Meningeal inflammatory response and fibrous tissue remodeling around intracortical implants: An in vivo two-photon imaging study.

Meningeal inflammation and encapsulation of neural electrode arrays is a leading cause of device failure, yet little is known about how it develops over time or what triggers it. This work characterizes the dynamic changes of meningeal inflammatory cells and collagen-I in order to understand the meningeal tissue response to neural electrode implantation. We use in vivo two-photon microscopy of CX3CR1-GFP mice over the first month after electrode implantation to quantify changes in inflammatory cell behavior as well as meningeal collagen-I remodeling. We define a migratory window during the first day after electrode implantation hallmarked by robust inflammatory cell migration along electrodes in the meninges as well as cell trafficking through meningeal venules. This migratory window attenuates by 2 days post-implant, but over the next month, the meningeal collagen-I remodels to conform to the surface of the electrode and thickens. This work shows that there are distinct time courses for initial meningeal inflammatory cell infiltration and meningeal collagen-I remodeling. This may indicate a therapeutic window early after implantation for modulation and mitigation of meningeal inflammation.

Single-shot detection of mid-infrared spectra by chirped-pulse upconversion with four-wave difference frequency generation in gases.

Single-shot detection of ultrabroadband mid-infrared spectra was demonstrated by using chirped-pulse upconversion technique with four-wave difference frequency generation in gases. Thanks to the low dispersion of the gas media, the bandwidth of the phase matching condition of the upconversion process becomes very broad and the entire mid-infrared spectrum spanning from 200 to 5500 cm(-1) was upconverted by using a 10 ps chirped pulse to visible wavelength radiation, which was detected with a conventional visible dispersive spectrometer. This method was demonstrated by the successful measurement of infrared absorption spectra of organic polymer films.

Reduction of neurovascular damage resulting from microelectrode insertion into the cerebral cortex using in vivo two-photon mapping.

Penetrating neural probe technologies allow investigators to record electrical signals in the brain. The implantation of probes causes acute tissue damage, partially due to vasculature disruption during probe implantation. This trauma can cause abnormal electrophysiological responses and temporary increases in neurotransmitter levels, and perpetuate chronic immune responses. A significant challenge for investigators is to examine neurovascular features below the surface of the brain in vivo. The objective of this study was to investigate localized bleeding resulting from inserting microscale neural probes into the cortex using two-photon microscopy (TPM) and to explore an approach to minimize blood vessel disruption through insertion methods and probe design. 3D TPM images of cortical neurovasculature were obtained from mice and used to select preferred insertion positions for probe insertion to reduce neurovasculature damage. There was an 82.8 +/- 14.3% reduction in neurovascular damage for probes inserted in regions devoid of major (>5 microm) sub-surface vessels. Also, the deviation of surface vessels from the vector normal to the surface as a function of depth and vessel diameter was measured and characterized. 68% of the major vessels were found to deviate less than 49 microm from their surface origin up to a depth of 500 microm. Inserting probes more than 49 microm from major surface vessels can reduce the chances of severing major sub-surface neurovasculature without using TPM.

Melatonin in Glycyrrhiza uralensis: response of plant roots to spectral quality of light and UV-B radiation.

Melatonin (N-acetyl-5-methoxytryptamine) is known to be synthesized and secreted by the pineal gland in vertebrates. Evidence for the occurrence of melatonin in the roots of Glycyrrhiza uralensis plants and the response of this plant to the spectral quality of light including red, blue and white light (control) and UV-B radiation (280-315 nm) for the synthesis of melatonin were investigated. Melatonin was extracted and quantified in seed, root, leaf and stem tissues and results revealed that the root tissues contained the highest concentration of melatonin; melatonin concentrations also increased with plant development. After 3 months of growth under red, blue and white fluorescent lamps, the melatonin concentrations were highest in red light exposed plants and varied depending on the wavelength of light spectrum in the following order red >> blue > or = white light. Interestingly, in a more mature plant (6 months) melatonin concentration was increased considerably; the increments in concentration were X4, X5 and X3 in 6-month-old red, blue and white light exposed (control) plants, respectively. The difference in melatonin concentrations between blue and white light exposed (control) plants was not significant. The concentration of melatonin quantified in the root tissues was highest in the plants exposed to high intensity UV-B radiation for 3 days followed by low intensity UV-B radiation for 15 days. The reduction of melatonin under longer periods of UV-B exposure indicates that melatonin synthesis may be related to the integrated (intensity and duration) value of UV-B irradiation. Melatonin in G. uralensis plant is presumably for protection against oxidative damage caused as a response to UV irradiation.

Plant-environment interactions: Accumulation of hypericin in dark glands of Hypericum perforatum.

BACKGROUND AND AIMS: Hypericum perforatum is a perennial herbaceous plant and an extract from this plant has a significant antidepressant effect when administered to humans. The plant is characterized by its secretory glands, also known as dark glands, which are mainly visible on leaves and flowers. The current study evaluates the influence of several environmental factors and developmental stages of the plant on the accumulation and synthesis of hypericin and pseudohypericin (Hy-G), the major bioactive constituents, in H. perforatum plants. METHODS: The appearance of dark glands on different parts of the plant, under several environmental conditions, was monitored by microscopy. Hy-G concentrations were quantified by high-performance liquid chromatography. KEY RESULTS: A significant presence of dark glands accompanying the highest concentrations of Hy-G was observed in the stamen tissues more than in any other organ of H. perforatum. A linear relationship between the number of dark glands and net photosynthetic rate of the leaf and Hy-G concentration in the leaf tissue was also established. A very high concentration of Hy-G was measured in the dark-gland tissues, but in the tissues without any dark glands it was almost absent. The presence of emodin, a precursor of Hy-G, at a high concentration in the dark-gland tissues, and its absence in the surrounding tissues was also observed, suggesting that the site of biosynthesis of Hy-G is in the dark-gland cells. A significantly low concentration of Hy-G (occasionally non-detectable) was measured in the xylem sap of the stem tissues. The dark-gland tissues collected from leaves, stems or flowers contained similar concentrations of Hy-G. CONCLUSIONS: The concentration of Hy-G in various organs of H. perforatum plants is dependent on the number of dark glands, their size or area, not on the location of the dark glands on the plant. The study provides the first experimental evidence that Hy-G is synthesized and accumulates in dark glands.

Temperature stress can alter the photosynthetic efficiency and secondary metabolite concentrations in St. John's wort.

Temperature stress is known to cause many physiological, biochemical and molecular changes in plant metabolism and possibly alter the secondary metabolite production in plants. The hypothesis of the current study was that temperature stress can increase the secondary metabolite concentrations in St. John's wort. Plants were grown under controlled environments with artificial light using cool white fluorescent lamps and CO2 enrichment and 70-day-old plants were subjected for 15 days to different temperature treatments of 15, 20, 25, 30 and 35 degrees C before harvested. Major aim of the study was to increase the major secondary metabolites in St. John's wort by applying temperature stress and to evaluate the physiological status of the plant especially the photosynthetic efficiency and peroxidase activity of the leaf tissues exposed to different temperatures under precisely controlled environmental factors. Results revealed that relatively high (35 degrees C) or low (15 degrees C) temperatures reduced the photosynthetic efficiency of the leaves of St. John's wort plants and resulted in low CO2 assimilation. Net photosynthetic rates and the maximal quantum efficiency of PSII photochemistry of the dark adopted leaves (phi(p)max) decreased significantly in the leaves of plants grown under 35 or 15 degrees C temperature treatments. High temperature (35 degrees C) treatment increased the leaf total peroxidase activity and also increased the hypericin, pseudohypericin and hyperforin concentrations in the shoot tissues. These results provide the first indication that temperature is an important environmental factor to optimize the secondary metabolite production in St. John's wort and controlled environment technology can allow the precise application of such specific stresses.

Photoautotrophic culture of Coffea arabusta somatic embryos: photosynthetic ability and growth of different stage embryos.

Coffea arabusta somatic embryos were cultured and development of stomata, rate of CO2 fixation or production, chlorophyll content and chlorophyll fluorescence were studied in embryos at different stages of development. Cotyledonary and germinated embryos have photosynthetic capacity, although pretreatment at a high photosynthetic photon flux (PPF) (100 micromol m(-2) s(-1)) for 14 d increased photosynthetic ability. Except in a very small number of cases, stomata did not develop fully in precotyledonary stage embryos and were absent in torpedo stage embryos. Low chlorophyll content (90-130 microg g(-1) fresh mass) was noted in torpedo and precotyledonary stage embryos compared with cotyledonary and germinated embryos (300-500 microg g(-1) fresh mass). Due to the absence of stomata and low chlorophyll content in the torpedo and precotyledonary stage embryos, the photosynthetic rate was low and, in some cases, CO2 production was observed. These data suggest that the cotyledonary stage is the earliest stage that can be cultured photoautotrophically to ensure plantlet development. When grown photoautotrophically (in a sugar-free medium with CO2 enrichment in the culture headspace and high photosynthetic photon flux), torpedo and precotyledonary stage embryos lost 20-25% of their initial dry mass after 60 d of culture. However, in cotyledonary and germinated embryos, the dry mass of each embryo increased by 10 and 50%, respectively. By using a porous supporting material, growth (especially root growth) was increased in cotyledonary stage embryos. In addition, photoautotrophic conditions, high PPF (100-150 micromol m(-2) s(-1)) and increased CO2 concentration (1100 micromol mol(-1)) were found to be necessary for the development of plantlets from cotyledonary stage embryos.

Photoautotrophic culture of Coffea arabusta somatic embryos: development of a bioreactor for large-scale plantlet conversion from cotyledonary embryos.

Somatic embryos were developed from in vitro-grown leaf discs of Coffea arabusta in modified Murashige and Skoog medium under 30 micromol m(-2) s(-1) photosynthetic photon flux (PPF). Cotyledonary stage embryos were selected from the 14-week-old cultures and were placed under a high (100 micromol m(-2) s(-1) PPF for 14 d. These pretreated embryos were grown photoautotrophically in three different types of culture systems: Magenta vessel; RITA-bioreactor (modified to improve air exchange); and a specially designed temporary root zone immersion bioreactor system (TRI-bioreactor) with forced ventilation. The aims of the study were to achieve large-scale embryo-to-plantlet conversion, and to optimize growth of plantlets under photoautotrophic conditions. The plantlet conversion percentage was highest (84 %) in the TRI-bioreactor and lowest in the modified RITA-bioreactor (20 %). Growth and survival of converted plantlets following 45 d of photoautotrophic culture in each of the three culture systems were studied. Fresh and dry masses of leaves and roots of plantlets developed in the TRI-bioreactor were significantly greater than those of plantlets developed in the modified RITA-bioreactor or Magenta vessel. The net photosynthetic rate, chlorophyll fluorescence and chlorophyll contents were also highest in plantlets grown in the TRI-bioreactor. Normal stomata were observed in leaves of plantlets grown in the TRI-bioreactor, whereas they could be abnormal in plantlets from the modified RITA-bioreactor. Survival of the plants after transfer from culture followed a similar pattern and was highest in the group grown in the TRI-bioreactor, followed by plants grown in the modified RITA-bioreactor and Magenta vessel. In addition, ex vitro growth of plants transferred from the TRI-bioreactor was faster than that of plants from the other culture systems.

Thrombin suppresses endothelial nitric oxide synthase and upregulates endothelin-converting enzyme-1 expression by distinct pathways: role of Rho/ROCK and mitogen-activated protein kinase.

An imbalance of nitric oxide and endothelin plays an important role in cardiovascular disease. Thrombin exerts profound effects on endothelial function. The present study investigated the molecular mechanisms by which thrombin regulates endothelial nitric oxide synthase (eNOS) and endothelin-converting enzyme (ECE)-1 expression in human endothelial cells. Incubation of human umbilical vein endothelial cells with thrombin (0.01 to 4 U/mL) for 15 to 24 hours markedly downregulated eNOS and increased ECE-1 protein level in a dose-dependent manner. Thrombin also decreased eNOS mRNA and increased ECE-1 mRNA level. In mRNA stability assay, thrombin shortened the half-life of eNOS mRNA but not that of ECE-1 mRNA. Activation of protease-activated receptor 1 by the agonist (SFLLRN, 10 to 100 micromol/L) had no effect on eNOS expression but increased ECE-1 level as thrombin. Thrombin activated Rho A and extracellular signal-regulated kinase (ERK)1 and ERK2. Inhibition of Rho A by C3 exoenzyme (20 microgram/mL) and ROCK by Y-27632 (10 micromol/L) prevented the downregulation of eNOS expression by thrombin. Y-27632 also prevented the reduction in NOS activity induced by prolonged incubation with thrombin. On the other hand, inhibition of ERK1 and ERK2 activation by PD98059 (50 micromol/L) prevented the upregulation of ECE-1 expression by thrombin as well as the increase in ECE activity and ET-1 accumulation in the medium. Treatment of rat aorta with thrombin overnight impaired endothelium-dependent relaxations but not endothelium-independent relaxations. Thus, thrombin suppresses eNOS and upregulates ECE-1 expression via Rho/ROCK and ERK pathway, respectively. These effects of thrombin may be important for endothelial dysfunction in cardiovascular disease, particularly during acute coronary episodes.

HMG-CoA reductase inhibition improves endothelial cell function and inhibits smooth muscle cell proliferation in human saphenous veins.

OBJECTIVES: This study examined effects of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor cerivastatin on human saphenous vein (SV), endothelial cells (EC) and smooth muscle cells (SMC). BACKGROUND: Venous bypass graft failure involves EC dysfunction and SMC proliferation. Substances that improve EC function and inhibit SMC proliferation would be of clinical relevance. METHODS: Both EC and SMC were isolated from SV. Endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production were analyzed by immunoblotting and porphyrinic microsensor. The SMC proliferation was assayed by 3H-thymidine incorporation. Protein kinases and cell cycle regulators were analyzed by immunoblotting. RESULTS: Cerivastatin (10(-9) to 10(-6) mol/liter) enhanced eNOS protein expression and NO release (about two-fold) in EC in response to Ca2+ ionophore (10(-6) mol/liter). This was fully abrogated by the HMG-CoA product mevanolate (2 x 10(-4) mol/liter). In SMC, platelet-derived growth factor (5 ng/ml) enhanced 3H-thymidine incorporation (298 +/- 23%, n = 4), activated cyclin-dependent kinase (Cdk2), phosphorylated Rb and down-regulated p27Kip1 (but not p21CiP1). Cerivastatin reduced the 3H-thymidine incorporation (164 +/- 11%, p < 0.01), inhibited Cdk2 activation and Rb phosphorylation, but did not prevent p27Kip1 down-regulation, nor p42mapk and p70S6K activation. Mevalonate abrogated the effects of cerivastatin on Cdk2 and Rb but only partially rescued the 3H-thymidine incorporation (from 164 +/- 11% to 211 +/- 13%, n = 4, p < 0.01). CONCLUSIONS: In humans, SVEC inhibition of HMG-CoA/mevalonate pathway contributes to the enhanced eNOS expression and NO release by cerivastatin, whereas in SMC, inhibition of this pathway only partially explains cerivastatin-induced cell growth arrest. Inhibition of mechanisms other than p42mapk and p70S6K or Cdk2 are also involved. These effects of cerivastatin could be important in treating venous bypass graft disease.

A combination of vermiculite and paper pulp supporting material for the photoautotrophic micropropagation of sweet potato.

A mixture of vermiculite (hydrous silicates) and paper pulp (waste product of paper industry) was used as a supporting material for the in vitro photoautotrophic micropropagation of plantlets. Sweet potato was used as a model plant to find out the appropriate proportion of vermiculite and paper pulp for the optimum growth of the plantlets. The plantlets grown in the conventional supporting material, agar, were used as the control. The study revealed that in all aspects, the plantlets grown in vermiculite mixed with 30% (w/w) paper pulp exhibited the highest growth performance. The shoot and root fresh mass were x2.7 greater than those in agar (control); the leaf, stem and root dry mass were also greater and at least two fold in this treatment compared with those in the control. The net photosynthetic rate per plantlet was highest in this treatment, and on day 20 it was 15.3 µmol CO(2) h(-1) as compared with 9.8 µmol CO(2) h(-1) in the control. The growth of both shoots and roots decreased gradually with the increase or decrease of percentage of paper pulp in the supporting material. In general, the growth was significantly poorer in the plantlets grown in 100% vermiculite than that in vermiculite mixed with 30% paper pulp but still greater than in the control. The porosity of the supporting materials increased with the increase in the percentage of paper pulp in the supporting material. After transplanting to the ex vitro condition the survival percentage did not vary significantly (90-100%) among the treatments, except in control where it was only 73%. The number of unfolded leaves and the stem height were similar among the treatments except those in the control.

Increased expression of L-type calcium channels in vascular smooth muscle cells at spastic site in a porcine model of coronary artery spasm.

Coronary artery spasm is caused primarily by increased contractility of vascular smooth muscle. Excessive Ca2+ entry into vascular smooth muscle cells (VSMCs) may be one of the key mechanisms for the spasm, but no study has ever directly examined the possible alterations of Ca2+ channels in the spastic coronary artery. Here we show that L-type Ca2+ channels are excessively expressed at the spastic site of the coronary artery. In a porcine model of coronary spasm with balloon injury, both receptor-mediated stimulation of L-type Ca2+ channels by serotonin and direct stimulation of the channels by Bay K 8644 (a dihydropyridine Ca2+ channel agonist) repeatedly induced coronary spasm in vivo, which was abolished by pretreatment with nifedipine, a dihydropyridine Ca2+ channel antagonist. In a single VSMC freshly dispersed from coronary arteries in vitro, patch-clamp experiments showed that current density of L-type Ca2+ channel current was significantly increased in VSMCs from the spastic site compared with that from the control site even when the channels were maximally stimulated by Bay K 8644. There was no difference in the sensitivity of the channels to Bay K 8644. These results indicate that functionally available L-type Ca2+ channels are excessively expressed at the spastic site of the coronary artery in our porcine model, suggesting that increased expression of L-type Ca2+ channels and concomitant increase in Ca2+ entry into VSMCs through the channels may contribute, at least in part, to the pathogenesis of coronary artery spasm.

Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm.

OBJECTIVE: We recently demonstrated in our swine model of coronary artery spasm that enhanced myosin light chain (MLC) phosphorylations (both MLC mono- and diphosphorylations) play a central role in the pathogenesis of the spasm. However, the molecular mechanism for and the phosphorylation sites for the enhanced MLC phosphorylations were unknown. In the present study, we addressed these points using hydroxyfasudil, a novel inhibitor of protein kinases, which we found preferentially inhibits Rho-kinase. METHODS: The specificity of the inhibitory effects of hydroxyfasudil on Rho-kinase, MLCK, MRCK beta and PKC were examined by kinase assay in vitro. The left porcine coronary artery was chronically treated with interleukin-1 beta (IL-1 beta, 2.5 micrograms). Two weeks after the operation, coronary artery vasomotion was examined both in vivo and in vitro. MLC phosphorylations were examined by Western blot analysis and the sites for the phosphorylations by anti-phosphorylated MLC antibodies that identified the monophosphorylation site as Ser19 and diphophorylation sites as Ser19/Thr18 of MLC. RESULTS: Inhibitory effects of hydroxyfasudil was at least 100 times more potent for Rho-kinase as compared with other protein kinases tested. Intracoronary serotonin (10 micrograms/kg) caused coronary hyperconstriction at the IL-1 beta-treated site in vivo, which was dose-dependently inhibited by hydroxyfasudil (p < 0.01). The coronary segment taken from the spastic site also showed hypercontractions to serotonin in vitro, which were again dose-dependently inhibited by hydroxyfasudil (p < 0.01). Western blot analysis showed that MLC monophosphorylation was significantly greater in the spastic segment than in the control segment, while MLC diphosphorylation was noted only at the spastic segment (p < 0.01). The sites for the mono- and diphosphorylated MLC were identified as the monophosphorylated site Ser19 and diphosphorylated sites Ser19/Thr18 of MLC, respectively. Both types of MLC phosphorylations at the spastic segment were markedly inhibited by hydroxyfasudil (p < 0.01). CONCLUSION: These results indicate that hydroxyfasudil-sensitive Rho-kinase-mediated pathway appears to mediate the enhanced MLC phosphorylations (on Ser19 and Ser19/Thr18 residues) and plays a central role in the pathogenesis of coronary artery spasm.

Intramural delivery of a specific tyrosine kinase inhibitor with biodegradable stent suppresses the restenotic changes of the coronary artery in pigs in vivo.

OBJECTIVES: This study was designed to examine whether or not intramural delivery of ST638 (a specific tyrosine kinase inhibitor) with biodegradable stent can suppress the restenotic changes of the coronary artery in vivo. BACKGROUND: Clinical and animal studies demonstrated that restenosis after coronary intervention results from a combined effect of neointimal formation and geometric remodeling (decrease in total cross-sectional area). Thus, the most effective strategy to prevent the restenosis appears to inhibit both the neointimal formation and geometric remodeling by antiproliferative agent and stent, respectively. We have previously shown that ST638 markedly suppresses the restenotic changes of the porcine coronary artery when applied from the adventitial site. METHODS: A poly-L-lactic acid biodegradable stent was coated with either ST638 (0.8 mg) or equimolar of its inactive metabolite, ST494. A pair of these stents were implanted alternatively in the left anterior descending or circumflex coronary artery in pigs (n=6). Three weeks after the procedure, coronary stenosis was assessed by angiography followed by histological examination. RESULTS: Coronary stenosis was significantly less at the ST638 stent site than at the ST494 stent site (47+/-5% vs. 25+/-4%, p < 0.01). Histological examination also showed that the extent of neointimal formation and that of geometric remodeling were significantly less at the ST638 stent site than at the ST494 stent site (p < 0.05). CONCLUSIONS: These results indicate that intramural delivery of a specific tyrosine kinase inhibitor with biodegradable stent overcomes the proliferative stimuli caused by balloon injury, the stent itself, and the drug coating on the stent, resulting in the suppression of the restenotic changes of the coronary artery in vivo. This strategy might also be useful in the clinical setting in humans.

Effects of light intensity and air velocity on air temperature, water vapor pressure, and CO2 concentration inside a plant canopy under an artificial lighting condition.

In order to characterize environmental variables inside a plant canopy under artificial lighting in the CELSS, we investigated the effects of light intensity and air velocity on air temperature, water vapor pressure, and CO2 concentration inside a plant canopy. Under a PPF of 500 micromoles m-2 s-1, air temperature was 2-3 degrees C higher, water vapor pressure was 0.6 kPa higher, and CO2 concentration was 25-35 micromoles mol-1 lower at heights ranging from 0 to 30 mm below the canopy than at a height 60 mm above the canopy. Increasing the PPF increased air temperature and water vapor pressure and decreased CO2 concentration inside the canopy. The air temperature was lower and the CO2 concentration was higher inside the canopy at an air velocity of 0.3 m s-1 than at an air velocity of 0.1 m s-1. The environmental variables inside the canopy under a high light intensity were characterized by higher air temperature, higher vapor pressure, and lower CO2 concentration than those outside the canopy.

Vasculoprotective role of inducible nitric oxide synthase at inflammatory coronary lesions induced by chronic treatment with interleukin-1beta in pigs in vivo.

BACKGROUND: We recently developed a porcine model in which chronic, local treatment with interleukin-1beta (IL-1beta) causes coronary arteriosclerotic changes and hyperconstrictive responses. Inflammatory cytokines are known to induce inducible NO synthase (iNOS) in the vascular smooth muscle. This study was designed to examine whether or not the production of NO by iNOS has a protective or deleterious effect on the coronary artery in vivo. METHODS AND RESULTS: A segment of the porcine coronary artery was aseptically wrapped with cotton mesh absorbing IL-1beta suspension. We inhibited both eNOS and iNOS activity by cotreatment with L-NAME (a nonspecific inhibitor of NOS) and iNOS activity alone by aminoguanidine (a selective inhibitor of iNOS). Immunostaining showed that iNOS was absent in the normal coronary artery, whereas it was highly expressed 1 day after the application of IL-1beta and thereafter downregulated until 14 days. In contrast, eNOS was well maintained throughout the study period. Two weeks after the operation, hyperconstrictive responses to intracoronary serotonin and neointimal formation were noted at the IL-1beta-treated site, and both responses were significantly greater at the site cotreated with either L-NAME or aminoguanidine. CONCLUSIONS: These results indicate that iNOS is transiently induced in vivo in response to local inflammation and that NO produced by iNOS exerts an inhibitory effect against the cytokine-induced proliferative/vasospastic changes of the coronary artery in vivo.

Platelet activating factor causes hyperconstriction at the inflammatory coronary lesions in pigs in vivo.

BACKGROUND: Although platelet activating factor (PAF) is an important vasoactive substance released from activated leukocytes, platelets and endothelial cells, little is known about its effect at the inflammatory coronary lesions in vivo. OBJECTIVE: To examine the coronary vasomotor responses to PAF at the inflammatory lesions in our swine model with interleukin-1 beta (IL-1 beta) in vivo. METHODS: Under aseptic conditions, the proximal segment of the porcine left coronary artery was dissected and wrapped with cotton mesh absorbing IL-1 beta. Two weeks after the operation, coronary vasomotion in response to intracoronary administration of 0.3 and 1 microgram/kg PAF, 1, 3, and 10 micrograms/kg serotonin, 1, 3 and 10 micrograms/kg histamine, and 5 and 50 micrograms/kg prostaglandin F2 alpha was examined by coronary arteriography. RESULTS: At the IL-1 beta-treated site, PAF, serotonin and histamine, but not prostaglandin F2 alpha, caused hyperconstriction (n = 8). A synergy of the vasoconstricting effects of PAF and serotonin was also noted (n = 6). Administration of TCV-309, a selective PAF receptor antagonist, abolished the hyperconstrictive responses to PAF but not those to other agonists (n = 6). The PAF-induced coronary hyperconstrictions were significantly inhibited by administrations of the protein kinase C inhibitors staurosporine and sphingosine, but not by administrations of ryanodine, thapsigargin, or indomethacin (n = 4 each). CONCLUSIONS: These results indicate that PAF causes hyperconstriction at the inflammatory coronary lesions in vivo by itself as well as in a synergistic manner with serotonin and that the PAF-induced hyperconstrictions are substantially mediated by a protein kinase C-dependent pathway in vivo.

Regulation of fibrillar collagen gene expression and protein accumulation in volume-overloaded cardiac hypertrophy.

BACKGROUND: Interstitial collagen accumulation has been extensively demonstrated to be increased at both mRNA and protein levels in pressure-overloaded cardiac hypertrophy. However, few data are available regarding the effects of volume overload on myocardial collagens. METHODS AND RESULTS: To determine whether the alterations of collagens may occur in volume-overloaded cardiac hypertrophy, we measured collagen types I and III mRNA levels and protein accumulation in left ventricular (LV) myocardium of rats at 3, 7, and 28 days after the creation of an aortocaval (AC) shunt. Eccentric LV hypertrophy was produced in rats with AC shunting. Northern blot analysis on RNA extracted from LV tissue indicated that the steady state mRNA levels for both type I and III collagen were persistently upregulated in AC shunt rats compared with sham-operated operated control rats. In contrast, the biochemical collagen protein concentration and morphometric collagen volume fraction were comparable between sham-operated control and AC shunt rats at any study time point. Furthermore, the immunohistochemical staining of types I and III collagen and Sirius red staining on myocardial tissue sections revealed no significant alterations in the distribution or density of fibrillar collagens in AC shunt rats. Tissue collagenase activity was not different between control and AC shunt rats after 28 days. CONCLUSIONS: Cardiac volume overload increases LV collagen mRNA as does pressure overload. However, in contrast to pressure-overloaded hypertrophy, the upregulation of collagen transcriptional activity does not result in subsequent myocardial fibrosis in volume-overloaded hypertrophy due to AC shunting. Therefore, the upregulation of collagen gene expression and protein accumulation might be different in pressure-overloaded and volume overloaded hypertrophy.

Tyrosine kinase inhibitor markedly suppresses the development of coronary lesions induced by long-term treatment with platelet-derived growth factor in pigs in vivo.

Platelet-derived growth factor (PDGF) plays an important role in the development of coronary atherosclerosis. However, it remains to be examined what morphologic and functional changes are induced in vivo by the long-term treatment with PDGF itself or what pharmacologic interventions could suppress those changes in vivo. Our study was designed to address these points. We examined the effects of long-term treatment with PDGF on the porcine coronary artery in vivo. Under aseptic conditions, the proximal segments of the left porcine coronary artery were gently wrapped with cotton mesh absorbing sepharose beads either with or without recombinant human PDGF-AA or -BB. Two weeks after the operation, coronary hyperconstrictions to intracoronary serotonin or histamine were noted at the sites treated with PDGF-AA or -BB. Histologically, neointimal formation and geometric remodeling (reduction of the total vessel area) were noted at the PDGF-treated sites. These functional and histologic changes of the coronary artery induced by PDGF were markedly inhibited by cotreatment with ST 638, a specific inhibitor of tyrosine kinases. A Western blot analysis showed that ST 638 markedly suppressed the PDGF-induced tyrosine phosphorylations in the coronary segment. These results indicate that long-term treatment with PDGF induces neointimal formation, geometric remodeling, and vasospastic responses in vivo, for all of which, activation of tyrosine kinases is substantially involved.

Enhanced myosin light chain phosphorylations as a central mechanism for coronary artery spasm in a swine model with interleukin-1beta.

BACKGROUND: Although coronary artery spasm plays an important role in a wide variety of ischemic heart diseases, the intracellular mechanism for the spasm remains to be clarified. We examined the role of myosin light chain (MLC) phosphorylations, a key mechanism for contraction of vascular smooth muscle, in our swine model with interleukin-1beta (IL-1beta). METHODS AND RESULTS: IL-1beta was applied chronically to the porcine coronary arteries from the adventitia to induce an inflammatory/proliferative lesion. Two weeks after the operation, intracoronary serotonin repeatedly induced coronary hyperconstrictions at the IL-1beta-treated site both in vivo and in vitro, which were markedly inhibited by fasudil, an inhibitor of protein kinases, including protein kinase C and MLC kinase. Western blot analysis showed that during serotonin-induced contractions, MLC monophosphorylation was significantly increased and sustained in the spastic segment compared with the control segment, whereas MLC diphosphorylation was noted only in the spastic segment. A significant correlation was noted between the serotonin-induced contractions and MLC phosphorylations. Both types of MLC phosphorylation were markedly inhibited by fasudil. In addition, MLC diphosphorylation was never induced by a simple endothelium removal in the normal coronary artery, whereas enhanced MLC phosphorylations in the spastic segment were noted regardless of the presence or absence of the endothelium. CONCLUSIONS: These results indicate that enhanced MLC phosphorylations in the vascular smooth muscle play a central role in the pathogenesis of coronary spasm in our swine model.