK) pathway activation and vascular endothelial growth factor (VEGF) release resulting in reduced tumor cell response. Here, we examined the association of this mechanism with the induction of reactive oxygen species (ROS) under irradiation (IR). METHODS: Intracellular ROS after IR were measured. We modeled radiation-induced ROS by exposure of two SCC lines to H2 O2 and evaluated the impact of irradiation and ROS on ERK phosphorylation by Western blot, immunohistochemistry, and ELISA. RESULTS: We found elevated pERK levels after treatment with IR and H2 O2 , which could be distinctly suppressed by U0126. Immunohistochemistry and ELISA revealed increased intracellular VEGF levels after H2 O2 application. CONCLUSIONS: Our data show that irradiation-induced ROS activate the MAPK pathway and release of VEGF. As VEGF is known to be released after cellular distress resulting in cytoprotection, the described mechanism is potentially of importance for therapy success.

Impact of ozone exposure on prostaglandin release in nasal polyps.

A dysregulation of the cyclooxygenases and a leukotriene/prostaglandin imbalance are assumed to be part of the pathogenesis of the aspirin (ASA) intolerance syndrome. Ozone is an air pollutant with known proinflammatory effects on exposed epithelia, however, its impact on the expression of the cyclooxygenases 1 and 2 (cox1/2) and prostaglandin E(2) (PGE(2)) in the nasal mucosa is not well known. Therefore, we analyzed cox expression and PGE(2) levels after ozone exposure in nasal mucosa and in nasal polyps considering ASA intolerance. Isolated epithelial nasal cells from control subjects without chronic rhinosinusitis (CRS), and those from patients with nasal polyps with and without ASA intolerance were cultured and exposed in vitro to ozone. Cox1/2 expression levels were analyzed by immunohistochemistry and PGE(2) release by ELISA. After ozone exposure cox1/2 expression remained unchanged in all the three groups. PGE(2) release was lowered in cell cultures from controls and from polyps of ASA tolerant but not in those of ASA intolerant patients after ozone exposure. In the latter, PGE(2) expression remained unchanged. Our in vitro data suggest that aspirin tolerant patients with polyps might be more affected by ozone compared to aspirin intolerant ones. The potential clinical impact of impaired PGE(2) expression caused by ozone on the functions of respiratory epithelia remains to be clarified.

Cold Atmospheric Plasma Reduces Vessel Density and Increases Vascular Permeability and Apoptotic Cell Death in Solid Tumors.

Cold atmospheric plasma (CAP) has demonstrated promising anti-cancer effects in numerous in vitro and in vivo studies. Despite their relevance for the treatment of solid tumors, effects of CAP on tumor vasculature and microcirculation have only rarely been investigated. Here, we report the reduction of vessel density and an increase in vascular permeability and tumor cell apoptosis after CAP application. Solid tumors in the chorioallantoic membrane of chicken embryos were treated with CAP and evaluated with respect to effects of CAP on embryo survival, tumor size, and tumor morphology. Furthermore, intratumoral blood vessel density, apoptotic cell death and the tumor-associated microcirculation were investigated and compared to sham treatment. Treatment with CAP significantly reduced intratumoral vessel density while increasing the rate of intratumoral apoptosis in solid tumors. Furthermore, CAP treatment increased vascular permeability and attenuated the microcirculation by causing vessel occlusions in the tumor-associated vasculature. These effects point out the potential of CAP as a promising and yet underrated therapeutic modality for addressing the tumor vasculature in the treatment of solid tumors.

Extracts of Rheum palmatum and Aloe vera Show Beneficial Properties for the Synergistic Improvement of Oral Wound Healing.

Various local and systemic factors compromise oral wound healing and may lead to wound dehiscence, inflammation, or ulcers. Currently, there is a lack of topical therapeutical options. Thus, this study aimed to investigate the effect of Aloe vera (AV) and Rheum palmatum root (RPR) on oral wound healing capacity in vitro. The effect of AV and RPR on human primary fibroblast viability and migration was studied by measuring metabolic activity and gap closure in a scratch assay. Furthermore, cell cycle distribution and cytoskeletal features were analyzed. Antimicrobial activity against the oral pathogen Porphyromonas gingivalis was evaluated by broth microdilution assay. AV and RPR increased fibroblast migration after single agent treatment. Synergistic effects of the plant extract combination were observed regarding cellular migration which were confirmed by calculation of the phenomenological combination index (pCI), whereas the cell cycle distribution was not influenced. Furthermore, the combination of AV and RPR showed synergistic antibacterial effects as determined by the fractional inhibitory concentration index. This study demonstrated that the combination of AV and RPR can promote the migration of human primary fibroblasts in vitro and exert antimicrobial efficacy against P. gingivalis, suggesting these compounds for the topical treatment of wound healing disorders.

Analysis of differentially expressed proteins in oral squamous cell carcinoma by MALDI-TOF MS.

PURPOSE: To explore the presence of differentially expressed proteins in OSCC for discrimination of tumour and normal mucosa to establish potential biomarkers and therapeutic targets. EXPERIMENTAL DESIGN: Paired protein samples of 12 individuals (tongue cancer and non-cancerous mucosa) were separated by two-dimensional polyacrylamid gel electrophoresis. The protein patterns were compared pairwise and protein spots were quantified. We identified about 70 regulated proteins which we subsequently identified by MALDI-TOF mass spectrometry. RESULTS: Cancerous and non-cancerous tissues could be most precisely distinguished by a panel of proteins. They include the heat shock proteins (hsp)70 and 90, keratins (ck) 5, 6, 13, 14, 16, 17 and 19, beta globin, alpha-2-actin, stratifin, tropomyosin, calreticulin precursor, beta-2-tubulin, galectin7, thioredoxin, involucrin, adenylyl-cyclase-associated protein, disulfide isomerase-associated protein, thyrosine 3-monooxygenase, MYL2 and the s100 calcium binding protein. MYL3, cardiac muscle alpha actin 1 proprotein and transferrin were under-represented in OSCC. Six biomarkers, ck6 und ck13, beta globin, alpha-2-actin, hsp70 and hsp90 discriminated best between cancerous and non-cancerous oral tissues. All over-expressed proteins were analysed by STRING-analysis to highlight experimentally determined and computationally predicted interactions between the proteins. Especially involucrin, hsp70, calreticulin precursor, stratifin, (ck) 5, 6, 14, 19, tyrosine 3-monooxygenase, beta-2-tubulin and disulfide isomerase associated protein showed multiple relations. CONCLUSION: We identified six proteins which are differentially expressed in most OSCC compared to healthy tissues. Of those, by string analysis, multiple interaction partners are assumed for hsp70. This protein is supposed to be the most promising candidate as marker molecule and target for OSCC therapy.

Zinc Oxide Nanoparticles Can Intervene in Radiation-Induced Senescence and Eradicate Residual Tumor Cells.

Despite recent advancements in tumor therapy, metastasis and tumor relapse remain major complications hindering the complete recovery of many cancer patients. Dormant tumor cells, which reside in the body, possess the ability to re-enter the cell cycle after therapy. This phenomenon has been attributed to therapy-induced senescence. We show that these cells could be targeted by the use of zinc oxide nanoparticles (ZnO NPs). In the present study, the properties of tumor cells after survival of 16 Gy gamma-irradiation were investigated in detail. Analysis of morphological features, proliferation, cell cycle distribution, and protein expression revealed classical hallmarks of senescent cells among the remnant cell mass after irradiation. The observed radiation-induced senescence was associated with the increased ability to withstand further irradiation. Additionally, tumor cells were able to re-enter the cell cycle and proliferate again after weeks. Treatment with ZnO NPs was evaluated as a therapeutical approach to target senescent cells. ZnO NPs were suitable to induce cell death in senescent, irradiation-resistant tumor cells. Our findings underline the pathophysiological relevance of remnant tumor cells that survived first-line radiotherapy. Additionally, we highlight the therapeutic potential of ZnO NPs for targeting senescent tumor cells.

Zinc Oxide Nanoparticles Exhibit Favorable Properties to Promote Tissue Integration of Biomaterials.

Due to the demographic change, medicine faces a growing demand for tissue engineering solutions and implants. Often, satisfying tissue regeneration is difficult to achieve especially when co-morbidities hamper the healing process. As a novel strategy, we propose the incorporation of zinc oxide nanoparticles (ZnO NPs) into biomaterials to improve tissue regeneration. Due to their wide range of biocompatibility and their antibacterial properties, ZnO NPs are already discussed for different medical applications. As there are versatile possibilities of modifying their form, size, and function, they are becoming increasingly attractive for tissue engineering. In our study, in addition to antibacterial effects of ZnO NPs, we show for the first time that ZnO NPs can foster the metabolic activity of fibroblasts as well as endothelial cells, both cell types being crucial for successful implant integration. With the gelatin sponge method performed on the chicken embryo's chorioallantoic membrane (CAM), we furthermore confirmed the high biocompatibility of ZnO NPs. In summary, we found ZnO NPs to have very favorable properties for the modification of biomaterials. Here, incorporation of ZnO NPs could help to guide the tissue reaction and promote complication-free healing.

Surface Modification of Porous Polyethylene Implants with an Albumin-Based Nanocarrier-Release System.

BACKGROUND: Porous polyethylene (PPE) implants are used for the reconstruction of tissue defects but have a risk of rejection in case of insufficient ingrowth into the host tissue. Various growth factors can promote implant ingrowth, yet a long-term gradient is a prerequisite for the mediation of these effects. As modification of the implant surface with nanocarriers may facilitate a long-term gradient by sustained factor release, implants modified with crosslinked albumin nanocarriers were evaluated in vivo. METHODS: Nanocarriers from murine serum albumin (MSA) were prepared by an inverse miniemulsion technique encapsulating either a low- or high-molar mass fluorescent cargo. PPE implants were subsequently coated with these nanocarriers. In control cohorts, the implant was coated with the homologue non-encapsulated cargo substance by dip coating. Implants were consequently analyzed in vivo using repetitive fluorescence microscopy utilizing the dorsal skinfold chamber in mice for ten days post implantation. RESULTS: Implant-modification with MSA nanocarriers significantly prolonged the presence of the encapsulated small molecules while macromolecules were detectable during the investigated timeframe regardless of the form of application. CONCLUSIONS: Surface modification of PPE implants with MSA nanocarriers results in the alternation of release kinetics especially when small molecular substances are used and therefore allows a prolonged factor release for the promotion of implant integration.

Zinc oxide nanoparticles for therapeutic purposes in cancer medicine.

The importance of zinc as a trace metal in the human body has long been overlooked. We now gradually discover that the impact of zinc on the health of our body might be as far-reaching as that of iron. Concurrently, nanomaterials containing zinc, in particular zinc oxide nanoparticles (ZnO NPs), are becoming increasingly attractive as innovative agents for medical applications. Zinc oxide is characterized by a good biocompatibility which allows the exploitation of its antibacterial, antifungal, antiviral, and anti-cancer qualities in a therapeutic setting. This perspective outlines the current state of knowledge concerning the interaction of zinc oxide nanoparticles with eukaryotic cells and the human body. Furthermore, it sheds light on the importance of zinc under physiological conditions. This helps to place the in vivo behavior of zinc oxide nanoparticles in the proper context. We evaluate the potential of zinc oxide nanoparticles as innovative anti-tumor agents by summarizing important results of current studies in this field and discuss the proposed mechanisms that give zinc oxide nanoparticles a selective toxicity for tumor cells.

Monitoring of tumor growth and vascularization with repetitive ultrasonography in the chicken chorioallantoic-membrane-assay.

The chorioallantoic-membrane (CAM)-assay is an established model for in vivo tumor research. Contrary to rodent-xenograft-models, the CAM-assay does not require breeding of immunodeficient strains due to native immunodeficiency. This allows xenografts to grow on the non-innervated CAM without pain or impairment for the embryo. Considering multidirectional tumor growth, limited monitoring capability of tumor size is the main methodological limitation of the CAM-assay for tumor research. Enclosure of the tumor by the radiopaque eggshell and the small structural size only allows monitoring from above and challenges established imaging techniques. We report the eligibility of ultrasonography for repetitive visualization of tumor growth and vascularization in the CAM-assay. After tumor ingrowth, ultrasonography was repetitively performed in ovo using a commercial ultrasonographic scanner. Finally, the tumor was excised and histologically analyzed. Tumor growth and angiogenesis were successfully monitored and findings in ultrasonographic imaging significantly correlated with results obtained in histological analysis. Ultrasonography is cost efficient and widely available. Tumor imaging in ovo enables the longitudinal monitoring of tumoral development, yet allowing high quantitative output due to the CAM-assays simple and cheap methodology. Thus, this methodological novelty improves reproducibility in the field of in vivo tumor experimentation emphasizing the CAM-assay as an alternative to rodent-xenograft-models.

Phosphoproteome Profiling Reveals Multifunctional Protein NPM1 as part of the Irradiation Response of Tumor Cells.

To fight resistances to radiotherapy, the understanding of escape mechanisms of tumor cells is crucial. The aim of this study was to identify phosphoproteins that are regulated upon irradiation. The comparative analysis of the phosphoproteome before and after irradiation brought nucleophosmin (NPM1) into focus as a versatile phosphoprotein that has already been associated with tumorigenesis. We could show that knockdown of NPM1 significantly reduces tumor cell survival after irradiation. NPM1 is dephosphorylated stepwise within 1 hour after irradiation at two of its major phosphorylation sites: threonine-199 and threonine-234/237. This dephosphorylation is not the result of a fast cell cycle arrest, and we found a heterogenous intracellular distribution of NPM1 between the nucleoli, the nucleoplasm, and the cytoplasm after irradiation. We hypothesize that the dephosphorylation of NPM1 at threonine-199 and threonine-234/237 is part of the immediate response to irradiation and of importance for tumor cell survival. These findings could make NPM1 an attractive pharmaceutical target to radiosensitize tumor cells and improve the outcome of radiotherapy by inhibiting the pathways that help tumor cells to escape cell death after gamma irradiation.

Zinc overload mediated by zinc oxide nanoparticles as innovative anti-tumor agent.

The predicted global cancer burden is expected to surpass 20 million new cancer cases by 2025. Despite recent advancement in tumor therapy, a successful cancer treatment remains challenging. The emerging field of nanotechnology offers great opportunities for diagnosis, imaging, as well as treatment of cancer. Zinc oxide nanoparticles (ZnO NP) were shown to exert selective cytotoxicity against tumor cells via a yet unknown mechanism, most likely involving the generation of reactive oxygen species (ROS). These nanoparticles are a promising therapeutic opportunity as zinc is a nontoxic trace element and its application in medically-related products is considered to be safe. We could show that ZnO NP can exert cytotoxic effects on several human tumor cell lines. There can be found ZnO NP concentrations which selectively damage tumor cells while human fibroblasts do not sustain lasting damage. Cytotoxicity is attributable to the release of zinc ions from the nanoparticles outside the cells as well as to a direct cell-nanoparticle interaction. This involves uptake of the particles into the tumor cells. With a silica shell the cytotoxicity can be delayed which can help in the future for a safe transport in the blood stream. Cellular damage finally cumulates in apoptotic cell death via zinc overload within 48 h after treatment with ZnO NP. A therapeutical perspective could be the targeted accumulation of ZnO NP at the tumor side to induce local zinc overload that substantially damages the tumor cells with no or low side effects. We suggest further studies to explore the potential of ZnO NP as an innovative anti-tumor agent.

Fluorescein- and EGFR-Antibody Conjugated Silica Nanoparticles for Enhancement of Real-time Tumor Border Definition Using Confocal Laser Endomicroscopy in Squamous Cell Carcinoma of the Head and Neck.

Intraoperative definition of tumor free resection margins in head and neck cancer is challenging. In the current proof-of-principle study we evaluated a novel silica nanoparticle-based agent for its potential use as contrast enhancer. We synthesized silica nanoparticles with an average size of 45 nm and modified these particles with the fluorescence stain fluorescein isocyanate (FITC) for particle detection and with epidermal growth factor receptor (EGFR)-targeting antibodies for enhanced tumor specificity. The nanoparticles exhibited good biocompatibility and could be detected in vitro and in vivo by confocal laser scanning microscopy. Additionally, we show in an ex vivo setting that these modified nanoparticles specifically bind to tumor samples and could be detected using a handheld confocal fluorescence endomicroscope. From a clinical point of view, we believe that this method could be used for tumor border contrast enhancement and for better intraoperative definition of R-0 tumor resection.

Early stress response of human nasal respiratory epithelia after exposure to 1-methoxypropanol-2.

To evaluate the impact of 1-methoxypropanol-2 (MEP) for the stimulation of an inflammatory response in human respiratory mucosa, we exposed 22 primary cell cultures of nasal respiratory epithelia of healthy individuals to MEP concentrations at the level of the German MAK-value (100 ppm) and to the 10-fold concentration (1000 ppm). After 4 and 24h we analyzed the transcription of TNF-alpha, IL-1beta, IL-6, IL-8, MCP-1, GMCSF, Cox-1 and Cox-2 by quantitative PCR as well as the release of the respective cytokines by ELISA. At both MEP concentrations we observed a significant increase of TNF-alpha-, IL-1beta-, IL-6- and Cox-2-transcripts after 4h. After 24h cytokine transcription of TNF-alpha, IL-1beta and IL-6 was normalized, but Cox-2 remained elevated. On the protein level IL-1beta as well as granulocyte macrophages colony stimulating factor (GM-CSF) were decreased after 4h or 24h and uniquely IL-8 levels were increased after 4h. Our data suggest that MEP induces the transcription of genes encoding proinflammatory cytokines and mediators but largely not translation of those. Considering these in vitro data, existing exposure limits seem to be safe with respect to inflammatory responses of the upper respiratory tract. However, the effects of long-term exposures to MEP should be watched closely.

Impact of vascular endothelial growth factor release on radiation resistance.

There is increasing evidence of an angiogenic response of irradiated tumors resulting in decreased radiation sensitivity. However, little is known about the contribution of tumor vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF)-release induced by irradiation to the individual level of resistance. In this in vitro study, we analysed the VEGF- and bFGF-release of six epithelial tumor cell lines before and after irradiation and correlated these data to the corresponding irradiation resistance. Two head and neck squamous cell carcinoma (HNSCC), two renal cell carcinoma (RCC), and two ovarian cancer (OC) cell lines were each exposed to 2 or 6 Gy single dose using a 137Cs-source. Non-irradiated controls were processed in parallel. Survival rates were assessed by colony assays as a measure of resistance. The released VEGF and bFGF was quantified by ELISA assays. Additionally, the expression of VEGF and its respective receptors (FLK, FLT, and NRP1) was visualized by immunohistochemistry. VEGF-release was significantly increased (p<0.05) in all cell lines after irradiation. Release was most prominent in the RCC cell lines, less in the HNSCC cell lines and lowest in the OC cell lines. Radiation resistance correlated to the absolute level of released VEGF after irradiation as well as to its relative increase (r>0.9, p<0.01). bFGF levels were not correlated to resistance. VEGF and all three VEGF-receptors were detected in all cell lines analyzed supporting the concept of an autocrine protective mechanism. We suggest that tumor cell survival after irradiation may be enhanced by released VEGF and that the level of released VEGF directly corresponds to the resistance of the tumor to irradiation.

mRNA-induction and cytokine release during in vitro exposure of human nasal respiratory epithelia to methyl methacrylate.

BACKGROUND: Methyl methacrylate (MMA) has been reported to cause histopathological changes in rodent nasal epithelium after inhalation challenges. Data in humans are lacking. METHODS: In this in vitro design 22 primary cell cultures taken from inferior turbinate tissue of healthy individuals were exposed to MMA concentrations of 50 ppm (German MAK-value) and 200 ppm. mRNA expression and cytokine release of inflammatory mediators were quantified after 4h and after 24h. Controls were exposed to synthetic air. Q-PCR analysis was performed for TNF-alpha, IL-1beta, IL-6, IL-8, MCP-1, GMCSF, Cox-1 and Cox-2. ELISA assays were performed from culture supernatants for TNF-alpha, IL-1beta, IL-6, IL-8, MCP-1 and GMCSF. RESULTS: Acute inductions of mRNA after 4h were observed for TNF-alpha, IL-1beta, IL-6, IL-8 and MCP-1 at 50 ppm. ELISA analysis of the described parameters did not reveal any significant upregulations at both concentrations after both 4h and 24h. CONCLUSIONS: The obtained data suggest that exposure of human respiratory epithelia in vitro to 50 ppm and to 200 ppm of MMA does not induce lasting upregulation of the inflammatory mediators measured in this study. The exposure limit of 50 ppm appears safe following these results obtained from human respiratory epithelia.

Expression of vascular endothelial growth factor and basic fibroblast growth factor in sporadic vestibular schwannomas correlates to growth characteristics.

HYPOTHESIS: Expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) may have an impact on the growth characteristics of sporadic vestibular schwannomas (VSs). BACKGROUND: Vestibular schwannoma is a benign, slow-growing neoplasm that accounts for 6% of all intracranial tumors. The biological backgrounds for neoplastic growth and especially for the various growth patterns of VS remain largely unknown. Because several angiogenic and cytotrophic factors have been described to be involved in the growth of malignant tumors, we initiated this study to examine 2 major representatives of such growth factors in VS and their possible correlation to the growth characteristics of sporadic VSs. METHODS: Surgical specimens from 17 patients with sporadic VS were examined, and the expression of 2 major angiogenic and neurotrophic factors, bFGF and VEGF, was quantitatively analyzed at the mRNA and protein levels. The microvessel density (MVD) was defined by CD31 staining. RESULTS: All tumors showed expression of bFGF and VEGF at both the mRNA and protein levels. The mRNA expression and the protein expression of both growth factors correlated positive to tumor volume, to tumor growth index, and to MVD. CONCLUSION: The bFGF and VEGF mRNA expression and the bFGF and VEGF protein expression in sporadic VS correlates to the tumour volume, to the tumor growth index, and to the MVD. This might indicate an angiogenic and neurotrophic influence of these factors and a possible involvement in the growth of sporadic VS.