Effects of dystrophin isoforms on signal transduction through neural retina: genotype-phenotype analysis of duchenne muscular dystrophy mouse mutants.

Duchenne and Becker muscular dystrophy patients have mutations in the dystrophin gene. Most show reduced b-wave amplitudes in the dark-adapted electroretinogram (ERG). We studied normal C57BL/6J mice and five X-linked muscular dystrophy strains with different dystrophin mutations to determine whether the location of the mutation within the gene affects the mouse ERG and to correlate such effects with dystrophin isoform expression. Amplitudes and implicit times were measured for a-waves, b-waves, and digitally filtered oscillatory potentials. mdx and mdxCv5 mice, with mutations near the amino terminus and lacking expression of Dp427, had ERGs similar to those of C57BL/6J mice. mdxCv2 and mdxCv4 mice, with mutations in the center of dystrophin and who do not express isoforms Dp427, Dp260, or Dp140 (mdxCv4), had increased b-wave and oscillatory potential implicit times. mdxCv3 mice, with a mutation near the carboxy terminus resulting in deficiency of all dystrophin isoforms, had increased b-wave and oscillatory potential implicit times and reduced scotopic b-wave amplitudes. Fitting the a-wave data to a transduction activation phase mathematical model showed normal responses for all phenotypes, suggesting that the b-wave delays are due to defects beyond the rod outer segment, most likely at the rod to on-bipolar cell synapse. The variation in the ERG phenotype with the position of the dystrophin gene mutation suggests that there are different contributions by each isoform to retinal electrophysiology. Although Dp427 and Dp140 isoforms do not appear to be important contributors to the ERG, lack of Dp260 and possibly Dp71 isoforms is associated with an abnormal ERG.

Expression of basic fibroblast growth factor in nasal polyps.

Basic fibroblast growth factor (bFGF) is a polypeptide that is mitogenic for a wide variety of cell types. We used Northern blot analysis and immunohistochemistry to determine if bFGF is expressed in the nasal polyp tissue; bFGF messenger RNA was detectable in the polyps examined by Northern blot analysis. Strong immunostaining for bFGF was found in blood vessels and along the basement membrane of the epithelial cell layers. Basal epithelial cells and some infiltrating mononuclear cells also stained for bFGF. Proliferating cell nuclear antigen colocalized with bFGF to basal epithelial cells, endothelial cells, and areas of focal epithelial metaplasia. The polyp tissue was double-labeled with a mouse monoclonal antitryptase, a specific mast cell marker, and anti-bFGF. A significant number (65% +/- 19%) of the bFGF-positive mononuclear cells in the polyp tissues were positive for tryptase. These findings suggest that bFGF may contribute to the endothelial and epithelial proliferation in nasal polyp tissues and that mast cells are one source of this growth factor.

Ultrastructural immunolocalization of basic fibroblast growth factor in mast cell secretory granules. Morphological evidence for bfgf release through degranulation.

We previously reported that mast cells (MCs) serve as a source of basic fibroblast growth factor (bFGF), a potent angiogenic and mitogenic polypeptide, suggesting that bFGF may mediate MC-related neovascularization and fibroproliferation. Unlike many other growth factors, bFGF lacks a classic peptide sequence for its secretion, and the mechanism(s) for its release remains controversial. Because MCs release a wide spectrum of bioactive products via degranulation, we hypothesized that MC degranulation may be a mechanism of bFGF release and used ultrastructural immunohistochemistry to test the hypothesis. We reasoned that if bFGF is released through degranulation, it should be localized to MC secretory granules. Human tissues with chronic inflammation and rat/mouse tissues with anaphylaxis were studied. In all tissue samples examined, positive staining (or immunogold particle localization) for bFGF in MCs was predominantly in the cytoplasmic granules. Moderate bFGF immunoreactivity was also found in the nucleus, whereas the cytosol and other subcellular organelles exhibited minimal immunogold particle localization. In contrast, no immunogold particle localization for bFGF was observed in lymphocytes or plasma cells. In rat/mouse lingual tissue undergoing anaphylaxis, immunogold particle localization for bFGF was found not only in swollen cytoplasmic granules but also in the extruded granules of MCs. Three different anti-bFGF antibodies gave similar immunogold particle localization patterns, whereas all controls were negative. These results provide morphological evidence suggesting that, despite the lack of a classic secretory peptide in its structure, bFGF is localized to the secretory granules in MCs and may be released through degranulation.

Fibroproliferation and mast cells in the acute respiratory distress syndrome.

BACKGROUND: Mast cells (MCs), which are a major source of cytokines and growth factors, have been implicated in various fibrotic disorders. To clarify the contribution of MCs to fibrogenesis, lung tissue from patients with the acute respiratory distress syndrome (ARDS) was examined during exudative through to fibroproliferative stages. METHODS: Lung tissue was obtained from 17 patients with ARDS who had pathological features of the early exudative stage (n = 6) or the later reparative stages (n = 11), from four patients with idiopathic pulmonary fibrosis, and from three patients with normal lung tissue. Immunohistochemical localisation of tryptase (found in all human MCs), chymase (found in a subset of human MCs), alpha-smooth muscle actin (identifies myofibroblasts), and procollagen type I was performed. RESULTS: Normal lung tissue exhibited myofibroblast and procollagen type I immunolocalisation scores each of < 5 and MC scores of 1. Increased scores were defined as myofibroblast and procollagen type I scores of > 10 and MC scores of > or = 2. Eighty percent of lung tissue samples from the early exudative stage of ARDS exhibited increased numbers of myofibroblasts, 50% had increased numbers of procollagen type I producing cells, while only 17% had increased numbers of MCs compared with control samples. All samples from the later reparative stages of ARDS had increased numbers of myofibroblasts and procollagen type I producing cells. Increased numbers of MCs were seen in 55% of samples from the reparative stages. There was no significant shift in MC phenotype in the ARDS samples. CONCLUSIONS: Increased numbers of myofibroblasts and procollagen type I producing cells were frequently found early in the course of ARDS. MC hyperplasia was unusual during this stage, but was often a feature of the later reparative stages. MCs do not appear to initiate fibroproliferation in ARDS.

Immunolocalization of bFGF in pterygia: association with mast cells.

PURPOSE: To examine the expression of basic fibroblast growth factor in pterygia and determine whether mast cells contain basic fibroblast growth factor (bFGF) in this disease process. METHODS: Formalin-fixed paraffin-embedded tissue samples of pterygia (n = 14) were used to study the expression of bFGF with immunohistochemistry. Seven samples were also double labeled with specific antibodies to bFGF and tryptase (a specific mast cell marker). RESULTS: In all tissue samples examined, bFGF was specifically localized in blood vessels, epithelium, and a subset of connective tissue cells. The majority of bFGF-positive connective tissue cells were also tryptase positive (90.4 +/- 3.6%). Nearly all the tryptase-positive cells (mast cells) in the pterygia tissues coexpressed bFGF (98 +/- 1.5%). CONCLUSIONS: These findings suggest that bFGF may have biologic effects on the epithelium and blood vessels in pterygia, with epithelial and endothelial cells as a possible source. In addition, mast cells contain bFGF in the pterygia tissues examined in this study. The mast cells may serve as an additional source of bFGF, possibly modulating a variety of cell types as well as the extracellular matrix in pterygia.

Immunolocalization of transforming growth factor-alpha and its receptor in the normal and hyperoxia-exposed neonatal rat retina.

PURPOSE: Transforming growth factor-alpha (TGF-alpha) is a mitogenic polypeptide for a variety of different cells types including retinal neurons and glial cells. We have examined the temporal and spatial expression of TGF-alpha and its receptor in the normal and hyperoxia-exposed neonatal rat retina to determine if the expression is consistent with a role in retinal development and response to retinal injury. METHODS: We have used immunohistochemistry to examine TGF-alpha and epidermal growth factor receptor (EGF-R) on postnatal days (1, 5, 10, 14, 18, and 25). To examine TGF-alpha and EGF-R expression after retinal injury we studied the retinas from rats which were exposed to 80% oxygen for 10 days and then recovered in room air. Immunolocalization of type IV collagen was performed to examine the retinal vasculature development after hyperoxia. RESULTS: The pattern of TGF-alpha and EGF-R expression in the neural retina evolved from a diffuse pattern on postnatal day 1 to restricted sites on postnatal day 14. The TGF-alpha immunoreactivity was consistent with localization in Müller cells on postnatal day 14. Both TGF-alpha and EGF-R patterns were altered in the retinas from rats that had been exposed to hyperoxia and recovered in room air for 4 days. The type IV confirmed immunostaining confirmed vaso-obliteration in the deep layer of retinal vessels after hyperoxia. CONCLUSIONS: Our findings of altered expression of TGF-alpha and EGF-R during retinal development suggests a biological function for this growth factor, possibly promoting retinal cell proliferation, differentiation, and survival. The altered immunolocalization of TGF-alpha and EGF-R in the hyperoxia-exposed retina suggest that TGF-alpha is likely involved in the retinal response to injury.

Altered immunohistochemical localization of basic fibroblast growth factor after bleomycin-induced lung injury.

Basic fibroblast growth factor (bFGF) is a potent inducer of growth and proliferation for many cell types involved in wound healing. Although bFGF has previously been identified in lung tissue, its role in the pathogenesis of pulmonary fibrosis is unknown. We investigated the distribution of bFGF after bleomycin-induced lung injury in the rat in hope of learning how bFGF might participate in the process of lung injury, repair and fibrosis. Increased immunoreactive bFGF was found in the extracellular matrix after bleomycin and co-localized to a marker of active cell proliferation. This suggests that bFGF may participate in directing cell proliferation following lung injury. In addition, a marked increase in the number of mast cells with strong reactivity for bFGF was found at days 14 and 21 after bleomycin. These cells may represent a source of bFGF during the fibroproliferative stage after lung injury.

Mast cells are a major source of basic fibroblast growth factor in chronic inflammation and cutaneous hemangioma.

Mast cells play an essential role during development of inflammation after chemical and immunological insults and have been implicated in tissue fibrosis and angiogenesis. The exact contribution of mast cells to these conditions is largely unknown. In this study, we found that a potent angiogenic and mitogenic polypeptide, basic fibroblast growth factor (bFGF), is localized to the majority of mast cells from normal skin and lung and in tissue samples characterized by fibrosis, hyperplasia, and neovascularization. Using specific antibodies to mast cell tryptase, tissue macrophage, and bFGF, we demonstrate that cytoplasmic bFGF immunoreactivity is localized to 96.8 +/- 9.6% of tryptase-positive cells in human fibrotic lung tissue (n = 10), 82.3 +/- 6.9% of tryptase-positive cells in rheumatoid synovia (n = 6), and 93.1 +/- 4.8% of tryptase-positive cells in skin hemangioma (n = 5). Moreover, these tryptase-positive cells comprise a major portion (86 to 97%) of nonvascular cells exhibiting cytoplasmic bFGF staining in these tissues. In contrast, macrophage-like cells contribute less than 10% of the bFGF-positive cells in the same samples. The specificity of the immunostaining results was supported by the finding that cultured human mast cells (HMC-1) express both bFGF mRNA and protein. Our data indicate that mast cells, a primary source of heparin, also serve as a significant source of a heparin-binding growth factor, bFGF, in these disease processes. These observations suggest that mast cells may contribute to these pathological conditions by releasing this polypeptide.

Increased expression of basic fibroblast growth factor in hyperoxic-injured mouse lung.

Basic fibroblast growth factor (bFGF) is a mitogenic polypeptide for a wide variety of cell types and has been immunolocalized in the rodent and human lung. We investigated the mRNA and protein expression of bFGF in hyperoxic-injured adult mouse lungs using northern blot analysis and immunohistochemistry. Mice (6-8 weeks) were continuously exposed to 80% oxygen up to 4 days. Levels of bFGF mRNA were increased from room air control on days 3 and 4 of hyperoxia. mRNA levels of acidic fibroblast growth factor (aFGF), fibronectin, and transin/stromelysin were also examined in this injury model. Similar to bFGF, the fibronectin and transin/stromelysin mRNA levels were increased after 3 days of hyperoxia. In contrast, the aFGF mRNA levels were gradually reduced on each day of hyperoxia. A rabbit polyclonal anti-bFGF antibody was used to determine the distribution and levels of expression in the hyperoxic-injured lungs. The room air control and day 1 hyperoxic-exposed lungs exhibited staining for bFGF in the basement membranes of the blood vessels, airways, and alveoli. Patchy but intense alveolar staining was prominent on day 4 of hyperoxia. The bFGF immunoreactivity of blood vessels and airways was unaffected by the hyperoxia exposure. These results suggest that bFGF may play a role in the alveolar response to hyperoxic-induced injury by virtue of the altered mRNA levels and protein distribution in this injury model.

Structure and absolute configuration of a high affinity 5-HT3 receptor antagonist, (5aS,9aS)-N-[(3S)-1-azabicyclo[2.2.2]octan-3-yl]-2-chloro- 5a,6,7,8,9,9a-hexahydro-4-dibenzofurancarboxamide hydrochloride.

The absolute configuration was established as (S,S,S) by the R-factor test and by careful measurement of 197 enantiomorph-sensitive Friedel pairs of reflections. The determination also confirms the absolute stereochemistry of (-)-3-aminoquinuclidine, a compound used in the preparation of the title material. The cyclohexane/tetrahydrofuran ring fusion is cis. The quinuclidine moiety has almost perfect threefold symmetry; the front and rear halves are twisted about this axis by 15 degrees. Quinuclidine-N--H ... Cl- and amide-N--H ... Cl- hydrogen bonds link screw-dyad-related molecules along the b axis.

Differential action of plasma and albumin on transcapillary exchange of anionic solute.

We tested two hypotheses to account for the reduction in coupling of anionic solute to water flow (solvent drag) in microvessels during perfusion with plasma compared with albumin. Solvent drag is determined by both hydraulic conductivity (Lp) and solute reflection coefficient (sigma). Accordingly, decreased solvent drag during plasma perfusion must be the result of an increase in sigma (hypothesis 1) or reduction of Lp (hypothesis 2) or some combination of both mechanisms. These hypotheses were assessed by measuring Lp, sigma, and diffusive solute permeability (Psd) to the anionic protein alpha-lactalbumin in frog mesenteric exchange microvessels during plasma or albumin perfusion. The solute permeability coefficient to alpha-lactalbumin (Ps alpha-lactalbumin) was lower during exposure to plasma than bovine serum albumin (BSA) [(Ps alpha-lactalbumin)plasma/(Ps alpha-lactalbumin)BSA = 0.31 +/- 0.11 (means +/- SE, n = 9)]. Solute reflection coefficient to alpha-lactalbumin (sigma alpha-lactalbumin) was 0.69 +/- 0.02 in plasma and 0.34 +/- 0.03 in BSA (n = 7). Lp was not significantly influenced by perfusate protein composition (Lp plasma/Lp BSA = 1.02 +/- 0.11; n = 20). These data lead to the conclusion that the actions of plasma are to confer charge selectivity for anionic solute and, to a lesser extent, modify the porous pathways of the microvessel wall. Taken together, these results indicate that porous pathways contribute significantly to macromolecular flux in plasma-perfused vessels.

Transvascular albumin and IgG flux in skin and skeletal muscle following plasmapheresis.

The extravascular uptake for labeled albumin and IgG and the extravascular masses for endogenous albumin and IgG were measured in skin and skeletal muscle from anesthetized rabbits following 24 hr of intermittent plasmapheresis. An amount of protein equivalent to the total intravascular protein mass was removed. There was a significant reduction in the extravascular mass for albumin in both tissues and for IgG in skin. The shift of albumin out of the extravascular space of skin and skeletal muscle could account for 95% of the vascular replacement of albumin. The extravascular uptake for the labeled proteins was measured as the 1-hr extravascular distribution space at plasma concentration divided by time and expressed as a plasma clearance. The plasma volume in the tissue samples was estimated from the 3-min distribution space for labeled transferrin. Following plasmapheresis the rate of extravascular uptake for both labeled proteins was greater than that for control or sham-operated animals, suggesting an increase in transvascular protein permeability. The transvascular fluxes for native albumin and IgG were not increased due to the decrease in the plasma concentrations. The results were consistent with the major mechanism for a shift of plasma proteins being due to a decrease in plasma protein concentration and a subsequent increase in lymph flow instead of a decrease in transvascular protein permeability.

Effect of albumin on hydraulic conductivity of pulmonary artery endothelial monolayers.

We tested the hypothesis that albumin reduces the vascular wall hydraulic conductivity by an interaction with the endothelium. The system consisted of luminal and abluminal chambers separated by a microporous filter onto which was grown a confluent monolayer of ovine pulmonary artery endothelial cells. The abluminal chamber filtrate was collected for timed periods during increases in transendothelial pressures of 5, 10, 15, and 20 cmH2O. The transendothelial water flux was linearly related to the hydrostatic pressure. Hydraulic conductivity (Lp) was determined from the slope of this relationship per unit surface area. In the absence of albumin, Lp of the endothelium and the filter was 14.8 +/- 3.8 x 10(-5) cm.s-1.cmH2O-1. The addition of either 2.5 or 5.0 mg/ml ovine serum albumin to the medium reduced Lp values similarly to 2.0 +/- 0.3 x 10(-5) and 2.5 +/- 1.1 x 10(-5) cm.s-1.cmH2O-1, respectively. Removal of albumin from the media reversed the effect of albumin on Lp. The filter Lp value of 3.2 +/- 0.3 x 10(-3) cm.s-1.cmH2O-1 was unaffected by albumin. Endothelial Lp value did not decrease with 5.0 mg/ml of 70-kDa neutral dextran. Albumin decreased Lp in the presence of epsilon-amino-caproic acid to the same extent as albumin alone, suggesting that the positively charged lysine sites on albumin did not mediate the effect. The results indicate that albumin decreases Lp due to an interaction between albumin and the endothelial cell.

Initial equilibration of albumin and IgG in rabbit hind paw skin and lymph.

To determine if the interstitial distribution of plasma proteins influences transport from plasma to lymph, the initial plasma-to-extravascular and plasma-to-lymph equilibrations for 131I-labeled rabbit albumin and 125I-labeled rabbit IgG were compared in hind paw heel skin of anesthetized New Zealand White rabbits. Plasma specific activities were maintained constant with time using continuous infusions. The plasma space in skin samples taken at the end of the experiment was estimated by a 3-min accumulation of 59Fe-labeled bovine transferrin. Albumin and IgG concentrations in plasma, lymph, and skin were determined by rocket electroimmunoassay. The lymph specific activity relative to that of plasma for the larger sized IgG was significantly greater (P less than 0.01) than that for albumin during the initial 8 hr of equilibration. The tissue specific activity relative to that of plasma for IgG was significantly less (P less than 0.01) than that for albumin. The distribution space at lymph concentration for both proteins was 1 ml/g dry skin wt, indicating similar excluded volumes. These data are consistent with the hypothesis that convective movement of plasma proteins through the interstitium affects lymph equilibration while diffusion affects tissue equilibration.

Role of albumin arginyl sites in albumin-induced reduction of endothelial hydraulic conductivity.

We determined the effect of albumin on endothelial hydraulic conductivity (Lp) and the contributions of the positively charged arginyl and lysinyl residues of albumin in mediating the effect. Studies were made using monolayers of cultured sheep pulmonary artery endothelial cells grown to confluence on polycarbonate filters. Water flux was measured as transendothelial hydrostatic pressure was varied from 5 to 20 cm H2O. Lp was calculated from the slope of the relationship of water flux versus pressure. The Lp of endothelial monolayers perfused with albumin-free Hanks Balanced Salt Solution (HBSS) was compared to perfusion with HBSS containing either native albumin, or albumin in which the arginyl residues were modified by a condensation reaction with 1,2-cyclohexanedione (CHD-albumin), or albumin in which the lysinyl residues were modified by a substitution reaction with succinic anhydride (SC-albumin). Baseline Lp at 2.5 mg/ml native albumin was 1.6 +/- 0.1 X 10(-6) cm/s/cm H2O compared to the filter Lp after removing cells of 3.0 +/- 0.3 X 10(-4) cm/s/cm H2O. Endothelial Lp increased by 60% when albumin concentration was decreased from 2.5 mg/ml to 0.5 mg/ml (P less than 0.05), but did not change with an increase in concentration to 10 mg/ml. Albumin-free buffer and CHD-albumin increased endothelial Lp by 2.2 +/- 0.3-fold and 1.9 +/- 0.3-fold, respectively (P less than 0.05). All endothelial Lp values were restored to baseline when the native albumin concentration was returned to 2.5 mg/ml. Excess l-arginine (2 X 10(-3) M) inhibited the effect of native albumin and increased endothelial Lp 1.5 +/- 0.02-fold (P less than 0.05), but excess l-lysine (4 X 10(-3) in the presence of native albumin had no effect on Lp. None of the perfusates altered the filter Lp value. Neutral dextran (70 kD), in contrast to native albumin, had no effect on endothelial Lp. These results indicate that albumin reduces the hydraulic conductivity of endothelial monolayers in a concentration-dependent fashion and that the arginyl residues of albumin are required for the response. The effect of albumin may be mediated by a charge interaction of albumin with the endothelium.

Initial equilibration of albumin in rabbit hindpaw skin and lymph.

We compared initial plasma to extravascular and plasma to lymph equilibrations of 125I-labeled rabbit albumin in hindpaw heelskin of anesthetized rabbits during control conditions, increased venous pressure (IVP), and intra-arterial infusions of papaverine or bradykinin. Plasma tracer concentrations were maintained constant during the experiments. Control prenodal lymph specific activity relative to plasma specific activity (L*/P*) was significantly greater (P less than 0.01) than extravascular specific activity relative to plasma specific activity (M/P) following 3, 5.5, and 11 h of tracer infusion. Papaverine increased lymph flow and lymph albumin flux (LCL) 2.4-fold, without significantly changing the extravascular albumin mass (MA). IVP increased LCL 2.5-fold and decreased MA 25%. L*/P* at 3 h was twice control following papaverine. M*/P* at 3 h was not significantly affected by papaverine or IVP. Bradykinin increased LCL 10-fold and MA 2.3-fold. L*/P* and M*/P* were 3.1 and 4.3 times control, respectively. The data are consistent with extravascular albumin distributing into two pools that equilibrate with plasma at different rates. Papaverine and IVP affect the faster pool, whereas bradykinin affects both pools.

Albumin and IgG in skin and skeletal muscle after plasmapheresis with saline loading.

The acute effect of removing plasma equivalent to 1.7% body wt and replacing it with saline equivalent to 10% body wt on the extravascular distribution of water, albumin, and immunoglobulin G (IgG) in skin and skeletal muscle was studied in anesthetized rabbits. The plasma protein concentration decreased by 43%. Prenodal lymph was collected from hindpaw skin or skeletal muscle. The extracellular and plasma volumes in excised tissue samples were measured using 51Cr-labeled ethylenediaminetetraacetic acid and 125I-labeled albumin, respectively. The protein spaces were calculated from measurements of endogenous albumin and IgG concentrations using immunochemical techniques. Lymph flow from both tissues increased more than twice control, whereas the lymph total protein concentration decreased to less than one-half control. Three to six hours after the saline infusion, the skin interstitial volume was 30% greater than control, whereas the extravascular masses of albumin and IgG were 20% greater than control. For muscle, the interstitial volume was twice the control value, whereas the extravascular masses of albumin and IgG were not significantly altered. There was a large decrease in the lymph protein concentration after acute plasmapheresis. However, there was not an acute decrease in the extravascular albumin or IgG masses from skin or skeletal muscle. This may be due to the presence of the collagen matrix and edema fluid.

Extravascular distribution of albumin and IgG during high-permeability edema in skin.

The effects of local intra-arterial infusions of bradykinin on the interstitial distribution of albumin and gamma-immunoglobulin G (IgG) was studied in hind paw skin of anesthetized rabbits. After 4 h of a continuous bradykinin infusion, lymph flow in a prenodal popliteal lymphatic was five times base line. The lymph-to-plasma concentration ratio for total protein was increased by 34%. Samples of heel skin were taken at the end of the experiment. The extracellular and plasma spaces in the tissue samples were measured using 51Cr-labeled ethylenediaminetetraacetic acid and 125I-labeled human serum albumin, respectively. The protein spaces were calculated from measurements of endogenous albumin and IgG concentrations using rocket electroimmunoassay. After bradykinin, the interstitial volume was 28% greater than control, whereas the extravascular masses of albumin and IgG were twice as great as the control values. Based on lymph concentration, both proteins distributed in 50% of the interstitial volume during control. Following bradykinin, both proteins distributed in a greater fraction of the interstitium. However, the excluded volume, equal to 0.9 ml/g dry wt, was not altered after bradykinin. This may indicate an increase in free-fluid channels with no hydration of the interstitial gel matrix.