Transglutaminase-mediated fibronectin multimerization in lung endothelial matrix in response to TNF-alpha.

Exposure of lung endothelial monolayers to tumor necrosis factor (TNF)-alpha causes a rearrangement of the fibrillar fibronectin (FN) extracellular matrix and an increase in protein permeability. Using calf pulmonary artery endothelial cell layers, we determined whether these changes were mediated by FN multimerization due to enhanced transglutaminase activity after TNF-alpha (200 U/ml) for 18 h. Western blot analysis indicated that TNF-alpha decreased the amount of monomeric FN detected under reducing conditions.

Increased histamine release and granulocytes within the thalamus of a rat model of Wernicke's encephalopathy.

The current study examined the possible role of increased histamine release and granulocyte activity in the vascular changes that precede the onset of necrotic lesions with the thalamus of the pyrithiamine-induced thiamine deficiency (PTD) rat model of Wernicke's encephalopathy (WE). An increase in histamine release and the number of granulocytes was observed in lateral thalamus on day 9 and in medial thalamus on day 10 of PTD treatment, a duration of thiamine deficiency associated with perivascular edema in this brain region. Within the hippocampus, histamine release was significantly increased on day 9, declined to control levels on days 10-12, and was significantly elevated on days 12-14.

Increased recycling of (alpha)5(beta)1 integrins by lung endothelial cells in response to tumor necrosis factor.

Tumor necrosis factor (alpha) (TNF-(alpha) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-(alpha) may influence cell-matrix interactions by altering the clustering as well as internalization of the (&agr;)5(beta)1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-(alpha) caused an increase in the intracellular staining of (alpha)5(alpha)1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery.

Hepatic fibronectin matrix turnover in rats: involvement of the asialoglycoprotein receptor.

Fibronectin (Fn) is a major adhesive protein found in the hepatic extracellular matrix (ECM). In adult rats, the in vivo turnover of plasma Fn (pFn) incorporated into the liver ECM is relatively rapid, i.e.

A 70-kDa amino-terminal fibronectin fragment supports gelatin binding to macrophages and decreases gelatinase activity.

We previously reported that a macrophage response that increased binding to 125I-radiolabeled soluble denatured collagen (gelatin) was induced by preincubation of macrophage with a 70-kDa amino-terminal fibronectin fragment and soluble nonlabeled gelatin [S. F. Penc, F.

Circulating cellular fibronectin may be a natural ligand for the hepatic asialoglycoprotein receptor: possible pathway for fibronectin deposition and turnover in the rat liver.

It has been postulated that the in vivo removal of many plasma glycoproteins after desialylation is mediated by their interaction with a specific endocytic receptor on hepatocytes called the asialoglycoprotein receptor (ASGP-R), which is known to have a high affinity for specific carbohydrate residues, such as galactose. However, this mechanism has never been proven in vivo, nor has a naturally occurring ligand for the ASGP-R been identified. We investigated the influence of the terminal galactose residues on plasma fibronectin (pFn) on its liver deposition and turnover in adult rats, using neuraminidase to remove sialic acid residues to expose galactose residues.

Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters.

A neutral protease with an estimated Mr of about 26 kD and responsible for cleavage ofmyosin LC2 was isolated from hamster skeletal muscle. Complementary DNAs were generated by RT-PCR using total hamster muscle RNA and degenerate oligonucleotide primers based on the sequences of two internal peptides. The nucleotide sequences of the resultant cDNAs were subsequently determined and the complete amino acid sequence of the protease deduced.

ATP-MgCL2 reduces intestinal permeability during mesenteric ischemia.

ATP-MgCl2 has been demonstrated to have beneficial attributes in numerous models of organ ischemia. In this study we examined whether ATP-MgCl2 could decrease permeability in ischemic segments of rat ileum. Ileal segments (nonischemic and ischemic) from the same rat were cannulated and perfused, and the plasma to lumen clearance of 51Cr-EDTA was measured.

Surfaces modified with covalently-immobilized adhesive peptides affect fibroblast population motility.

Cell population motility and adhesion of rat skin fibroblasts were evaluated on aminophase glass modified with covalently-immobilized biologically active peptides, specifically, either arginine glycine-aspartic acid-serine (RGDS) or tyrosine-isoleucine-glycine-serine-arginine-glycine (YIGSRG). Fibroblast population motility was decreased and adhesion was increased on substrates modified with covalently immobilized RGDS peptide compared to substrates with the covalently immobilized non-adhesive peptides arginine-glycine-glutamic acid-serine and arginine-aspartic acid-glycine-serine. Fibroblast motility was not significantly changed on substrates modified with covalently-immobilized YIGSRG peptide; however, fibroblast adhesion was decreased on that substrate.

A role for a 70-kDa amino-terminal fibronectin fragment in supporting soluble gelatin interactions with rat peritoneal macrophages.

Seventy-kilodalton amino-terminal and 180-kDa cell-binding fibronectin fragments were used to determine which fibronectin domains support soluble gelatin interactions with macrophages. At each time measured, intact and 180-kDa fibronectin supported significantly larger quantities of cell-associated gelatin than control levels (P < 0.05).

Premortem biodistribution of radioactivity in the rat: measurement of blood and tissue activity of tracers used in clinical imaging studies.

Radioactive tracers are used in nuclear medicine imaging studies to detect sites of human disease. Use of animal models helps to establish tracer biodistribution kinetics and, thus, is critical to the early testing of radiopharmaceuticals. We developed a method to characterize the premortem temporal, spatial, and compartmental biodistribution of tracer molecules in the rat and used this method to study three tracers of potential value in detecting thromboembolic disease.

Vascular clearance and organ uptake of G- and F-actin in the rat.

This study comparatively evaluated the kinetics of removal and organ distribution of circulating G- and F-actin. Both F- and G-actin were cleared in two phases (fast component with a t1/2 of 3-5 min and a slow component with a t1/2 of hours). There was no effect of dose on either the fast- or slow-compartment clearance kinetics at the doses tested (5-100 micrograms/100 g body wt).

Hepatic removal of 125I-DLT gelatin after burn injury: a model of soluble collagenous debris that interacts with plasma fibronectin.

The decline of plasma fibronectin after surgery, trauma, and burn, as well as during severe sepsis after injury, appears to limit hepatic Kupffer cell phagocytic activity. Intravenous infusion of gelatin-coated particles to simulate blood-borne particulate collagenous tissue debris in the circulation after injury also depletes plasma fibronectin. We used soluble gelatin conjugated with 125I-labeled dilactitol tyramine (DLT-gelatin) as a model of soluble collagenous tissue debris.

Collagen-induced rat platelet reactivity is enhanced in whole blood in both the presence and absence of dense granule secretion.

Collagen induced aggregation, ATP secretion and thromboxane (TxB2) generation of storage pool deficient platelets were compared to normal platelets of closely related rat strains. Platelet function was monitored in citrated-platelet-rich-plasma (PRP) and citrated whole blood. Wistar (W) and fawn-hooded (FH) rat strains and their F2 hybrids were utilized.

Rebound elevation of fibronectin after tissue injury and ischemia: role of fibronectin synthesis.

Plasma fibronectin (pFn) stimulates macrophage phagocytosis of tissue debris; pFn deposition in tissues may influence vascular integrity. Although the acute depletion of pFn after surgery and/or injury has been described, less attention has been given to the rebound hyperfibronectinemia presumably "triggered" by the early pFn depletion. Using a model that compartmentalized the site of tissue injury and thus attenuated the initial pFn depletion, we studied this rebound elevation of pFn in anesthetized rats (250-350 g) after the surgical trauma of groin dissection alone (sham group) or surgery coupled with 4 h of hindlimb ischemia (experimental group).

Liver and spleen phagocytic depression after peripheral ischemia and reperfusion.

Liver and spleen phagocytic clearance of blood-borne microparticulate tissue debris and products of intravascular coagulation after trauma and surgical injury is an important mechanism to limit the deposition of debris in the pulmonary vascular bed. Plasma fibronectin (pFn) modulates this clearance process. We evaluated the effect of a localized peripheral ischemia and reperfusion injury on liver and spleen phagocytic function.

Pulmonary microvascular endothelial cells constitutively release xanthine oxidase.

The generation of oxidants in reperfused ischemic tissues by xanthine oxidase (XO) may contribute to tissue damage. We exposed bovine pulmonary microvascular endothelial (BPMVE) cells to hypoxia and subsequent reoxygenation and examined alterations in intracellular and extracellular XO activities. BPMVE cells incubated 24 h under hypoxic conditions (less than 1% O2) showed a twofold increase in intracellular xanthine dehydrogenase activity and a smaller increase in intracellular XO activity compared to normoxic BPMVE.

Incorporation of circulating fibronectin into various tissues during sepsis: colocalization with endogenous tissue fibronectin.

We studied the plasma clearance and tissue incorporation of intravenously infused purified human plasma fibronectin into various tissues during a period of acute lung vascular injury induced by lethal postoperative bacteremia in sheep. Lung, liver, spleen, and heart tissue were examined for both endogenous sheep tissue fibronectin as well as the experimentally infused human fibronectin using dual-label immunofluorescence. Awake sheep (n = 4) received a postoperative iv infusion of 5 x 10(9) live Pseudomonas over a 60-min infusion interval.

Lectin binding to gp60 decreases specific albumin binding and transport in pulmonary artery endothelial monolayers.

The effect of albumin binding to cultured bovine pulmonary artery endothelial cell (BPAEC) monolayers on the transendothelial flux of 125I-labelled bovine serum albumin (BSA) was examined to determine its possible role on albumin transcytosis. The transport of 125I-BSA tracer across BPAEC grown on gelatin- and fibronectin-coated filters (0.8 microns pore diam.

Serum albumin decreases transendothelial permeability to macromolecules.

We examined the effects of serum albumin and other serum proteins on the fluxes of tracer 125I-albumin (MW 69 kDa) and 125I-haptoglobin (MW 100 kDa) across the pulmonary artery endothelial monolayer in vitro to test the role of serum proteins in modulating the endothelial barrier function. Replacement of control complete culture medium (20% fetal calf serum in DMEM) with DMEM alone increased the transendothelial 125I-albumin clearance rate (a measure of 125I-albumin permeability) by 83% of the control value. Repletion with 50% calf serum or with 2.

Blood-borne fragments of fibronectin after thermal injury.

Fibronectin is an adhesive protein that can promote phagocytosis and endothelial cell adhesion. Plasma fibronectin declines following burn in animals and patients, potentially due to its complexing with circulating collagenous debris as well as its rapid binding to sites of tissue injury. Such depletion of fibronectin initiates an opsonic deficiency of the plasma.

High-affinity binding of fibronectin to cultured Kupffer cells.

Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique.

Increased endothelial albumin permeability mediated by protein kinase C activation.

We examined the effects of activation of endothelial protein kinase C (PKC) of the endothelial barrier function. Exposure of confluent bovine pulmonary artery endothelial cell monolayers to phorbol 12-myristate 13-acetate (PMA) resulted in concentration-dependent (10(-8)-10(-6) M) increases in PKC activity and in the transendothelial flux of 125I-albumin. Exposure of the endothelium to 1-oleoyl 2-acetyl glycerol (OAG) also increased the transendothelial flux of 125I-albumin in a concentration-dependent manner.

Blood-borne collagenous debris complexes with plasma fibronectin after thermal injury.

Plasma fibronectin augments the clearance of blood-borne foreign and effete complexes by mononuclear phagocytes. The release of a "gelatin-like" ligand into plasma after thermal injury has been reported. We quantified the release of this collagenous debris from thermally injured skin, and its potential interaction with soluble fibronectin in plasma using anesthetized rats.

Determination of Kupffer cell Fc receptor function in vivo following injury.

This study was carried out to determine whether Kupffer cell Fc receptor function is depressed after injury. Three approaches to the determination of Fc receptor function were evaluated: IgG-coated erythrocytes (EIgG) were used as the receptor probe with a perfused liver system, EIgG were used as the receptor probe in vivo, and small aggregates of IgG (AIgG) were used as the receptor probe in vivo. Nearly half of the injected dose of EIgG was taken up by the perfused liver (nonrecirculating, serum-free system).