Characterization of 3 different types of aquaporins in Carcinus maenas and their potential role in osmoregulation.

Intertidal crustaceans like Carcinus maenas shift between an osmoconforming and osmoregulating state when inhabiting full-strength seawater and dilute environments, respectively. While the bodily fluids and environment of marine osmoconformers are approximately isosmotic, osmoregulating crabs inhabiting dilute environments maintain their bodily fluid osmolality above that of their environment by actively absorbing and retaining osmolytes (e.g., Na+, Cl-, urea) while eliminating excess water. Few studies have investigated the role of aquaporins (AQPs) in the osmoregulatory organs of crustaceans, especially within brachyuran species. In the current study, three different aquaporins were identified within a transcriptome of C. maenas, including a classical AQP (CmAQP1), an aquaglyceroporin (CmGLP1), and a big-brain protein (CmBIB1), all of which are expressed in the gills and the antennal glands. Functional expression of these aquaporins confirmed water transport capabilities for CmAQP1, CmGLP1, but not for CmBIB1, while CmGLP1 also transported urea. Higher relative CmAQP1 mRNA expression within tissues of osmoconforming crabs suggests the apical/sub-apically localized channel attenuates osmotic gradients created by non-osmoregulatory processes while its downregulation in dilute media reduces the water permeability of tissues to facilitate osmoregulation. Although hemolymph urea concentrations rose upon exposure to brackish water, urea was not detected in the final urine. Due to its urea-transport capabilities, CmGLP1 is hypothesized to be involved in a urea retention mechanism believed to be involved in the production of diluted urine. Overall, these results suggest that AQPs are involved in osmoregulation and provide a basis for future mechanistic studies investigating the role of AQPs in volume regulation in crustaceans.

Impact of heatwaves and environmental ammonia on energy metabolism, nitrogen excretion, and mRNA expression of related genes in the indicator model system Daphnia magna.

Due to increasing anthropogenic impacts, heatwaves and prolonged exposure to elevated concentrations of ammonia (HEA) may occur in aquatic environments as a single stressor or a combination thereof, potentially impacting the physiology of exposed animals. In the current study, common water fleas Daphnia magna were exposed for one week to either a 5°C increase in temperature, an increase of 300 µmol l-1 total environmental ammonia, or to both of these stressors simultaneously. Exposure to elevated temperature caused a decrease in MO2, ammonia excretion rates, a downregulation of mRNA coding for key Krebs cycle enzymes and the energy consuming Na+/K+-ATPase and V-type H+-ATPase, as well as the energy distributing crustacean hyperglycemic hormone Rh-protein. High environmental ammonia inflicted a lesser inhibitory effect on the energy metabolism of Daphnia, but initiated ammonia detoxification processes via urea synthesis evident by elevated urea excretion rates and a mRNA upregulation of arginase. Effects observed under the combined stressors resembled largely the effects seen after acclimation to elevated temperature alone, potentially due to the animals' capability to efficiently detoxify critical ammonia loads. The observed physiological effects and potential threats of the environmental stressor are discussed in detail.

Effect of supplementing a fibrous diet with a xylanase and ß-glucanase blend on growth performance, intestinal glucose uptake, and transport-associated gene expression in growing pigs.

The present study evaluated supplemental carbohydrase effect on performance, intestinal nutrient uptake, and transporter mRNA expressions in growing pigs offered a high-fiber diet manufactured with distillers dried grains with solubles (DDGS). Twenty-four pigs (22.4 ± 0.7 kg BW) were randomly assigned to 1of 3 nutritionally adequate diets (8 pigs per diet) based on corn and soybean meal (SBM) with either 0 (control) or 30% DDGS (high fiber [HF]). The third diet was supplemented with a xylanase and ß-glucanase blend (XB) in addition to the 30% DDGS (HF+XB). Parameters determined were ADFI, ADG, G:F, plasma glucose and plasma urea nitrogen (PUN) concentrations, jejunal tissue electrophysiological properties, and mRNA expressions of the sodium-dependent glucose transport 1 (SGLT1) and cationic AA transporter, bo,+AT, in the jejunal and ileal tissues. In addition, mRNA expressions of the short-chain fatty acid transporters, monocarboxylate transporter 1 (MCT1) and sodium-coupled monocarboxylate transporter, and mucin genes were quantified in the ileum. Feed intake, plasma glucose, and jejunal tissue electrophysiological properties were not affected (P > 0.05) by diet. However, control-fed pigs had superior growth rate and feed efficiency and higher PUN (P < 0.05) than HF- and HF+XB-fed pigs. The HF diet increased (P < 0.05) SGLT1 mRNA expression in the jejunum and decreased (P < 0.05) bo,+ mRNA expression in the ileum. The XB supplementation also increased bo,+ mRNA expression in the ileum relative to HF-fed pigs. Additionally, MCT1 mRNA expression was greater (P < 0.05) in the ileum of the HF- and HF+XB-fed pigs. In the present study, XB supplementation influenced nutrient transporter mRNA expression, although it was not accompanied by improved pig performance.

Effect of dietary phytic acid on performance and nutrient uptake in the small intestine of piglets.

An experiment was conducted with piglets to determine the effect of dietary phytic acid supplementation on performance, electrophysiological properties of jejunum mounted in Ussing chambers, sodium-dependent glucose transporter 1 (SGLT1) protein expression in jejunum, and plasma glucose and Na concentrations. Sixteen piglets with an average initial BW of 7.40 ± 0.36 kg were randomly assigned to 2 experimental diets with 8 piglets per diet. The diets were casein-cornstarch-based and were either unsupplemented or supplemented with 2% phytic acid (as Na phytate). The basal diet was formulated to meet the recommendation of NRC (1998) for energy, AA, minerals, and vitamins for piglets. The experiment lasted for 21 d, and at the end, BW gain and feed consumption were determined, and blood samples were collected for determination of plasma glucose and Na concentrations. The piglets were then euthanized to determine jejunal electrophysiological properties (transmural potential difference and short-circuit current) and SGLT1 protein expression. Phytic acid supplementation reduced ADG (P = 0.002), ADFI (P = 0.017), and G:F (P = 0.001) from 316.1 to 198.2 g, 437.4 to 360.3 g, and 0.721 to 0.539 g/g, respectively. Phytic acid supplementation also tended to reduce (P = 0.088) potential difference (-3.80 vs. -2.23 mV) and reduced (P = 0.023) short-circuit current from 8.07 to 0.1 µA/cm(2). However, phytic acid supplementation did not affect SGLT1 protein, and blood plasma glucose and Na concentrations. In conclusion, dietary phytic acid reduced growth performance and transmural short-circuit current in the jejunum of piglets. The reduced transmural short-circuit current in the jejunum by phytic acid implies reduced active Na transport in the jejunum by the phytic acid. Therefore, it seems that dietary phytic acid reduces growth performance of pigs partly through reduced capacity of the small intestine to absorb Na.

D-4F, an apoA-1 mimetic, decreases airway hyperresponsiveness, inflammation, and oxidative stress in a murine model of asthma.

Asthma is characterized by oxidative stress and inflammation of the airways. Although proinflammatory lipids are involved in asthma, therapies targeting them remain lacking. Ac-DWFKAFYDKVAEKFKEAFNH(2) (4F) is an apolipoprotein (apo)A-I mimetic that has been shown to preferentially bind oxidized lipids and improve HDL function. The objective of the present study was to determine the effects of 4F on oxidative stress, inflammation, and airway resistance in an established murine model of asthma. We show here that ovalbumin (OVA)-sensitization increased airway hyperresponsiveness, eosinophil recruitment, and collagen deposition in lungs of C57BL/6J mice by a mechanism that could be reduced by 4F. OVA sensitization induced marked increases in transforming growth factor (TGF)ß-1, fibroblast specific protein (FSP)-1, anti-T15 autoantibody staining, and modest increases in 4-hydroxynonenal (4-HNE) Michael's adducts in lungs of OVA-sensitized mice. 4F decreased TGFß-1, FSP-1, anti-T15 autoantibody, and 4-HNE adducts in the lungs of the OVA-sensitized mice. Eosinophil peroxidase (EPO) activity in bronchial alveolar lavage fluid (BALF), peripheral eosinophil counts, total IgE, and proinflammatory HDL (p-HDL) were all increased in OVA-sensitized mice. 4F decreased BALF EPO activity, eosinophil counts, total IgE, and p-HDL in these mice. These data indicate that 4F reduces pulmonary inflammation and airway resistance in an experimental murine model of asthma by decreasing oxidative stress.

Expression of Ca2+-ATPase and Na+/Ca2+-exchanger is upregulated during epithelial Ca2+ transport in hypodermal cells of the isopod Porcellio scaber.

It is thought that a plasma membrane Ca(2+)-transport ATPase (PMCA) and a Na(+)/Ca(2+)-exchange (NCE) mechanism are involved in epithelial Ca(2+) transport (ECT) in a variety of crustacean epithelia. The sternal epithelium of the terrestrial isopod Porcellio scaber was used as a model for the analysis of Ca(2+)-extrusion mechanisms in the hypodermal epithelium. Using RT-PCR, we amplified a cDNA fragment of 1173 bp that encodes a protein sequence possessing 72% identity to the PMCA from Drosophila melanogaster and a cDNA fragment of 791 bp encoding a protein sequence with 50% identity to the NCE from Loligo opalescens. Semiquantitative RT-PCR revealed that the expression of both mRNAs increases from the non-Ca(2+)-transporting condition to the stages of CaCO(3) deposit formation and degradation. During Ca(2+)-transporting stages, the expression of PMCA and NCE was larger in the anterior sternal epithelium (ASE) than in the posterior sternal epithelium (PSE). The results demonstrate for the first time the expression of a PMCA and a NCE in the hypodermal epithelium of a crustacean and indicate a contribution of these transport mechanisms in ECT.

Molecular characterization of V-type H(+)-ATPase (B-subunit) in gills of euryhaline crabs and its physiological role in osmoregulatory ion uptake.

The vacuolar-type H(+)-ATPase (V-ATPase) has been implicated in osmoregulatory ion uptake across external epithelia of a growing variety of species adapted to life in fresh water. In the present study, we investigated whether the V-ATPase may also function in a euryhaline species that tolerates brackish water (8 salinity) but not fresh water, the shore crab Carcinus maenas. cDNA coding for the regulatory B-subunit of the V-ATPase was amplified and sequenced from C. maenas gills and partially sequenced from four other crab species. Two isoforms differing in the 3'-untranslated region were found in C. maenas. In this species, the abundance of B-subunit mRNA was greater in the respiratory anterior gills than the ion-transporting posterior gills and was not increased by acclimation to dilute salinity. Immunocytochemical analysis showed that the B-subunit protein is not targeted to the apical membrane but is distributed throughout the cytoplasmic compartment. Physiological studies of isolated perfused gills indicated that the V-ATPase inhibitor bafilomycin had no effect on transepithelial potential difference. Thus, in contrast to the freshwater-tolerant Chinese crab Eriocheir sinensis, in which the V-ATPase appears to play an important osmoregulatory role, the V-ATPase in C. maenas probably functions in acidification of intracellular organelles but not in transbranchial NaCl uptake.

Na(+)+K(+)-ATPase in gills of the blue crab Callinectes sapidus: cDNA sequencing and salinity-related expression of alpha-subunit mRNA and protein.

Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na(+)+K(+)-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' and 5' untranslated regions of the mRNA coding for the alpha-subunit of Na(+)+K(+)-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The alpha-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83 % identity to those of other arthropods and 74-77 % identity to those of vertebrate species. Quantitative RT-PCR analysis showed high levels of alpha-subunit mRNA in posterior gills 6-8 compared with anterior gills 3-5. Western blots of gill plasma membranes revealed a single Na(+)+K(+)-ATPase alpha-subunit protein band of the expected size. The posterior gills contained a much higher level of alpha-subunit protein than the anterior gills, in agreement with previous measurements of enzyme activity. Immunocytochemical analysis showed that the Na(+)+K(+)-ATPase alpha-subunit protein detected by alpha5 antibody is localized to the basolateral membrane region of gill epithelial cells. Transfer of blue crabs from 35 to 5 per thousand salinity was not accompanied by notable differences in the relative proportions of alpha-subunit mRNA and protein in the posterior gills, suggesting that the enhanced Na(+)+K(+)-ATPase enzyme activity that accompanies the hyperosmoregulatory response may result from post-translational regulatory processes.

Expression of arginine kinase enzymatic activity and mRNA in gills of the euryhaline crabs Carcinus maenas and Callinectes sapidus.

Phosphagen kinases catalyze the reversible dephosphorylation of guanidino phosphagens such as phosphocreatine and phosphoarginine, contributing to the restoration of adenosine triphosphate concentrations in cells experiencing high and variable demands on their reserves of high-energy phosphates. The major invertebrate phosphagen kinase, arginine kinase, is expressed in the gills of two species of euryhaline crabs, the blue crab Callinectes sapidus and the shore crab Carcinus maenas, in which energy-requiring functions include monovalent ion transport, acid-base balance, nitrogen excretion and gas exchange. The enzymatic activity of arginine kinase approximately doubles in the ion-transporting gills of C. sapidus, a strong osmoregulator, when the crabs are transferred from high to low salinity, but does not change in C. maenas, a more modest osmoregulator. Amplification and sequencing of arginine kinase cDNA from both species, accomplished by reverse transcription of gill mRNA and the polymerase chain reaction, revealed an open reading frame coding for a 357-amino-acid protein. The predicted amino acid sequences showed a minimum of 75 % identity with arginine kinase sequences of other arthropods. Ten of the 11 amino acid residues believed to participate in arginine binding are completely conserved among the arthropod sequences analyzed. An estimation of arginine kinase mRNA abundance indicated that acclimation salinity has no effect on arginine kinase gene transcription. Thus, the observed enhancement of enzyme activity in C. sapidus probably results from altered translation rates or direct activation of pre-existing enzyme protein.

Active osmoregulatory ion uptake across the pleopods of the isopod Idotea baltica (Pallas): electrophysiological measurements on isolated split endo- and exopodites mounted in a micro-ussing chamber.

The mechanism of active, osmoregulatory ion uptake was investigated in the pleopods of the marine isopod Idotea baltica (Pallas). Using isolated split half-podites of isopods acclimated to brackish water (20 salinity) mounted in a micro-Ussing chamber and symmetrically superfused with identical haemolymph-like salines, a mean short-circuit current I(sc) of -445 microA cm(-)(2) was measured in endopodites 3-5, corresponding to an inwardly directed transcellular movement of negative charge. Application of ouabain (5 mmol l(-)(1)) to the basolateral superfusate resulted in the almost total abolition of the I(sc) (reduced from -531 to -47 microA cm(-)(2)), suggesting that the Na(+)/K(+)-ATPase is the driving force for active, electrogenic uptake of NaCl. In contrast, mean I(sc) values close to zero were found in preparations of all exopodites and in endopodites 1 and 2. The specific activities of Na(+)/K(+)-ATPase corresponded with these results. Specific activities were highest in posterior endopodites 3-5 and depended on ambient salinity. In all other rami, the activities were much lower and independent of ambient salinity. Activities in posterior endopodites 3-5 were lowest in isopods acclimated to 30 salinity (2-4 micromol P(i )mg(-)(1 )protein h(-)(1)), increased in individuals kept in 20 salinity (8.4 micromol P(i )mg(-)(1 )protein h(-)(1)) and were highest in isopods acclimated to 15 salinity (18.2 micromol P(i )mg(-)(1 )protein h(-)(1)). When specimens were transferred from 30 to 40 salinity, Na(+)/K(+)-ATPase activity increased in the posterior endopodites. The electrophysiological and Na(+)/K(+)-ATPase activity measurements show that active electrogenic ion transport in this species occurs almost exclusively in posterior endopodites 3-5. The endopodite of the fifth pleopod of I. baltica exhibited a microscopic structure remarkably similar to that described for the lamellae of the phyllobranchiae of brachyurans. It is composed of two opposed epithelial monolayers of ionocytes, each covered by cuticle. Bundles of pillar cells are located within the ionocyte layers, which are separated by a fenestrated lamellar septum of connective tissue. The results obtained in this study indicate that endopodites 3-5 play the main role in osmoregulatory ion uptake of the isopod I. baltica. Moreover, the Na(+)/K(+)-ATPase is the only driving force behind active electrogenic ion uptake across the epithelial cells.

Ischemia-induced coronary collateral growth is dependent on vascular endothelial growth factor and nitric oxide.

BACKGROUND: We hypothesized that ischemia-induced expression of vascular endothelial growth factor (VEGF) and the production of NO stimulate coronary collateral growth. METHODS AND RESULTS: To test this hypothesis, we measured coronary collateral blood flow and VEGF expression in myocardial interstitial fluid in a canine model of repetitive myocardial ischemia under control conditions and during antagonism of NO synthase. Collateralization was induced by multiple (1/h; 8/d), brief (2 minutes) occlusions of the left anterior descending coronary artery for 21 days. In controls, collateral blood flow (microspheres) progressively increased to 89+/-9 mL. min(-1). 100 g(-1) on day 21, which was equivalent to perfusion in the normal zone. Reactive hyperemic responses (a measure of the severity of ischemia) decreased as collateral blood flow increased. In N(G)-nitro-L-arginine methyl ester (L-NAME)- and L-NAME+nifedipine-treated dogs, to block the production of NO and control hypertension, respectively, collateral blood flow did not increase and reactive hyperemia was robust throughout the occlusion protocol (P<0.01 versus control). VEGF expression (Western analyses of VEGF(164) in myocardial interstitial fluid) in controls peaked at day 3 of the repetitive occlusions but waned thereafter. In sham-operated dogs (instrumentation but no occlusions), expression of VEGF was low during the entire protocol. In contrast, VEGF expression was elevated throughout the 21 days of repetitive occlusions after L-NAME. Reverse transcriptase-polymerase chain reaction analyses revealed that the predominant splice variant expressed was VEGF(164). CONCLUSIONS: NO is an important regulator of coronary collateral growth, and the expression of VEGF is induced by ischemia. Furthermore, the induction of coronary collateralization by VEGF appears to require the production of NO.

Effects of transforming growth factor-beta and mechanical strain on osteoblast cell counts: an in vitro model for distraction osteogenesis.

Factors known to regulate bone production during distraction osteogenesis include mechanical strain on bone forming cells and up-regulation of transforming growth factor-beta (TGF-beta) during the distraction, or strain phase of distraction osteogenesis. In the present study, an in vitro model was used to evaluate the functional effect of exogenous TGF-beta1 on mitogenesis in murine-derived MC3T3 osteoblasts during the period of active mechanical strain. The first hypothesis to be tested was that mitogenic suppression of MC3T3 osteoblasts by TGF-beta1 is further enhanced when these cells are also subjected to mechanical strain. To test this hypothesis, MC3T3 osteoblasts were seeded on flexible and rigid membranes. These were subjected to cyclic, vacuum-induced strain, simulating physiologic stress loads. After 24 hours, all cells were transferred to media containing TGF-beta1, and strain was continued for an additional 48 hours. The study was repeated by using two doses of TGF-beta1. This study demonstrated that final cell counts were significantly decreased in the presence of TGF-beta1 in both the nonstrained and strained groups (p < 0.0001). The final cell count in the strained group was significantly less than that in the nonstrained group (p < 0.0001) for both concentrations of TGF-beta1 tested, confirming the initial hypothesis. The second hypothesis to be tested was that alteration in the mitogenic response of MC3T3 osteoblasts after strain is not directly due to autocrine factors produced by the strained osteoblasts. To test this hypothesis, a proliferation assay was performed on nonconfluent MC3T3 osteoblasts by using conditioned media collected from strained and nonstrained osteoblasts. This study demonstrated no significant differences in cell counts after addition of conditioned media collected from strained versus nonstrained cells, confirming the latter hypothesis. The present study demonstrates the functional significance of mechanical strain on osteoblast cell counts. Furthermore, this may help to explain the temporal relationship observed during the early distraction (strain) phase of distraction osteogenesis in rodent models in which peak up-regulation of TGF-beta1 gene expression correlates with peak suppression of osteoblast function as measured by gene expression of extracellular matrix proteins.

Repetitive coronary artery occlusions induce release of growth factors into the myocardial interstitium.

Our objective was to delineate the temporal sequence of mitogenic activity in myocardial interstitial fluid (IF) during enhancement of collateral growth. Collateral development in chronically instrumented dogs was induced by eight 2-min coronary occlusions/day for 21 days. Collateralization was assessed by measurement of blood flow in the region distal to a total coronary occlusion. Myocardial IF was obtained periodically from an intramyocardial catheter, and mitogenic activity was assessed by proliferative response of cultured endothelial cells (EC) and vascular smooth muscle cells (VSMC) to the IF. Three experiments were conducted to test that the mitogenic activity is induced by protein growth factors: 1) protein digestion of the myocardial IF with Pronase-coupled latex beads; 2) heat inactivation (boiling) of the IF; and 3) neutralization of the mitogenic activity with antibodies for basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Blood flow was reconstituted to baseline levels during occlusion after 3 wk of repetitive coronary occlusions. After initiation of occlusion the mitogenic activity of the myocardial IF on VSMC and EC increased up to days 12-14 and was reduced on days 19-23. Pronase treatment and heat inactivation blocked the mitogenic effect. Treatment with antibodies for bFGF and VEGF neutralized the proliferative response to the myocardial IF at specific times. bFGF antibody inhibited the mitogenic effect significantly on days 12-14. VEGF antibody neutralized the mitogenicity of the myocardial IF on day 7, days 12 and 13, and days 19 and 20 significantly. We conclude that myocardial IF harvested from ischemic myocardium is highly mitogenic up to 2 wk after initiation of repetitive coronary occlusions. After 3 wk of ischemia, the degree of mitogenic activity for VSMC and EC was decreased from peak levels. The antibodies could not neutralize the mitogenic effect of the myocardial IF during this time period. These results suggest that mitogens are expressed during various stages of collateral development in a time-dependent manner, that the mitogens are proteinaceous in nature, and that bFGF and VEGF are released into the myocardial IF.

Coordinate expression of the insulin-like growth factor system after microembolisation in porcine heart.

OBJECTIVES: Coronary microembolisation in the pig heart induces angiogenesis in a model of sterile inflammation due to focal necrosis. We have recently shown in this model that insulin-like growth factor I (IGF-I) is involved in inflammation-linked angiogenic processes due to its enhanced transcription after 72 h of ischaemia by infiltrating monocytes in areas of microsphere-induced focal necrosis where capillary sprouting could be detected. To obtain further insights into this process we studied by means of Northern blot analysis and in situ hybridisation the gene expression of other members of the IGF family, i.e. the six IGF binding proteins (IGFBPs), the insulin receptor, and the type I IGF receptor. METHODS: Myocardial injury was induced by injection of 25 microns non-radioactive microspheres into the left circumflex artery (LCx) in pigs that were killed after 3-24, 72, or 168 h of microembolisation. Tissue was collected from a non-ischaemic control area and the LCx region of the same heart for further analysis. RESULTS: We found decreased IGFBP-5 (2.7-fold; P < 0.02) mRNA concentrations after 72 h of microembolisation in ischaemic tissue versus control tissue from the same heart, preceded by a 1.9-fold elevated level of IGFBP-3 mRNA at 3-24 h (P < 0.05). IGFBP-6 was increased in ischaemic tissue at all time points studied. In situ hybridisation identified myocytes as the main producers of IGFBP-3 and IGFBP-6 mRNA. The mRNA levels of IGFBP-2, IGFBP-4, the insulin receptor, and the type I IGF receptor were constitutively expressed but did not change after microembolisation. Neither in heart nor in other organs studied transcripts of IGFBP-1 could be detected. Furthermore, we demonstrated that mRNA of the other components of the IGF system was expressed in almost all porcine organs except liver. CONCLUSION: These results indicate a coordinate gene expression of the IGF system in microembolised porcine myocardium, compatible with a role of IGF-I, IGFBP-3, IGFBP-5, and IGFBP-6 in inflammation-linked angiogenesis and/or repair processes.

Importance of monocytes/macrophages and fibroblasts for healing of micronecroses in porcine myocardium.

In porcine heart, embolization of small coronary arteries with microspheres in 25 microns in diameter induces collateral capillary vessel growth by angiogenesis in and around focal necrosis. By histological analysis the inflammatory infiltrates in this porcine tissue were characterized by numerous monocytes/macrophages and fibroblasts as well as neutrophils and numerous capillaries, some in mitosis. The aim of the present study, therefore, was to clarify the role of monocytes/macrophages and fibroblasts in angiogenesis and in repair in ischemic porcine myocardium. Using a human acidic fibroblast growth factor (aFGF) cDNA probe for in situ hybridisation labeling for aFGF mRNA was seen in monocytes and macrophages only, beginning at day 1, with a maximum at 3 and 7 days, and minimal labeling at 4 weeks. We have also shown, with a specific antibody and fluorescence microscopy, that tumur necrosis factor alpha (TNF alpha) follows the same time sequence and that it is produced by monocytes/macrophages. The number of capillaries in infiltrates at 3 and 7 days as revealed by the lectin Dolichus Biflorus Agglutinin was high and declined at 4 weeks. In situ hybridisation using a rat cDNA probe for fibronectin showed the increased production of fibronectin mRNA in fibroblasts. To describe the expression of fibronectin and the collagens I, III, VI immunohistochemistry was used. A comparison showed that fibroblasts produced fibronectin mRNA starting at day 3, but the protein was only maximally expressed at day 7 and 4 weeks. Collagen I, III, VI expression was highest at 1-4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of extracellular matrix proteins and the role of fibroblasts and macrophages in repair processes in ischemic porcine myocardium.

In the experimental model of coronary microembolization in pig hearts, the processes of wound healing and scar formation were studied. Methods employed were: electron microscopy, immunohistochemistry using monoclonal antibodies (against fibronectin, laminin, collagen I, III, and VI, chondroitin sulfate, and vimentin), and in situ hybridization with radioactively labeled RNA (histones, fibronectin) or cDNA (acidic fibroblast growth factor) probes. The following time course for expression of various proteins and their mRNAs was established: Mitotic activity was significant at 3 d as well as expression of fibronectin mRNA. Cellularity comprising blood borne cells and macrophages was high. At 7 d, fibronectin, laminin and collagen VI accumulation were pronounced, vimentin positive cells were numerous. At 4 weeks, collagen expression was prominent, but interstitial cells were still present. It is concluded that healing after myocardial necrosis passes through the classical phases of wound healing, i.e., granulation tissue formation and final scar formation. Different extracellular matrix proteins show a differing time course of expression, tumor necrosis factor-alpha (TNF-alpha) and acidic fibroblast growth factor (aFGF) produced by macrophages may be involved in inflammatory processes and angiogenesis. Scar formation is not yet completed at 4 weeks after injury.

The extracellular matrix in human cardiac tissue. Part II: Vimentin, laminin, and fibronectin.

In continuation of our previous work describing the localization of the collagens type I, III, VI, and IV the present study describes the localization of vimentin, laminin, and fibronectin in human myocardium obtained as left ventricular needle biopsies during cardiac surgery. Myocardium from normal pigs served for comparison. Monoclonal antibodies against the various proteins were used on frozen sections, labeled with fluorescein and viewed in the fluorescence microscope. Vimentin, the intermediate filament of mesenchymal cells, is present in fibroblasts, fibrocytes, and endothelial cells. Laminin is observed in the basal membrane of myocytes, smooth muscle and endothelial cells. The staining intensity for the B1-chain is higher in and around myocytes as compared with the B2-chain antibody, but more blood vessels were stained with the latter. The antibody against the A-chain only stained the basal lamina of vascular cells but not that of myocytes. Fibronectin was localized homogeneously throughout the extracellular space as matrix material in which the cellular elements and the various other proteins such as collagens are embedded. Intracellular staining in myocytes (T-tubules) was commonly observed. Both parts of this study show the distribution of extracellular proteins in normal human cardiac tissue and are intended to be the basis for investigations of pathological changes in diseased human myocardium.