Polyamines in Pulmonary Vascular Biology Jack W Olson and Mark N Gillespie

1. Introduction

Polyamines are essential for cell growth and development. They regulate many functions, including cell division, migration, ion channel regulation, apoptosis, and the cellular synthesis of DNA, RNA, and proteins. A recent review on the roles of polyamines in the lung emphasized studies on respiratory cell biology and polyamine uptake (1). The primary goal of this chapter is to review evidence that polyamines contribute to pheno-typical changes in pulmonary vascular cells that underlie the pathogenesis of pulmonary arterial hypertension. Because arginases can regulate polyamines, their potential role in the pathogenesis of pulmonary hypertension and asthma also will be reviewed. The data suggest polyamines may be future therapeutic targets for pulmonary hypertension, although clinical trials measuring polyamines and their regulation are lacking.

2. Overview of Pulmonary Hypertension

Chronic pulmonary arterial hypertension is frequently a fatal disease in humans (reviewed in refs. 2-4). Its pathobiology is multifactorial and is characterized by progressive narrowing of the small pulmonary arteries. The narrowing is due primarily to vasoconstriction, thrombosis, and most importantly, remodeling of the small pulmonary arteries that causes increased pulmonary vascular resistance and, ultimately, right heart failure. Remodeling occurs in all three layers of the vessel wall and is due in part to proteolysis and the accumulation of extracellular matrix proteins, plus increased cell size and number. Resident vessel wall cells—endothelial, smooth muscle, and fibroblasts—and circulating platelets and inflammatory cells participate in the narrowing process. For example, endothelial cells frequently are injured or dysfunctional. They can display phenotypical abnormalities in proliferation, survival, and neovascularization, and also inappropriate secretion of factors regulating vasodilation, vasoconstriction, growth, and coagulation. Pulmonary arterial smooth muscle cells increase in cell size and number and secrete excessive amounts of extracellular matrix proteins. Mutations or polymorphisms in genes regulating pulmonary arterial smooth muscle or endothelial cell proliferation, apoptosis and differentiation have

From: Polyamine Cell Signaling: Physiology Pharmacology and Cancer Research Edited by: J.-Y. Wang and R. A. Casero, Jr. © Humana Press Inc., Totowa, NJ

been identified in some patients with inherited or idiopathic pulmonary arterial pulmonary hypertension (2-4).

Although no animal model completely reproduces the pathology of human pulmonary arterial hypertension, the monocrotaline (MCT) and chronic hypoxia rat models mimic several key features and are the most frequently studied (reviewed in refs. 2-4). Although most studies on the role of polyamines in these rat models have focused on the regulation of polyamines by ornithine decarboxylase (ODC) and polyamine transport, only a few studies have examined the contribution of arginine and arginases.

3. Polyamines and MCT-Induced Pulmonary Hypertension

A single subcutaneous injection of MCT, a plant pyrrolizidine alkaloid, causes pulmonary thrombosis, inflammatory cell infiltration, vasoconstriction, and perivascular edema within 1 to 4 d. Although lung endothelial injury is evident within hours, the progressive vascular remodeling process leading to the development of sustained pulmonary hypertension and right ventricular hypertrophy (RVH) develops over 2-3 wk. The pathology is not reversible and is ultimately fatal. Lung endothelial cell injury and subsequent dysfunctional endothelium are considered the primary events initiating MCT-caused pulmonary hypertension in rats. MCT-injured endothelial cells exhibit altered proliferative, survival, and barrier properties, and they elicit formation of throm-botic lesions, a proinflammatory environment, increased proteolytic activity, altered cellular phenotypes, and extracellular matrix changes. MCT-treated lungs accumulate cytokines, growth factors, and vasoactive peptides. Vascular smooth muscle cells and pericytes respond to MCT by switching to phenotypes that migrate, proliferate, and produce an abnormal extracellular matrix. Vascular cell fate also is altered by increased proteinase activity and subsequent changes in the extracellular matrix.

We reasoned that polyamines may be causally related to these events and, therefore hypothesized that elevated lung contents of polyamines and activities of ODC and S-adenosylmethionine decarboxylase (AdoMetDC) would precede development of pulmonary hypertension and RVH. Figure 1 summarizes data showing time-dependent increases in lung ODC and AdoMetDC activities and polyamine contents. Lung ODC activity was increased approximately eightfold on d 1 and remained elevated through d 7 (5). Lung putrescine levels were increased from d 7 through 21, and both spermidine and spermine were first elevated at d 10 after MCT administration (6). This sustained

Fig. 1. (Opposite page) Time-dependent changes in lung ornithine decarboxylase (ODC), AdoMetDC, polyamine contents, mean pulmonary artery pressure (Ppa), and right ventricular hypertrophy (RVH) after a single subcutaneous dose of monocrotaline (MCT). RVH was quantitated as the ratio of the right ventricular free wall weight (RV) to the weight of the left ventricle plus septum (LV+S). Controls were given the MCT vehicle (0.9% NaCl) and studied at each time point. Data reported are the fold change from control rats (mean ± the standard error). Control values were: ODC activity = 8.3 ±1.5 nmoles/total lung weight/60 min; AdoMetDC activity = 2.6 ± 0.25 nmoles/total lung weight/60 min; putrescine = 53.8 ± 5.2 nmoles/total lung weight; spermidine = 930 ± 24 nmoles/total lung weight; spermine = 431 ± 15 nmoles/total lung weight; Ppa = 17.5 ± 0.5 mm Hg; RVH = 0.27 ± 0.005 RV/LV+S. Asterisk (*) indicates data differ from controls at p < 0.05. n = 9 rats in each group. (Adapted from refs. 6 and 9.)

Fig. 1.

elevation of lung polyamine contents and ODC and AdoMetDC activities substantially preceded the development of RVH and pulmonary hypertension, which were first evident at d 14 and 16, respectively (5,6). These changes in lung enzyme activity and contents of polyamines were dependent on the dose of MCT. Furthermore, polyamine catabolism was modulated by MCT (7). Both A^-acetylspermidine contents and the activity of spermidine/spermine acetyltransferase, an enzyme controlling the back conversion of spermidine and spermine to putrescine, were increased dose- and time-dependently by MCT. Neither A1-acetylspermine nor A1-acetylputrescine could be detected in lungs from control rats or from rats treated with MCT. The previously described changes in whole lung contents of polyamines were hypothesized to occur in lung vascular cells because they are primary cellular targets of MCT. As predicted, left pulmonary arterial segments from the first to the sixth intrapulmonary branch had increased contents of putrescine and spermidine at 7 and 21 d after MCT treatment, whereas spermine content was increased at 21 d (8).

If increased ODC activity is necessary for MCT-caused development of pulmonary hypertension, then inhibition of ODC activity should block development of hypertensive vascular disease in MCT-treated rats. Figure 2 shows that continuous treatment with DFMO (a-difluoromethylornithine), a highly specific, enzyme-activated, irreversible inhibitor of ODC activity, prevented the increase in lung putrescine and spermidine contents without significantly altering spermine content (9,10). Importantly, DFMO completely prevented the MCT-increased pulmonary arterial pressure and right ventricle hypertrophy (6,16). The development of lung perivascular edema at d 7 was also blunted by DFMO, whereas the proposed first step of MCT pathology—that is, the hepatic biotransformation of MCT to its active pyrrolic metabolites—was not influenced by DFMO treatment (9). DFMO also prevented the primary causes of MCT-increased pulmonary artery pressure:pulmonary vascular vasoconstriction, thrombosis, and arterial medial thickening (9,11) (Fig. 2). Because arterial medial thickening is primarily from increased cell size and number plus accumulation of extracellular matrix proteins, DFMO might target these events. In this context, DFMO prevented MCT-increased DNA synthesis (3H-thymidine incorporation into whole-lung DNA) and thereby may limit vascular cell growth and division (10). DFMO also could prevent MCT-caused alterations in the extracellular matrix, such as the accumulation of fibronectin (FN), elastin, collagen, and tenascin-C (2). FN, which can stimulate multiple signaling events and alter cell fate, is deposited in large amounts in airways, parenchyma, and pulmonary arteries of rats administered MCT (12). In addition, preliminary studies suggest MCT-increased ODC activity may generate FN variants favoring lung remodeling by enhancing rates of FN gene transcription and exon-specific alternative splicing (13). DFMO treatment completely prevented the MCT-stimulated splicing of the FN IIIA exon and expression of FN messenger RNA (mRNA) and FN protein in rat lungs. These results support the concept that elevated ODC activity promotes MCT-induced remodeling of the pulmonary vasculature in part through enhancing FN splicing and expression.

Another event contributing to MCT-caused vascular structural changes is pulmonary endothelial cell apoptosis. Preliminary studies have been initiated to examine the roles of ODC in the MCT-caused apoptotic response in rat lungs and cultured rat pulmonary

Fig. 2. Continuous DFMO treatment prevents monocrotaline (MCT)-caused changes in rat lung polyamine contents and pulmonary arterial medial thickening (MT) and pressure (Ppa) and right ventricular hypertrophy (RVH). MT was determined in 50- to 100-^m pulmonary arteries as the percent of external diameter (ratio of smooth muscle layer thickness to the diameter between external elastic lamina). Control and DFMO-only rats were given the MCT vehicle (0.9% NaCl) and studied 21 d later. DFMO only and MCT+DFMO rats received 2% DFMO in the drinking water from 4 d before MCT until the study was terminated at 21 d after administration of MCT. Data reported are the fold change from control rats (mean ± the standard error). Control values were: putrescine = 24 ±9 nmoles/left lung weight; spermidine = 293 ± 47 nmoles/left lung weight; spermine = 154 ±24 nmoles/left lung weight; MT = 8 ± 0.6% of external diameter; Ppa = 11 ± 2.2 mm Hg; RVH = 0.30 ± .01 RV/LV+S. Asterisk (*) indicates data differ significantly from control or DFMO only groups at p < 0.05. n = 6 rats in each group. (Adapted from refs. 9 and 15.)

Fig. 2. Continuous DFMO treatment prevents monocrotaline (MCT)-caused changes in rat lung polyamine contents and pulmonary arterial medial thickening (MT) and pressure (Ppa) and right ventricular hypertrophy (RVH). MT was determined in 50- to 100-^m pulmonary arteries as the percent of external diameter (ratio of smooth muscle layer thickness to the diameter between external elastic lamina). Control and DFMO-only rats were given the MCT vehicle (0.9% NaCl) and studied 21 d later. DFMO only and MCT+DFMO rats received 2% DFMO in the drinking water from 4 d before MCT until the study was terminated at 21 d after administration of MCT. Data reported are the fold change from control rats (mean ± the standard error). Control values were: putrescine = 24 ±9 nmoles/left lung weight; spermidine = 293 ± 47 nmoles/left lung weight; spermine = 154 ±24 nmoles/left lung weight; MT = 8 ± 0.6% of external diameter; Ppa = 11 ± 2.2 mm Hg; RVH = 0.30 ± .01 RV/LV+S. Asterisk (*) indicates data differ significantly from control or DFMO only groups at p < 0.05. n = 6 rats in each group. (Adapted from refs. 9 and 15.)

artery endothelial cells (PAECs) (14). Immunohistochemical analyses revealed that PAECs within the lungs of MCT-treated rats had increased immunostaining for ODC, c-myc, and caspase-3, a key apoptotic enzyme, at d 1,4, and 7 after MCT administration. DFMO treatment blocked the increase in PAEC caspase-3 immunostaining. Western blot analysis of lungs confirmed that DFMO prevented the MCT-caused activation of caspase 3 and degradation of poly (ADP-ribose) polymerase. In cultured rat PAECs, ODC activity was increased 4, 24, and 72 h after addition of MCT pyrrole, the active metabolite of MCT. This increase in ODC activity was due in part to c-myc-enhanced ODC transcription. MCT pyrrole also initiated an apoptotic response from 4 to 72 h as indicated by increased annexin-V staining, caspase-3 activity, and DNA laddering. DFMO treatment blocked MCT-increased annexin-V staining and partially blocked and delayed caspase-3 activity and DNA laddering. These data suggest increased ODC activity contributes to MCT-caused PAEC apoptosis and that DFMO targets one of the earliest stages in the development of pulmonary hypertension.

If increased lung contents of polyamines are required for the development of MCT-caused pulmonary hypertension, the protective effects of DFMO should be reversible by elevating lung polyamine contents through supplementation with exogenous polyamines or ornithine. In this regard, the protection afforded by DFMO was reversed by chronic coadministration of ornithine, the substrate for putrescine biosynthesis by ODC (15). The rats receiving the combination treatment of MCT plus DFMO and ornithine had lung polyamine contents at levels normally associated with MCT treatment alone. This confirms that DFMO was acting as a specific inhibitor of polyamine biosynthesis in MCT-induced pulmonary hypertension. Using the same protocol, Hacker (10) independently showed ODC and polyamines are essential for the development and progression of MCT-induced pulmonary hypertension.

These experiments provided evidence that continuous DFMO treatment, when initiated at the time of MCT administration, prevents MCT-caused hypertensive vascular disease. These studies did not address whether DFMO can cause regression or prevent progression of the disease after it is established, a question relevant for therapy of humans with pulmonary hypertension. In this context, DFMO treatment, when started at a time well after the early onset of MCT-induced pneumotoxicity, retained at least some of its protective effects (15). DFMO treatment initiated at d 10 after MCT— when lung polyamine contents are elevated, and perivascular edema, inflammatory cell infiltration, vasoconstriction, and vascular remodeling are evident—significantly blunted, but did not completely prevent, the MCT-caused RVH and increases in putrescine and spermidine contents. It thus remains unanswered whether complete regression of MCT-induced pulmonary hypertension can be achieved by treatment regimens that reduce polyamine lung contents to control levels. Perhaps a regimen that combines DFMO plus a polyamine uptake blocker will be required. In this context, MCT can enhance polyamine uptake in cultured vascular cells, although it is not known if lung polyamine transport is altered in MCT-treated rats.

4. Polyamines and Chronic Hypoxia-Induced Pulmonary Hypertension

Although MCT and chronic hypoxia elicit pulmonary hypertension in rats, there are notable differences between these models (2,3,16). Compared with MCT, vascular endothelial injury, and thrombosis, recruitment of circulating leukocytes and perivas-cular inflammation are less pronounced in hypoxic pulmonary hypertension. In addition, pulmonary vasoconstriction is more significant, type II pneumocyte toxicity is not apparent, and the time to develop pulmonary hypertension is compressed in hypoxic rats. Although it is the cause of death in MCT-treated rats, pulmonary hypertension naturally regresses after hypoxic animals are returned to a normoxic environment.

Because of the differences between the two models, we tested whether the polyamines are mediators of chronic hypoxia-induced pulmonary vascular remodeling.

As with MCT, hypoxia-increased lung polyamine contents temporally preceded development of pulmonary artery hypertension (17-19). Specifically, lung contents of spermidine and spermine increased within 1 d and putrescine within 4 d after continuous exposure to hypoxia (simulated altitude: 4570 m) and these changes occurred before increases in pulmonary arterial pressure and development of RVH (Fig. 3). Unexpectedly, and opposite to MCT-treated rats, lung ODC activity was reduced below control levels from 4 to 14 d of exposure. This response to hypoxia seemed to be specific to ODC activity because hypoxic lungs had sustained increases in AdoMetDC and spermidine/spermine acetyltransferase activities and A^-acetylspermidine contents. Additional studies tested whether polyamine transport contributed to hypoxia-increased lung polyamine contents. Putrescine transport kinetics were assessed in isolated, salt solution-perfused lungs from rats previously exposed to normoxia or hypoxia for 7 d, a time when ODC was reduced to its lowest level. Accelerated uptake from the vascular compartment plus decreased efflux from the lung contributed to the elevated lung putrescine contents since hypoxia-increased the apparent Km and capacity for putrescine uptake and the T^ for loss of putrescine from a slowly effluxing pool. These data support the concept that hypoxia-increased lung polyamine contents are not from ODC activity but instead, result from elevated AdoMetDC and spermidine/spermine acetyltransferase activities coupled with accentuated uptake and decreased efflux of putrescine.

Despite the ability of hypoxia to decrease total lung ODC activity, DFMO attenuated the hypoxia-increased lung contents of putrescine and spermidine, and blunted the increases in pulmonary arterial pressure and medial thickness (17,18) (Fig. 4). DFMO did not alter the hypoxia-evoked increase in hematocrit, vasoconstriction, and RVH. These observations suggest that depression of polyamine biosynthesis with DFMO blunts the sustained increase in pulmonary arterial pressure by attenuating hypoxia-induced medial thickening. Although the DFMO-sensitive resident cell types within the rat lung remain unidentified, DFMO may target the recruitment of nonresident lung cells to the pulmonary circulation because these cells likely participate in the hypoxia-caused vascular remodeling process (16).

To begin to identify lung resident cell types in which hypoxia induces polyamine transport in lungs, rat lung and rat main pulmonary arterial explants were incubated with 14C-spermidine in either normoxic or hypoxic environments for 24 h (20). Autoradiographic evaluation revealed 14C-spermidine localized prominently to conduit, muscularized, and partially muscularized pulmonary arteries in hypoxic, but not normoxic, lung tissue and localized to both intimal and medial arterial cells of hypoxic main pulmonary arterial explants, but the increase was most evident in the smooth muscle cells. Experiments using main pulmonary arterial explants denuded of endothe-lium and in addition, cultured pulmonary artery smooth muscle (PASMCs) cells and PAECs showed that endothelial-derived factors are required for hypoxia-enhanced spermidine uptake in the underlying smooth muscle. Rat PASMCs cultured in nor-moxic environment were found to express two discrete transporter systems: one for

Fig. 3. Time-dependent changes in lung ODC, AdoMetDC, polyamine contents, mean pulmonary artery pressure, and right ventricular hypertrophy after continuous exposure to hypoxia (10% environmental oxygen). Control rats were exposed to room air (normoxia) and studied at each time point. Data reported are the fold change from control rats (mean ± the standard error). Control values were: ODC activity = 2.0 ± 0.12 nmoles/total lung weight/60 min; AdoMetDC

Fig. 3. Time-dependent changes in lung ODC, AdoMetDC, polyamine contents, mean pulmonary artery pressure, and right ventricular hypertrophy after continuous exposure to hypoxia (10% environmental oxygen). Control rats were exposed to room air (normoxia) and studied at each time point. Data reported are the fold change from control rats (mean ± the standard error). Control values were: ODC activity = 2.0 ± 0.12 nmoles/total lung weight/60 min; AdoMetDC

Fig. 4. Continuous DFMO treatment blunts chronic hypoxia-caused changes in rat lung polyamine contents and pulmonary arterial medial thickening (MT) and pressure (Ppa) and right ventricular hypertrophy (RVH). Rats were exposed to either normoxia or hypoxia for 21 d and 2% DFMO in the drinking water was given continuously starting 2 d before hypoxia. Control values were: putrescine = 20 ±0.1 nmoles/left lung weight; spermidine = 270 ± 24 nmoles/left lung weight; spermine = 144 ±6 nmoles/left lung weight; MT = 4.7 ± 0.36% of external diameter; Ppa = 15 ± 0.4 mm Hg; RVH = 0.25 ± .02 RV/LV+S. *Data differ significantly from normoxia group at p < 0.05; **data differ significantly from normoxia and hypoxia groups at p < 0.05; #data differ from DFMO at p < 0.05. n = 6 rats in each group. (Adapted from ref. 18.)

Fig. 4. Continuous DFMO treatment blunts chronic hypoxia-caused changes in rat lung polyamine contents and pulmonary arterial medial thickening (MT) and pressure (Ppa) and right ventricular hypertrophy (RVH). Rats were exposed to either normoxia or hypoxia for 21 d and 2% DFMO in the drinking water was given continuously starting 2 d before hypoxia. Control values were: putrescine = 20 ±0.1 nmoles/left lung weight; spermidine = 270 ± 24 nmoles/left lung weight; spermine = 144 ±6 nmoles/left lung weight; MT = 4.7 ± 0.36% of external diameter; Ppa = 15 ± 0.4 mm Hg; RVH = 0.25 ± .02 RV/LV+S. *Data differ significantly from normoxia group at p < 0.05; **data differ significantly from normoxia and hypoxia groups at p < 0.05; #data differ from DFMO at p < 0.05. n = 6 rats in each group. (Adapted from ref. 18.)

putrescine and another for all three polyamines (21). Intriguingly, hypoxia caused a selective, time-dependent induction of putrescine transport—neither spermidine nor spermine uptake were enhanced by hypoxia. Inhibition of hypoxia-stimulated putrescine uptake by a polyamine-specific transport inhibitor suppressed hypoxia-induced p38

activity = 3.7 ± 0.2 nmoles/total lung weight/60 min; putrescine = 16 ± 0.9 nmoles/left lung weight; spermidine = 223 ±16 nmoles/left lung weight; spermine = 130 ±9 nmoles/left lung weight; Ppa = 15.4 ± 1.0 mm Hg; RVH = 0.26 ± .001 RV/LV+S. Asterisk (*) indicates data differ from controls at p < 0.05. n = 5-6 rats in each group. (Adapted from refs. 17and 19.)

mitogen-activated protein kinase activation. These important observations suggest that a specific increase in the putrescine uptake pathway is necessary for hypoxia-induced activation of p38 mitogen-activated protein kinase—a growth regulatory protein kinase involved in cell adaptation to hypoxia. Further studies seem warranted to determine whether treatment with a polyamine uptake blocker can prevent development of hypoxia-induced pulmonary hypertension in rats.

Hypoxia not only regulates polyamine transport in both rat lung and cultured lung vascular cells, but also inhibits cultured PASMC (22,23) and PAEC (24) ODC activity, suggesting hypoxia decreases ODC activity in these cell types in the rat lung. Hypoxia time dependently inhibited ODC activity and mRNA content in cultured rat PAECs without altering antizyme contents. This finding is not particularly surprising because hypoxia inhibits ODC and augments polyamine transport in PAECs, whereas antizyme can inhibit both ODC and transport. The hypoxia-induced decrease in ODC activity was prevented by two strategies known to suppress proteasome-mediated ODC degradation: treatment with the proteasome inhibitor lactacystin or use of PAECs expressing a truncated ODC protein incapable of interacting with the proteasome. Both strategies prevented hypoxia-stimulated polyamine transport. These data suggest that in cultured rat PAECs, hypoxia-decreased ODC activity may be a stimulus initiating enhanced polyamine transport. Whether this apparently unusual regulatory process occurs in the hypoxic lung will require additional studies.

The mechanisms by which chronic hypoxia inhibits ODC activity and at the same time elevates polyamine contents in lung and cultured vascular cells appear to be unusual for the lung. In the other published studies on the regulation of lung polyamine content, enhanced ODC activity was a key determinant of polyamine content. For example, increased ODC activity accompanies increased polyamine content in postnatal lung development (25), repair of hyperoxia-injured lungs (26), and MCT-induced pulmonary hypertension. Furthermore, DFMO forestalls postnatal lung development (25), inhibits repair of hyperoxic lung injury (26), and prevents development of hypertensive pulmonary vascular disease in MCT-treated rats. Angiogenesis associated with growth of postnatal lungs (25) and repair of hyperoxic lungs (26) was significantly reduced by DFMO, further implicating polyamines as being required for pulmonary endothelial cell proliferation. Hypoxia may be a stimulus that has unique and complex polyamine regulatory properties.

5. Arginases and Pulmonary Hypertension

Arginase activity also regulates vascular cell polyamine contents. Arginases hydrolyze arginine to ornithine and thereby can regulate putrescine biosynthesis (27). Alternatively, arginases compete with nitric oxide (NO) synthase for L-arginine to modulate the production of NO (27-30), and by directly inhibiting ODC and AdoMetDC activity, NO can reduce polyamine biosynthesis (31,32). In the case when arginase activity is enhanced, vascular smooth muscle (33,34) and endothelial cell (35,36) putrescine contents and proliferation rates are increased. When pulmonary artery endothelial cell arginase activity is inhibited, NO production is enhanced (37) and, in turn, NO inhibition of polyamine biosynthesis can prevent vascular cell proliferation

(31-33). In this context, the decreased NO production by injured or dysfunctional endothelial cells is believed to contribute to the inappropriate proliferation of both vascular and nonresident circulating cells in some vascular diseases, such as pulmonary hypertension.

The excessive lung vasoconstriction and vascular cell proliferation in pulmonary hypertensive humans is believed to be due partly to low NO production (2-4,38,39) and may occur because of elevated lung arginase activity (40). Although the expression of lung NO synthase enzymes in patients with pulmonary hypertension was normal, these patients had low quantities of arginine and high serum arginase activity (40). Immunohistochemistry revealed high arginase contents localized to the endothelium of hypertensive pulmonary arteries and arterioles. Compared with controls, PAECs isolated from human hypertensive lungs had higher arginase II protein expression and activity, and produced lower amounts of NO. It seems reasonable to postulate the combination of high arginase activity plus low NO may lead to excessive production of polyamines that subsequently promote vascular cell proliferation. Lung polyamine contents and their regulatory enzymes need to be evaluated in pulmonary hypertensive patients.

A few studies have evaluated L-arginine as potential therapy for pulmonary hypertensive patients because their NO levels are decreased. Overall, arginine therapy has had limited and varied success when administered acutely or for 1 wk, although long-term arginine therapy has not been studied in humans with pulmonary hypertension (39). Another study reported that 10 patients with pulmonary hypertension secondary to sickle cell disease had decreased serum L-arginine contents and tended to have increased serum arginase activity, and 5 d of oral arginine therapy reduced pulmonary artery pressure (41). In rat models of pulmonary hypertension, chronic oral L-arginine reduced pulmonary vascular remodeling and hypertension in hypoxia-exposed (42) and MCT-treated rats (42,43), whereas D-arginine was not effective (42). Although not examined in the above studies, L-arginine-enhanced NO production may have inhibited both ODC and AdoMetDC activity, and thereby reduced the pulmonary hypertension caused by hypoxia and MCT.

Arginases and polyamines may have important roles in the development of other lung diseases, such as fibrosis and asthma. Although nonvascular cell types are the primary targets in these diseases, remodeling of the lung vasculature occurs during the pathogenic process. In a rat model of pulmonary fibrosis, lung arginases I and II and ODC were increased and nitrogen oxides were decreased during the injury and repair response to hyperoxia (44). Furthermore, immunostaining for arginase I and II was increased in perivascular regions. As previously described, DFMO inhibits the angiogenesis-associated repair of hyperoxic lungs (26). The bleomycin mouse model of lung fibrosis has sustained increases in lung arginase and ODC mRNA contents (45). In studies on asthma, arginase is increased in asthmatic lungs from humans (46-48) and experimentally induced allergic asthma in guinea pigs (49) and mice (46). Of particular interest, putrescine was increased about twofold in allergen-challenged mouse lungs, although ODC mRNA was not changed from controls (46). Lungs of human asthmatics have not been examined for changes in polyamines or ODC, although asthmatics were reported to have elevated serum polyamine contents (50). Although asthma is a disease primarily of the airways, neovascularization of the airways occurs. In this regard, large increases of arginase I mRNA were found in perivascular and peribronchial regions in the lungs of asthmatic mice (46). Polyamines in the pulmonary circulation and airways may be important in asthma pathobiology and therapy and need further study.

6. Conclusions

It is evident that polyamines have important roles in the two most commonly studied animal models of pulmonary hypertension, although the mechanisms regulating the increased lung polyamine contents are clearly different. Increased lung ODC activity appears to be essential for the early pathogenic stages in the rat MCT model and to be important for the later stages of vascular remodeling. During the early stages, ODC activity regulates the responses of endothelial cells to injury. These responses likely include endothelial cell apoptosis, proliferation, barrier properties, and secretion of factors that create an inflammatory and thrombotic environment. In the later stages, ODC activity seems to contribute to the thickening of the medial and adventitial layers of the remodeling vasculature. In contrast to the MCT model, the role of ODC activity in the chronic hypoxia model is unclear because ODC activity is inhibited in response to hypoxia and, DFMO treatment reduces, but does not block, the pathological responses. Enhanced polyamine uptake and decreased efflux appear to be the predominant mechanisms, whereby hypoxia increases lung polyamine contents. Pulmonary arterial smooth muscle and endothelial cells seem to be primary targets of polyamine regulation in hypertensive pulmonary vascular disease. Based on these animal studies, polyamines regulate many pathogenic stages of pulmonary hypertension, and multiple mechanisms, including synthesis, degradation, and transport, control the contents of polyamines. The mechanisms regulating polyamines provide targets for multiple potential therapeutic strategies for pulmonary hypertension. Unfortunately, data supporting the importance of polyamines in pulmonary hypertension in humans are lacking, although increased arginase activity in pulmonary artery endothelial cells indicates polyamine contents may be elevated. Clinical studies are needed to evaluate lung polyamine contents and their regulation in patients with pulmonary hypertension. This new information will be critical for identifying whether polyamines are potential targets for the therapy of pulmonary hypertension.

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POLYAMINES IN CELLULAR SI GNALIN G OF Apoptosi S, Carcin OGENESI S, AND CAN CER Theraph Y

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