David W. Lipke

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.

Lymphocyte Subsets in Cord Blood of Preterm Infants: Effect of Antenatal Steroids

The objective of this study was to evaluate prospectively the influence of gestational age (GA) and short-term antenatal steroids on total lymphocyte count and lymphocyte subsets in cord blood from preterm infants. Two-color flow cytometric analyses of lymphocyte subsets were performed on cord blood collected from 67 infants. These infants were grouped according to GA: group I (term, n = 19); group II (GA 33–37 weeks, n = 25); group III (GA <33 weeks, n = 23). The mean absolute lymphocyte counts (ALC) in groups I, II and III were 5.6 ± 2.5 × 103/µl, 4.3 ± 1.5 × 103/µl and 3.5 ± 1.8 × 103/µl respectively. The mean values for CD4+ lymphocytes in groups I, II and III were 2.7 ± 0.8 × 103/µl, 2.0 ± 0.8 × 103/µl and 1.6 ± 0.9 × 103/µl respectively. Mean values for CD8+ lymphocytes were 0.9 ± 0.3 × 103/µl, 0.6 ± 0.3 × 103/µl and 0.5 ± 0.3 × 103/µl respectively. With decreasing GA, there was a statistically significant decrease in ALC (p = 0.0035), CD4+ lymphocytes (p = 0.0013) and CD8+ lymphocytes (p = 0.0064). We then evaluated the effect of antenatal steroids, now routinely administered to women with preterm onset of labor to facilitate fetal lung maturation, and found that after adjusting for GA, infants of women on antenatal steroids had significantly fewer ALC (p = 0.0001), CD4+ lymphocytes (p = 0.02) and CD25+ lymphocytes (p = 0.03). In this population of infants, the decreased number of lymphocytes seen at younger GAs is associated with antenatal steroid use.

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.

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.

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.

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.

Lung angiotensin receptor binding characteristics during the development of monocrotaline-induced pulmonary hypertension.

Alterations in lung angiotensin converting enzyme (ACE) activity in monocrotaline (MCT)-induced pulmonary hypertension in rats have suggested a pathophysiologic role for angiotensin II (AII) in pulmonary vascular remodeling. ACE inhibitors suppress MCT-induced pulmonary hypertension; however, losartan, an angiotensin type 1 (AT1) receptor antagonist, was without impact. The present study examined AII receptor binding characteristics by radioligand binding during the development of MCT-induced pulmonary hypertension. Saturation binding isotherms for [125I]AII binding to membrane preparations from rat lung were performed at 4, 10, and 21 days following a single injection of MCT (60 mg/kg) or saline vehicle. Right ventricular hypertrophy, an index of pulmonary hypertension, increased at 21 days post-MCT. Saturation binding isotherms revealed a single, high affinity site for [125I]AII binding in lung membranes from MCT-treated and control rats, with no change in receptor affinity or density during the development of pulmonary hypertension. Competition displacement binding demonstrated that the AT1 receptor predominates in lung membranes from control rats, with no alterations in AII receptor subtype distribution following MCT treatment. In summary, these results suggest that the AT1 receptor subtype predominates in rat lung and does not contribute to the development of MCT-induced pulmonary hypertension.

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.

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.

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 ofde novopolyamine synthesis further increased spermidine uptake in MCT‐treated cells. The depletion of cellular polyamine contents through the blockade of bothde novopolyamine 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‐KB. 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.