Porcine Pulmonary Artery Endothelial Cells Research Articles

오디 추출물의 혈관신생 억제 효과

본 연구에서는 PPAECs 세포를 이용하여 오디 추출물에 의한 세포의 이동과 발아에 미치는 효과 및 MMPs와 TIMPs의 분비에 미치는 효과를 조사하여 다음과 같은 결과를 얻었다. PPAECs 세포의 이동에 미치는 오디 추출물의 효과를 조사한 결과, 오디 추출물 농도 80 <TEX>${\mu}g/ml$</TEX>에서 대조군에 비해 이동이 약 36% 억제되었다. 혈관내피세포의 발아 역시, 오디추출물 농도 80 <TEX>${\mu}g/ml$</TEX>에서 PPAECs 세포의 발아가 대조군에 비해 약 50% 억제되었다. PPAECs 세포의 MMPs 및 TIMPs 분비에 미치는 오디 추출물의 효과를 조사한 결과, 오디 추출물 농도 80 <TEX>${\mu}g/ml$</TEX>에서 MMP-2의 분비는 약 45% 감소되었으며, MMP-9의 분비는 약 33% 감소되었다. TIMP-1은 오디 추출물 농도 80 <TEX>${\mu}g/ml$</TEX>에서 분비가 약 51% 억제되었으나, TIMP-2는 오디 추출물 농도 80 <TEX>${\mu}g/ml$</TEX>에서 분비가 약 150% 증가되었다. 이상의 결과를 요약하면, 오디 추출물에 의해 유도된 혈관내피세포의 이동 및 발아 억제는 부분적으로 MMP-2 및 MMP-9의 분비 감소에 의한 것이라 여겨진다. Endothelial cells release proteinases to degrade the extracellular matrix for their migration and sprouting in vivo. In the present study, we investigated the inhibitory effect of mulberry extracts on the migration and sprouting activity of porcine pulmonary artery endothelial cells (PPAECs). Mulberry extracts significantly inhibited migration and sprouting of PPAECs. Mulberry extracts inhibited expressions and secretions of matrix metalloproteinase-2 (MMP-2) and MMP-9. Our results suggest that the inhibitory effects of mulberry extracts on migration and sprouting of PPAECs are, in part, associated with the decreased secretion of MMP-2 and MMP-9.

Alumina nanoparticles induce expression of endothelial cell adhesion molecules

Nanotechnology is a rapidly growing industry that has elicited much concern because of the lack of available toxicity data. Exposure to ultrafine particles may be a risk for the development of vascular diseases due to dysfunction of the vascular endothelium. Increased endothelial adhesiveness is a critical first step in the development of vascular diseases, such as atherosclerosis. The hypothesis that alumina nanoparticles increase inflammatory markers of the endothelium, measured by the induction of adhesion molecules as well as the adhesion of monocytes to the endothelial monolayer, was tested. Following characterization of alumina nanoparticles by transmission electron microscopy (TEM), electron diffraction, and particle size distribution analysis, endothelial cells were exposed to alumina at various concentrations and times. Both porcine pulmonary artery endothelial cells and human umbilical vein endothelial cells showed increased mRNA and protein expression of VCAM-1, ICAM-1, and ELAM-1. Furthermore, human endothelial cells treated with alumina particles showed increased adhesion of activated monocytes. The alumina particles tended to agglomerate at physiological pH in serum-containing media, which led to a range of particle sizes from nano to micron size during treatment conditions. These data show that alumina nanoparticles can elicit a proinflammatory response and thus present a cardiovascular disease risk.

PCB77‐induced adhesion molecule expression in primary endothelial cells is mediated through caveolae signaling

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that can activate endothelial cells thus leading to atherogenic events. Induction of vascular adhesion molecules is a common feature in inflammatory environments, leading to the early development of atherosclerosis. We have investigated PCB77‐induced up‐regulation of adhesion molecules through caveolae‐regulated signaling in primary mouse aortic endothelial cells (MAECs) and porcine pulmonary artery endothelial cells (PAECs). PCB77 significantly increased the expression of vascular cell adhesion molecule‐1 (VCAM‐1) and intracellular adhesion molecule‐1 (ICAM‐1) measured by both Western blot and RT‐PCR in a dose dependent manner in both MAECs and PAECs. In addition, the adhesion of monocytes to PCB77‐activated endothelial cells was increased in a dose‐dependent pattern. Phosphorylation studies of caveolin‐1 and related kinases were conducted in endothelial cells containing either functional or siRNA silenced caveolin‐1 protein. Activation of these caveolae‐associated signaling pathways was decreased in absence of functional caveolae. These data provide the evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and toxicity induced by persistent environmental pollutants such as coplanar PCBs. (Supported by grants from NIH/NIEHS (P42ES07380), the University of Kentucky AES and KRF‐2007‐357‐F00045)

Endothelial Alpha-1-Antitrypsin Attenuates Cigarette Smoke Induced Apoptosis In Vitro

Background: Deficiency of the antiprotease alpha-1-antitrypsin (AAT) and exposure to cigarette smoke (CS) contribute to the development of early onset emphysema. CS-induced apoptosis of alveolar cells including endothelial cells plays critical role in the lung destruction. AAT deficiency is associated with increased lung tissue destruction as well. We hypothesize that AAT protects lung alveoli from noxious environmental stimuli such as CS-induced apoptosis. Methods: Porcine pulmonary artery endothelial cells (PAEC) were exposed to CS in the presence or absence of AAT (20μ M). AAT internalization and markers for apoptosis were assessed by confocal microscopy. Flow cytometry was performed in parallel to quantify the number of AAT-loaded and apoptotic cells. Results: We demonstrated that exogenous AAT accumulated in PAEC and protected cells from CS-induced apoptosis. AAT-loaded CS-exposed cells exhibited increased amounts of chaperone HSP-70 in their cytosol and less apoptosis inducing factor in their nuclei compared to AAT-untreated, CS-exposed cells. Conclusions: Our results suggest that AAT is taken up by endothelial cells via two mechanisms and that intracellular AAT may have a protective role in CS-induced endothelial apoptosis. This may open new insights into the field of endothelial serpins as agents capable of protecting the vasculature from environment-derived noxious substances.

Equilibrium and non-equilibrium charge-dependent quantification of endothelial cell hydrogel scaffolds

Using equilibrium swelling and non-equilibrium membrane potential measurements, this study assesses the charge density in two representative series of polyelectrolyte hydrogels and examines the morphological and proliferative responses of endothelial cells as a function of the prepared charge offset. The neutral monomers 2-hydroxyethylmethacrylate (HEMA) and poly(ethylene glycol) dimethacrylate (n = 1,000) (PEGDMA) were copolymerized with either the acidic monomer 2-sulfoethyl methacrylate (SEMA) or the basic monomer methacryloxy ethyltrimethylammonium chloride (MAETAC) to make membranes with pregelation charge offset concentrations varying from 0 to +/-200 mM. A thermodynamic analysis of swelling and membrane potential measurements quantified the hydrogel charge density state following equilibration at different ion strengths. Porcine pulmonary artery endothelial cells were seeded on samples of each HEMA and PEGDMA copolymer and the amount of cell coverage was measured over a 4-day period. Cellular attachment and proliferation increased with increasing proportions of charged monomers and showed a threshold pattern of attachment and growth on the positively charged HEMA-MAETAC copolymer hydrogels with increasing proportions of initially prepared charge. The series of PEGDMA copolymer hydrogels remained relatively resistant to cellular attachment and proliferation over the range of prepared charges considered in this study.

Optoelectric biosensor using indium-tin-oxide electrodes

The use of an optically thin indium-tin-oxide (ITO) electrode is presented for an optoelectric biosensor simultaneously recording optical images and microimpedance to examine time-dependent cellular growth. The transmittance of a 100 nm thick ITO electrode layer is approximately the same as the transmittance of a clean glass substrate, whereas the industry-standard Au(47.5 nm)/Ti(2.5 nm) electrode layer drops the transmittance to less than 10% of that of the glass substrate. The simultaneous optoelectric measurements permit determining the correlation of the cell-covered area increase with the microimpedance increase, and the example results obtained for live porcine pulmonary artery endothelial cells delineate the quantitative and comprehensive nature of cellular attachment and spreading to the substrate, which has not been clearly perceived before.

Simultaneous dynamic optical and electrical properties of endothelial cell attachment on indium tin oxide bioelectrodes

This study quantifies the dynamic attachment and spreading of porcine pulmonary artery endothelial cells (PPAECs) on optically thin, indium tin oxide (ITO) biosensors using simultaneous differential interference contrast microscopy (DICM) and electrical microimpedance spectroscopy. A lock-in amplifier circuit monitored the impedance of PPAECs cultivated on the transparent ITO bioelectrodes as a function of frequency between 10 Hz and 100 kHz and as a function of time, while DICM images were simultaneously acquired. A digital image processing algorithm quantified the cell-covered electrode area as a function of time. The results of this study show that the fraction of the cell-covered electrode area is in qualitative agreement with the electrical impedance during the attachment phase following the cell settling on the electrode surface. The possibility of several distinctly different states of electrode coverage and cellular attachment giving rise to similar impedance signals is discussed.

Transdifferentiation of pulmonary arteriolar endothelial cells into smooth muscle‐like cells regulated by myocardin involved in hypoxia‐induced pulmonary vascular remodelling

Myocardin gene has been identified as a master regulator of smooth muscle cell differentiation. Smooth muscle cells play a critical role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH) and pulmonary vascular remodelling (PVR). The purpose of this study was to investigate the change of myocardin gene expression in the pulmonary vessels of hypoxia-induced PH affected by Sildenafil treatment and the involvement of endothelial cells transdifferentiation into smooth muscle cells in the process of hypoxia-induced PH and PVR. Myocardin and relative markers were investigated in animal models and cultured endothelial cells. Mean pulmonary artery pressure (mPAP) was measured. Immunohistochemistry and immunofluorescence were used to show the expression of smooth muscle alpha-actin (SMA), in situ hybridization (ISH) and reverse transcription polymerase chain reaction (RT-PCR) were performed respectively to detect the myocardin and SMA expression at mRNA levels. Small interfering RNA (siRNA) induced suppression of myocardin in cultured cells. We confirmed that hypoxia induced the PH and PVR in rats. Sildenafil could attenuate the hypoxia-induced PH. We found that myocardin mRNA expression is upregulated significantly in the hypoxic pulmonary vessels and cultured cells but downregulated in PH with Sildenafil treatment. The porcine pulmonary artery endothelial cells (PAECs) transdifferentiate into smooth muscle-like cells in hypoxic culture while the transdifferentiation did not occur when SiRNA of myocardin was applied. Our results suggest that myocardin gene, as a marker of smooth muscle cell differentiation, was expressed in the pulmonary vessels in hypoxia-induced PH rats, which could be downregulated by Sildenafil treatment, as well as in hypoxic cultured endothelial cells. Hypoxia induced the transdifferentiation of endothelial cells of vessels into smooth muscle-like cells which was regulated by myocardin.

An endothelial cell compatible biosensor fabricated using optically thin indium tin oxide silicon nitride electrodes

This study describes the fabrication and performance of an endothelial cell compatible, optically thin, indium tin oxide (ITO) microimpedance biosensor. The biosensor was constructed by sputtering a thin insulating layer of silicon nitride (Si 3N 4) onto a 100 nm thick ITO layer. Indium tin oxide electrodes were formed by chemically etching 250 or 500 μm diameter holes through the Si 3N 4 insulating layer. The exposed ITO electrode was electrically connected to an ITO counter electrode, approximately 2 cm 2 in area, via a 400 μL well containing cell culture media. A lock-in amplifier circuit monitored the impedance of porcine pulmonary artery endothelial cells (PPAECs) cultivated on the electrodes as a function of frequency, between 10 and 100 kHz, and as a function of time, at 5.62 kHz. The ITO–Si 3N 4 microelectrodes provided consistent and repeatable impedance measurements to the attachment and spreading of PPAECs. In addition, the ITO–Si 3N 4 electrodes were recyclable, robust, resistant to ethanol sterilization, and had a high optical transmittance. Most importantly, the ITO–Si 3N 4 electrodes allowed optical access for dynamic cellular attachment imaging. The 5.62 kHz time dependent cellular impedance response to the drug Cytochalasin D further demonstrated the feasibility of using this electrode configuration for dynamic cellular impedance studies.

Use of recombinant calpain-2 siRNA adenovirus to assess calpain-2 modulation of lung endothelial cell migration and proliferation

In this study, we developed an adenoviral vector harboring calpain-2 siRNA expression unit in which sense and anti-sense strands composing the siRNA duplex were connected by a loop and transcribed into a siRNA in porcine pulmonary artery endothelial cells (PAEC). We screened one efficient adenoviral vector Ad/si-m187 and found that Ad/si-m187 successfully exerted a gene knockdown effect on calpain-2 mRNA transcription and protein expression levels. The protein content of calpain-2 was reduced by 30%-80% in PAEC infected with Ad/si-m187 in comparison to a control adenoviral vector Ad/si-luc. The mRNA levels of calpain-2 were measured by real-time PCR and were decreased by 60%-100% and in a dose dependent manner. In correspondence to silencing calpain-2 gene expression, calpain-2 activity was decreased significantly. We further evaluated the role of calpain-2 in endothelial cell migration and proliferation. PAEC infected with Ad/si-m187 displayed impaired migration and cell proliferation in comparison to cells infected with control adenoviral vector (Ad/si-luc). These results indicate that adenoviral vector harboring calpain-2 siRNA expression unit is a valuable tool to study the biology of calpains and that calpain-2 plays an important role in lung endothelial cell migration and proliferation.

Nitric oxide decreases the stability of DMPO spin adducts

The effect nitric oxide (NO*) on the stability of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) adducts has been investigated using EPR spectroscopy. We report that the DMPO/HO* adduct, generated by porcine pulmonary artery endothelial cells in the presence of H2O2 and DMPO, or by a Fenton system (Fe(II)+H2O2) is degraded in the presence of the NO*-donor, 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) or by bolus addition of an aqueous solution of NO*. A similar effect of DEANO was observed on other DMPO adducts, such as DMPO/*CH3 and DMPO/*CH(CH3)OH, generated in cell-free systems. Measurements of the loss of DMPO/HO* in the presence of DEANO in aerated and oxygen-free buffers showed that in both of these settings the process obeys first-order kinetics and proceeds with similar efficacy. This indicates that direct interaction of the nitroxide with NO*, rather than with NO2* (formed from NO* and O2 in aerated media), is responsible for destruction of the spin adduct. These results suggest that the presence of NO* may substantially affect the quantitative determination of DMPO adducts. We also show that NO2* radicals, generated by a myeloperoxidase/H2O2/nitrite system, also degrade DMPO/HO*. Because DMPO is frequently used to study generation of superoxide and hydroxyl radicals in biological systems, these observations indicate that extra caution is required when studying generation of these species in the presence of NO* or NO2* radicals.

Superoxide auto-augments superoxide formation and upregulates gp91 phox expression in porcine pulmonary artery endothelial cells: Inhibition by iloprost

Central to the aetiology of Acute Respiratory Distress Syndrome (ARDS) is superoxide, the principal source of which is nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). To test whether superoxide may influence NADPH oxidase expression directly, the effect of incubation of superoxide with porcine pulmonary arterial endothelial cells on the expression of gp91 phox (a catalytic subunit of NADPH oxidase) and superoxide formation was investigated. Since iloprost has been purported to be potentially effective in treating ARDS, the effect of iloprost on superoxide-mediated effects was also studied. Pulmonary artery endothelial cells were incubated with xanthine/xanthine oxidase which generates superoxide, or tumour necrosis factor α (TNFα) or thromboxane A 2 analogue, U46619 (± superoxide dismutase [SOD] or catalase or iloprost) for 16 h. Cells were then washed and superoxide formation assessed spectrophometrically and gp91 phox expression using Western blotting. The role of NADPH oxidase was also studied in the above settings using apocynin, an NADPH oxidase inhibitor. Superoxide, TNFα and U46619 elicited an increase in the formation of superoxide and induced gp91 phox expression in pulmonary artery endothelial cells following a 16 h incubation an effect blocked by the continual presence of SOD and apocynin but not catalase. Apocynin completely inhibited superoxide formation induced with xanthine/xanthine oxidase after the 16 h incubation. Rotenone and allopurinol were without effect. Iloprost inhibited the formation of superoxide and gp91 phox expression. These data demonstrate that superoxide upregulates gp91 phox expression in pulmonary artery endothelial cells and thus augments superoxide formation, an effect blocked by iloprost. This constitutes a novel mechanism by which vascular superoxide creates a self-perpetuating cascade that may be of importance to the etiology of ARDS and other vasculopathies.

Chronic Ethanol Exposure Stimulates Endothelial Cell Nitric Oxide Production through PI-3 Kinase-and hsp90-dependent Mechanisms

Chronic ethanol (EtOH) ingestion increases the incidence of the Acute Respiratory Distress Syndrome (ARDS), a severe form of acute lung injury characterized by endothelial and epithelial barrier dysfunction. The regulated production of nitric oxide (NO) by the endothelium plays a central role in normal vascular function, and alterations in NO production have been implicated in barrier dysfunction. Although previous reports examined the impact of acute EtOH stimulation on endothelial NO production, this study extends those observations to clarify mechanisms of chronic EtOH-mediated alterations in endothelial nitric oxide synthase (eNOS) expression and NO production. Porcine pulmonary artery endothelial cells (PAEC) were treated with EtOH (0.04-0.16%, w/v) for 72 hr in sealed chambers to prevent evaporation. NO release and eNOS expression were determined to examine the effect of chronic EtOH stimulation on endothelial NO metabolism. While there was no change in the extent of phosphorylated eNOS at ser, chronic EtOH stimulation caused dose-dependent increases in NO production and increased eNOS expression, effects that were attenuated by the transcriptional inhibitor, alpha-amanitin (AA), and wortmannin, a specific phosphatidylinositol 3 kinase (PI3 K) inhibitor. EtOH stimulation also increased eNOS interaction with heat shock protein (hsp90), a molecular chaperone known to enhance eNOS activity. Geldanamycin, an hsp90 inhibitor, attenuated chronic EtOH-mediated increases in NO production. These results indicate that chronic EtOH exposure increases endothelial NO production by increasing eNOS protein levels through PI3 K-dependent up regulation of eNOS gene transcription and by increasing interactions between eNOS and hsp90. These findings clarify mechanisms by which chronic EtOH stimulation modulates vascular endothelial function and suggest new targets for investigation and intervention in EtOH-induced alterations in susceptibility to lung injury.

Hypoxia down-regulates secretion of MMP-2, MMP-9 in porcine pulmonary artery endothelial and smooth muscle cells and the role of HIF-1

Primary cell culture, techniques of gene transfection, gelatin zymography, and Western blot were used to investigate the effect of hypoxia on the secretion of MMP-2 and MMP-9 in pulmonary artery endothelial cells (PAEC) and smooth muscle cells (PASMC), and the role of HIF-1. Our results showed that (1) after exposure to hypoxia for 24 h, the protein content and activity of MMP-2 in the PAEC medium as well as these of MMP-2 and MMP-9 in PASMC medium (P < 0. 01) decreased significantly in contrast to those in normoxic group (P < 0.05); (2) after transfection of wild type EPO3'-enhancer, a HIF-1 decoy, the content and activity of MMP-2 and MMP-9 in hypoxic mediums became higher than those in normoxic group (P < 0.01), while transfection of mutant EPO3'-enhancer didn't affect the hypoxia-induced down-regulation. It is concluded that hypoxia could inhibit the secretion and activity of MMP-2 and MMP-9 in PAEC and PASMC, which could be mitigated by the transfection of EPO3'-enhancer and that HIF-1 pathway might contribute to hypoxia-induced down-regulation of MMP-2 and MMP-9.

Prednisolone augments superoxide formation in porcine pulmonary artery endothelial cells through differential effects on the expression of nitric oxide synthase and NADPH oxidase.

1. Prednisolone, a potent anti-inflammatory drug, has proved ineffective in treating acute respiratory distress syndrome (ARDS). ARDS is associated with superoxide (O(2)(*-)) generation, which negates nitric oxide (NO). NO also downregulates NADPH oxidase and inhibits O(2)(*-) formation. A possible reason for the lack of effect of prednisolone may due to an inhibition of eNOS expression. In order to test this proposal, the effect of prednisolone on O(2)(*-) formation and the expression of gp91(phox) (catalytic subunit of NADPH oxidase) and eNOS in pig pulmonary artery (PA) segments and PA endothelial cells (PAECs) and PA vascular smooth muscle cells (PAVSMCs) was investigated. 2. PA segments and cells were incubated with prednisolone and tumour necrosis factor-alpha (TNF-alpha) for 16 h. O(2)(*-) formation was measured spectrophometrically and gp91(phox) and eNOS expression by Western blotting. The role of the NO-cGMP axis was studied using morpholinosydnonimine hydrochloride, the diethylamine/NO complex (DETA-NONOate), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ) and the stable cGMP analogues, 8-bromo cGMP and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP). NO release was studied using a fluorescence assay and O(2)(*-)-NO interactions with a nitrite/nitrate assay. 3. Prednisolone elicited significant increase in O(2)(*-) formation in intact PA segments and PAECs, but not PAVSMCs, in a concentration-dependent manner. In endothelium-denuded segments, prednisolone slightly enhanced O(2)(*-) release. TNF-alpha further increased prednisolone-enhanced O(2)(*-) formation in intact PA segments and PAECs. NADPH oxidase inhibitor, apocynin, inhibited O(2)(*-) formation. Increased O(2)(*-) release and gp91(phox) expression in PAECs elicited by prednisolone was blocked by SIN-1 (3-morpholinosydnonimine hydrochloride), DETA-NONOate, 8-pCPT-cGMP and 8-bromo cGMP. The effects of SIN-1 on gp91(phox) expression were reversed by ODQ. Finally, eNOS protein expression was significantly reduced by prednisolone. 4. Prednisolone increases O(2)(*-) in porcine PAECs through a downregulation of endogenous eNOS expression. Since the NO-cGMP axis inhibits gp91(phox) expression, the resultant decrease in endogenous NO formation then augments NADPH oxidase activity, which in turn results in increased O(2)(*-) formation. Since O(2)(*-) promotes inflammation, this mechanism may explain why prednisolone is ineffective in treating ARDS. Therapeutically, the coadministration of an NO donor may render prednisolone more effective in treating ARDS.

Effects of nitric oxide on sGC redox states

Soluble guanylyl cyclase (sGC), the best-established nitric oxide receptor is a heterodimeric heme-protein synthesizing cGMP upon NO binding to its Fe(II)heme. cGMP in turn plays an important role in the regulation of vascular tone and platelet aggregation. The redox state of the sGC heme is fundamental for the activation by NO. A novel class of sGC activators, such as the recently described BAY 58-2667, are however NO-independent and target sGC in its Fe(III) and heme-free state. Given the therapeutic potential of such sGC activators, the mechanisms that modulate the sensitivity of sGC towards these agonists is of great interest. In this study we found, that treatment of primary porcine pulmonary artery endothelial cells with the NO donor DETA-NO (100 μmol/l) leads to a downregulation of DEA-NO stimulated and an up-regulation of BAY 58-2667 stimulated sGC activities (pmol/106 cells) [DEA-NO: 4.83 ± 0.11 vs. 3.02 ± 0.08***, BAY 58-2667: 0.77 ± 0.01 vs. 1.91 ± 0.04***; control vs. 2 h DETA-NO pre-treatment]. Furthermore, we observed a down-regulation of sGC (α1/β1) protein levels upon treatment with DETA-NO in a doseand time-dependent manner. Interestingly, the decrease in protein levels (maximal at t = 72 h: 56.7 ± 7% of control for sGC α1 and 44.4 ± 8% of control for sGC β1) occured subsequent to the apparent reduction in NO stimulatable sGC activity (maximal at t = 2 h). Downregulation of cellular cGMP responses has recently been attributed to upregulation of cGMP breakdown via enhanced PDE5 activity. Thus we tested the effects of the cGMP analogue 8-Br-cGMP (125/250/500 μmol/l), the mixed NO-independent sGC activator and PDE inhibitor YC-1 (10/50/100 μmol/l) and the selective phosphodiesterase-5 inhibitor zaprinast (50/100/200 μmol/l) on sGC protein levels. In contrast to DETA-NO, none of these modulators altered sGC protein expression arguing against a cGMP/PDE dependent mechanism. To determine if endogenously produced NO mimicked the effects of DETA-NO on sGC we utilised two models. First, porcine endothelial cells were pretreated with the endothelial NO synthase (eNOS) inhibitor L-NAME (100 μmol/l from 2 to 72 hours) and, second, aortas from eNOS deficient mice were investigated. L-NAME induced changes in endothelial NO and BAY 58-2667 stimulated sGC activity, that were reciprocal to those observed with DETA-NO treatment. While sGC expression and the response to DEA-NO were enhanced, the response to BAY 58-2667 was un-altered. Concurrent to these findings, in aortic homogenates from eNOS -/mice, an increased DEA-NO induced sGC response was observed as compared to wild type controls. In conclusion, our data suggests endogenous regulation of the sGC redox state via NO. The decrease in the cGMP response to DEA-NO and the increase to BAY 58-2667 induced cGMP production following DETA-NO pretreatment, indicates a change in the redox state of the sGC heme from a ferrous to ferric or even heme free. Furthermore, these changes in the redox state of sGC, seem to induce a protein degradation of sGC (α1/β1). Hence, desensitization of the NO/cGMP-pathway is heterogenous, occurring either as a result of PDE-5 upregulation or, as in pulmonary endothelial cells, via a PDE-5-independent mechanism involving a change in sGC redox state and protein stability. from 2nd International Conference of cGMP Generators, Effectors and Therapeutic Implications Potsdam, Germany, 10–12 June, 2005

Involvement of matrix metalloproteinase-9 in platelet-activating factor-induced angiogenesis

Platelet-activating factor (PAF) augments angiogenesis by promoting the synthesis of various angiogenic factors, via the activation of NF-κB. In this study, we investigated the role of the matrix metalloproteinase (MMP)-9, in PAF-induced angiogenesis. PAF increased mRNA expression, protein synthesis, and MMP-9 activity in ECV304 cells, in a NF-κB-dependent manner. PAF increased MMP-9 promoter activity in ECV304, which was inhibited by WEB2107, and NF-κB inhibitors. Transfected NF-κB subunits, p65 or/and p50, increased luciferase activity in the reporter plasmid MMP-9, resulting in an increase not only of MMP-9 luciferase activity, but also of mRNA expression in MMP-9. MMP-9 or NF-κB inhibitors significantly inhibited PAF-induced angiogenesis, in a dose-dependent manner, in an in vivo mouse Matrigel implantation model. In a parallel to the Matrigel implantation study, MMP-9 or NF-κB inhibitors inhibited PAF-induced sprouting of porcine pulmonary arterial endothelial cells. These data indicate that NF-κB-dependent MMP-9 plays a key role in PAF-induced angiogenesis.

Molecular cloning, characterization, and chromosomal assignment of porcine cationic amino acid transporter-1

We have cloned and characterized the gene encoding the porcine cationic amino acid transporter, member 1 (CAT-1) (HGMW-approved gene symbol SLC7A1) from porcine pulmonary artery endothelial cells. The porcine SLC7A1 encodes 629 deduced amino acid residues showing a higher degree of sequence similarity with the human counterpart (91.1%) than with the rat (87.3%) and mouse (87.6%) counterparts. Confocal microscopic examination of porcine CAT-1–GFP-expressing HEK293 cells revealed that porcine CAT-1 localizes on the plasma membrane. Amino acid uptake studies in Xenopus oocytes injected with cRNA encoding this protein demonstrated transport properties consistent with system y +. Radiation hybrid mapping data indicate that the porcine SLC7A1 maps to the distal end of the short arm of pig chromosome 11 (SSC11). This map location is consistent with the known conservation of genome organization between human and pig and provides further confirmation that we have characterized the porcine orthologue of the human SLC7A1.

Effects of 3,4-dihydroxyacetophenone on cytosolic calcium in pulmonary artery endothelial and smooth muscle cells during acute hypoxia.

The effects of 3, 4-Dihydroxyacetophenone (3, 4-DHAP) on cytosolic free calcium [Ca2+]i in pulmonary artery endothelia (PAECs) and smooth muscle cells (PASMCs) during acute hypoxia were studied. Porcine pulmonary artery endothelial and smooth muscle cells (PASMCs) were cultured primarily, and they were divided into 4 groups: groups incubated under normoxia or hypoxia and those with or without treatment with 3,4-DHAP. The [Ca2+]i of both PAECs and PASMCs was measured by determining the fluorescence of fura 2 AM on spetrofluorometer. Our results showed that hypoxia caused significant elevation of [Ca2+]i, in both PAECs and PASMCs, 3,4-DHAP could attenuate the hypoxic elevation of [Ca2+]i only in PASMCs but not in PAECs. It is concluded that 3,4-DHAP decreases the hypoxic elevation of [Ca2+]i in PASMCs. This might contribute to its inhibitory effect on hypoxic pulmonary vasoconstriction.

Nitric oxide-induced persistent inhibition and nitrosylation of active site cysteine residues of mitochondrial cytochrome-c oxidase in lung endothelial cells

Persistent inhibition of cytochrome-c oxidase, a terminal enzyme of the mitochondrial electron transport chain, by excessive nitric oxide (NO) derived from inflammation, polluted air, and tobacco smoke contributes to enhanced oxidant production and programmed cell death or apoptosis of lung cells. We sought to determine whether the long-term exposure of pulmonary artery endothelial cells (PAEC) to pathophysiological concentrations of NO causes persistent inhibition of complex IV through redox modification of its key cysteine residues located in a putative NO-sensitive motif. Prolonged exposure of porcine PAEC to 1 mM 2,2'-(hydroxynitrosohydrazino)-bis-ethanamine (NOC-18; slow-releasing NO donor, equivalent to 1-5 microM NO) resulted in a gradual, persistent inhibition of complex IV concomitant with a reduction in ratios of mitochondrial GSH and GSSG. Overexpression of thioredoxin in mitochondria of PAEC attenuated NO-induced loss of complex IV activities, suggesting redox regulation of complex IV activity. Sequence analysis of complex IV subunits revealed a novel putative NO-sensitive motif in subunit II (S2). There are only two cysteine residues in porcine complex IV S2, located in the putative motif. Immunoprecipitation and Western blot analysis and "biotin switch" assay demonstrated that exposure of PAEC to 1 mM NOC-18 increased S-nitrosylation of complex IV S2 by 200%. Site-directed mutagenesis of these two cysteines of complex IV S2 attenuated NO-increased nitrosylation of complex IV S2. These results demonstrate for the first time that NO nitrosylates active site cysteines of complex IV, which is associated with persistent inhibition of complex IV. NO inhibition of complex IV via nitrosylation of NO-sensitive cysteine residues can be a novel upstream event in NO-complex IV signaling for NO toxicity in lung endothelial cells.