Oxygen radical-induced mitochondrial DNA damage and repair in pulmonary vascular endothelial cell phenotypes.

Mitochondrial (mt) DNA is damaged by free radicals. Recent data also show that there are cell type-dependent differences in mtDNA repair capacity. In this study, we explored the effects of xanthine oxidase (XO), which generates superoxide anion directly, and menadione, which enhances superoxide production within mitochondria, on mtDNA in pulmonary arterial (PA), microvascular (MV), and pulmonary venous (PV) endothelial cells (ECs). Both XO and menadione damaged mtDNA in the EC phenotypes, with a rank order of sensitivity of (from most to least) PV > PA > MV for XO and MV = PV > PA for menadione. Dimethylthiourea and deferoxamine blunted menadione- and XO-induced mtDNA damage, thus supporting a role for the iron-catalyzed formation of hydroxyl radical. Damage to the nuclear vascular endothelial growth factor gene was not detected with either XO or menadione. PAECs and MVECs, but not PVECs, repaired XO-induced mtDNA damage quickly. Menadione-induced mtDNA damage was avidly repaired in MVECs and PVECs, whereas repair in PAECs was slower. Analysis of mtDNA lesions at nucleotide resolution showed that damage patterns were similar between EC phenotypes, but there were disparities between XO and menadione in terms of the specific nucleotides damaged. These findings indicate that mtDNA in lung vascular ECs is damaged by XO- and menadione-derived free radicals and suggest that mtDNA damage and repair capacities differ between EC phenotypes.

Regulation of ornithine decarboxylase activity and polyamine transport by agmatine in rat pulmonary artery endothelial cells.

Agmatine, a product of arginine decarboxylation in mammalian cells, is believed to govern cell polyamines by inducing antizyme, which in turn suppresses ornithine decarboxylase (ODC) activity and polyamine uptake. However, since agmatine is structurally similar to the polyamines, it is possible that it exerts antizyme-independent actions on polyamine regulatory pathways. The present study determined whether agmatine inhibited ODC activity and polyamine transport in rat pulmonary artery endothelial cells (PAECs) by an antizyme-dependent mechanism. Agmatine caused time-dependent reductions in ODC activity, which occurred before increases in antizyme. Interventions that suppressed proteasome function caused large increases in ODC activity but failed to attenuate inhibitory effects of agmatine. When agmatine was present in the culture medium, 14C-polyamine uptake was competitively inhibited as evidenced by substantial elevations in K(m) values. If PAECs were incubated with agmatine for periods sufficient to increase antizyme, there were modest decreases in V(max) for putrescine and spermidine but not for spermine. These effects of agmatine on polyamine transport were insensitive to protein synthesis inhibition. Collectively, our findings show that agmatine decreases ODC activity and polyamine transport in PAECs, but a causal role for antizyme in these actions of agmatine is difficult to establish. Nevertheless, these observations are consistent with a model in which PAECs express both antizyme-1 and -2, but only the latter contributes to agmatine-mediated suppression of ODC activity.

Free radical production in hypoxic pulmonary artery smooth muscle cells.

This study used an inexpensive and versatile environmental exposure system to test the hypothesis that hypoxia promoted free radical production in primary cultures of rat main pulmonary artery smooth muscle cells (PASMCs). Production of reactive species was detected by fluorescence microscopy with the probe 2', 7'-dichlorodihydrofluorescein, which is converted to the fluorescent dichlorofluorescein (DCF) in the presence of various oxidants. Flushing the airspace above the PASMC cultures with normoxic gas (20% O(2), 75% N(2), and 5% CO(2)) resulted in stable PO(2) values of approximately 150 Torr, whereas perfusion of the airspace with hypoxic gas (0% O(2), 95% N(2), and 5% CO(2) ) was associated with a reduction in PO(2) values to stable levels of approximately 25 Torr. Hypoxic PASMCs became increasingly fluorescent at approximately 500% above the normoxic baseline after 60 min. Hypoxia-induced DCF fluorescence was attenuated by the addition of the antioxidants dimethylthiourea and catalase. These findings show that PASMCs acutely exposed to hypoxia exhibit a marked increase in intracellular DCF fluorescence, suggestive of reactive oxygen or nitrogen species production.

Cellular disposition of transported polyamines in hypoxic rat lung and pulmonary arteries.

The polyamines putrescine, spermidine (SPD), and spermine are a family of low-molecular-weight organic cations essential for cell growth and differentiation and other aspects of signal transduction. Hypoxic pulmonary vascular remodeling is accompanied by depressed lung polyamine synthesis and markedly augmented polyamine uptake. Cell types in which hypoxia induces polyamine transport in intact lung have not been delineated. Accordingly, rat lung and rat main pulmonary arterial explants were incubated with [(14)C]SPD in either normoxic (21% O(2)) or hypoxic (2% O(2)) environments for 24 h. Autoradiographic evaluation confirmed previous studies showing that, in normoxia, alveolar epithelial cells are dominant sites of polyamine uptake. In contrast, hypoxia was accompanied by prominent localization of [(14)C]SPD in conduit, muscularized, and partially muscularized pulmonary arteries, which was not evident in normoxic lung tissue. Hypoxic main pulmonary arterial explants also exhibited substantial increases in [(14)C]SPD uptake relative to control explants, and autoradiography revealed that enhanced uptake was most evident in the medial layer. Main pulmonary arterial explants denuded of endothelium failed to increase polyamine transport in hypoxia. Conversely, medium conditioned by endothelial cells cultured in hypoxic, but not in normoxic, environments enabled hypoxic transport induction in denuded arterial explants. These findings in arterial explants were recapitulated in rat cultured main pulmonary artery cells, including the enhancing effect of a soluble endothelium-derived factor(s) on hypoxic induction of [(14)C]SPD uptake in smooth muscle cells. Viewed collectively, these results show in intact lung tissue that hypoxia enhances polyamine transport in pulmonary artery smooth muscle by a mechanism requiring elaboration of an unknown factor(s) from endothelial cells.

Interactions between agmatine and polyamine uptake pathways in rat pulmonary artery endothelial cells.

Agmatine, a product of arginine metabolism in vascular endothelial cells, is structurally similar to the natural polyamines, putrescine, spermidine and spermine. To test the hypothesis that agmatine and polyamines interacted at the level of the polyamine transporter, we determined if polyamines competed with agmatine for import and whether interventions modulating polyamine import exerted coordinate effects on agmatine uptake. Multiple lines of evidence were obtained to suggest that agmatine enters pulmonary artery endothelial cells (PAECs) via the polyamine transporter, though its intracellular disposition after uptake appears different from the natural polyamines.

Regulation of gadd153 mRNA expression by hypoxia in pulmonary artery smooth muscle cells.

Hypoxia causes pulmonary hypertension and induces oxygen radicals in pulmonary artery smooth muscle cells (PASMCs). Since oxidative stress regulates gaddl53 expression, we examined gaddl53 mRNA in PASMCs cultured in a hypoxic environment. Gadd153 mRNA content was increased in PASMCs cultured for 24 hours in 1% oxygen. This increase was not abrogated by inhibition of protein synthesis. To explore the signaling pathways mediating hypoxic regulation of gaddl53 mRNA, the impact of calcium channel blockade by verapamil, G protein inhibition by pertussis toxin, and protein kinase C (PKC) down-regulation, was examined. Although none of these interventions reduced basal expression of gaddl53 mRNA in PASMCs, all of them suppressed the induction by hypoxia. In contrast, antioxidants had no effect. These observations indicate hypoxia induces gaddl53 expression in PASMCs through common signaling pathways.

Capillary filtration coefficient, vascular resistance, and compliance in isolated mouse lungs.

Although many recently produced transgenic mice possess gene alterations affecting pulmonary vascular function, there are few baseline measurements of vascular resistance and permeability. Therefore, we excised the lungs of C57/BL6 mice and perfused them with 5% bovine serum albumin in RPMI-1640 culture medium at a nominal flow of 0.5 ml/min and ventilated them with 20% O(2)-5% CO(2)-75% N(2). The capillary filtration coefficient, a sensitive measurement of hydraulic conductivity, was unchanged over 2 h (0.33 +/- 0.03 ml. min(-1). cmH(2)O(-1). 100 g(-1)) in a control group ventilated with low peak inflation pressures (PIP) but increased 4. 3-fold after high PIP injury. Baseline pulmonary vascular resistance was 6.1 +/- 0.4 cmH(2)O. ml(-1). min. 100 g(-1) and was distributed 34% in large arteries, 18% in small arteries, 14% in small veins, and 34% in large veins on the basis of vascular occlusion pressures. Baseline vascular compliance was 5.4 +/- 0.3 ml. cmH(2)O(-1). 100 g(-1) and decreased significantly with increased vascular pressures. Baseline pulmonary vascular resistance was inversely related to both perfusate flow and microvascular pressure and increased to 202% of baseline after infusion of 10(-4) M phenylephrine due to constriction of large arterial and venous segments. Thus isolated mouse lung vascular permeability, vascular resistance, and the longitudinal distribution of vascular resistance are similar to those in other species and respond in a predictable manner to microvascular injury and a vasoconstrictor agent.

Pulmonary mechanical and immunologic dysfunction in a murine model of AIDS.

Human immunodeficiency virus-infected patients occasionally exhibit alveolar septal wall thickening and decreases in gas diffusion capacity, but the mechanism underlying these abnormalities is unknown. The present study evaluated septal wall thickness and gas exchange properties in a murine model of the acquired immunodeficiency syndrome and determined whether there were alterations in lung lymphocyte deposition and activation that could contribute to changes in respiratory structure and function. Although alveolar septal wall thickness did not differ from control at 1, 2, and 4 wk postimmunosuppressive virus infection, at 8 wk after infection, septal wall thickness was substantially increased. Immunohistochemical evaluation at this time revealed marked increases in the septal wall deposition of fibronectin and collagen type IV. Pulmonary function tests on anesthetized mice with virus-induced septal wall thickening demonstrated that, although total lung capacity, compliance, and functional residual capacity were unaltered, diffusion capacity for carbon monoxide was significantly impaired. A diffuse nonspecific interstitial pneumonitis was present in lungs of immunodeficient mice, and flow cytometry indicated that both lymphocytes and macrophages were activated. Reverse transcriptase-polymerase chain reaction analysis of lung lymphocytes demonstrated enhanced mRNA expression for several cytokines known to affect lung structure. These results show that impaired gas exchange occurs in a murine model of acquired immunodeficiency syndrome and suggest that such alterations may be mediated by elaboration of cytokines from activated lung lymphocytes and macrophages.

Pulmonary lymphoid cell activation and cytokine expression in murine AIDS-associated interstitial pneumonitis.

Limited information is available about the pathogenesis of acquired immune deficiency syndrome (AIDS)-associated idiopathic interstitial pneumonitis, a common noninfectious complication of human immunodeficiency virus (HIV) infection. Infection of C57B1/6 mice with LP-BM5 retrovirus, a murine model of AIDS, leads to development of a diffuse interstitial pneumonitis that displays many features of human AIDS-associated interstitial pneumonitis. To further characterize the cellular and molecular features of this lung disease, the temporal development of cellular infiltration, cytokine expression, and virus replication were evaluated in lung tissue of virus-infected mice. Persistent expression of viral RNA was detectable in lungs as early as 1 wk after infection. Infiltration of the lungs by CD4+ and CD8+ T cells, by IgG+ and IgA+ B cells, and by macrophages was observed by 4 wk after infection and continued through 8 wk of infection. Histologically, cellular infiltration was most pronounced in peribronchial and perivascular regions, whereas inflammation of alveolar septae and alveolar spaces was minimal. In contrast to normals, T cells from infected lungs were immunodeficient in that they failed to proliferate in response to the mitogen concanavalin A (ConA). However, evaluation of cytokine mRNA expression by interstitial lung lymphoid cells indicated that cells from infected lungs were chronically activated, in that elevated expression of interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) was observed throughout the course of infection. Similarly, expression by interstitial lung lymphoid cells of mRNA for the proinflammatory cytokine IL-1 and the fibrogenic cytokine transforming growth factor-beta (TGF-beta) was also increased following infection. These results indicate that retrovirus-induced immunodeficiency in mice is associated with infiltration and chronic activation of lymphoid cells in the lungs. Furthermore, simultaneous expression of IL-10, IFN-gamma, and TGF-beta suggests that cytokine-expressing cells in infected lungs may be unresponsive to inhibitory and antiinflammatory effects of IL-10 and/or TGF-beta, thus contributing to chronicity of inflammation in this disorder.

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.

Regulation of ornithine decarboxylase by hypoxia in pulmonary artery smooth muscle cells.

The polyamines are a family of low-molecular-weight organic cations that play essential intracellular regulatory roles in cell growth and differentiation. Elevations in cellular polyamine contents necessary for most physiological and pathological events in the lung appear to be driven by increase de novo synthesis. In contrast, increases in lung cell polyamines required for hypoxic pulmonary vascular disease can be attributed to augmented transmembrane polyamine transport which may, in turn, be the result of hypoxia-related decreases in the activity of the initial and generally rate-limiting enzyme in de novo polyamine synthesis, ornithine decarboxylase (ODC). To begin to define the unusual mechanism whereby hypoxia governs polyamine regulatory pathways, the present study examined the impact of varying severity and durations of hypoxic exposure on ODC activity and mRNA content in cultured bovine main pulmonary artery smooth muscle cells (PASMC). The effect of hypoxia on the activity of another rate-limiting enzyme in polyamine synthesis, S-adenosylmethionine decarboxylase (AdoMet-DC), also was examined. Hypoxia caused time-dependent decreases in ODC and AdoMet-DC activities that were related to the severity of hypoxic exposure. Similarly, ODC mRNA content also was depressed by hypoxic exposure. The relationship between the decline in ODC activity and mRNA content was roughly linear. To determine whether hypoxia impairs ODC mRNA stability, two different inhibitors of transcription and Northern analyses were used to follow the decay in ODC mRNA abundance in hypoxic and normoxic PASMC. Densitometric scanning of Northern analysis indicated that ODC mRNA stability did not differ between hypoxic and normoxic PASMC. These results suggest that the reduction in ODC activity provoked by hypoxia in cultured bovine PASMC can be ascribed in part to a diminished transcriptional rate rather than to alterations in mRNA stability.

The potential of a novel polyamine transport inhibitor in cancer chemotherapy.

The polyamines, putrescine (PUT), spermidine (SPD) and spermine (SPM), are a family of low molecular weight organic cations that are essential for cell growth, differentiation and neoplastic transformation. The marked compensatory increase in extracellular polyamine influx may be a reason for the unsatisfactory clinical chemotherapeutic effect of polyamine synthesis blockers like difluoromethylornithine (DFMO). In this study, a polymeric conjugate of SPM (poly-SPM) that blocks the import of polyamines into mammalian cells was used to test the potential therapeutic exploitation of the polyamine transport system in anticancer therapy. Our results indicate that a temperature-dependent polyamine transport system is expressed in two human cancer cell lines, MES-SA uterine sarcoma cells, K562 leukemic cells and their respective multiple drug resistance (MDR) positive counterparts, Dx5 and K562/R7 cells. The V(max) values for 14C-PUT and 14C-SPD uptake were significantly higher in MES-SA than in Dx5 cells, whereas the respective Km values were significantly lower. Addition of 20 microM poly-SPM reduced both the uptake of 14C-polyamines and the cellular polyamine contents in both cancer cell lines. In addition, the poly-SPM conjugate evoked a concentration-dependent cytotoxicity in MES-SA and K562 cells and their MDR-positive variants. Presence of aminoguanidine, an amine oxidase blocker, failed to alter the IC50 values generated with poly-SPM, which indicates that this polymer is not a substrate for amine oxidase. Moreover, coadministration of 25 microM SPD reversed the cytotoxic effect exerted by poly-SPM on both the MES-SA and Dx5 cells as reflected by an increase in their IC50 values. Relative to parental cells, the MDR-positive variants exhibited a lower 14C-polyamine uptake rate and were more resistant to the cytotoxic effect of poly-SPM. Pretreatment with 1 mM DFMO for 24 hr significantly increased polyamine transport, but failed to reduce intracellular SPM contents or exert a cytotoxic effect in both cancer cell lines. On other hand, the combination of DFMO and poly-SPM produced a greater depletion of polyamine content accompanied by a higher cytotoxicity than either agent alone. These results provide the first direct evidence that pharmacologic interruption of polyamine uptake may be an effective approach to cancer therapy. In addition, it appears that expression of MDR influences polyamine transport and renders cells more resistant to the cytotoxic effects of SPM polymer.

Reversal of doxorubicin, etoposide, vinblastine, and taxol resistance in multidrug resistant human sarcoma cells by a polymer of spermine.

We have previously described the synthesis of a cytotoxic polymeric conjugate of spermine (Poly-SPM) which is able to inhibit the transport of polyamines (spermine, spermidine, and putrescine) into normal and malignant cells. Recent studies examining the toxicity of Poly-SPM in parental and multidrug resistant (MDR) cancer cells have revealed a cross-resistance in the MDR variant Dx5 to the toxic effects of the conjugate in the MDR-positive cells. There were also differences in spermine and putrescine uptake rates between parental and MDR-positive with the MDR-positive cells having a lower Vmax and a higher Km. The ability of this Poly-SPM to reverse MDR was examined in MDR variants (Dx5 cells) of the human sarcoma cell line MES-SA. The cells express high levels of the mdr1 gene product, P-glycoprotein, and are 25-to 60-fold resistant to doxorubicin (DOX), etoposide (VP-16), vinblastine (VBL), and taxol (TAX). Cytotoxicity was measured by the MTT [3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Poly-SPM (50 microM) lowered the drug concentration IC50 values in the Dx5 cells by 37-fold with VBL, 42-fold with DOX, 29-fold with VP-16, and 25-fold with TAX when compared to the control IC50 values without Poly-SPM. This reversal of resistance was concentration dependent, decreasing 17-fold with DOX, 6.1-fold with VBL, 19-fold with VP-16, and 5-fold with TAX when 25 microM Poly-SPM was used. No modulation was observed in the parental cell line MES-SA, which does not express the mdr1 gene. Poly-SPM had no influence on the IC50 of non-MDR chemotherapeutic agents such as cisplatin. The modulation studies correlated with the ability of Poly-SPM to reverse the cellular accumulation defect of [3H]-VBL and [3H]-TAX in the Dx5 but not MES-SA cells. Pretreatment of the Dx5 cell with alpha-difluoromethylornithine (DFMO at 2 and 5 microM) for 24 h increased the function of the MDR transporter to further decrease the cellular accumulation of VBL and TAX when compared to untreated cells. DFMO pretreatment is known to upregulate the polyamine transporter(s). These findings show that, in addition to inhibiting polyamine transport, Poly-SPM reverses MDR in Dx5 cells, suggesting a potential relationship between the polyamine influx transporter and the MDR efflux pump. This potential functional link between the polyamine influx transporter(s) and the MDR efflux transporter (P-glycoprotein) offers a novel approach to inhibiting this form of drug resistance.

Inhibition of pulmonary surfactant biophysical activity by cationic polyamino acids.

PURPOSE: The purpose of this study is to investigate the interaction of cationic polyamino acids, polylysine and polyarginine, with rat pulmonary surfactant at the air/water interface. METHODS: Surface pressure measurements of rat pulmonary surfactant in the presence and absence of polyamino acids were carried out in both dynamic and static modes. RESULTS: In dynamic cycle studies, compression and expansion of adsorbed surfactant films in the presence of the cationic polyamino acids resulted in a delayed attainment of the plateau surface pressure. In area studies of spread surfactant films at constant surface pressure, cationic polyamino acids in the subphase resulted in an increase in film area. Increased film area was also observed when a polyamino acid was injected beneath films of dipalmitoyl-phosphatidylcholine/phosphatidylglycerol. In the presence of the cationic polyamino acids, the equilibrium surface pressure (at constant film area) of pulmonary surfactant was elevated in a concentration- and molecular weight-dependent manner. CONCLUSIONS: These data indicate that the model cationic peptides interact with surfactant lipid, possibly electrostatically with phosphatidylglycerol. It is concluded that the surface activity of pulmonary surfactant is significantly inhibited by the presence of the polycations, possibly by the formation of a mixed lipid/polyamino acid film.

A novel polymeric spermine conjugate inhibits polyamine transport in pulmonary artery smooth muscle cells.

The polyamines putrescine, spermidine and spermine (SPM) are low molecular weight organic cations that play essential intracellular regulatory roles in cell growth and differentiation. Whereas both de novo polyamine synthesis and transmembrane transport regulate cell polyamine contents, exploitation of pathways as pharmacologic targets has been limited by the lack of agents which specifically block polyamine transport. We now report the synthesis and biologic activity of novel polymeric glutaraldehyde conjugates of putrescine, spermidine and SPM which act at the cell membrane to inhibit polyamine uptake in cultured bovine pulmonary artery smooth muscle cells. Each conjugate caused dose-related inhibition of [14C]polyamine transport in pulmonary artery smooth muscle cells with the polymeric SPM conjugate being most effective in inhibiting the uptake of all three polyamines. Polymeric SPM failed to impair uptake of neutral or charged amino acids or to associate with pulmonary artery smooth muscle cells in a temperature-dependent manner. The polymeric SPM conjugate caused substantial decreases in cell polyamine contents which were associated with concentration-dependent cytotoxicity. Spectroscopic analyses of the polymeric SPM conjugate indicated that its molecular weight was 25 +/- 0.5 kDa, which is equivalent to approximately 90 monomeric--HN(CH2)3NH(CH2)4NH(CH2)3NH(CH2)5--units. These findings indicate that reduced polymeric glutaraldehyde conjugates of the polyamines may function as specific inhibitors of polyamine transport and thus provide a basis for examination of polyamine transport as a pharmacologic target in disorders characterized by dysregulated cell growth and differentiation.

Basic fibroblast growth factor alterations during development of monocrotaline-induced pulmonary hypertension in rats.

The chemical signaling pathways which orchestrate lung cell responses in hypertensive pulmonary vascular disease are poorly understood. The present study examined temporal alterations in lung basic Fibroblast Growth Factor (bFGF) in a well characterized rat model of monocrotaline (MCT)-induced pulmonary hypertension. By immunohistochemical analysis, there were progressive increases in bFGF in airway, vascular and gas exchange regions of MCT-treated rat lungs. Increases in bFGF preceded the onset of right ventricular hypertrophy at day 21 after MCT administration. Enhanced bFGF immunostaining was observed as early as day 4 in focal areas of the parenchyma, and by day 14 there was enhanced bFGF staining in alveolar macrophages, neutrophils and alveolar septa, which persisted through day 21. In conducting airways, there was elevated bFGF immunostaining in the smooth muscle cell (SMC) layer by days 4 and 7 and in the ciliated epithelium and its basement membrane at days 14 and 21. Cells morphologically similar to Clara cells in the luminal surfaces of bronchioles stained intensely on days 14 and 21. In the nucleus and cytoplasm of medial SMCs within pulmonary arteries, there was a progressive increase in bFGF staining starting at day 4. Lung bFGF mRNA was increased slightly at days 1, 4 and 7, while lung bFGF protein, as judged by western blot analysis, was increased at days 14 and 21 compared to controls. The present results, considered in teh light of teh documented roles of bFGF in vascular cell migration, growth and synthesis of extracellular matrix components, suggest that bFGF may contribute to the structural remodeling processes underlying the development of chronic pulmonary hypertension in MCT-treated rats.

Monocrotaline alters type II pneumocyte morphology and polyamine regulation.

Lungs from monocrotaline (MCT)-treated rats exhibit altered polyamine metabolism and content. One of the prominent morphological abnormalities in MCT-treated lungs is a decrease in population density of type II pneumocytes. Against this background, the present study tested the hypothesis that failure to maintain normal population density of type II pneumocytes is associated with MCT-induced derangements in polyamine biosynthesis and/or transmembrane polyamine transport. After a 24-hr treatment, cultured type II pneumocytes exhibited numerous vacuoles at the highest dose of 3.2 mM MCT but not at the lower dose of 1.6 mM MCT. Intracellular spermidine content was significantly reduced at the highest dose of MCT. Relative to controls, the abundance of mRNA for both ornithine decarboxylase, and S-adenosylmethionine decarboxylase, key regulatory enzymes in polyamine synthesis, was not altered. However, the activities of both of these enzymes were dramatically reduced. Increased mRNA for the catabolic polyamine enzyme, spermine/spermidine-N1-acetyltransferase (SAT), paralleled significant increases in SAT activity. MCT also caused a concentration-related inhibition of spermidine uptake in type II cells, characterized by a fourfold decrease in Vmax with little change in Km. These results show that MCT alters type II pneumocyte polyamine regulatory mechanisms and may help explain the decreased population density of type II pneumocytes in MCT-treated rats.