Production and inhibition of the gelatinolytic matrix metalloproteinases in a human model of vein graft stenosis.
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OBJECTIVES: human vein graft stenoses are caused by intimal hyperplasia, a process which is characterised by extensive degradation and accumulation of extracellular matrix. This study investigated the role of the matrix metalloproteinases (MMPs) - the principal physiological mediators of extracellular matrix degradation - in the development of intimal hyperplasia in cultured human long saphenous vein. DESIGN: experimental study. MATERIALS AND METHODS: paired venous segments with the endothelium intact or denuded were cultured in standard conditions for 14 days. At the termination of culture, MMPs were extracted from one half of the tissue, whilst the remainder of the vein was prepared for histological examination. RESULTS: stereologic analysis revealed that the endothelium intact veins developed a significantly thicker neointima when compared to the denuded venous segments (20 micron v. 0 micron, p=0.006). Quantification of MMPs by substrate gel enzymography demonstrated that the development of a neointima was associated with increased production of the gelatinolytic MMP-9 (p=0. 03) in intact veins. Immunocytochemistry showed that the MMP-9 localised to the internal elastic lumina, which suggested a role in facilitating smooth-muscle-cell migration into the intima. The role of MMPs-2 and -9 in intimal hyperplasia was further investigated by culturing intact venous segments with a therapeutic concentration of doxycycline--a potent MMP inhibitor. These experiments demonstrated that a therapeutic dose of doxycycline significantly reduced neointimal thickness (control 21 micron, doxycycline 10 mg/l-5.5 micron), in conjunction with a significant reduction in the production of MMP-9. CONCLUSIONS: these data suggest that elevated levels of MMPs may play a significant role in the development of human intimal hyperplasia and that inhibition of these enzymes may offer a potential therapeutic strategy for the prevention of hyperplastic lesions. (+info)
Differential modulation of caffeine- and IP3-induced calcium release in cultured arterial tissue.
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To investigate the Ca2+-dependent plasticity of sarcoplasmic reticulum (SR) function in vascular smooth muscle, transient responses to agents releasing intracellular Ca2+ by either ryanodine (caffeine) or D-myo-inositol 1,4,5-trisphosphate [IP3; produced in response to norepinephrine (NE), 5-hydroxytryptamine (5-HT), arginine vasopressin (AVP)] receptors in rat tail arterial rings were evaluated after 4 days of organ culture. Force transients induced by all agents were increased compared with those induced in fresh rings. Stimulation by 10% FCS during culture further potentiated the force and Ca2+ responses to caffeine (20 mM) but not to NE (10 microM), 5-HT (10 microM), or AVP (0.1 microM). The effect was persistent, and SR capacity was not altered after reversible depletion of stores with cyclopiazonic acid. The effects of serum could be mimicked by culture in depolarizing medium (30 mM K+) and blocked by the addition of verapamil (1 microM) or EGTA (1 mM) to the medium, lowering intracellular Ca2+ concentration ([Ca2+]i) during culture. These results show that modulation of SR function can occur in vitro by a mechanism dependent on long-term levels of basal [Ca2+]i and involving ryanodine- but not IP3 receptor-mediated Ca2+ release. (+info)
Differential regulation of extracellular matrix molecules by mechanical strain of fetal lung cells.
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We have previously shown that an intermittent mechanical strain regimen (5% elongation, 60 cycles/min, 15 min/h) that simulates fetal breathing movements stimulated fetal rat lung cell proliferation. Because normal lung growth requires proper coordination between cell proliferation and extracellular matrix (ECM) remodeling, we subjected organotypic cultures of fetal rat lung cells (day 19 of gestation, term = 22 days) to this strain regimen and examined alterations in ECM gene and protein expression. Northern analysis revealed that mechanical strain reduced messages for procollagen-alpha1(I) and biglycan and increased the levels of mRNA for collagen-alpha1(IV) and -alpha2(IV), whereas laminin beta-chain mRNA levels remained constant. Regardless of mRNA changes, mechanical strain increased the protein content of type I and type IV collagen as well as of biglycan in the medium. Mechanical strain did not affect gene expression of several matrix metalloproteinases (MMPs), such as MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), and MMP-3 (stromelysin-1). Neither collagenase nor gelatinase (A and B) activities in conditioned medium were affected by mechanical strain. Tissue inhibitor of metalloproteinase activities in conditioned medium remained unchanged during the 48-h intermittent mechanical stretching. These data suggest that an intermittent mechanical strain differentially regulates gene and protein expression of ECM molecules in fetal lung cells. The observed increase in matrix accumulation appears to be mainly a result of an increased synthesis of ECM molecules and not of decreasing activity of degradative enzymes. (+info)
Expression and action of transforming growth factor beta (TGFbeta1, TGFbeta2, and TGFbeta3) during embryonic rat testis development.
(60/6597)
The objective of the current study was to determine the role of transforming growth factor beta (TGFbeta) during seminiferous cord formation and embryonic testis development. The expression pattern of mRNA for TGFbeta isoforms was evaluated during testis development through a quantitative reverse transcription-polymerase chain reaction (QRT-PCR) procedure. Expression of mRNA for TGFbeta1 was highest at postnatal day 0 (P0) and P10. In contrast, TGFbeta2 was high at embryonic day 15 (E15), declined at E16, and showed a transient increase at P0 through P3 of testis development. Interestingly, expression of mRNA for TGFbeta3 was high during embryonic development and then declined after P3. Immunohistochemical localization of TGFbeta1 and TGFbeta2 demonstrated expression in Sertoli cells at E14 and in the seminiferous cords at P0. Selective interstitial cells expressed high concentrations of TGFbeta1 and TGFbeta2 in P0 testis. TGFbeta3 was expressed in selective cells at the junction of the E14 testis and mesonephros. The cells expressing TGFbeta3 in the testis appeared to be preperitubular cells that resided around the seminiferous cords. TGFbeta3 was localized to gonocytes in P0 testis. TGFbeta1 was found to have no influence on seminiferous cord formation in embryonic organ cultures of E13 testis. In contrast, growth of both E13 and E14 embryonic organ cultures was inhibited by TGFbeta1 and resulted in reduced testis size (40% of controls) with fewer cords present. A P0 testis cell culture and thymidine incorporation assay were used to directly examine the effects of recombinant TGFbeta1. TGFbeta1 alone had no influence on thymidine incorporation in P0 testis cell cultures when compared to controls. Interestingly, TGFbeta1 inhibited epidermal growth factor (EGF), and 10% calf serum stimulated P0 testis cell growth but not FSH-stimulated growth. Therefore, TGFbeta1 appears to inhibit testis growth in both the embryonic and early postnatal periods. The hormonal regulation of TGFbeta expression was measured using P0 testis cell cultures and a QRT-PCR procedure for each TGFbeta isoform. High concentrations of EGF stimulated expression of mRNA for TGFbeta1 after 24 h but suppressed expression of TGFbeta3. In contrast, there was no effect of FSH on TGFbeta isoform expression. In summary, TGFbeta regulates embryonic and P0 testis growth through inhibiting the actions of positive growth factors such as EGF. In addition, EGF but not FSH appears to regulate TGFbeta isoform expression. Combined observations from the present study demonstrate that TGFbeta isoforms are differentially expressed and appear to be regulators of testis growth during the embryonic and early postnatal periods. (+info)
Aging-dependent depression in the kinetics of force development in rat skinned myocardium.
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Normal aging of the rodent heart results in prominent prolongation of the twitch. We tested the hypothesis that increased expression of beta-myosin heavy chain (MHC), as occurs in the normal aging process in the rodent heart, contributes to the prolongation of the twitch by depressing the kinetics of cross-bridge interaction. Using 3-, 9-, 21-, and 33-mo-old male Fischer 344 x Brown Norway F1 hybrid rats, we examined both the rate of tension development (kCa) and unloaded shortening velocity in chemically skinned myocardium. Although kCa in all four age groups was dependent on the level of Ca2+ activation, both submaximal and maximal kCa were significantly slower in 9-, 21-, and 33-mo-old rats relative to 3-mo-old rats. Furthermore, unloaded shortening velocity was significantly reduced in 9-, 21-, and 33-mo-old rats compared with 3-mo-old rats. Collectively, these data strongly suggest that the aging-related increase in beta-MHC expression results in a progressive slowing of cross-bridge interaction kinetics in skinned myocardium, which most likely contributes to the overall aging-dependent reduction in myocardial functional capacity. (+info)
Changes in intracellular Na+ and pH in rat heart during ischemia: role of Na+/H+ exchanger.
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The role of the Na+/H+ exchanger in rat hearts during ischemia and reperfusion was investigated by measurements of intracellular Na+ concentration ([Na+]i) and intracellular and extracellular pH. Under our standard conditions (2-Hz stimulation), 10 min of ischemia caused no significant rise in [Na+]i but an acidosis of 1.0 pH unit, suggesting that the Na+/H+ exchanger was inactive during ischemia. This was confirmed by showing that the Na+/H+ exchange inhibitor methylisobutyl amiloride (MIA) had no effect on [Na+]i or on intracellular pH during ischemia. However, there was a short-lived increase in [Na+]i of 8.2 +/- 0.6 mM on reperfusion, which was reduced by MIA, showing that the Na+/H+ exchanger became active on reperfusion. To investigate the role of metabolic changes, we measured [Na+]i during anoxia. The [Na+]i did not change during 10 min of anoxia, but there was a small, transient rise of [Na+]i on reoxygenation, which was inhibited by MIA. In addition, we show that the Na+/H+ exchanger, tested by sodium lactate exposure, was inhibited during anoxia. These results show that the Na+/H+ exchanger is inhibited during ischemia and anoxia, probably by an intracellular metabolic mechanism. The exchanger activates rapidly on reperfusion and can cause a rapid rise in [Na+]i. (+info)
Dynamics of tissue oxygenation in isolated rabbit heart as measured with near-infrared spectroscopy.
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We investigated the role of myoglobin (Mb) in supplying O2 to mitochondria during transitions in cardiac workload. Isovolumic rabbit hearts (n = 7) were perfused retrogradely with hemoglobin-free Tyrode solution at 37 degrees C. Coronary venous O2 tension was measured polarographically, and tissue oxygenation was measured with two-wavelength near-infrared spectroscopy (NIRS), both at a time resolution of approximately 2 s. During transitions to anoxia, 68 +/- 2% (SE) of the NIRS signal was due to Mb and the rest to cytochrome oxidase. For heart rate steps from 120 to 190 or 220 beats/min, the NIRS signal decreased significantly by 6.9 +/- 1.3 or 11.1 +/- 2.1% of the full scale, respectively, with response times of 11.0 +/- 0.8 or 9.1 +/- 0.5 s, respectively. The response time of end-capillary O2 concentration ([O2]), estimated from the venous [O2], was 8.6 +/- 0.8 s for 190 beats/min (P < 0.05 vs. NIRS time) or 8.5 +/- 0.9 s for 220 beats/min (P > 0.05). The mean response times of mitochondrial O2 consumption (VO2) were 3.7 +/- 0.7 and 3.6 +/- 0.6 s, respectively. The deoxygenation of oxymyoglobin (MbO2) accounted for only 12-13% of the total decrease in tissue O2, with the rest being physically dissolved O2. During 11% reductions in perfusion flow at 220 beats/min, Mb was 1.5 +/- 0.4% deoxygenated (P < 0.05), despite the high venous PO2 of 377 +/- 17 mmHg, indicating metabolism-perfusion mismatch. We conclude that the contribution of MbO2 to the increase of VO2 during heart rate steps in saline-perfused hearts was small and slow compared with that of physically dissolved O2. (+info)
Ser16 prevails over Thr17 phospholamban phosphorylation in the beta-adrenergic regulation of cardiac relaxation.
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Phospholamban is a critical regulator of sarcoplasmic reticulum Ca2+-ATPase and myocardial contractility. To determine the extent of cross signaling between Ca2+ and cAMP pathways, we have investigated the beta-adrenergic-induced phosphorylation of Ser16 and Thr17 of phospholamban in perfused rat hearts using antibodies recognizing phospholamban phosphorylated at either position. Isoproterenol caused the dose-dependent phosphorylation of Ser16 and Thr17 with strikingly different half-maximal values (EC50 = 4.5 +/- 1.6 and 28. 2 +/- 1.4 nmol/l, respectively). The phosphorylation of Ser16 induced by isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine correlated to increased cardiac relaxation (r = 0.96), whereas phosphorylation of Thr17 did not. Elevation of extracellular Ca2+ did not induce phosphorylation at Thr17; only in the presence of a submaximal dose of isoproterenol, phosphorylation at Thr17 increased eightfold without additional effects on relaxation rate. Thr17 phosphorylation was partially affected by ryanodine and was completely abolished in the presence of 1 micromol/l verapamil or nifedipine. The data indicate that 1) phosphorylation of phospholamban at Ser16 by cAMP-dependent protein kinase is the main regulator of beta-adrenergic-induced cardiac relaxation definitely preceding Thr17 phosphorylation and 2) the beta-adrenergic-mediated phosphorylation of Thr17 by Ca2+-calmodulin-dependent protein kinase required influx of Ca2+ through the L-type Ca2+ channel. (+info)