Initiation of reepithelialization upon wounding is still poorly understood. To enhance this understanding, we focus here on changes in the adhesive state of desmosomes of cultured Madin-Darby canine kidney cells in response to wounding of confluent cell sheets. Previous results show that desmosomal adhesion in Madin-Darby canine kidney cells changes from a calcium-dependent state to calcium independence in confluent cell sheets. We show that this change, which requires culture confluence to develop, is rapidly reversed upon wounding of confluent cell sheets. Moreover, the change to calcium dependence in wound edge cells is propagated to cells hundreds of micrometers away from the wound edge. Rapid transition from calcium independence to calcium dependence also occurs when cells are treated with phorbol esters that activate PKC. PKC inhibitors, including the conventional isoform inhibitor Gö 6976, cause rapid transition from calcium dependence to calcium independence, even in subconfluent cells. The cellular location of the ␣ isoform of PKC correlates with the calcium dependence of desmosomes. Upon monolayer wounding, PKC␣ translocates rapidly to the cell periphery, becomes Triton X-100 insoluble, and also becomes concentrated in lamellipodia. The PKC␣ translocation upon wounding precedes both the increase in PKC activity in the membrane fraction and the reversion of desmosomes to calcium dependence. Specific depletion of PKC␣ with an antisense oligonucleotide increases the number of cells with calcium-independent desmosomes. These results show that PKC␣ participates in a novel signaling pathway that modulates desmosomal adhesion in response to wounding.

The retinal vasculature of postmortem normal human and diabetic eyes was studied using an immunohistochemical technique in conjunction with confocal laser scanning microscopy. The technique, which stained for von Willebrand factor, allowed both large areas of the retinal vasculature to be visualised and abnormalities to be studied in detail without disturbing the tissue architecture. Only one microaneurysm, defined as any focal capillary dilation, was observed in 10 normal eyes but numerous microaneurysms were seen in 4 out of 5 diabetic retinas ; counts varied between 0 and 26 per 0.41 mm# sample area. Microaneurysms were classified into 3 categories according to morphology : saccular, fusiform and focal bulges. Most were saccular, these having no preferred orientation. The majority of microaneurysms were associated with just 2 vessels suggesting they were unlikely to develop at vascular junctions. The majority were observed to originate from the inner nuclear layer and were therefore in the deeper part of the inner retinal capillary plexus. Variation in the staining of microaneurysms may correlate with endothelial dysfunction seen clinically as dye leakage during fluorescein angiography.

Platelets are a major source of transforming growth factor-beta1 (TGF-beta1) in the circulation as they release latent growth factor in response to activation. We report here that human platelets, when stimulated with thrombin, activated a significant proportion of the latent TGF-beta released. Latent TGF-beta activation was independent of cytokine release, since activation was delayed compared to platelet degranulation. Activation occured in releasates and did not require the continuous presence of platelets. Classical mechanisms of latent TGF-beta activation were not involved, since activation was not affected by gene deletion and/or inhibitors of the known TGF-beta activators/co-factors, thrombospondin-1 (TSP-1), mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR), plasminogen/plasmin, or several other candidate proteases. In contrast, latent TGF-beta activation was significantly inhibited by the furin inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone and L-hexaarginine. We show that platelets contain a furin-like enzyme which is released upon platelet activation. We conclude that, following activation, platelets release and activate latent TGF-beta1 via mechanisms involving the release and activity of a furin-like proprotein convertase. This novel mechanism of latent TGF-beta activation might represent an important mediator and therapeutic target of platelet TGF-beta1 functions, for example, in early wound repair, fibrosis, or arteriosclerosis.

In humans, the greater omentum is a fatty peritoneal fold that extends from the greater curvature of the stomach to cover most abdominal organs. It performs many functions, which include acting as a reservoir of resident peritoneal inflammatory cells, a storage site for lipid, and a regulator of fluid exchange in and out of the peritoneal cavity. Most importantly, the omentum readily adheres to areas of inflammation and peritoneal damage, often leading to adhesion formation. Despite its clinical importance, the omentum remains an understudied organ, and discrepancies exist as to its exact morphology. This study uses a combination of phase contrast microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) to elucidate the structure of the greater omentum of both human and mouse and determine whether it possesses a typical surface mesothelial cell lining similar to other serosa. Results indicated that both human and murine omenta were of similar structure and composed of two distinct types of tissue, one adipose-rich and the other translucent and membranous. The adipose-rich regions were well-vascularised and covered by a continuous mesothelial cell layer except at the sites of milky spots. In contrast, translucent areas were poorly vascularised and contained numerous fenestrations of varying size. The possible function and developmental origin of these gaps is unclear; however, their role in promoting omental adhesion formation and in the successful use of omental graft material is discussed.

The formation of the endoderm during primitive streak stages in avian embryos was studied by combining several of the following techniques for each embryo. These included microsurgery, time-lapse filming, use of chick-quail chimaeras, tritiated thymidine autoradiography and a novel technique for identifying the morphology of the cells after small pieces of tissue from known areas had been maintained in culture for 24 h. Using these techniques we have confirmed that the ventral layer of the early chick embryo receives contributions from both the marginal and the central regions of the area pellucida. The former seems to consist of yolky cells derived from the germ wall, whilst the latter consists of smaller, less yolky cells derived from the more dorsal layers of the embryo. The movement of the lower layer anteriorly during these stages appears to be dependent upon mechanical constraints imposed upon it by the expanding tissue in more caudal regions. The extent of each of the two contributions to the lower layer was determined as a function of stage and presence or absence of a lower layer, and the findings are discussed in the light of the existing literature.

Chronic venous ulcers, an example of abnormal wound healing, show chronic inflammation with defective matrix deposition which together with the underlying vascular pathology, result in delayed healing. L-arginine is known to be metabolized by one of two pathways: nitric oxide synthase (NOS), producing nitric oxide (NO), or arginase, producing ornithine. NO is involved in many pathological conditions including vascular and inflammatory disorders. This study therefore investigated the distribution, level and activity of NOS and arginase in chronic venous ulcers in comparison with normal skin, using immunocytochemistry, western blotting, and enzyme assays. The results demonstrated an increased distribution of both NOS and arginase in chronic venous ulcer tissue compared with normal skin, with inflammatory cells and vascular endothelial cells as the main sources. These data were confirmed by western blot analysis, which showed increased levels of both enzymes in chronic venous ulcers. Moreover, there was significantly increased activity of both total NOS (p<0.04) and inducible NOS (p<0.05) in chronic venous ulcer tissue compared with normal skin, and significantly increased activity of arginase (p<0.01) in chronic venous ulcer tissue in comparison with normal skin. NO is known to combine with hydroxyl free radicals forming peroxynitrite, a potent free radical which causes tissue destruction. NO overexpression in chronic venous ulcers may be involved directly or indirectly (through production of peroxynitrite) in the pathogenesis and delayed healing of chronic venous ulcers, through its effects on vasculature, inflammation, and collagen deposition. Arginase is known to enhance matrix deposition. Thus, increased levels of arginase in chronic venous ulcers could contribute to the pathogenesis of lipodermatosclerosis associated with chronic venous insufficiency, predisposing to the formation of chronic venous ulcers and also to matrix cuff formation around blood vessels.

Chronic venous ulcers are an example of abnormal wound healing showing chronic inflammation which together with the underlying vascular pathology results in delayed healing. Prostaglandins are among the most important mediators of inflammation. They have proinflammatory effects, predominantly by affecting the vasculature. Cyclooxygenase (COX) is the rate-limiting enzyme in prostanoid synthesis. It is present in two isoforms: COX-1 (constitutive cyclooxygenase) which is produced in the body to maintain normal haemostatic functions, and COX-2 (inducible cyclooxygenase), which is induced during inflammation in response to cytokines. Using immunoenzymatic labelling and western blot analysis, this study has shown that both COX-1 and COX-2 were up-regulated in chronic venous leg ulcers by comparison with normal human skin. De novo appearance of COX-2 in chronic venous ulcers was demonstrated, which is not seen in normal human skin. The main cellular sources of both COX isoforms are macrophages and endothelial cells. COX-2 is also produced by mast cells and fibroblasts. A COX radioimmunoassay showed up-regulation of COX activity in chronic venous ulcers compared with normal skin (p<0.05). Up-regulation of COX-1 in chronic venous leg ulcers could produce prostacyclin, which contributes to angiogenesis. Thus, inhibition of COX-1 by non-steroidal anti-inflammatory drugs (NSAIDs) could increase the local ischaemia and hypoxia associated with chronic venous ulcers. On the other hand, up-regulation of COX-2 is most likely responsible for the persistent inflammation in chronic venous leg ulcers. COX-2 selective inhibitors could therefore be effective in the treatment of chronic venous ulcers.

Previous immunocytochemical analysis showed that the base of venous ulcers was deficient in fibronectin compared with surrounding "normal" dermis. Here, we investigate whether impaired synthetic ability of ulcer fibroblasts could underlie this observation. Ulcer fibroblasts, established in culture from biopsies of the edge of chronic venous leg ulcers, were compared with normal fibroblasts grown from biopsies of site-and age-matched normal skin for their ability to synthesize matrix molecules. Collagen and fibronectin synthesis were measured following metabolic labeling, as collagenase susceptible counts and counts with gelatin affinity, respectively. More collagen was produced by normal fibroblasts than ulcer fibroblasts, both when the cells were cultured on plastic and in collagen gels. In fibronectin synthesis, however, there was no major difference between the two cell types on either substratum. The hypoxic environment to which ulcer fibroblasts are exposed may have caused the intrinsic differences in collagen synthesis by the two fibroblast types. When we tested the effect of culturing cells under hypoxic conditions, both cell types produced less collagen, especially normal fibroblasts grown in a collagen gel, but there was no effect of hypoxia on fibronectin synthesis. We conclude that venous ulcer edge-derived fibroblasts have an impaired ability to synthesize collagen in vitro, but synthesize fibronectin normally. Therefore, the low level of fibronectin found in venous ulcers is not likely to be due to the inability of ulcer cells to produce it or to the response to hypoxic conditions but may be due to the degradation of synthesized fibronectin by proteases present in these ulcers.