Tenascin-C modulates adhesion of cardiomyocytes to extracellular matrix during tissue remodeling after myocardial infarction.

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays important roles in tissue remodeling. TNC is not normally expressed in adults but reappears under pathologic conditions. The present study was designed to clarify the contribution of TNC to ventricular remodeling after myocardial infarction. We examined the expression of TNC after experimental myocardial infarction in the rat by immunohistochemistry and in situ hybridization. Within 24 hours of permanent coronary ligation, interstitial fibroblasts in the border zone started to express TNC mRNA. The expression of TNC was down-regulated on Day 7 and was no longer apparent by Day 14 after infarction. During the healing process, TNC protein and TNC-producing cells were found at the edges of the residual myocardium. Some of the TNC-producing cells were immunoreactive for alpha-smooth muscle actin. In culture, TNC increased the number of cardiomyocytes attached to laminin but inhibited the formation of focal contacts at costameres. The results indicate that during the acute phase after myocardial infarction, interstitial cells in the border zone synthesize TNC, which may loosen the strong adhesion of surviving cardiomyocytes to connective tissue and thereby facilitate tissue reorganization.

Inhibitory effects of vesnarinone in the progression of myocardial damage in experimental autoimmune myocarditis in rats.

OBJECTIVES: Vesnarinone, a positive inotropic and immunomodulatory agent, diminishes nitric oxide (NO) levels by suppressing the induction of inducible NO synthase (iNOS) expressed in cytokine-stimulated macrophages and cardiomyocytes. We examined whether vesnarinone exerts inhibitory effects on the progression of myocardial damage in experimental autoimmune myocarditis in rats through suppression of iNOS. METHODS: Myocarditis was induced in 30 Lewis rats by injection of porcine cardiac myosin and vesnarinone was orally administered to 20 of the 30 rats. On day 21 after immunization (the climax of inflammation), the hemodynamics were examined and the severity of myocarditis was evaluated by determining the area ratio (%) [affected/entire area] of myocardial lesions in histological sections. Levels of serum CK-MB, NOx (NO2(-)+NO3-), TNF-alpha and IL-1 beta, and cyclic GMP, iNOS mRNA, TNF-alpha and IL-1 beta in heart tissues were determined. Expression of iNOS and TNF-alpha protein were examined by immunohistochemical methods. RESULTS: Histopathological examination revealed extensive myocardial destruction and massive infiltration of inflammatory cells in the vesnarinone-untreated rats. The area ratio of the lesions in the treated rats was significantly lower than that in the untreated ones. Levels of CK-MB, NOx, cyclic GMP, cytokines and iNOS mRNA were significantly lower in the vesnarinone-treated rats. Infiltrating macrophages and cardiomyocytes in the untreated rats showed much higher levels of expression of iNOS and TNF-alpha than those in the vesnarinone-treated rats. CONCLUSIONS: Vesnarinone may prove to be useful in the treatment of myocarditis by attenuating NO production through suppression of iNOS induced by cytokines.

Thermal preconditioning protects rat cardiac muscle cells from doxorubicin-induced apoptosis.

Doxorubicin (DOX=adriamycine), an effective chemotherapeutic agents for cancers, has severe cardiotoxicity. In the paresent study, we examined the protective effect of thermal preconditioning (TP) against apoptosis of rat cardiac muscle cells induced by DOX. Treatment with DOX (10 microM) for 24 hrs resulted in apoptosis of cardiac muscle cells, which was evaluated by examining "DNA ladder" formation and TUNEL staining. The number of TUNEL-positive cells was significantly decreased in cells subjected to TP by incubation at 42 degrees C for 30 min, 24 hrs prior to DOX-treatment. Antisense oligonucleotides of the heat shock protein (HSP) 70 blunted this effect. These results indicate that DOX-induced apoptosis in cardiac muscle cells is prevented by TP, at least in part, via a HSP70-mediated mechanism.

Nitric oxide induces apoptotic death of cardiomyocytes via a cyclic-GMP-dependent pathway.

Recently, we have reported that excess amounts of nitric oxide (NO) produced by inducible NO synthase are involved in the development of myocardial damage in rats with induced myocarditis. However, there remain many problems to be solved concerning its mechanism of action. In this study, we examined whether NO induces apoptotic cell death in cardiomyocytes. Cultured neonatal rat cardiomyocytes were exposed to S-nitroso-N-acetylpenicillamine (SNAP) and (+/-)-E-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamine (NOR 3), as NO donors, or 8-bromo-cyclic GMP (cGMP), an analog of cGMP which functions as a second messenger in cells stimulated by NO. DNA fragmentation was confirmed by electron microscopy, by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method, and by agarose gel electrophoresis. Exogenously supplied SNAP or NOR 3 induced cardiomyocyte apoptosis in a dose- and time-dependent manner. Cardiomyocytes exposed to SNAP displayed typical features of apoptosis as demonstrated by electron microscopy. Treatment of the cells with 8-bromo-cGMP also induced apoptosis. In cardiomyocytes, SNAP-induced apoptosis was completely blocked by a PKG inhibitor (KT5823) and by a soluble guanylate cyclase inhibitor (ODQ) and was suppressed by hemoglobin and was completely blocked by ZVAD-FMK, a caspase inhibitor. These results show that NO-mediated apoptosis of cardiomyocytes is cGMP dependent and that caspases are involved in this process.

The Fas/Fas ligand system is involved in the pathogenesis of autoimmune myocarditis in rats.

The mechanisms responsible for myocardial injury and cell death in myocarditis are still unclear. We examined whether myocardial cell death occurs via apoptosis in myosin-induced autoimmune myocarditis in rats and whether the Fas/Fas ligand (FasL) system plays a role in this apoptosis. On days 14, 17, 21, and 35 after immunization with porcine heart myosin, some cardiomyocytes and infiltrating lymphocytes were found to be apoptotic on in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay, but none was on day 60 and in control rats. Apoptotic indices peaked at day 17, and laddering of genomic DNA from the affected myocardium was observed on days 17 and 21 on agarose gel electrophoresis. The expression of Fas mRNA and protein was detected on days 17 and 21 in some cardiomyocytes and infiltrating lymphocytes by Northern blot analysis and immunohistochemistry, respectively. In addition, FasL was detected in some infiltrating lymphocytes on days 14, 17, and 21 by both in situ hybridization and immunostaining, and FasL-positive lymphocytes were mainly CD4+ cells. Some rats were injected with anti-Fas Ab (0.1 mg/kg) or anti-FasL Ab (0.1 mg/kg), and subsequently, inflammatory lesions exhibited less severe than did untreated rats with myocarditis. These findings suggest that cell death via apoptosis of cardiomyocytes and lymphocytes is one of the mechanisms of myocardial injury in autoimmune myocarditis, and that the Fas/FasL system might play a role in the induction of this apoptosis.

Participation of nitric oxide and peroxynitrite in the development of myocardial tissue damage in acute myocardial infarction.

Many factors, including superoxides, contribute to tissue damage in acute myocardial infarction (AMI). Excess nitric oxide (NO) produced by inducible NO synthase (iNOS) has also been reported to participate in myocardial injury associated with AMI, but its exact role remains unclear. To elucidate the role of NO and peroxynitrite in the pathogenesis of myocardial injury associated with AMI, we examined the expression of iNOS in the autopsied specimens of the left ventricle obtained from 15 patients with AMI and five with old MI by immunohistochemistry using an anti-iNOS polyclonal antibody. The distribution of nitrotyrosine was also examined immunohistochemically. In patients who died from 12 hours to 3 weeks after the infarction, positive immunoreactivity for iNOS was observed in residual myocytes, macrophages, and vascular endothelial cells in the peri-infarcted area. Degenerating myocytes in that area in all of that group showing positive staining for iNOS were also stained positive for anti-nitrotyrosine antibody selfsame. These findings were not observed in the myocardial specimens obtained from patients who died within 12 hours after the onset of AMI, showing a minimal number of inflammatory cells, or in the specimens from patients with an old myocardial infarction, which showed scar tissue and no cellular infiltration. Inducible NOS and nitrotyrosine were expressed in damaged myocardium from patients with AMI, suggesting that the NO radical and peroxynitrite are involved in the pathogenesis of myocardial damage.

Distribution of inducible nitric oxide synthase in ulcerative colitis.

OBJECTIVE: To investigate reports that the biosynthesis of nitric oxide (NO) is increased in the colonic mucosa of patients with active ulcerative colitis (UC), which suggests that serum NOx levels may be an important indicator of UC activity. METHODS: To determine the role of NO within the colon, we purchased polyclonal antibodies against human-inducible NO synthase (iNOS). We then examined the distribution of iNOS-reactive cells in UC colon tissues. RESULTS: In specimens from 12 UC patients, iNOS-positive neutrophils and macrophages were observed at the base of the ulcer but not in distant areas in the active stage. iNOS expression in colon mucosa was virtually absent during the inactive stage of UC and within the colon of patients with non-UC colitis. CONCLUSIONS: We conclude that NO in colonic mucosa may play a potential role in the pathogenesis of UC.

Nitric oxide contributes to the progression of myocardial damage in experimental autoimmune myocarditis in rats.

BACKGROUND: Excess amounts of NO produced by an inducible NO synthase (iNOS) in response to cytokines may be cytotoxic and can be destructive to tissue. We investigated the role of NO in the development of myocardial damage and the effects of aminoguanidine (AG), an inhibitor of iNOS, on experimental autoimmune myocarditis in rats. METHODS AND RESULTS: Autoimmune myocarditis was induced in 20 Lewis rats by injection of porcine cardiac myosin. Ten of the 20 rats were administered AG. The severity of myocarditis was evaluated by measuring the size of myocarditic lesion and serum levels of CK-MB. Serum NO levels were determined using the Cd/Cu method. Tissue specimens were immunohistochemically examined for iNOS and nitrotyrosine. Histopathological study revealed extensive myocardial destruction and massive inflammatory cell infiltration in AG-untreated rats but only focal mononuclear cell infiltration in AG-treated rats. The mean percent areas of inflammatory lesions in the untreated and treated rats were 56 +/- 13% and 3 +/- 2%, respectively (P < .001). NO levels were 102 +/- 23 and 25 +/- 9 IU/L, respectively (P < .01). CK-MB levels were 68 +/- 13 and 16 +/- 13 nmol/L, respectively (P < .01). Superoxide production as measured with an ex vivo monitoring system was also significantly decreased in the treated rats. Nitrotyrosine relating to the generation of peroxynitrite was detected through immunostaining in the inflammatory lesions of untreated rats but not in those of treated rats. CONCLUSIONS: Excess amounts of NO produced by iNOS appear to contribute to the progression of myocardial damage in myocarditis. AG may prove to be useful in the treatment of myocarditis.

Calcitonin gene-related peptide (CGRP) and hypertrophy of cardiomyocytes.

We studied whether calcitonin gene-related peptide (CGRP), a neuropeptide secreted from the sensory nervous supply to the myocardium, induces hypertrophy of cardiomyocytes in culture. CGRP increased the cell surface area of neonatal rat cardiomyocytes; the surface area of the cells was almost doubled by treatment with CGRP for 48 h. Furthermore, CGRP up-regulated mRNA expression for skeletal alpha-actin and atrial natriuretic peptides, which are genetic markers for cardiac hypertrophy. These results indicate that CGRP is a potent hypertrophic factor for cardiomyocytes.

Hypertrophic cardiomyopathy in Noonan syndrome.

Noonan syndrome, a well-known multiple congenital anomalies syndrome, is frequently accompanied by cardiovascular diseases including hypertrophic cardiomyopathy (HCM). The incidence of HCM in Noonan syndrome is approximately 20-30% and one-third of cases reveal ventricular outflow obstruction. HCM in Noonan syndrome is occasionally associated with a congenital heart defect, whereas classic HCM seldom accompanies cardiac malformations. Asymmetric septal hypertrophy and symmetric septal hypertrophy (concentric hypertrophy) can be observed both in HCM with Noonan syndrome and in classic HCM, but apical hypertrophy has not been reported in Noonan syndrome yet, although it appears in classic HCM. Congestive heart failure is the major cause of death in patients with HCM in Noonan syndrome, but cases of sudden death have also been reported. The histopathologic findings of ventricular myocardial tissue in HCM with Noonan syndrome are similar to those in classic HCM.

Engineering of artificial cell adhesion proteins by grafting the Arg-Gly-Asp cell adhesive signal to a calpastatin segment.

A new artificial cell adhesive protein was engineered by grafting the Arg-Gly-Asp (RGD) sequence, the minimal recognition signal of fibronectin for interaction with integrins, to a calpastatin segment by in vitro mutagenesis. The mutagenized protein showed cell adhesive activity in addition to calpain inhibitory activity. The RGD signal grafted to the calpastatin segment was recognized by the vitronectin receptor but not by the fibronectin receptor.

Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography.

A method for the enantiomeric analysis of amino acids of mammalian tissues is described. An excellent resolution of D- and L-enantiomers of common protein amino acids was achieved by employing a combination of thin-layer chromatography and high-performance liquid chromatography. D-Enantiomers and L-enantiomers of glutamate, aspartate, glutamine, asparagine, serine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine and histidine, as well as glycine were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide. The amino acid diastereomers were separated by two-dimensional thin-layer chromatography. Each amino acid diastereomer was then analysed by reversed-phase high-performance liquid chromatography for the resolution of D- and L-enantiomers. Very sharp peaks were obtained using a conventional octadecylsilyl-bonded column, and the possibility of analysing these amino acids (except tyrosine and histidine) in subnanomole amounts was indicated. The method was used to demonstrate the presence of D-enantiomers of alanine, proline and serine in mouse kidney.

Construction and characterization of a fusion protein with epidermal growth factor and the cell-binding domain of fibronectin.

An efficient expression system was constructed for C-EGF, a fusion protein made of a fragment of the cell-binding domain of human fibronectin (FN) bound with epidermal growth factor (EGF). C-EGF was produced in Escherichia coli HB101 cells carrying the recombinant plasmid pCE102 as inclusion bodies, which were solubilized and refolded after purification. C-EGF had both cell-adhesive and EGF activities, so it might be more effective than EGF in therapeutic applications. This fusion system would be useful for the construction of a recombinant drug delivery system for cells that have fibronectin receptors (integrins).

Antigenic diversity of the S-layer proteins from pathogenic strains of Aeromonas hydrophila and Aeromonas veronii biotype sobria.

The antigenic relatedness of paracrystalline surface array proteins with subunit molecular weights of approximately 52,000 from isolates of Aeromonas hydrophila and Aeromonas veronii biotype sobria belonging to a single heat-stable serogroup was examined. Enzyme-linked immunosorbent assay and immunoblotting with two different polyclonal antisera against surface exposed and non-surface-exposed epitopes of the S-layer protein from A. hydrophila TF7 showed that the S-layer proteins of the mesophilic aeromonads were antigenically diverse. NH2-terminal amino acid sequence analysis of four antigenically different proteins showed that while the proteins were structurally related, they differed in primary sequence. Absorption experiments with heterologous live cells showed that cross-reactive epitopes were in non-surface-exposed regions of the S-layer proteins, while absorption with homologous live cells showed that the immunodominant epitopes of the S-layer protein of strain TF7 were strain specific and exposed on the surface of the native, tetragonal array produced by this strain. Proteolytic digestion of the TF7 S-layer protein with trypsin, chymotrypsin, or endoproteinase Glu-C produced an amino-terminal peptide of approximate Mr 38,000 which was refractile to further proteolytic cleavage under nondenaturing conditions. This peptide carried the immunodominant surface-exposed region of the protein, and chemical cleavage with cyanogen bromide further mapped the portion of these surface-exposed epitopes to a peptide of approximate Mr 26,000, part of which maps within the Mr 38,000 protease-resistant NH2-terminal peptide.

The presence of free D-alanine, D-proline and D-serine in mice.

The presence of free D-alanine, D-proline and D-serine was demonstrated in mammalian tissues, using a mutant mouse strain lacking D-amino acid oxidase. In the experiment, free amino acids from the kidney and serum were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA) to diastereomers, separated by two-dimensional thin-layer chromatography (TLC), and analysed by reversed-phase high-performance liquid chromatography (HPLC) for the resolution of D- and L-isomers. D/L ratios of alanine, proline and serine were obtained based on the peak areas of HPLC.

Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.

Role of type III homology repeats in cell adhesive function within the cell-binding domain of fibronectin.

Recombinant fibronectin (FN) fragments and their mutant proteins were produced to elucidate the role of type III homology repeats in cell adhesive activity within the cell-binding domain of FN. Cell adhesive activity of the 11.5-kDa fragment, the cell attachment site of the cell-binding domain, was less than 0.1% that of native FN despite the presence of the Arg-Gly-Asp-Ser sequence. The activity increased as type III homology repeats were added to the N terminus of the 11.5-kDa fragment, and a 52-kDa fragment with four additional type III repeats had almost the same activity of native FN. Deletion of Arg-Gly-Asp from the fully active fragments completely abolished the cell adhesive activity. Deletion of one or two repeats from the 52-kDa fragment affected the extent of the cell adhesive activity, the degree of the effect being inversely correlated with the distance of the deletion from the type III repeat containing Arg-Gly-Asp-Ser. Rearrangement of type III repeats caused much loss of activity. These results suggest that the number and kinds of type III repeats and their correct alignment rather than the putative synergistic site decide the extent of the specific cell adhesive activity.

Inhibition of lung metastasis by synthetic and recombinant fragments of human fibronectin with functional domains.

We have investigated the antimetastatic effect of synthetic or recombinant peptides containing the functional domains of fibronectin on experimental and spontaneous lung metastases of murine tumor cells. CS1 peptide which is present within type III homology connecting segment (IIICS) as well as C-274 (cell-binding domain) were able to inhibit experimental lung metastasis when co-injected intravenously (iv) with B16-BL6 melanoma cells, while H-271 (heparin-binding domain) could not. In the spontaneous metastasis model, multiple iv administrations of CS1 or C-274 after surgical excision of primary tumors caused a significant reduction of metastatic colonies in the lung. Both CS1 and C-274 significantly inhibited cell adhesion and migration to fibronectin-coated substrates when added freely in solution. CS1 peptide also inhibited the cell adhesion and migration to laminin-coated substrates, but C-274 did not. H-271 did not have any inhibitory effect on cell adhesion or migration to either of the substrates. Similarly, CS1 inhibited tumor invasion to both Matrigel/fibronectin- and Matrigel/laminin-coated filters, whereas C-274 inhibited the invasion to only Matrigel/fibronectin-coated filter. These results indicate that CS1 peptide of fibronectin, lacking the Arg-Gly-Asp-containing domain, actively inhibits tumor metastases in spontaneous and experimental metastasis models. The use of such a peptide might offer a promising therapeutic approach for combatting or preventing cancer metastasis.

Modulation of haptotactic migration of metastatic melanoma cells by the interaction between heparin and heparin-binding domain of fibronectin.

We utilized recombinant fibronectin polypeptides with cell-binding domain and heparin-binding domains (referred to as C-274 and H-271, respectively) and their fusion polypeptide (CH-271) to examine the role of sulfated polysaccharide heparin and/or the functional domains of fibronectin in modulating tumor cell behavior. Both C-274 and CH-271 polypeptides with cell-binding domains promoted the adhesion and migration of B16-BL6 melanoma cells, whereas H-271 did not. Heparin bound to the immobilized polypeptides with heparin-binding domain (H-271, CH-271, and a mixture of C-274 and H-271 or fibronectin) but did not affect the tumor cell adhesion to the substrates. At the same time, heparin or two monoclonal antibodies against the heparin-binding domain were able to inhibit the haptotactic migration to CH-271 or fibronectin, though not to C-274 or a mixture of C-274 and H-271. This suggests that although heparin did not affect tumor cell adhesion to the cell-binding domain near the heparin-binding domain in CH-271 or fibronectin, it did lead to a modulation of cell motility. It seems likely that the regulatory mechanism may depend on interaction between heparin-like molecules on the cell surface and the heparin-binding domain in fibronectin, rather than on simple steric hindrance or on the masking of the cell-binding domain caused by the binding of heparin to heparin-binding domain.

Characterization of a functional domain of human calpastatin.

Expression plasmids were constructed from the cDNA of human calpastatin to examine the contribution to the inhibition of calpain of highly conserved sequences in each of four repetitive domains. A series of deletion derivatives of domain 1 proteins, truncated at either the amino or carboxy terminus, were produced in E. coli. Deletion from the amino terminus past the amino terminal conserved sequence decreased the inhibition. When the middle conserved sequence, the M-sequence, was further deleted, no inhibition was detected, but deletion from the carboxy terminus past the carboxy terminal conserved sequence did not decrease the inhibition until the M-sequence was reached. Nuclear magnetic resonance and circular dichroism spectra showed that domain 1 has an unfolded structure. Peptides that contained the M-sequence and some neighboring sequences were synthesized to measure the minimum size of the inhibitory peptide, which was the M-sequence with the next six residues on the amino terminal side.