Seth L. Schor

The association between tumour progression and vascularity in the oral mucosa.

Tumourigenesis in experimental models is associated with the formation of new blood vessels (angiogenesis). Recent studies have suggested that tumour angiogenic activity may be inferred in histological sections by measuring the density of the vasculature. The purpose of this study was to determine whether the transition from normal to dysplastic and neoplastic tissue in the oral mucosa is accompanied by quantitative or qualitative changes in the vascularity of the tissue, and how the estimate of vascularity is influenced by the vessel marker and method of assessment. A total of 100 specimens of normal oral mucosa, dysplastic lesions, and squamous cell carcinomas were examined. Sections were immunostained with the pan‐endothelial antibodies to von Willebrand Factor (vWF) and CD31, or with an antibody to the αvβ3 integrin, previously reported to be a marker of angiogenic vessels. Vascularity was quantitated by two different methods: highest microvascular density (h‐MVD) and microvascular volume, as determined by point counting (MVV). The results showed that vascularity, measured by the MVV method using antibodies to either vWF or CD31, increased significantly (P<0·0001) with disease progression from normal oral mucosa, through mild, moderate, and severe dysplasia to early and late carcinoma (76 paraffin‐embedded tissues examined). In contrast, h‐MVD did not discriminate between dysplastic lesions and carcinoma. A similar percentage of the total vessel volume (MVV) and density (h‐MVD) were positive for αvβ3 in 24 frozen tissues examined, including normal oral mucosa. It is concluded that there is a close association between vascularity and tumour progression in the oral mucosa. Morphometric analysis reflecting microvascular volume is more informative than the currently popular analysis of microvascular density. The expression of αvβ3 in the vasculature of oral tissues does not necessarily reflect the presence of angiogenic vessels.

Apoptosis, proliferation, and angiogenesis in oral tissues. Possible relevance to tumour progression

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. Copyright

The contraction of collagen matrices by dermal fibroblasts

Floating collagen gel cultures containing human foreskin fibroblasts have been observed to undergo a rapid contraction process. The initial rate of contraction (i.e., within the first 2 hr) was observed to be a linear function of cell number within the concentration range of 10(5)-10(6) cells/gel. Observation of thick, deresined sections of such contracting gels in the SEM, as well as observation of thin sections in the TEM, suggest that the fibroblasts exert a tension upon the surrounding collagen fibers. These observations further indicate that the fibroblasts migrate from the interior regions of the gel matrix and eventually form a monolayer of cells encapsulating the contracted collagen disc. These observations are discussed in terms of the possible mechanisms involved in gel contraction.

Motogenic Sites in Human Fibronectin Are Masked by Long Range Interactions

Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains 4–5FnI and 3FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes 3FnIII and disrupts the observed 4–5FnI-3FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites.

The Role of the Fibronectin IGD Motif in Stimulating Fibroblast Migration

The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 106 times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN.

The association between epithelial proliferation and disease progression in the oral mucosa

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.

The synthesis of subendothelial matrix by bovine aortic endothelial cells in culture

Bovine aortic endothelial cells cultured on collagenous or plastic substrata continuously synthesize and deposit a subendothelial matrix, independently of whether the cells are in the logarithmic or the stationary phase of growth. This subendothelial matrix contains fibrillar and amorphous elements comparable with those observed in the subendothelium in vivo. Deposition of subendothelial matrix on a collagen gel substratum both started earlier and progressed at approximately double the rate than that on denatured collagen. The relative composition of the subendothelial matrix was assessed by sequential incubation with trypsin, elastase and collagenase (Jones et al., 1979). The subendothelial matrix deposited on collagen gels by early confluent cultures and late post-confluent cultures differed in their enzyme sensitivity. These age-related changes in the enzyme sensitivity of the subendothelial matrix were characteristic for each cloned cell population examined. Comparable variations in the composition of the subendothelial matrix were not observed when the cells were cultured on plastic or gelatin-coated dishes; the subendothelial matrix deposited on these two substrata contained considerably more trypsin-sensitive material and less elastase and collagenase-sensitive material than the matrix deposited on native collagen gels. Age-related changes in the enzyme sensitivity of the subendothelial matrix deposited on collagen gels was found to be a function of the time elapsed since confluence and it was not related to the time elapsed since plating or to the number of cells present.

The effects of a collagenous extracellular matrix on fibroblast membrane organization: An ESR spin label study☆

Abstract Human skin fibroblasts, both in suspension and cultured within a three-dimensional collagen matrix have been examined by electron spin resonance ESR using the probe 5-doxyl stearic acid. The order of the plasma membrane was found to be strongly influenced by the collagen matrix, being greater for cells within the collagen gel than in suspension. The collagen cultures used in this study were either left attached to the walls of the plastic culture dish (‘attached’ gels) or dislodged and allowed to float freely in the culture medium (‘floating’ gels). Membrane order increased with time in attached gels, reaching a steady value after 2–3 h. A further increase in order was observed when floating gels were prepared 24 h later. Cell morphology within the collagen gel culture was observed to vary considerably, with time and mode of culture. Increased order, over that observed in suspension, was also found for cells attached to other substrata. The data indicate that the increase in membrane order observed in cells embedded within a three-dimensional collagen gel matrix compared with cells in suspension does not correlate with a particular cell morphology in the gel, but rather appears to result from the establishment of adhesive interactions with the surrounding collagen fibres.

Expression of Vascular Endothelial Growth Factor in Normal and Tumour Oral Tissues Assessed with Different Antibodies

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.

Fetal-like fibroblasts: their production of migration-stimulating factor and role in tumor progression.

Cancer pathogenesis is a multistep process involving the occurrence of an initiating genetic lesion and a series of subsequent events collectively referred to as progression [1,2]. In spite of the general acceptance of this model, little information is currently available regarding either the frequency at which initiating genetic lesions occur or the proportion of initiated cells that proceed on to develop into a clinically recognizable malignancy. Our assessment of the literature suggests that initiating events may be a relatively common occurrence and that only a small proportion of these cells actually go on to produce clinical tumors. Seen in this context, the various events that contribute to tumor progression play a critical role in determining the kinetics of disease development and may thereby furnish a potential target for intervention in individuals believed to be at elevated risk. Recent studies have indicated that the gradual acquisition during tumor progression of more ‘aggressive’ phenotypic characteristics, such as diminished growth control [3], local invasion [4], and metastasis [5], also involve the accumulation of genetic lesions within the emerging population of (pre-) neoplastic cells. The primary role of genetic lesions in the process of cancer development is further suggested by the occurrence of well-defined hereditary syndromes characterized by increased susceptibility to specific types of cancer (e.g., carcinoma of the breast) [6]. The identification and mapping of the relevant genes is currently being pursued with great vigor [7].