Oxidative stress mediates monocrotaline-induced alterations in tenascin expression in pulmonary artery endothelial cells.

Oxidative stress may be involved in monocrotaline (MCT)-induced endothelial cell injury and upregulation of extracellular matrix proteins in the pulmonary vasculature. To test this hypothesis, cytotoxicity, expression and distribution of tenascin (TN) as well as cellular oxidation were determined in porcine pulmonary artery endothelial cells (PAECs) exposed to MCT and/or to an oxygen radical scavenger, dimethylthiourea (DMTU). Relative to controls, treatment with 2.5 mM MCT for 24 hr produced cytotoxicity as evidenced by changes in cellular morphology, cell detachment, hypertrophy, reduction in cellular proliferation and severe cytoplasmic vacuolization. Parallel studies showed that MCT markedly altered the expression and distribution of TN in PAEC as determined by immunocytochemistry. Western analysis showed that MCT increased cellular TN content and promoted the appearance of an additional, smaller TN isoform. Northern analysis demonstrated an increase in the steady-state level of TN-specific mRNA in response to MCT treatment. Exposure to MCT also increased the synthesis of cell-associated and media-associated TN as determined by immunoprecipitation. In addition, MCT increased the intensity of cellular oxidative stress as measured by 2,7-dichlorofluorescein fluorescence. Co-treatment with DMTU prevented MCT-induced cytotoxicity, alterations in TN distribution and content, and reduced the increase in DCF fluorescence. These results suggest that MCT-induced cytotoxicity and upregulation of TN are mediated, at least in part, by induction of cellular oxidative stress.

Coarctation induces alterations in basement membranes in the cardiovascular system.

A coarctation hypertensive rat model was used to examine the effects of elevated blood pressure on basement membrane component synthesis by cardiac myocytes and aorta using immunohistochemistry and Northern blot analysis. Carotid arterial pressure increased immediately on coarctation, and left ventricular hypertrophy was maximal within 5 days. In immunohistochemical studies, fibronectin and laminin were increased and the basement membrane chondroitin sulfate proteoglycan decreased in both the subendothelial space and smooth muscle cell basement membranes of the aorta above the clip compared with controls, whereas only fibronectin was elevated in the aorta below the clip. No change in basement membrane staining intensity for the cardiac myocytes was observed. Alterations in steady-state mRNA levels for fibronectin and laminin in the aorta paralleled those observed by immunohistochemical analysis with regard to protein and tissue type affected as well as intensity of the changes. However, changes in mRNA levels (but not protein deposition) for perlecan and type IV collagen were also observed in aortas from hypertensive rats compared with controls. Increases in steady-state mRNA levels for all basement membrane components in the heart and vasculature peaked before maximal cardiac hypertrophy (5 days). These studies indicate that alterations in basement membrane component deposition in the hypertrophied vasculature occur at both transcriptional and translational levels and suggest that the cell attachment glycoproteins fibronectin and laminin may be important factors in the vascular response to elevated transmural pressure.

Proteoglycans and endothelial barrier function: effect of linoleic acid exposure to porcine pulmonary artery endothelial cells.

Certain fatty acids induce changes in endothelial barrier function which may be mediated by alterations in normal proteoglycan synthesis/metabolism. To test this hypothesis, pulmonary artery derived endothelial cells were treated with media supplemented with linoleic acid (18:2), and/or a known proteoglycan synthesis inhibitor, beta-D-xyloside. Independent exposure to 1 mM beta-D-xyloside or 90 microM 18:2 increased albumin transfer, i.e., decreased barrier function, when compared with control cultures. 18:2 and beta-D-xyloside increased albumin transfer additively, suggesting that the mechanisms by which 18:2 and beta-D-xyloside alter the proteoglycan metabolism are different. Compared with the control group, treatment with 18:2 inhibited proteoglycan synthesis, decreased anionic properties of heparan sulfate proteoglycans in the cell monolayers and caused the release of a unique chondroitin sulfate proteoglycan into the culture media. Treatment with beta-D-xyloside caused an increased incorporation of radioactive sulfate into glycosaminoglycans but inhibited proteoglycan synthesis. These results suggest that the fatty acid- and beta-D-xyloside-induced impairment in endothelial barrier function may involve changes in the synthesis, release and physicochemical properties of proteoglycans.

Oxidized lipid-mediated alterations in proteoglycan metabolism in cultured pulmonary endothelial cells.

Compared to cholesterol or linoleic acid (18:2), oxidized lipids such as cholestan-3 beta, 5 alpha, 6 beta-triol (triol) and hydroperoxy linoleic acid (HPODE) markedly impair endothelial barrier function in culture [Hennig and Boissonneault, 1987; Hennig et al. 1986]. Because proteoglycans contribute to vascular permeability properties, the effects of cholesterol and 18:2 and their oxidation products, triol and HPODE, on endothelial proteoglycan metabolism were determined. While cholesterol was without effect, a concentration-dependent decrease in cellular proteoglycans (measured by 35S incorporation) was observed after exposure to triol. Compared to control cultures, cholesterol reduced mRNA levels for the proteoglycans, perlecan and biglycan. Triol had a similar effect on biglycan but not an perlecan mRNA levels. Compared to 18:2, 1,3 and 5 microM HPODE depressed cellular proteoglycans. Perlecan mRNA levels were reduced more by HPODE when compared to 18:2. Biglycan mRNA levels were reduced by 3 microM, but not by 5 microM HPODE. These data demonstrate that oxidized lipids such as triol and HPODE can decrease cellular proteoglycan metabolism in endothelial monolayers and alter mRNA levels of major specific proteoglycans in a concentration-dependent manner. This may have implications in lipid-mediated disruption of endothelial barrier function and atherosclerosis.

Role of ATP and sodium in polyamine transport in bovine pulmonary artery smooth cells.

Increased polyamine transport may be a key mechanism driving elevations in lung cell polyamine content necessary for the development of chronic hypoxic pulmonary hypertension. Bovine pulmonary artery smooth muscle cells (PASMCs) in culture exhibit two carriers for polyamines, a non-selective one shared by the three polyamines, putrescine (PUT), spermidine (SPD), and spermine (SPM), and another that is selective for SPD and SPM. Hypoxia appears to up-regulate both carriers. In this study, we examined the role of ATP and the Na+ gradient in regulating polyamine transport in control PASMCs and in PASMCs with polyamine transport augmented by culture under hypoxic conditions (Po2: 15-30 torr). Inhibition of ATP synthesis with dinitrophenol+iodoacetate profoundly reduced polyamine uptake in both control and hypoxic PASMCs. Putrescine uptake was somewhat more sensitive to iso-osmotic replacement of extracellular Na+ with choline chloride or sucrose than were SPD or SPM in both hypoxic and standard cells, but under no conditions did Na+ replacement substantially alter polyamine uptake. Treatment of PASMCs with ouabain, a Na(+)-K+ ATPase inhibitor, or with gramicidin, a Na+ ionophore, minimally attenuated polyamine transport, whereas the Na+/K+ ionophore monensin increased polyamine uptake in standard, but not in hypoxic, cells. In general, the reduction in the extracellular Na+ content or ionophore-induced increases in Na+ permeability had a greater suppressive effect on polyamine transport in hypoxic cells than in standard cells, suggestive of the induction of Na(+)-dependent polyamine carriers by hypoxia. These observations indicate that the activities of the two putative polyamine transport pathways in standard PASMCs, as well as their up-regulation by hypoxia, require ATP synthesis. In addition, it appears that polyamine transport in PASMCs is composed of two components: one a prominent sodium-independent transporter and the other a relatively minor component that is sodium dependent. The latter may be activated by hypoxic exposure in combination with the induction of new polyamine carriers.

Alterations of growth factor transcripts in rat lungs during development of monocrotaline-induced pulmonary hypertension.

Although pathologic and hemodynamic changes in monocrotaline (MCT)-induced pulmonary hypertension have been studied extensively, relatively little is known about the inter- and intracellular signaling mechanisms underlying such alterations. As a first step to delineating signaling mechanisms governing adverse structural alterations in the hypertensive lungs, we examined changes in the steady-state levels of mRNAs encoding several growth factors including transforming growth factors (TGF), platelet-derived growth factors (PDGF), vascular endothelial cell growth factor (VEGF) and endothelin (ET) as a function of time in MCT-induced pulmonary hypertension in rats. These studies demonstrated a very diverse pattern of growth factor gene expression in response to MCT administration. In general, alterations in the steady-state levels of mRNAs encoding the growth factors preceded the onset of MCT-induced pulmonary hypertension. TGF-beta 1, -beta 2 and -beta 3 transcripts were seen to be elevated, whereas that of TGF-alpha and PDGF-A remained unchanged. Transcripts for PDGF-B and ET were increased in the early stages but declined to less than controls in the latter stages of MCT-induced hypertension. In contrast, levels of VEGF mRNA decreased to less than controls as the disease progressed. Viewed collectively, the diverse pattern of expression suggests that alterations in the levels of the growth factor transcripts may have a significant role in the development of pulmonary hypertensive disease and may be relevant to the pathological and structural changes in MCT-induced pulmonary hypertension.

RGD-containing peptides induce endothelium-dependent and independent vasorelaxations of rat aortic rings.

Peptides containing the extracellular matrix peptide cell attachment sequence RGD possess potent, endothelium-dependent vasorelaxant properties. In the present study, the ability of RGD-containing peptides to cause vasorelaxation in the presence and absence of a functional endothelium was examined in rat aortic rings along with the ability of RGD-containing peptides to increase cGMP production in these vessels. The active RGD-containing peptide GRGDNP induced rapid relaxation in endothelium-intact, norepinephrine contracted rat aortic rings. When the endothelium was removed, RGD-containing peptides produced a slow relaxation of contracted rings which took approx. 40 min to reach maximum relaxation. Control RGD peptides were without effect either in the presence or absence of a functional endothelium. While acetylcholine and sodium nitroprusside stimulated cGMP production in endothelium-intact and denuded aortic segments, neither the control RGD peptide nor the active GRGDNP increased cGMP in these vessels when compared to controls upon either short (30 s) or long (45 min) incubation times. These data indicate that relaxations of rat aortic rings in response to RGD-containing peptides occur both in the presence and absence of an intact endothelium and that cGMP is likely not the sole mediator of these responses.

Role of fatty acids and eicosanoids in modulating proteoglycan metabolism in endothelial cells.

Endothelial cell dysfunction is considered to be a critical event in the etiology of atherosclerosis. Thus, the preservation of endothelial structure and function are a prerequisite for normal control of vascular permeability properties, mediation of both inflammatory and immunologic responses and the general 'communication' between blood-borne cells and abluminal tissues. Many of these properties can be influenced by proteoglycans present in vascular tissues. There is evidence that selected lipids can be atherogenic by altering endothelial proteoglycan metabolism. Little is known about the role of fatty acids in modulating proteoglycan composition in endothelial cells. Data suggest, however, that linoleic acid in particular can adversely alter proteoglycan metabolism, which may be related to an imbalance in eicosanoid synthesis patterns. These events could be sufficient to disrupt normal endothelial barrier function, initiate smooth muscle migration and proliferation, and result in other metabolic dysfunctions associated with the etiology of vascular diseases such as atherosclerosis. Thus, the focus of this review is on fatty acids and eicosanoids as they may alter proteoglycan metabolism of vascular tissues and in particular of the endothelium.

Temporal alterations in basement membrane components in the pulmonary vasculature of the chronically hypoxic rat: impact of hypoxia and recovery.

The hypoxic model of pulmonary hypertension was used to examine temporal alterations in the deposition of the basement membrane (BM) and components of fibronectin, laminin, and Type IV collagen within vascular, airway, and gas exchange compartments of the lung. Because hypoxic pulmonary hypertension is a reversible model of hypertension, changes in fibronectin and laminin synthesis/deposition in the recovering lung were also examined. Long-term hypoxic exposure produced decreases in body weight, increased right ventricular and lung dry weights and elevations in pulmonary arterial pressure. Immunohistochemical analysis revealed consistent and progressive increases in the deposition of fibronectin and laminin, but not type IV collagen, in the subendothelial and medial BMs of large and small pulmonary arteries, but not in airways or lung parenchyma. These changes were observed by day 4 of hypoxia and were most prominent in the conducting vasculature. Northern analysis showed a biphasic pattern of alterations in steady-state levels of BM component mRNA in hypoxic rats with early reductions at days 4 and 7 followed by increases at day 12. Recovery from 12 days of hypoxia resulted in regression of pulmonary hypertension and right ventricular hypertrophy but not increased lung weight. Immunohistochemical analysis of fibronectin, laminin, and type IV collagen levels in the vasculature showed a temporal regression to levels that were not remarkably different from time-matched controls at day 30 of recovery. Northern analysis of lungs from hypoxic-recovery rats revealed increased steady-state levels of mRNA for fibronectin, laminin, and type IV collagen at all time points. These data indicate that long-term hypoxic exposure elicits marked alterations in the synthetic capacity and deposition of the important cell attachment BM glycoproteins fibronectin and laminin. In addition, recovery from hypoxia appears to be characterized by a lack of increased fibronectin and laminin levels in the conducting vasculature, suggesting a marked and rapid reorganization of the vascular BMs on both hypoxic exposure and recovery from hypoxia.

A novel technique for visualizing the intracellular localization and distribution of transported polyamines in cultured pulmonary artery smooth muscle cells.

The use of a combination of monofluorescein adducts of spermidine (FL-SPD) and spermine (FL-SPM) with confocal laser scanning microscopy (CLSM) provides a useful means for monitoring the fate and time-dependent changes in the distribution of transported polyamines within living cells. Polyamine-fluorescein adducts were synthesized from fluorescein isothiocyanate and the appropriate polyamine. Monofluorescein polyamine adducts (ratio 1:1) were isolated using thin layer chromatography, and the structure and molecular weight of the monofluorescein polyamine adducts were confirmed using NMR and mass spectroscopy, respectively. The covalent linkage of the fluorescent adduct moiety to SPD and SPM did not influence their rate of uptake by bovine pulmonary artery smooth muscle cells (PASMC). Similar to 14C-SPD and 14C-SPM, the rate of uptake of 14C-FL-SPD and 14C-FL-SPM in PASMC was temperature-dependent. Treatment for 24 h with difluoromethylornithine (DFMO), a selective blocker of the enzyme ornithine decarboxylase and an inducer of the polyamine transport system, significantly increased the cellular uptake of 14C-FL-SPD and 14C-FL-SPM compared to that of control cells. When compared to control cells, treatment of PASMC with the pyrrolizidine alkaloid monocrotaline for 24 h also significantly increased the cellular uptake of 14C-FL-SPD and 14C-FL-SPM. On the other hand, 24 h treatment of PASMC with a polymer of SPM, a selective blocker of the polyamine transport system, or with free spermine, markedly reduced the cellular accumulation of 14C-FL-SPD and 14C-FL-SPM. After a 20-min treatment of PASMC with FL-SPD or FL-SPM, CLSM revealed that adduct fluorescence was localized in the cytoplasm of living cells. Treatment with DFMO increased the cytoplasmic accumulation of both FL-SPD and FL-SPM. In addition, the fluorescence observed in the cytoplasm of chinese hamster ovary cells (CHO) was significantly higher than that detected in the cytoplasm of their polyamine transport deficient variants (CHOMGBG). The results of this study provide the first evidence of the utility of a novel method for visualizing the uptake, distribution, and cellular localization of transported polyamines in viable cultured mammalian cells.

Increased proteoglycan synthesis by the cardiovascular system of coarctation hypertensive rats.

Proteoglycan (PG) synthesis in the cardiovascular system of coarctation hypertensive rats was examined by in vivo and in vitro labeling of glycosaminoglycans with 35SO4 in rats made hypertensive for short (4 days) and longer (14 days) durations. With in vivo labeling, only tissues directly exposed to elevated pressure (left ventricle, LV and aorta above the clip, AOR increases) exhibited elevated PG synthesis after 4 days of hypertension. By 14 days, tissues both exposed to (LV and AOR increases) and protected from elevated pressure (right ventricle and kidney) exhibited elevated PG synthetic rates. Slight elevations in the proportion of galactosaminoglycans were observed with a concurrent proportional decrease in heparan sulfate PGs. Using the in vitro labeling procedure, no significant increases in PG synthesis were observed in any tissue at either 4 days or 14 days of hypertension. These data indicate that: 1) coarctation hypertension stimulates PG production that is dependent initially on increased pressure and later, on additional non-pressure related factors, 2) these other factors are responsible for enhanced PG production in tissues not directly exposed to pressure overload, 3) pressure and/or these other factors are essential for enhanced PG production in coarctation hypertension, and 4) synthesis of all GAG types appears to be affected.

Enzymes of the kallikrein-kinin system in rainbow trout.

Evidence for a kallikrein-kinin system (KKS) in fish is incomplete. In the present study, components of the KKS were identified in rainbow trout. Tissues were assayed for kallikrein-like esterolytic activity using three synthetic kallikrein substrates (TAME, VGAN, and PPAN), and the presence of kallikrein substrate (kininogen) in trout plasma was estimated by bradykinin (BK) radioimmunoassay of plasma activated with trypsin (T). Formation of pressor-depressor substances in vivo by porcine glandular kallikrein (GK) and T was measured after intra-arterial injection into unanesthetized trout. Gill and kidney contained kallikrein activity (TAME and VGAN assays); little activity was observed with PPAN. Aprotinin inhibited gill activity (TAME assay). T liberated 42 +/- 3 (SE) ng (n = 10) of immunoreactive BK per milliliter of plasma. Injection of GK in vivo reduced plasma kininogen levels for over 24 h. GK produced pressor responses only in fish pretreated with the angiotensin-converting enzyme (ACE) inhibitor captopril. This effect was mediated partly through stimulation of alpha-adrenergic receptors. T produced slight pressor responses that were captopril insensitive. These results show that trout possess elements of the KKS system including kallikrein-like enzymatic activity, kininogen, receptor-mediated vascular sensitivity to kallikrein products, and kininolytic activity consistent with ACE (kininase II).

A specific inhibitor of mammalian kallikrein, Phe-Phe-Arg-chloromethyl ketone, inhibits the production of vasoactive substances from trout plasma by kallikrein and blocks endogenous kallikrein-like activity in trout gills.

The cardiovascular effects of the kallikrein-kinin system (KKS) have not been completely characterized in lower vertebrates. In the present study, a specific, irreversible kallikrein inhibitor, Phe-Phe-Arg-chloromethyl ketone (PPACMK) was used to examine: 1. the role of the KKS in blood pressure regulation in vivo; 2. the nature of the pressor substance formed by the action of kallikrein on trout plasma in vivo and in vitro; and 3. the presence of kallikrein in trout gills and kidney. Dorsal aortic cannulated rainbow trout were used for in vivo blood pressure assays and two colorimetric serine-protease assays were used to examine tissue kallikrein activity. PPACMK alone had no effect on blood pressure in vivo. Pretreatment of porcine kallikrein with PPACMK inhibited the enzyme's pressor effect in trout by 80% and significantly attenuated the synthesis of vasopressor substance(s) from heat-treated trout plasma in vitro. Approximately 30% of gill serine protease activity was inhibited by pretreatment with PPACMK; no PPACMK-sensitive kallikrein activity was observed in the kidney. Salt water adaptation did not affect kallikrein-specific activity in the gill. These results show that the salmonid KKS does not appear to be involved in the regulation of systemic blood pressure. Further, the formation of pressor substances from trout plasma in vivo and in vitro is due to kallikrein activity and the peptides generated are similar. A kallikrein, similar to the mammalian enzyme, is found in trout gills, suggesting that the salmonid KKS may be a local mediator of gill function.

Generation of vasoactive substances in trout and rat plasma by trypsin and kallikrein.

In the preceding study we demonstrated kallikrein-like enzymatic activity in trout tissues and showed that kallikrein incubated with trout plasma (T60K) produces a vasopressor substance(s). The present study further examines the effects of T60K in fish and mammals in vitro and in vivo. T60K produced a dose-dependent pressor response in both trout and rats, whereas kallikrein-activated rat plasma (R60K) was pressor in trout and depressor in rats. Captopril did not affect the response of rats to T60K or R60K. Phenoxybenzamine attenuated the T60K response in trout but not in rats, thus T60K effects in trout are partially mediated through catecholamines. Blockade of angiotensin II (ANG II) receptors in rats with [Sar1,Ala8]-ANG II abolished the pressor effects of T60K. T60K produced dose-dependent contractions in isolated trout and rabbit arteries; ANG II was ineffective in trout arteries. T60K-contracted trout arteries were relaxed by atrial natriuretic peptide and forskolin, whereas diltiazem and sodium nitroprusside were without effect. [Sar1,Ala8]ANG II inhibited T60K-induced contractions of rabbit arteries and relaxed rings previously contracted with T60K. The active component of T60K has a molecular weight less than 10,000, is heat stable, and is inactivated by peptidases. It is immunologically different than mammalian angiotensins but binds to and displaces radiolabeled ANG II from ANG II receptors. These results suggest that kallikrein forms a vasoactive substance(s) in trout plasma that is neither bradykinin nor ANG II but is similar to the latter in its pharmacological effects.

Vascular effects of kinins in trout and bradykinin metabolism by perfused gill.

In the preceding studies we have shown that elements of a kallikrein-kinin system (KKS) are present in trout. The present study examines the cardiovascular effects of intra-arterial kinin injection and the ability of perfused gills or gill homogenates to metabolize bradykinin or the pressor substance generated in trout plasma by glandular kallikrein (T60K). Bradykinin (BK), t-kinin, kallidin, and Met-kallidin produced pressor responses in vivo. BK responses were unaffected by alpha-adrenergic blockade or cyclooxygenase inhibition. Perfused gills extracted approximately 40% of a [3H]BK bolus, however, metabolites were not recovered from the effluent perfusate. Gill homogenates completely metabolized [3H]BK and inactivated the pressor substance T60K. Captopril reduced BK and T60K metabolism by gill homogenates. The present study demonstrates that kinins have pressor effects in trout that are not mediated through adrenergic or prostanoid-derived mechanisms. The results also suggest that trout do not release endothelium-derived relaxing factors in response to kinin injection. The gill is able to metabolize BK and T60K, although, with respect to BK, this process appears to involve intracellular hydrolysis and may be only partially dependent on angiotensin-converting enzyme. Inactivation of BK by gill tissue may be fundamentally different from kinin metabolism by the mammalian lung.

Characterization of angiotensin-converting enzyme in the gills of rainbow trout,Salmo gairdneri (Richardson).

Several physical and chemical parameters of angiotensin-converting enzyme (ACE) were determined using a spectrophotometric assay of gill tissue homogenates from rainbow trout. This assay is based on the evolution of free hippuric acid via enzymatic cleavage of histidyl-leucine from the synthetic substrate hippuryl-l-histidyl-l-leucine (HHL). Piscine ACE exhibited enzymatic and kinetic properties similar to those reported for the partially purified mammalian enzyme. Proteolytic activity was both temperature and pH dependent and demonstrated hyperbolic kinetics with an apparent Km of 2.5 mM. Hydrolysis of HHL was activated by Cl(-) at concentrations between 20 mM and 100 mM. Captopril (1 × 10(-6) M) and MK-422 (1 × 10(-6) M) blocked trout gill ACE activity, however, EDTA was inhibitory only at high concentrations (1 × 10(-3) M). These results demonstrate that trout ACE is functionally similar to mammalian ACE and that the spectro-photometric assay for ACE developed by Cushman and Cheung can be applied to analysis of converting enzyme activity in fish tissue homogenates.

Temporal alterations in specific basement membrane components in lungs from monocrotaline-treated rats.

The present study utilized the monocrotaline (MCT) model of pulmonary hypertension in rats to examine temporal alterations in steady-state levels of basement membrane (BM) component mRNA and deposition of protein using Northern analysis and immunohistochemistry, respectively. MCT (60 mg/kg, subcutaneous) produced sustained increases in lung dry tissue mass by 7 days, right ventricular mass by 14 days, and pulmonary arterial pressure by 21 days after administration. mRNA levels specific for laminin (LM) were elevated as early as 1 day after MCT treatment, while mRNA for all BM components examined except type IV collagen were increased in lungs from MCT-treated rats by day 4. Differences in LM, perlecan (PN), and type IV collagen-specific mRNAs from lung tissue between MCT-treated and control rats disappeared by day 14. In contrast, fibronectin (FN) mRNA remained elevated in lung tissue from MCT-treated rats from day 4 onward. Increases in immunolocalizable FN and LM in the vasculature, and PN and type IV collagen in gas exchange areas, were observed 4 days after MCT treatment compared with controls. These changes generally became more pronounced by 21 days after MCT administration, at which time the parenchyma of MCT-treated rats also demonstrated increases in immunolocalizable FN, LM, and BM-chondroitin sulfate proteoglycan (BM-CSPG). The pulmonary vasculature additionally showed increases in type IV collagen, PN, and BM-CSPG in MCT-treated rats compared with controls by 21 days. These observations suggest that the accumulation of specific BM components in the pulmonary vasculature and parenchyma may contribute to the pathogenesis and maintenance of MCT-induced hypertensive pulmonary vascular disease.

Tumor necrosis factor reduces proteoglycan synthesis in cultured endothelial cells.

Tumor necrosis factor (TNF)-induced disruption of vascular endothelial barrier function may be due in part to alterations in proteoglycan metabolism. To test this hypothesis, confluent endothelial cell monolayers were exposed for 24 h to 500 or 1,000 U of TNF per milliliter of culture medium together with 20 microCi Na2 35SO4. HPLC anion-exchange separation of proteoglycans secreted into media of control as well as TNF-treated cultures revealed one major peak (representing 95% of total radioactivity) and one minor peak (representing 5% of total radioactivity), which eluted at 0.6 and 0.9 M NaCl, respectively. One single peak was obtained from control as well as TNF-treated endothelial cell monolayers and eluted at 1.2 M NaCl. TNF treatment did not change the total quantity of radioactive proteoglycans secreted into the media but significantly decreased the amount of proteoglycans in endothelial cell monolayers. However, TNF treatment did not alter the size or glycosaminoglycan (GAG) composition of the proteoglycans either in the media or in the cell monolayers. In addition, mRNA levels of specific proteoglycans, perlecan and biglycan, were measured upon TNF treatment, using Northern analysis. TNF treatment caused a dose-dependent decrease in mRNA levels for the core proteins of perlecan, a major heparan sulfate proteoglycan (HSPG), and biglycan in endothelial cultures. These results suggest that TNF decreases production of proteoglycans and alters normal endothelial cell proteoglycan metabolism which may be sufficient to impair endothelial barrier function.

Polyamine regulatory processes and oxidative stress in monocrotaline-treated pulmonary artery endothelial cells.

Alterations in polyamine metabolism may be a critical mechanism of monocrotaline (MCT)-induced structural remodeling of the pulmonary vasculature. In the present study, the hypothesis that MCT, through the induction of oxidative stress, modulates cellular polyamine regulatory mechanisms which in turn might be involved in the upregulation of fibronectin production in pulmonary artery endothelial cells (PAEC) was examined. A 24-h treatment with MCT significantly increased PAEC polyamine concentrations as compared to vehicle-treated cells. In addition, exposure to MCT caused an increase in abundance of ornithine decarboxylase (ODC) mRNA, upregulation of ODC activity and enhancement of spermidine import into PAEC. Inhibition of de novo polyamine synthesis further increased spermidine uptake in MCT-treated cells. The depletion of cellular polyamine contents through the blockade of both de novo polyamine biosynthesis and polyamine transport prevented MCT-induced increases in the medium level of fibronectin. In addition, PAEC treatment with MCT stimulated cellular oxidative stress as determined by increased levels of thiobarbituric acid reactive substances, enhanced dichlorofluorescein fluorescence and activation of NF-kappa B. A co-treatment with dimethylthiourea, an oxygen radical scavenger, prevented MCT-induced increases in cellular oxidation and attenuated disturbances in polyamine metabolism. These data suggest that MCT can stimulate polyamine regulatory processes in PAEC possibly through an increase in cellular oxidative stress. The present study may have significant implication in understanding mechanisms of MCT-induced pulmonary hypertension and remodeling of pulmonary vasculature.

Eflornithine alters changes in vascular responsiveness associated with coarctation hypertension.

This study examined the temporal effects of the polyamine synthesis inhibitor eflornithine (alpha-difluoromethylornithine) on vascular responses to KCI, norepinephrine, sodium nitroprusside and acetylcholine in aortic rings from coarctation hypertensive rats. Coarctation hypertension reduced the contractile response of aortic rings to KCI and norepinephrine, increased sensitivity (reduced the EC50 value) to norepinephrine and attenuated relaxation to acetylcholine by 14 days of hypertension. Treatment of coarctation hypertensive rats with eflornithine resulted in a normalization of the contractile intensity to KCI and norepinephrine and relaxations to acetylcholine by 14 days of hypertension. Responses to sodium nitroprusside were similar in all groups at all time points. Hyperresponsiveness to norepinephrine produced by coarctation of the aorta was not affected by eflornithine. These studies indicate that normalization of vascular function can occur in the presence of significantly elevated blood pressure upon chronic administration of eflornithine. This functional normalization correlates with eflornithine-mediated regression of structural abnormalities normally associated with pressure overload hypertension.