Interactions of thrombospondin with extracellular matrix proteins: selective binding to type V collagen.

Thrombospondin (TS), a protein first described in platelets, was recently shown to be synthesized and secreted by endothelial cells, fibroblasts, and smooth muscle cells. The presence of TS in the extracellular matrix of cultured cells has prompted us to examine the associations of this protein with matrix macromolecules. Interactions of TS with both matrix and serum proteins were tested using an enzyme-linked immunosorbent assay. With this assay we assessed the binding of TS in solution to proteins adsorbed to polystyrene microtiter plates. Among collagens, platelet TS bound to type V but not to types I, III, or IV. This selective interaction was confirmed in experiments using proteins linked to cyanogen bromide-activated Sepharose. TS released from platelets in response to thrombin activation, as well as that secreted by endothelial cells in culture, bound to type V but not to type I collagen-Sepharose. No binding was observed to denatured type V collagen-Sepharose. The binding region for type V collagen was located in a chymotrypsin-produced fragment of TS with chains of Mr = 70,000, after reduction. Interactions of TS with a number of other proteins, including fibronectin, fibrinogen, and laminin, could be demonstrated using the enzyme-linked immunosorbent assay technique but the interpretation of these findings is difficult since comparable binding to protein-Sepharose was not always observed. Our findings suggest that both the extravascular distribution and function of TS in vivo may involve an interaction with type V collagen.

Thrombospondin: synthesis and secretion by cells in culture.

Thrombospondin, a high molecular weight glycoprotein secreted by platelets in response to activation by thrombin, has been identified by immunofluorescence in bovine aortic endothelial cells, human foreskin fibroblasts, and human aortic smooth muscle cells. Immunofluorescence patterns were found to be similar using antisera raised to thrombospondins purified either from bovine aortic endothelial cells or from human platelets. Radioimmune precipitation of pulse-labeled cellular proteins confirmed the presence of thrombospondin in positively stained cells. A sensitive quantitative enzyme-linked immunosorbent assay (ELISA) was developed and used to determine that the accumulation of secreted thrombospondin was similar for endothelial cells and fibroblasts but was higher for smooth muscle cells. The presence of thrombospondin in a variety of cells suggests that its function may not be limited to an involvement in platelet interactions.

Relative contribution of leukotriene B4 to the neutrophil chemotactic activity produced by the resident human alveolar macrophage.

Human alveolar macrophages release chemotactic activity for neutrophils, providing a role for alveolar macrophages in regulating inflammation in the lung. As alveolar macrophages produce large amounts of leukotriene B4 (LTB4), a chemotactically active lipoxygenase product of arachidonic acid, we investigated the contribution of LTB4 to the total neutrophil chemotactic activity produced by these cells. Normal human alveolar macrophages were recovered by bronchoalveolar lavage from healthy volunteers and incubated either with the calcium ionophore A23187 for 1 h, or with opsonized zymosan particles or latex beads for 3 h. Nordihydroguaretic acid (NDGA), a relatively specific lipoxygenase inhibitor, blocked the release of neutrophil chemotactic activity after all three stimuli in a dose-dependent manner. This correlated with blockade of LTB4 production as measured by high performance liquid chromatography using freshly isolated alveolar macrophages, as well as blockade of [3H]LTB4 production by macrophages prelabeled with [3H]arachidonate. Molecular sieve chromatography using Sephadex G-50 confirmed that essentially all of the chemotactic activity in the stimulated macrophage supernatants co-eluted with authentic [3H]LTB4, and that NDGA completely blocked the chemotactic activity in the eluting fractions. Readdition of authentic LTB4 (1 X 10(-7) M) to the NDGA-blocked macrophage supernatants restored the chemotactic activity in the supernatants. The macrophage supernatants did not contain platelet-activating factor-like activity, as measured by the stimulation of [3H]serotonin release from rabbit platelets, and by high performance liquid chromatography. NDGA did not change the protein-secretion profiles of fresh alveolar macrophages, or of macrophages prelabeled with [35S]methionine. The complement (C) components C5adesarg were not detected in any of the supernatants by radioimmunoassay. Concentration of the supernatants by positive pressure filtration (5,000-D membrane) did not augment chemotactic activity in the stimulated supernatants or uncover chemotactic activity in the NDGA-blocked supernatants. As with the 3-h studies, when alveolar macrophages were incubated overnight with opsonized zymosan, all of the increase in chemotactic activity could also be blocked by NDGA. These data indicate that LTB4 is the predominant neutrophil chemotactic factor secreted by the normal resident human alveolar macrophage in response to two major types of stimuli, calcium fluxes across the cell membrane and the phagocytosis of opsonized particulates.

Phenotypic expression of collagen types in mesangial matrix of diabetic and nondiabetic rats.

This study was performed to evaluate the composition of the extracellular matrix of the mesangium in both diabetic and nondiabetic rats. Four groups of rats (n = 10 each) were studied. Nondiabetic rats were injected with saline (group 1) or insulin (3.5 U NPH daily) (group 2). Streptozocin-induced diabetic rats were similarly injected with saline (group 3) or insulin (group 4). Six weeks after initiation of study, glomerular diameter (micron) was increased in groups 2 (147 +/- 21), 3 (144 +/- 22), and 4 (150 +/- 7) compared with group 1 (104 +/- 12) (P less than .01). Glomerular hypertrophy was associated with an increase in the relative amount of mesangial matrix as determined by staining for fibronectin. By immunofluorescence microscopy (0-4+ scale), type I collagen antigen was not detected in the mesangium of any of the experimental groups. Staining for type V collagen and thrombospondin was similar between the experimental groups. Type III collagen antigen was not detected in the mesangium of control (group 1) or insulin-deficient diabetic rats (group 3); however, it was detected (2-3+) in the mesangium of both insulin-treated diabetic and nondiabetic rats (Mann-Whitney, P less than .01). Comparable intensity of staining (1+) for type IV collagen antigen was detected in the mesangium of animals from groups 1, 2, and 4; however, the staining intensity was markedly increased (3+) in insulin-deficient diabetic rats (group 3; Mann-Whitney, P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombospondin in early human wound tissue.

We examined partial thickness incised human wounds of 2, 3, 5, 7, and 14 days of age for the presence of thrombospondin by immunostaining and light microscopy. At 2, 3, 5, and 7 days after wounding, thrombospondin is present primarily at the cut edges of the lateral and deep margins of the wound. It appears to be cleared from these extracellular matrix sites, and is no longer detectable in those sites in most 14-day-old wounds. Thrombospondin staining is present, however, in increased amounts around the vascular channels within and adjacent to the 7- and 14-day wounds in increased amounts relative to vascular channels distant from the wound. Our observations are consistent with known in vitro data regarding the binding of thrombospondin to fibrin and components of the extracellular matrix, as well as with data showing that proliferating endothelial cells secrete more thrombospondin than quiescent endothelial cells. These data support the hypothesis that thrombospondin plays a role in the early organization of the extracellular matrix of wounds.

The repertoire of T cell antigen receptor beta-chain variable regions associated with psoriasis vulgaris.

We investigated whether the pattern of T-cell receptors expressed by T cells in inflamed psoriatic skin differed substantially from the pattern seen in T cells from the peripheral blood. A bias or restriction in the repertoire of T-cell receptors found in the lesional skin of different patients might imply that specific subsets of T cells were causally associated with initiating or maintaining the lesions. By using a polymerase chain reaction-based assay of T-cell receptor beta-chain variable region mRNA, we found that the patterns of beta-chain mRNAs displayed in 14 samples of lesional skin or six samples of noninvolved skin were not significantly less diverse than the patterns found in matched peripheral blood samples. There was no evidence that the active lesions of multiple patients showed overexpression of T cells expressing one or a few T-cell receptor forms. The pattern of T-cell receptors displayed in clinically normal skin from normal control individuals showed about the same diversity as normal blood. While these results may not exclude either classical antigen or superantigen-based T-cell activation mechanisms in active plaques, the absence of a simple pattern of Vbeta usage in different patients suggests than other aspects of T-cell biology including trafficking, proliferation, co-stimulation, or responses to cytokines must also be considered.

Insulin and insulin-like growth factor I binding to cultured rat glomerular mesangial cells.

The potential effects of insulin and insulin-like growth factor I (IGF-I) on mesangial cell (MC) metabolism and growth were examined. Radiolabeled insulin or IGF-I were incubated with cell membranes from rapidly proliferating (subconfluent) or nonproliferating (confluent) MC in the presence of increasing concentrations of unlabeled heterologous and homologous ligands (0-10(-6) M). Insulin binding to MC was specific and saturable, with Scatchard analysis of binding data showing the characteristic curvilinear plot. The predicted insulin binding maximum of 4.2 X 10(-12) M/100 micrograms protein for a theoretical high affinity site was consistent with a relatively low density of receptors, which were the same in proliferating and nonproliferating cell preparations. Specific binding of IGF-I to MC was also demonstrated. Binding data for membranes from proliferating cultures generated a linear Scatchard plot, which predicted a binding maximum of 3.5-9.7 X 10(-11) M/100 micrograms protein and a Kd of 2.0-3.2 X 10(-9) M. In contrast, membranes from nonproliferating cultures had no demonstrable specific binding of IGF-I. Covalent cross-linking of radiolabeled IGF-I to membranes from subconfluent cells demonstrated specific binding to a 145K membrane protein. A 95K membrane protein from a partially purified receptor preparation demonstrated autophosphorylation when incubated with 5 X 10(-9) M IGF-I. Incubation of MC with 10(-9) M IGF-I doubled cellular growth rates, an effect that could be duplicated only with high concentrations (10(-6) M) of insulin. These observations indicate that MC express predominantly receptors for IGF-I, and that growth stimulatory effects of physiological concentrations of IGF-I and pharmacological concentrations of insulin are probably mediated through the IGF-I receptor.

Insulin induces rapid and specific rearrangement of the cytoskeleton of rat mesangial cells in vitro.

Mesangial cells (MCs) grown without supplemental insulin (SI-MCs) express a quiescent phenotype and extracellular matrix (ECM) composition similar to MCs in vivo. In contrast, MCs routinely propagated in insulin (SI+MCs) are stimulated to proliferate, change their phenotype, and produce large amounts of collagens I and III. These effects of insulin may in part be mediated through cytoskeletal rearrangement. Differences in cytoskeletal arrangement were compared between SI-MCs and SI+MCs and 1 hr after addition of insulin (1 nM) or IGF-1 (100 nM) to SI-MCs. Cells were examined by light microscopy, electron microscopy, and immunostaining for specific cytoskeletal proteins and fibronectin. Insulin induced rapid rearrangement of stress fibers. Surface ruffling, actin aggregation, vimentin retraction, rearrangement of vinculin in focal adhesions, and fibronectin extraction were apparent. These direct effects of insulin on the SI-MC cytoskeleton occurred before insulin-induced changes in ECM composition. IGF-I induced cytoskeletal reorganization distinct from insulin. These observations demonstrate that insulin and IGF-I have unique effects on the MC cytoskeleton, which is turn may mediate secondary ligand effects on MCs.

Light microscopic immunolocation of thrombospondin in human tissues.

Affinity-purified antisera against thrombospondin were used to locate the presence of this glycoprotein in frozen sections of several human tissues by immunofluorescence techniques. Immunostaining was observed in the peritubular connective tissue and in basement membrane regions beneath glandular epithelium in skin and lung. Intense immunostaining was observed at the dermal-epidermal junction in skin and in small blood vessels throughout this tissue. Skeletal muscle exhibited positive staining with anti-thrombospondin antisera within interstitial areas. Immunostaining was confined to the luminal portions of large blood vessels such as aorta. In large blood vessels that contained lesions of atherosclerosis, immunostaining was observed throughout the lesion area and was especially prominent surrounding some of the lesion cells. These results indicate that thrombospondin is located within the matrix of a variety of human tissues and supports the suggestion that this glycoprotein is an endogenous component of some extracellular matrices.

Necrotizing sialometaplasia. A condition simulating malignancy.

Necrotizing sialometaplasia occurred in a 55-year-old woman. The ulcerated lesion on the hard palate was treated conservatively and resolved spontaneously in about three months. The histology of the lesion consisted of coagulative necrosis of the salivary gland lobules and prominent squamous metaplasia within adjacent viable lobules. Since this benign lesion has frequently been mistaken for mucoepidermoid carcinoma or squamous cell carcinoma, recognition of it may spare the patient a radical surgical procedure.

Dermatologic features of the superior vena cava syndrome.

BACKGROUND: The superior vena cava syndrome occurs when extrinsic compression or intraluminal occlusion impedes blood flow through this vessel. The most common underlying cause is a malignant neoplasm, especially a bronchogenic carcinoma. This article describes the cutaneous findings of this disorder. OBSERVATIONS: Among the earliest and most prominent features are numerous, dilated, vertically oriented, and tortuous cutaneous venules or veins above the rib cage margin. Recognition of this cutaneous sign allowed us to make a diagnosis of lung cancer in several patients. Other features include upper body edema and ruddiness or cyanosis, distended neck veins, proptosis, and conjunctival suffusion. CONCLUSIONS: Detecting the characteristic cutaneous features can lead to an early diagnosis of the superior vena cava syndrome. These skin changes usually represent indirect dermatologic signs of an underlying malignant neoplasm; for most patients, this syndrome is the initial manifestation of their cancer. The most common cause is a bronchogenic carcinoma, especially the small-cell variety, but others include lymphomas, primary mediastinal tumors, and metastases to the mediastinal lymph nodes from extrathoracic primary tumors, especially breast cancer. Treatment of the underlying malignant neoplasm and relief of the obstruction produce prompt improvement in the dermatologic findings.

Location and partial characterization of the heparin-binding fragment of platelet thrombospondin.

Purified platelet thrombospondin (TS) was subjected to proteolysis with a number of proteases including factors IXa, Xa, thrombin, elastase, trypsin, and chymotrypsin. All enzymes yielded fragments of TS which bound to heparin-Sepharose. Only chymotrypsin cleavage produced a single species of heparin-binding fragment, as analyzed by SDS-PAGE. This fragment had a chain molecular weight of 28,000, and contained no interchain disulfide bonds. Amino acid sequence analysis of the heparin-binding fragment and of TS revealed a single sequence, indicating that the fragment constitutes the amino-terminal domain of TS and that the three chains in TS are identical in this region.

Issues in validation of a dermatologic surgery simulator.

At the University of Washington, we have been developing a suturing simulator using novel finite element model techniques which allow real-time haptic feedback. The issues involved in measuring validity in a suturing model have not been examined in a systematic way. Very few studies exist on the surgical factors that lead to good sutures. We have examined published data on these factors as well as previously studied metrics in suture training. This information has been combined with a review of types of validity (e.g., face, construct, predictive and concurrent) and reliability that must be considered in assessing any surgical simulator.

Fast finite element modeling for surgical simulation.

Given the geometric complexity of anatomical structures, realistic real-time deformation of graphical reconstructions is prohibitively computationally intensive. Instead, real-time deformation of virtual anatomy is roughly approximated through simpler methodologies. Since the graphical interpolations and simple spring models commonly used in these simulations are not based on the biomechanical properties of tissue structures, these "quick and dirty" methods typically do not accurately represent the complex deformations and force-feedback interactions that can take place during surgery. Finite element (FE) analysis is widely regarded as the most appropriate alternative to these methods. Extensive research has been directed toward applying the method to modeling a wide range of biological structures, and a few simple FE models have been incorporated into surgical simulations. However, because of the highly computational nature of the FE method, its direct application to real-time force-feedback and visualization of tissue deformation has not been practical for most simulations. This limitation is primarily due to the overabundance of information provided by the standard FE approaches. If the mathematics is optimized to yield only the information essential for the surgical task, computation time can be drastically reduced. Parallel computation and preprocessing of the model before the simulation begins can also reduce the size of the problem and greatly increase computation speed. Such methodologies are being developed in a combined effort between the Human Interface Technology Laboratory (HIT Lab) and the Mechanical Engineering Department of the University of Washington. We have created computer demonstrations which support real-time interaction with simple finite element soft tissue models. In collaboration with the Division of Dermatology, a real-time skin surgery simulator is being developed using these fast FE methods.

Creating fast finite element models from medical images.

The procedure for creating a patient-specific virtual tissue model with finite element (FE) based haptic (force) feedback varies substantially from that which is required for generating a typical volumetric model. In addition to extracting geometrical and texture map data to provide visual realism, it is necessary to obtain information for supporting a FE model. Among many differences, FE-based VR environments require a FE model with appropriate material properties assigned. The FE equation must also be processed in a manner specific to the surgical task in order to maximize deformation and haptic computation speed. We are currently developing methodologies and support software for creating patient-specific models from medical images. The steps for creating such a model are as follows: 1) obtain medical images and texture maps of tissue structures; 2) extract tissue structure contours; 3) generate a 3D mesh from the tissue structure contours; 4) alter mesh based on simulation objectives; 5) assign material properties, boundary nodes and texture maps; 6) generate a fast (or real-time) FE model; and 7) support the tissue models with task-specific tools and training aids. This paper will elaborate on the above steps with particular reference to the creation of suturing simulation software, which will also be described.

Pustular tinea pedis.

Pustules are uncommon in tinea pedis and may suggest a bacterial infection. We describe a patient with large pustules on his feet that contained hyphae on Gram's stain of the pus and on a potassium hydroxide preparation of the pustule roof. Cultures were negative for bacteria, but grew Trichophyton rubrum.

Adult scurvy.

UNLABELLED: Unlike most animals, which form ascorbic acid by metabolizing glucose, humans require an exogenous source. Vitamin C occurs primarily in fruits and vegetables, and scurvy develops from inadequate consumption of these sources, usually because of ignorance about proper nutrition, psychiatric disorders, alcoholism, or social isolation. The earliest symptom of scurvy, occurring only after many weeks of deficient intake, is fatigue. The most common cutaneous findings are follicular hyperkeratosis, perifollicular hemorrhages, ecchymoses, xerosis, leg edema, poor wound healing, and bent or coiled body hairs. Gum abnormalities, which occur only in patients with teeth, include gingival swelling, purplish discoloration, and hemorrhages. Pain in the back and joints is common, sometimes accompanied by obvious hemorrhage into the soft tissue and joints. Syncope and sudden death may occur. Anemia is frequent, leukopenia occasional. Treatment with vitamin C results in rapid, often dramatic, improvement. (J Am Acad Dermatol 1999;41:895-906.) LEARNING OBJECTIVE: At the conclusion of this learning activity, participants should be familiar with the history, pathogenesis, clinical features, and treatment of scurvy in adults.