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Migration Stimulating Factor (MSF) is a 70 kDa truncated isoform of fibronectin (FN); its mRNA is generated from the FN gene by an unusual two-stage processing. Unlike full-length FN, MSF is not a matrix molecule but a soluble protein which displays cytokine-like activities not displayed by any other FN isoform due to steric hindrance. There are two isoforms of MSF; these are referred to as MSF+aa and MSF-aa, while the term MSF is used to include both.MSF was first identified as a motogen secreted by foetal and cancer-associated fibroblasts in tissue culture. It is also produced by sprouting (angiogenic) endothelial cells, tumour cells and activated macrophages. Keratinocytes and resting endothelial cells secrete inhibitors of MSF that have been identified as NGAL and IGFBP-7, respectively. MSF+aa and MSF-aa show distinct functionality in that only MSF+aa is inhibited by NGAL.MSF is present in 70-80% of all tumours examined, expressed by the tumour cells as well as by fibroblasts, endothelial cells and macrophages in the tumour microenvironment (TME). High MSF expression is associated with tumour progression and poor prognosis in all tumours examined, including breast carcinomas, non-small cell lung cancer (NSCLC), salivary gland tumours (SGT) and oral squamous cell carcinomas (OSCC). Epithelial and stromal MSF carry independent prognostic value. MSF is also expressed systemically in cancer patients, being detected in serum and produced by fibroblast from distal uninvolved skin. MSF-aa is the main isoform associated with cancer, whereas MSF+aa may be expressed by both normal and malignant tissues.The expression of MSF is not invariant; it may be switched on and off in a reversible manner, which requires precise interactions between soluble factors present in the TME and the extracellular matrix in contact with the cells. MSF expression in fibroblasts may be switched on by a transient exposure to several molecules, including TGFß1 and MSF itself, indicating an auto-inductive capacity.Acting by both paracrine and autocrine mechanisms, MSF stimulates cell migration/invasion, induces angiogenesis and cell differentiation and alters the matrix and cellular composition of the TME. MSF is also a survival factor for sprouting endothelial cells. IGD tri- and tetra-peptides mimic the motogenic and angiogenic activities of MSF, with both molecules inhibiting AKT activity and requiring αvß3 functionality. MSF is active at unprecedently low concentrations in a manner which is target cell specific. Thus, different bioactive motifs and extracellular matrix requirements apply to fibroblasts, endothelial cells and tumour cells. Unlike other motogenic and angiogenic factors, MSF does not affect cell proliferation but it stimulates tumour growth through its angiogenic effect and downstream mechanisms.The epithelial-stromal pattern of expression and range of bioactivities displayed puts MSF in the unique position of potentially promoting tumour progression from both the "seed" and the "soil" perspectives.

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Migration-stimulating factor (MSF), a soluble genetically truncated isoform of fibronectin, is a potent oncofoetal regulatory molecule. Its 2.1-kb message is generated from the fibronectin gene by a variant of standard alternative splicing involving premature intra-intronic cleavage. MSF is constitutively expressed by both epithelial and stromal cells during foetal development and in patients with cancer, but is generally not expressed in healthy adults. MSF affects the behaviour of a broad range of potential target cells (fibroblasts, vascular, and epithelial) in terms of stimulation of their migration/invasion, matrix remodelling and induction of angiogenesis. It also functions as an autocrine survival factor for the angiogenic endothelium. MSF expression by foetal and cancer patient cells adherent to an appropriate matrix may be persistently switched off by a transient exposure to TGF-beta1; conversely, MSF expression by adult dermal fibroblasts adherent to other matrices may be persistently switched on by a transient exposure to TGF-beta or various pharmacological agents known to alter gene expression by epigenetic mechanisms. The manifestation of MSF effects on target cells is similarly dependent on the inter-dependent signalling of soluble factors and matrix molecules. The significant association between elevated MSF expression and poor survival in patients with breast and oral cancer suggests that MSF may function as a driver of tumour progression. Accordingly, we suggest that the downregulation of MSF expression (eg, by siRNA or pharmacological agents) and/or inhibition of its bioactivities (by function-neutralising antibodies or MSF inhibitors) may provide a clinically efficacious means of improving treatment outcome in cancer patients.

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AIM: To quantify vascularity in periradicular granulomas using different endothelial markers, and assess its value as an index of angiogenesis by comparing granulomas with healthy periodontal ligament (PDL). To use oral tumours, compared with adjacent normal mucosa, as positive controls. METHODOLOGY: Paraffin-embedded sections were stained with antibodies to von Willebrand factor (vWF), a pan-endothelial marker, and CD105, a putative marker for angiogenic vessels. Vascularity was quantified by different methods reflecting vessel volume and density. RESULTS: Irrespective of the marker or method used, vascularity values were similar in periradicular granuloma and PDL. Both tissues were highly vascularized, with levels similar to those found in oral squamous cell carcinoma. Vascularity was significantly higher in the latter than in normal mucosa. Fewer vessels were positive for CD105 than for vWF in the normal mucosa, whereas similar numbers were found in the other tissues examined. CONCLUSIONS: A comparison of vascularity in oral tumours and normal oral mucosa provided evidence of angiogenesis in the former. Staining with CD105 added limited value to staining with vWF in these tissues. In contrast, a comparison of periradicular granuloma and PDL failed to demonstrate evidence of angiogenesis in the granuloma. As all vessels were similarly stained with vWF and CD105 in granuloma and PDL, a possible hypothesis is that all vessels are newly formed in these tissues. A more plausible alternative is that CD105 expression may reflect the metabolic activity or intrinsic characteristics of the tissues, rather than the presence of angiogenic vessels.

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The extracellular matrix profoundly affects cellular response to soluble motogens. In view of this critical aspect of matrix functionality, we have developed a novel assay to quantify chemo-regulated cell migration within biologically relevant 3-dimensional matrices. In this "sandwich" assay, target cells are plated at the interface between an upper and lower matrix compartment, either in the presence of an isotropic (uniform) or anisotropic (gradient) spatial distribution of test motogen. Cell migration in response to the different conditions is ascertained by quantifying their subsequent disposition within the upper and lower matrix compartments. The objective of this study has been to compare the motogenic activities of platelet-derived growth factor (PDGF-AB) and transforming growth factor-beta isoforms (TGF-beta1, -beta2 and -beta3) in the sandwich assay and the commonly employed transmembrane assay. As previously reported, dermal fibroblasts exhibited a motogenic response to isotropic and anisotropic distributions of all tested cytokines in the transmembrane assay. In contrast, only PDGF-AB and TGF-beta3 were active in the sandwich assay, each eliciting directionally unbiased (symmetrical) migration into the upper and lower type I collagen matrices in response to an isotropic cytokine distribution and a directionally biased response to an anisotropic distribution. TGF-beta1 and -beta2 were completely devoid of motogenic activity. These results are consistent with the reported differential bioactivities of PDGF and TGF-beta3 compared to TGF-beta1 and -beta2 in animal models of wound healing and suggest that the sandwich assay provides a means of obtaining physiologically relevant data regarding chemo-regulated cell migration.

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Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein implicated in the regulation of angiogenesis and tumour development. Our objectives were to ascertain the quantity and quality of RNA extracted from archival, formalin-fixed, paraffin embedded, oral tissues and their application in measuring the concentrations of TSP-1 mRNA in these tissues. We compared three techniques of isolation of RNA as well as related experimental variables. TSP-1 mRNA was measured in specimens of normal, dysplastic, and malignant oral tissues by real-time reverse transcriptase polymerase chain reaction (RT-PCR). RNA suitable for analysis by real-time RT-PCR was obtained by the three techniques tested, although the yield varied depending on the protocol used (range 0.2-3.6 microg/mm(3)). The mean (S.D.) concentrations of TSP-1 mRNA relative to 18S were 21.1 (7.2) in normal oral tissues (n=9), 11.0 (8.2) in dysplastic tissue (n=8) and 7.3 (5.3) in carcinomatous tissue (n=17). The difference between normal and carcinomatous specimens was significant (p=0.01). This reduction in expression of TSP-1 mRNA from normal to dysplasia to carcinoma may favour the angiogenic drive that accompanies the development of oral tumours.

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BACKGROUND: Chronic foot ulceration is a major source of morbidity in diabetic patients. Despite traditional comprehensive wound management, including vascular reconstruction, there remains a cohort of patients with non-responding wounds, often resulting in amputation. These wounds may benefit from molecular manipulation of growth factors to enhance the microcirculation. METHODS: A review of the current literature was performed using Pubmed, with secondary references obtained from key articles. RESULTS AND CONCLUSION: There has been a generally disappointing clinical outcome from growth factor trials, although topical platelet-derived growth factor has shown significant benefit and should be considered in non-healing, well perfused ulcers after failure of conventional wound care. The modulatory role of the extracellular matrix in the cellular response to growth factors and data from regenerative-type fetal wound healing are further areas of interest. The chemical induction of microvessel formation may become a future therapeutic option.

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In addition to the well documented role of cytokines in mediating tissue-level interactions, it is now clear that matrix macromolecules fulfil a complementary regulatory function. Data highlighted in the present review extend the repertoire of matrix signalling mechanisms, (1) introducing the concept of 'matrikines', these defined as proteinase-generated fragments of matrix macromolecules that display cryptic bioactivities not manifested by the native, full-length form of the molecule, and (2) indicating that a previously identified motogenic factor (migration stimulating factor [MSF]) produced by foetal and cancer patient fibroblasts is a genetically generated truncated isoform of fibronectin, which displays bioactivities cryptic in all previously identified fibronectin isoforms. These observations are discussed in the context of the contribution of a 'foetal-like' stroma to the progression of breast cancer.

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Expression of vascular endothelial growth factor (VEGF) in oral tissues was assessed using different antibodies. Quantitative and topographical differences were observed between paraffin and cryostat sections. Two polyclonal antibodies (PC36, PC37) differing in their cross-reactivity with VEGF121 (not recognized by PC36), were used to stain serial cryostat sections of normal oral mucosa (n = 8) and squamous cell carcinoma (n = 7). The expression of VEGF in the epithelium was overall higher with PC37 than with PC36, the difference being significant in normal oral mucosa (p = 0.001) but not in squamous cell carcinoma samples (p = 0.094). With PC36, VEGF expression was significantly higher in squamous cell carcinoma than in normal oral mucosa specimens, whereas the opposite was true with PC37. Our results suggest that the relative levels of isoform 121 to that of 165 (and possibly others) may be different in the tissues examined, with VEGF121 preferentially expressed in normal oral mucosa. Previously published conflicting results may, therefore, be due to the presence of variable ratios of VEGF isoforms in the tissues examined, combined with differences in the cross-reactivity of the antibodies used. VEGF isoforms 121, 165 and (for the first time) 189 were detected in two frozen oral tissues by polymerase chain reaction amplification. Quantification of specific VEGF isoforms will be required in future studies concerned with the clinical value of VEGF expression.

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The aim of this study was to assess whether vascular endothelial growth factor (VEGF) expression in oral tissues is associated with angiogenesis, disease progression or field cancerisation. Vascularity and VEGF immunoreactivity were quantified in 68 archival specimens including normal oral mucosa (NOM), dysplasia (DYS) and squamous cell carcinoma (SCC). Vascularity increased significantly with disease progression; it was also higher in NOM adjacent to SCC than in NOM from healthy tissue, suggesting an association with field cancerisation. VEGF expression in epithelial cells was evaluated using two antibodies and three indices. VEGF indices and vascularity were not directly correlated. The expression of VEGF was similar in all DYS and NOM specimens, whether or not adjacent to a concurrent lesion. A comparison of SCC with NOM or DYS led to opposite results, depending on the VEGF antibody and index used. We conclude that VEGF expression in the oral mucosa may play a physiological role, but does not appear to be associated with angiogenesis, field cancerisation or transition to dysplasia. Further studies concerned with tumour development require examining specific VEGF isoforms and standardisation of the methodology.

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Verrucae vulgaris (skin warts) are benign proliferative lesions which are generally associated with human papillomavirus type 2 (HPV-2) infection. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen able to induce angiogenesis and vasodilation. Our previous findings indicate that these two processes take place during the formation of skin warts. The purpose of this study was to determine whether VEGF expression in these lesions was associated with HPV infection, angiogenesis or vasodilation. To this end, paraffin-embedded specimens of skin warts which were either negative for HPV-1, -2, -3 and -4 (HPV-; n = 18), or positive for HPV-2 (HPV+; n = 21) were compared with histologically normal perilesional skin (n = 13). Serial sections were stained with antibodies to von Willebrand Factor (vWF) and to VEGF. Vascularity was quantified by point counting vWF-positive blood vessels. Small and large vessels were quantified separately, using a cut-off value of 50 microm diameter. VEGF expression in the epidermis was estimated by consensus of two independent observers according to three indices: (1) percentage of cells stained, (2) intensity of the staining, and (3) product of area and intensity (final score). Results were analysed by nonparametric tests. Similar levels of VEGF were found in specimens of normal skin, HPV- and HPV+ warts, irrespective of the index used. There was no significant correlation between VEGF expression and vascularity values for either small or large vessels. These results indicate that, on its own, VEGF expression is not associated with angiogenesis, vasodilation or HPV infection in skin warts. The presence of VEGF in normal skin suggests that it may play a role in tissue homeostasis.

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AIMS: High expression of the angiogenic factor vascular endothelial growth factor (VEGF) in tumours has been found to be associated with poor prognosis in some studies, but not in others. The aims of this study were to determine the prognostic value of VEGF in operable non-small cell lung cancer (NSCLC) and its possible association with vascularity. METHODS: Sections from 81 NSCLC archival specimens were stained with antibodies to von Willebrand factor (vWF) and VEGF. Vascularity was measured by the average density of vWF positive vessels. VEGF expression in tumour cells was assessed by consensus of two independent observers according to three indices, namely: (1) percentage of area stained, (2) intensity of staining, and (3) final score (product of area and intensity). RESULTS: VEGF immunoreactivity was present in all tumours and adjacent normal lung tissue. None of the three VEGF indices was associated with vascularity or the clinical parameters examined. Mean survival times were shorter in patients with high VEGF expression, but the difference was not significant. This applied to the full cohort of patients, or when analysed separately according to tumour type or stage. However, high VEGF expression was associated with poor survival in patients with high vascularity (p = 0.02). VEGF had no discriminant value among patients with low vascularity. Vascularity had no prognostic value, except for late stage patients (UICC stages II and IIIa combined; n = 36), where high vascularity was associated with longer survival (p = 0.01). CONCLUSIONS: VEGF on its own has no prognostic value in NSCLC, but may become a useful indicator when combined with vascularity. VEGF may play a physiological role in the normal lung.

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The inner lining of blood vessels, the endothelium, consists of a monolayer of endothelial cells (ECs), that present a free luminal surface and attach on their abluminal side to the underlying basement membrane (apart from a minimal amount of cell-cell overlap). A great deal of heterogeneity exists in the morphology of the endothelium and in the phenotype displayed by individual ECs. In spite of this, all ECs may be defined by two general criteria: anatomical location (i.e., luminal wall of blood vessels) and functionality (e.g., provision of a nonthrombogenic surface). In a mature resting vessel, the functionality and integrity of the endothelium is maintained under steady state conditions by the biosynthetic activity of the ECs, in conjunction with low levels of cell proliferation and motility. Significant changes in the motility of the endothelial cells, often accompanied by cell proliferation, occur during angiogenesis and in response to vessel injury.

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Angiogenesis is a complex morphogenetic process involving the coordinate migration of several cell types, including endothelial cells (EC), pericytes, and stromal fibroblasts (1-4). Angiogenesis is regulated by interactions between cells, soluble factors, and extracellular matrix components. The extracellular matrix (EM) in contact with vascular cells changes during angiogenesis in terms of its composition and structural organization. For example, ECs lining the lumen in a "resting" vessel are attached to a 2D substratum of specialized structure and composition (i.e., the basement membrane). Following exposure to angiogenic factors, endothelial cells migrate from their 2D environment into the surrounding 3D tissue stroma. Within this 3D macromolecular environment, the endothelial cells adopt an elongated "sprouting"rdo; phenotype and synthesise new EM components. Pericytes and fibroblasts are normally resident within a 3D macromolecular matrix, as provided by the vessel basement membrane and tissue stroma, respectively. Nevertheless, pericytes also form part of the newly formed vascular sprouts and fibroblasts surround and accompany these. In addition, vascular sprouts are commonly accompanied by inflammatory cells that produce proteases and cytokines, thereby contributing to further alterations in the composition of the microenvironment. The migration of ECs, pericytes, and adjacent fibroblasts during angiogenesis is directional. As new vessels move towards the source of angiogenic stimulus, they migrate into matrices of different and variable composition (e.g., during wound healing new vessels and fibroblasts invade a fibrin clot) (1-7).

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Tumour growth is accompanied by angiogenesis and reduced apoptosis in experimental animals. The aim of this study was to examine the prognostic value of apoptosis and the association between apoptosis and vascularity in non-small cell lung cancer (NSCLC). Following in-situ end-labelling of DNA, apoptotic cells were quantified by three different indices: as a percentage, either counting total cells (AI-tc) or point-counting (AI-pc), or as cells per area (AI-area). Blood vessels were stained with vWF antibody and vascularity was quantified by three methods. Median values for AI-tc, AI-pc and AI-area were 0.38, 0.32 and 10.7, respectively. High values were associated with improved survival, reaching statistical significance for AI-area (p < 0.05). All three apoptotic indices were significantly correlated with each other, but no correlation was found between indices of apoptosis and vascularity. As previously reported, vascularity had no prognostic value. These results indicate that, in NSCLC, vascularity is not informative, but apoptotic index may be a useful prognostic factor.

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Experimental animal models have demonstrated that angiogenesis is essential for tumour progression, whilst sustained tumour growth requires a positive balance between tumour cell proliferation and cell death (apoptosis). The aim of this study was to determine the relative contribution of apoptosis, proliferation, and angiogenesis to disease progression in the oral mucosa. Histological sections of 47 archival specimens were examined; these included four groups of oral tissues: normal mucosa (n=12), moderate dysplasia (n=11) severe dysplasia (n=6), and squamous cell carcinoma (n=18). Apoptotic cells were visualized by in-situ end-labelling of DNA, proliferative cells by staining with Ki-67 antibody, and blood vessels with von Willebrand factor (vWF) antibody. One-way analysis of variance showed that indices of apoptosis (AI), proliferation (PI), and angiogenesis (vascularity) increased significantly with disease progression from normal oral mucosa, through dysplasia, to carcinoma (p<0.0001 for every index). The increase from normal mucosa to moderate dysplasia was significant for PI and vascularity, but not for AI. In contrast, the increase from dysplasia to carcinoma was significant for AI and vascularity, but not for PI. These data suggest that disease progression in the oral mucosa is accompanied by angiogenesis and increases in both epithelial proliferation and apoptosis. Net epithelial growth results from proliferation starting earlier and proceeding at a higher rate than apoptosis.

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The aim of this study was to determine the ultrastructural characteristics of the microvasculature of healthy human dental pulp, with particular reference to pericytes. Pulp tissue was taken from healthy impacted third molars following extraction. Eight teeth were obtained from 17- to 25-year-old patients and pulp tissue was processed for examination using standard techniques for transmission electron microscopy. The pulp was rich in capillaries composed of endothelial and peri-endothelial cells in a 4: 1 ratio. Endothelial cells contained typical and abundant Weibel-Palade bodies. Three types of peri-endothelial cells were identified: pericytes, transitional cells and fibroblasts. Pericytes were embedded within the capillary basement membrane. Transitional cells were partly surrounded by basement membrane, but separated from the endothelium by collagen fibrils; fibroblasts were outside, but adjacent to the basement membrane and closely associated with collagen fibrils. Pericytes and transitional cells, but not peri-endothelial fibroblasts, contained low numbers of dense bodies similar to the endothelial Weibel-Palade bodies. Our observations are consistent with the hypothesis that, during normal tissue turnover, some pericytes may originate from endothelium and migrate away from the vessel wall to undergo transition to a fibroblastic phenotype.

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BACKGROUND: Angiogenesis is required for tumour growth. Since human papillomavirus (HPV) infection is associated with the development of neoplastic lesions, the aim of this study was to determine the possible association between HPV infection and angiogenesis in benign tumours. MATERIALS AND METHODS: Specimens of skin warts which were either negative for HPV types 1, 2, 3 and 4 (HPV-ve; n = 15), or positive for HPV2 (HPV+ve; n = 19) were compared with normal skin (NS, n = 10). Vascularity and inflammation were assessed in consecutive sections. vWF-positive blood vessels were classified as small or large using a cut-off value of 50 microns diameter. RESULTS: Vascularity values for small vessels increased significantly from NS to HPV-ve warts and from HPV-ve to HPV+ve warts. Large vessels were found only in warts and their abundance was not related to HPV status. No significant association was found between vascularity and inflammation or between vascularity values for small and large vessels. CONCLUSIONS: The development of skin warts is accompanied by angiogenesis and vasodilation and these two processes may be independently regulated. Further increased angiogenesis, but not vasodilation, is associated with the presence of HPV type 2 DNA.

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Although the IGD amino acid motif (iso-gly-asp) is a highly conserved feature of the fibronectin type I module, no biological activity has as yet been ascribed to it. We have previously reported that the gelatin-binding domain of fibronectin stimulates the migration of human skin fibroblasts into native, but not denatured, type I collagen substrata. Two IGD-containing type I modules are present within the gelatin-binding domain. The object of this study was to ascertain whether soluble synthetic peptides containing the IGD motif stimulate fibroblast migration. We found that IGD peptides stimulated fibroblast migration in the following order of activity: IGDS (as present in the ninth type I module) > IGDQ (as present in the seventh type I module) > IGD. The scrambled SDGI peptide and the well-characterised RGDS peptide were devoid of motogenic activity. The migratory response of fibroblasts to IGD-containing peptides consisted of two distinct phases: an initial period of peptide-mediated cell activation and a subsequent period of enhanced migration manifest in the absence of further IGD peptide. Cell activation was substratum-independent (occurring equally well on both native and denatured type I collagen substrata), whilst the manifestation of enhanced migration was persistent and substratum-dependent (being evident only by cells adherent to a native collagen substratum). Our data further indicated that cell activation (1) is elicited by a signal transduction cascade occurring within minutes of cell exposure to IGD-containing peptides, (2) is dependent upon integrin alphavbeta3 functionality, (3) involves the tyrosine phosphorylation of focal adhesion kinase (ppFAK125) and (4) is inhibited by signalling mediated through integrin alpha5beta1. The expression of migration stimulating activity by soluble IGD-containing peptides clearly distinguishes them from their RGD counterparts. This is the first identified biological activity of the highly conserved IGD motif and provides a rational platform for the development of a novel family of therapeutic compounds designed to stimulate cell migration in relevant clinical situations, such as impaired wound healing.

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The purpose of this study was to examine the possible association between epithelial proliferation and disease progression in the oral mucosa. Archival specimens of normal oral mucosa (n = 12), dysplasia (n = 17) and squamous cell carcinoma (n = 18) were sectioned and proliferating cells visualised by staining with Ki-67 antibody. The proliferative index of the epithelium (PI) was determined by total cell counts and point counting. Similar results were obtained using either method. Comparison of the three groups of tissues by one-way analysis of variance showed a significant increase in PI with increasing lesion severity (p < 0.001). The PI of both dysplasia and carcinoma groups was significantly higher than that of normal oral mucosa (p < 0.001). However, the difference between dysplasia and carcinoma groups was not significant. PI was not associated with tobacco or alcohol consumption. We therefore conclude that Ki-67 expression is an early marker of disease progression in the oral mucosa but, on its own, is not a good indicator of neoplastic transformation.

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The aim of this study was to test the hypotheses that (a) microvascular density (MVD) measured in histological sections of resected non-small cell lung carcinomas is an index of angiogenesis and (b) the measurement of MVD in a single block is representative of the overall MVD of the tumour. MVD was quantitated in one block per specimen of 60 lung tumours and nine normal lung tissues, and in 47 blocks taken from different regions of four tumours. Blood vessels were stained with antibody to von Willebrand Factor and MVD was quantitated using two methods: average density throughout the section (a-MVD) and density in the most vascularized area or 'hot spot' (h-MVD). Similar h-MVD values were found in tumours and in normal bronchus, whereas a-MVD was greater in the latter (P < 0.01). When 47 blocks from four tumours were analysed, inter-tumour variation was significant (P < 0.001) in spite of significant intra-tumour variation. The highest MVD value was not necessarily found in the periphery of the tumour. The four tumours were ranked into either two or four tiers according to their overall MVD. In 50 random selections of one block per tumour, the correct ranking was achieved in 68-74% of cases with the two-tier ranking and in 6-16% of cases with the four-tier ranking (h-MVD and a-MVD values respectively). These results suggest that elevated MVD values do not necessarily represent angiogenesis in non-small cell lung carcinomas. When only one block per tumour is examined, the chance of obtaining an accurate estimate of the vascularity of that tumour may be lower than 68%.

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