Fluvastatin Inhibits Hypoxic Proliferation and p38 ... - ATS Journals

Fluvastatin Inhibits Hypoxic Proliferation and p38 MAPK Activity in Pulmonary Artery Fibroblasts Christopher M. Carlin, Andrew J. Peacock, and David J. Welsh Scottish Pulmonary Vascular Unit, Western Infirmary, Glasgow, United Kingdom

The earliest structural change in hypoxia-induced pulmonary hypertension is increased proliferation of adventitial fibroblasts. This fibroproliferative response occurs in acute and chronic hypoxic models, is dependent on p38 mitogen-activated protein (MAP) kinase activation, is selective for the pulmonary circulation, and would seem an important therapeutic target. Simvastatin attenuates pulmonary vascular remodeling in animal models, but additional information regarding mechanisms of action, differential antiproliferative effects and dose responses of available statins is required for appropriate clinical trial design. Our objectives were to determine the effects of statins on acute hypoxia-induced proliferation and p38 MAP kinase activation in pulmonary and systemic artery fibroblasts, to assess the effects of cholesterol intermediates, prenyltransferase and related inhibitors, and to determine the statin’s mechanism of action. Atorvastatin, fluvastatin, and simvastatin inhibited adventitial fibroblast proliferation. At low doses (1 mM), this effect was selective for hypoxic (versus serum-induced) proliferation and was also selective for pulmonary (versus systemic) fibroblasts. Complete inhibition of hypoxia-induced p38 MAP kinase activity was achieved at this 1-mM dose. The lipophilic statins exhibited similar potency. The statin effect was reversed by geranylgeranyl pyrophosphate and mimicked by geranylgeranyl transferase and Rac1 inhibitors. Hypoxia-induced p38 MAP kinase activation and proliferation in pulmonary adventitial fibroblasts is dependent on a geranylgeranylated signaling protein, probably Rac1. One micromolar of fluvastatin exhibits a circulation- and stimulus-selective antiproliferative effect on pulmonary artery fibroblasts. The pharmacokinetics of fluvastatin would suggest that its antiproliferative effects may be useful in pulmonary hypertension associated with hypoxia. Keywords: pulmonary hypertension; hypoxia; adventitial fibroblast; p38 MAP kinase; HMG-CoA reductase

Severe pulmonary arterial hypertension (PAH) is an important complication of hypoxic lung diseases, with a poor prognosis and no validated treatment (1). The pathology and clinical features are similar to other forms of PAH (which can develop, for example, as a primary disease without obvious precipitant, as a familial disorder, following anorexigen ingestion, or in association with various systemic diseases) (2). Extensive structural changes in the pulmonary arteries—pulmonary vascular remodelling—are seen in PAH and severe hypoxia-associated pulmonary hypertension, and these changes are thought to be the principal mechanism responsible for elevated pulmonary vascular resistance in these conditions (3–5). The dogma that pulmonary vascular remodeling causes pulmonary hypertension in chronic hypoxia by luminal narrowing and vessel loss has recently been challenged (6, 7). Regardless of whether it is luminal narrowing by inward remodeling, loss of vessel disten-

(Received in original form January 17, 2007 and in final form April 30, 2007) Correspondence and requests for reprints should be addressed to Dr. David Welsh, Scottish Pulmonary Vascular Unit, Western Infirmary, Dumbarton Road, Glasgow G11 6NT, UK. E-mail: [email protected] Am J Respir Cell Mol Biol Vol 37. pp 447–456, 2007 Originally Published in Press as DOI: 10.1165/rcmb.2007-0012OC on June 7, 2007 Internet address: www.atsjournals.org

CLINICAL RELEVANCE New drugs for pulmonary hypertension are required. Animal model work has suggested benefit from statins. This study provides new insights into hypoxic signaling in pulmonary artery fibroblasts and information required for clinical trials of statins.

sibility, generalized reduction of the pulmonary vascular bed, or sustained vasoconstriction in structurally abnormal vessels that leads to the severe pulmonary hypertension, it seems clear that pulmonary vascular remodeling is an important process to understand, and an important target for new treatments for all forms of pulmonary hypertension. New treatments have improved the prognosis of PAH but, at best, they achieve only partial regression of the vascular abnormalities. Beneficial effects are likely to be transient and effects on severe hypoxia-associated PAH have not yet been fully validated. New drugs with anti-remodeling potential for all forms of PAH are required, with a full understanding of their mechanisms of action necessary if we are to judge how best to combine or substitute them for established agents. Though all vascular cells contribute to the progression of pulmonary vascular remodeling, we have focused on the role of the pulmonary adventitial fibroblast (PAF). Adventitial fibroblasts contribute significantly to vascular remodeling after vessel injury (8–10), particularly in the pulmonary circulation in response to hypoxic insult (3, 11–14) and in PAH. Although hypoxia is only one cause of pulmonary hypertension, its utility as a model is established: it is the model that has led to most current therapies. Study of the PAF’s response to hypoxia provides important insights into the pathophysiology of pulmonary vascular remodeling and direct information about one of the commonest forms of pulmonary hypertension. The pulmonary and systemic circulations respond differently to hypoxia, and differential responses of adventitial fibroblasts seem to be a key determinant of this circulation-specific response. We have previously shown that, in response to acute or chronic hypoxia, PAFs demonstrate increased proliferation. Although mitogen-activated protein (MAP) kinases are variously activated, this increased proliferation—whether triggered by acute or chronic hypoxia—is exclusively dependent on activation of p38 MAP kinase (15–18). In contrast, no activation of p38 MAP kinase or increased proliferation is seen in systemic adventitial fibroblasts. Though pharmacologic inhibition of p38 MAP kinase may be a circulation-selective treatment strategy for hypoxic/ fibroproliferative pulmonary hypertensive conditions, there are no clinically established direct p38 inhibitors, so we have sought to explore the signaling pathway(s) upstream of p38 to identify agents that may indirectly inhibit this pathway. Statin drugs have been shown to reverse pulmonary vascular remodeling in pulmonary hypertension animal models (19–24). This has led to suggestions that statin treatment may be valuable in human pulmonary hypertension, with some suggestion of benefit in a small case series (25). The mechanism(s) of action

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in animal models are, however, not understood. With an incomplete understanding of mechanisms of action and differential effects of the different statins, we are currently unable to predict how best to deploy these drugs and premature clinical trials would seem to carry a major risk. Statins have the potential to inhibit diverse cell signaling systems via inhibition of HMG-CoA reductase (Figure 1). We considered it possible that statin influenced pathways may be involved in the hypoxia-induced activation of p38 MAP kinase in PAFs and that investigation of this may provide insights into how statin therapy may be helpful in pulmonary hypertensive disorders. Using an acute hypoxic rat cell model, we compared the effects of different statins on serum and hypoxia-induced pulmonary and systemic adventitial fibroblast proliferation and p38 MAP kinase activation. We assessed the effect of cholesterol pathway intermediates and related inhibitors to determine the statin’s mechanism of action.

MATERIALS AND METHODS

ously described (26). Briefly, vessels were dissected free, opened into a flat sheet, and muscular tissue and endothelial cell layers were removed by gentle abrasion. The remaining tissue (adventitia) was then dissected into 1-mm3 portions, z 25 of which were evenly distributed over the base of a 25-cm2 culture flask containing 2 ml of Dulbecco’s modified Eagle’s medium (DMEM) with 20% fetal calf serum (FCS), penicillin/streptomycin (400 iu/ml and 400 mg/ml), amphotericin B (5 mg/ml), and L-glutamine (27 mg/ml). Explants were incubated in a humidified atmosphere of 5% CO2 in air at 37.88C. We have previously shown by staining for actin that this technique produces a pure culture of fibroblasts (27). Cells were subsequently maintained in DMEM containing 10% FCS, supplemented with antibiotics and L-glutamine as before, and used between passages 3 and 10.

Growth of Cells in a Hypoxic Environment Maintenance of cells in a nitrogen-supplemented, humidified, temperaturecontrolled incubator (Galaxy R; Wolf Laboratories, York, UK) allowed control of internal oxygen levels at 5%, while CO2 level was maintained at 5%. This achieved a tissue culture supernatant PO2 of 35 mm Hg for the hypoxic conditions, which we have validated in our previous work (26).

Materials Tissue culture flasks and media were from Gibco (Paisley, Renfrewshire, UK). Fetal calf serum was obtained from Imperial Laboratories (Andover, Hants, UK). FTase inhibitor 1, GGTI-2133, SB203580, simvastatin, pravastatin, hydroxyfasudil and NSC23766 were from Calbiochem (Nottingham, UK). Other statins were donated by the manufacturing pharmaceutical company (Atorvastatin, Pfizer, Sandwich, UK; Fluvastatin, Novartis, Horsham, UK). [3H]Thymidine was from Amersham Biosciences (Little Chalfont, UK). Rabbit polyclonal antibodies for p38 MAP kinase and its activated dual phosphorylated form (Thr180/Tyr182) were obtained from New England Biolabs (Hertfordshire, UK). All other reagents were obtained from Sigma (Poole, Dorset, UK).

Primary Culture of Rat Pulmonary Artery Fibroblasts and Rat Aortic Fibroblasts Fibroblasts from adult male Sprague-Dawley rats were obtained from freshly excised vessels using a modified explant technique, as previ-

[3H]Thymidine Incorporation, Cell Proliferation, and Viability Rat pulmonary artery fibroblasts (RPAFs) and aortic fibroblasts (RAFs) were grown in normoxic conditions to 60% confluence in 24well plates and then quiesced for 24 hours in serum-free medium. Cells were then cultured under conditions of normoxia or hypoxia, in the presence or absence of serum and experimental mediators, for a further 24 hours. Increased proliferation of RPAFs to hypoxia is seen with concentrations of serum 0.1 to 10% (18). For these experiments 1% serum was selected, as this maximizes differences between normoxic and hypoxic proliferation, allowing recognition of any small differences in the inhibitory effects of the different statins. Fibroblasts were pulsed with [3H]thymidine (0.1 mCi/well) 4 hours before the end of the 24hour experimental period with scintillation counting as previously described (15). To assess proliferation and viability, 24-hour experiments were repeated, and fibroblasts were removed from the wells by 0.05% trypsin and then counted using a Fischer hemocytometer/0.4% trypan blue.

Figure 1. Cholesterol biosynthesis pathway. A illustrates selected intermediates of the cholesterol biosynthesis pathway. Dotted arrows indicate indirect conversion steps. The normal structure and function of GTPase proteins (e.g., Ras, RhoA) and lipid rafts—shown in light gray boxes—require the ready intracellular provision of the indicated cholesterol pathway intermediates. Integrity of lipid rafts has been shown to be necessary for cellular responses to specific stimuli, including hypoxia. B shows the pathway annotated with available inhibitors (dark gray boxes). Statins inhibit the synthesis of mevalonate and thus have the potential to interfere with the indicated cell signaling pathways. Statins have also been shown to have effects on cell activity via direct interaction cell receptors (e.g., with the LFA-1 receptor on T lymphocytes). Prenyltransferase enzymes (farnesyl transferase or geranylgeranyl transferase I and II) modify selected GTPase proteins by attaching the specific isoprenoid molecule onto the protein. Specific inhibitors are available for farnseyl transferase (FTase I) and geranylgeranyl transferase I (GGTase I). Zaragozic acid (squalestatin) is a specific squalene synthesis inhibitor. When added to cell medium for a short period, methyl-b-cyclodextrin disrupts lipid rafts by absorbing membrane cholesterol. By using these inhibitors and selectively replacing cholesterol pathway intermediates, the specific signaling pathways involved in an individual cell response can be explored.

Carlin, Peacock, and Welsh: Statins and Hypoxic PAF Proliferation

Western Blot Analysis We have previously shown that hypoxic activation of p38 MAP kinase in RPAFs peaks at 16 hours, and accordingly selected this time point for these experiments (18). RPAFs were grown to 90% confluence in 6well plates and stimulated with 1% serum 6 mediators in normoxia or hypoxia for 16 hours. Protein was obtained from cell lysates, electrophoresed on 10% SDS-PAGE resolving gels under reducing conditions, and transferred to a PVDF membrane (Millipore, Watford, UK) as previously described (17). Blots were blocked and probed with rabbit anti-dual phosphorylated p38 MAP kinase antibody (to assess p38 activation) or p38 MAP kinase antibody (to assess total p38, ensuring equivalent protein loading) with an anti-rabbit HRP-linked antibody used for detection.

Statistics Proliferation results represent the mean of four experiments performed on cells from the same animal; graphs are presented as mean 6 SD. All results shown are representative, having been repeated in experiments from more than four different animals. Data were analyzed using Student’s t test, with a value of P , 0.05 indicating statistical significance.

RESULTS Effects of Fluvastatin on DNA Synthesis in Serum-Stimulated Pulmonary and Systemic Fibroblasts

Incremental doses of serum resulted in increased DNA synthesis in pulmonary and systemic fibroblasts (Figure 2). Addition of fluvastatin at a dose of 1 mM had no effect on the response to serum, but 10 mM completely inhibited serum-induced proliferation (P , 0.05). This inhibitory effect was not circulation specific. Atorvastatin and simvastatin had similar effects with identical statin dose responses (data not shown). Effects of Acute Hypoxia and Fluvastatin on DNA Synthesis and Cell Proliferation in Pulmonary and Systemic Fibroblasts

There was a substantial increase in DNA synthesis (Figure 3A) and cell proliferation (Figure 3B) of rat pulmonary artery fibroblasts (RPAFs) after 24 hours of acute exposure to hypoxia versus normoxia, in the presence of 1% serum (P , 0.05). As previously shown, hypoxia had no effect on proliferation in rat PAFs, in the absence of serum. A quantity of 0.1 mM fluvastatin had no effect. In contrast to the 10-mM dose required to inhibit

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serum-induced proliferation, 1 mM fluvastatin completely inhibited hypoxic proliferation in RPAF cells (P , 0.05). This effect was consistent across a range of serum concentrations (1–10%, data not shown). Fibroblasts from rat aorta (RAFs) proliferated in 1% serum but, in contrast to the RPAF cells, there was no increase in DNA synthesis (Figure 3A) or cell proliferation (Figure 3B) in hypoxic conditions. A quantity of 1 mM fluvastatin had no effect on serum-induced RAF proliferation. Hypoxia and fluvastatin (at doses up to 10 mM) did not affect RPAF or RAF cell viability: more than 95% of cells excluded trypan blue for all experimental conditions. Relative Effects of Different Statins on Hypoxia-Induced Proliferation in RPAFs

No significant differences in DNA synthesis were seen when comparing the effects of atorvastatin, simvastatin, and fluvastatin on RPAF cells in acute hypoxia (Figure 4). No effect on proliferation was seen at doses less than 0.5 mM. For these three lipophilic statins, proliferation was normalized to normoxic levels at doses of 1 mM or greater. With regard to this effect (selective inhibition of hypoxia-induced PAF proliferation), no difference in potency was identifiable across a narrow dose range. The hydrophilic statin pravastatin had no effect on fibroblast proliferation (to serum or hypoxia) at doses up to 50 mM. Inhibition of Hypoxia-Induced p38 MAP Kinase Activation and Proliferation by Low-Dose Fluvastatin Is Reversed by Geranylgeranyl Pyrophosphate

To assess which cell signaling system influenced by statins was being activated by hypoxia in RPAFs, we repeated the previous [3H]thymidine assays with sequential addition of the key cholesterol pathway intermediates (Figure 1). Mevalonate, squalene, farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP) had no effect on serum-normoxic or hypoxic proliferation when added without statin (Figure 5A). Hypoxic proliferation of RPAFs was blocked by 1 mM fluvastatin but restored by the addition of mevalonate, FPP, or GGPP (P , 0.05). Repletion with squalene had no effect.

Figure 2. Serum-induced proliferation in adventitial fibroblast cells is unaffected by 1 mM fluvastatin but inhibited by 10 mM fluvastatin (P , 0.001). Growth-arrested rat pulmonary artery fibroblastss (A) and rat aortic fibroblasts (B) were stimulated with graded doses of serum for 24 hours 6 fluvastatin 1 mM or 10 mM. DNA synthesis, as an index of cell proliferation, was assessed by [3H]thymidine uptake. Values shown are mean 6 SD from four replicate experiments on cells from the same animal; experiments were repeated on cells from more than four animals, and representative graphs are shown here.

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Figure 3. Hypoxic proliferation of rat pulmonary artery fibroblast cells is blocked by fluvastatin at a dose of 1 mM. Rat aortic fibroblast proliferation is unaffected by hypoxia or low-dose fluvastatin. Growth-arrested RPAFs and RAFs were stimulated in normoxia or 5% hypoxia (35 mm Hg) for 24 hours in the presence of 1% serum and fluvastatin 0.1 mM (F0.1) or 1 mM (F1). Cell proliferation was assessed by [3H]thymidine uptake (A) and by cell counting (B). Scintillation counts are expressed as a ratio versus the control count from the individual experiment. Acute hypoxia, in the presence of serum, significantly increased DNA synthesis and proliferation in RPAFs (P , 0.05), but had no effect on RAFs. Fluvastatin 1 mM dose had no effect on normoxic serum–induced proliferation in RPAFs or RAFs but blocked the effect of acute hypoxia on RPAFs. Fluvastatin 0.1 mM had no effect. Values shown are mean 6 SD from four replicate experiments on cells from the same animal; experiments were repeated on cells from more than four animals, and representative graphs are shown here (* value significantly greater than normoxia-1%serum, P , 0.05).

We next assessed the effect of fluvastatin and cholesterol intermediates on hypoxia-induced p38 MAP kinase phosphorylation. Protein was obtained from cells exposed to experimental conditions (1% serum, normoxia, or hypoxia 6 mediators) for 16 hours (time point at which p38 phosphorylation in RPAFs in hypoxia is maximal). Western blots using antibodies specific to p38’s active, dual phosphorylated form and to total p38 (to ensure equal protein loading) were obtained. As before, only minimal p38 phosphorylation was seen in serum-normoxic conditions. Hypoxia led to significant increases in phosphorylated p38 and this was completely inhibited by 1 mM of fluvastatin (Figure 5B). Cholesterol pathway intermediates had no influence on p38 phosphorylation when added to cells without statin. Repletion with mevalonate, FPP, and GGPP negated the inhibitory effect of fluvastatin on p38 MAP kinase phosphorylation, but addition of squalene again had no influence on the fluvastatin effect. The effects of fluvastatin and prenyl intermediates on p38 phosphorylation exactly mirrored the [3H]thymidine assay results.

Geranylgeranyl Transferase Inhibitor 1 Blocks Hypoxic-Induced Proliferation and p38 MAPK Phosphorylation in RPAFs

To further determine which cell signaling pathways (dependent on cholesterol pathway intermediates) were being activated by hypoxia, we studied the effects of prenyltransferase inhibitors, a squalene synthase inhibitor, and the cholesterol-depleting agent methyl-b-cyclodextrin (MBCD, Figure 1). These agents had no effect on serum-normoxic proliferation (Figure 6A). Hypoxic proliferation was completely inhibited by the geranylgeranyl transferase inhibitor. The farnesyl transferase inhibitor, zaragozic acid, and MBCD had no effect on hypoxic proliferation, despite the large cell culture doses used. The effects of these inhibitors on hypoxia-induced p38 MAP kinase phosphorylation matched the proliferation results (Figure 6B). The geranylgeranyl transferase inhibitor completely blocked hypoxia-induced p38 MAP kinase phosphorylation, while the other inhibitors had no effect.


451 Figure 4. Dose response and comparative effects of different statins on hypoxia-induced proliferation in rat pulmonary artery fibroblast cells. The effect of a range of doses of lipophilic statins (fluvastatin, atorvastatin, simvastatin) and a hydrophilic statin (pravastatin) on [3H]thymidine uptake in quiescent RPAFs stimulated with 1% serum and 24 hours of hypoxia (5%, 35 mm Hg) was assessed. Results are plotted as ratios of measured thymidine uptake versus thymidine uptake in control cells (mean of 8 experiments 6 SE). The mean 6 1SD [3H]thymidine uptakes of RPAFs stimulated with 1% serum in normoxia or hypoxia without statin are shown as shaded areas. DNA synthesis in RPAFs exposed to acute hypoxia was reduced to normoxic levels by lipophilic statins at doses > 1 mM. No difference in potency between the lipophilic statins could be identified. Pravastatin had no effect on RPAF proliferation.

A Rac GTPase Inhibitor, but Not a Rho Kinase Inhibitor, Blocks Hypoxic-Induced Proliferation and p38 MAPK Phosphorylation in RPAFs

Preceding results suggested a Rho-family GTPase signaling system as an intermediate upstream of p38 MAP kinase in acute hypoxic proliferative signaling in RPAFs. Based on previous observations, the RhoA-Rho kinase and Rac1-NADPH oxidase pathways seemed the most likely candidates (28, 29). As a preliminary assessment of these, we compared the effects of the Rho kinase inhibitor hydroxyfasudil and the recently described selective Rac1 GTPase guanine exchange factor–binding inhibitor NSC23766 (30). NSC23766 inhibited hypoxia-induced p38 MAP kinase phosphorylation and proliferation in RPAFs, with no effect on serum-normoxic proliferation (Figures 7A and 7B). Hydroxyfasudil had no effect, despite the high cell culture dose selected.

DISCUSSION In this study we have examined acute hypoxic proliferative signaling in pulmonary artery fibroblast cells and identified an obligatory role for a geranylgeranylated signaling protein, acting upstream of p38 mitogen-activated protein kinase. Inhibitor studies indicate that this geranylgeranylated signaling protein is likely to be Rac1. We found that relatively low doses of HMG CoA reductase inhibitors selectively blocked hypoxia-induced p38 MAP kinase phosphorylation and proliferation in rat pulmonary adventitial fibroblasts, via the inhibition of protein geranylgeranylation. The lipophilic statins had similar potencies in this in vitro study, but the hydrophilic statin pravastatin had no effect. We found the antiproliferative effects of low dose (1 mM) fluvastatin on adventitial fibroblasts to be selective for both the hypoxic stimulus and for the pulmonary circulation. Effects on the proliferative response to serum in pulmonary or systemic fibroblasts were only seen at higher doses (> 10 mM). This dosedependent selectivity suggests that signaling pathways activated by hypoxia in RPAFs are exquisitely dependent on products of the cholesterol biosynthesis pathway (and thus amenable to inhibition by low doses of statins). Differing sensitivities of cellular processes to statin inhibition has been previously demonstrated. This effect may reflect the rapid turnover of the prenylated (farnesylpyrophosphate- or geranylgeranylphosphate-conjugated) proteins involved in these pathways (31) and may also explain the relative lack of side effects with standard doses of statins, despite their effects on cell signaling pathways which would

seem essential to normal organ function (32). Activation of this statin-sensitive hypoxic signaling pathway must require synergy between serum and hypoxia, as no increase in proliferation or p38 MAP kinase activation was seen with hypoxia alone, in this cell type. Though it could be argued that a ‘‘pure’’ hypoxic signaling pathway has not been examined here, we believe that a serum-hypoxic pathway is more physiological: serum starvation is an artificial process that does not occur in vivo. A major obstacle in the treatment of pulmonary hypertension in the past has been an inability to selectively affect the pulmonary circulation. Our observations once again underscore the fundamental differences between the pulmonary and systemic circulations and suggest that—with appropriate dosing— statins may have a pulmonary selective anti-fibroproliferative effect. In addition, the hypoxic signaling pathways delineated in RPAFs here may be a significant determinant of the differential proliferative and remodeling responses of the pulmonary and systemic circulations to hypoxia. Inhibition of pulmonary vascular cell proliferation by statins has previously been reported, but effects were only recognized with high cell culture doses of statins (30–80 mM), which are much higher than those achieved with standard dosage of statins, in vivo, in humans (33, 34). Furthermore, in these previous studies, the pulmonary specificity of this antiproliferative effect was not studied: at these cell culture doses statins have nonspecific antiproliferative effects on virtually any cell studied, including systemic vascular cells (35), possibly via disruption of centromere assembly (36). It is notable that standard oral dosage of fluvastatin achieves peak circulating concentrations of 1 mM, a dose that had a positive and selective effect in our experiments. Considering the pharmacokinetics of statin drugs (Table 1), our study is the first to demonstrate that statins— specifically fluvastatin—may have an inhibitory effect on pulmonary vascular cell proliferation at cell culture doses that are within the concentration range achievable in the human pulmonary circulation, with oral statin dosing. Though this study was performed on rat cells in an acute in vitro cell model, we have previously shown that PAF proliferative and MAP kinase responses to hypoxia are conserved across species and are the same regardless of whether the trigger is acute or chronic hypoxia (15, 18, 21). It therefore seems appropriate to cautiously extrapolate these results to chronic hypoxic lung disease and the human pulmonary circulation. In contrast to other studies (37–39), we found no differences in vitro in the potency or effects of the lipophilic statins

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Figure 5. Cholesterol biosynthesis intermediates influence the inhibitory effects of fluvastatin on DNA synthesis and p38 MAP kinase phosphorylation in RPAFs exposed to acute hypoxia. Growth-arrested RPAFs were stimulated in normoxia (N) or 5% hypoxia (H, 35 mm Hg) in the presence of 1% serum 6 fluvastatin 1 mM (F1) or 10 mM (F10) and the cholesterol biosynthesis intermediates mevalonate (M, 1 mM), squalene (Sq, 2.5 mM), farnesyl pyrophosphate (FPP, 0.5 mM), and geranylgeranyl pyrophosphate (GGPP, 0.5 mM). (A) DNA synthesis was assessed by [3H]thymidine uptake. Fluvastatin inhibited hypoxic proliferation; this effect was reversed by mevalonate, FPP, or GGPP, but not by squalene. Cholesterol intermediates alone had no effect on normoxic or hypoxic proliferation. Scintillation counts are expressed as a ratio versus the control count from the individual experiement. Values shown are mean 6 SD from four replicate experiments on cells from the same animal; experiments were repeated on cells from more than four animals, and representative graphs are shown (* value significantly greater than normoxia-1%serum, P , 0.05; ** value significantly less than normoxia-1% serum, P , 0.05). (B) Cells grown to 90% confluence and quiesced in 6-well plates were stimulated for 16 hours. Levels of total (tp38) and phosphorylated (pp38) p38 MAP kinase in the cell lysates of normoxic (N) and hypoxic (H) cells were determined by Western blot analysis as described (see MATERIALS AND METHODS). Hypoxia-induced p38 phosphorylation was completely inhibited by fluvastatin 1 mM (F1). The inhibitory effect of fluvastatin was negated when mevalonate, FPP or GGPP were replaced but the statin effect was uninfluenced by squalene. Cholesterol intermediates alone did not influence p38 activation. Results shown are typical of experiments conducted on RPAF cells from four different animals.

atorvastatin, fluvastatin, and simvastatin. This is important because atorvastatin and simvastatin undergo significant hepatic first-pass metabolism and thus achieve much lower serum concentrations for a given oral dose, when compared with fluvastatin (Table 1). There are many barriers limiting direct comparisons between cell-culture doses in an in vitro study and doses in an in vivo animal study or in clinical use. In particular, active metabolites of simvastatin or atorvastatin may enhance their efficacy in vivo. In addition, in the work presented here, we have studied only fibroblast proliferation and p38 MAP kinase activation. Other potentially therapeutically useful effects of

statins (e.g., improved endothelial function, anti-inflammatory effects) have not yet been fully studied in the pulmonary circulation, and different statins and/or different doses may be relevant for these effects. No conclusions can currently be made about statin choice or dosage for clinical studies in human pulmonary hypertension; it does, however, seem clear that these factors should be carefully considered before embarking on large clinical trials of statins in pulmonary hypertension. The absence of any difference in potency at a cellular level in these experiments would suggest that pharmacokinetic considerations should be an important factor when selecting statin(s) for


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Figure 6. The effect of prenyltransferase inhibitors on acute hypoxia-induced DNA synthesis and p38 MAP kinase phosphorylation in rat pulmonary artery fibroblast cells. Growth-arrested RPAFs were stimulated for 24 hours, in normoxia or hypoxia (35 mm Hg), in the presence of 1% serum 6 fluvastatin (F, 1 mM); farnesyl transferase inhibitor, FTaseI1 (FTI, 5 mM); geranylgeranyltransferase inhibitor, GGTI-2133 (GGTI, 5 mM); or the squalene synthase inhibitor, zaragozic acid (ZA, 60 mM). Membrane cholesterol depletion was achieved by preincubating selected cells with methyl-b-cyclodextrin (MBCD, 1 mM) for 1 hour. (A) DNA synthesis was assessed by [3H]thymidine uptake. Scintillation counts are expressed as a ratio versus the control count from the individual experiement. Normoxic serum–induced proliferation was unaffected by inhibitors. Fluvastatin and the geranylgeranyl transferase inhibitor GGTI-2133 inhibited hypoxic-induced proliferation, but FTase inhibitor 1, zaragozic acid, methyl-b-cyclodextrin, and hydroxyfasudil had no effect. Values shown are mean 6 SD from four replicate experiments on cells from the same animal; experiments were repeated on cells from more than four animals, and representative graphs are shown (* value significantly greater than normoxia-1%serum, P , 0.05). (B) Cells grown to 90% confluence and quiesced in 6-well plates were stimulated for 16 hours. Levels of total (tp38) and phosphorylated (pp38) p38 MAP kinase in the cell lysates of normoxic (N) and hypoxic (H) cells were determined by Western blot analysis as described (see MATERIALS AND METHODS). Hypoxiainduced p38 phosphorylation was completely inhibited by the geranylgeranyl transferase inhibitor but was unaffected by the other inhibitors. Results shown are typical of experiments conducted on RPAF cells from four different animals.

clinical study. Drug interactions are also important, and statins have notable interactions with established pulmonary vascular therapies such as bosentan and warfarin. It is considered relevant that fluvastatin has a reduced potential for interactions when compared with the other lipophilic statins. We determined the mechanism by which statins inhibited acute hypoxia-induced p38 phosphorylation and proliferation in PAFs. We confirmed that the statin effect was mediated via HMG-CoA reductase inhibition, by demonstrating a reversal of the inhibitory effect with mevalonate, the immediate product of HMG-CoA reductase (Figure 1). The lack of effect seen with squalene and subsequently with a squalene synthase inhibitor (zaragozic acid) and cholesterol-depleting agent (MBCD) indicated that signaling systems dependent on intracellular cholesterol supplies or cell membrane cholesterol (e.g., lipid raft integrity) were not implicated in hypoxic-p38 MAP kinase–proliferative signaling in this cell type. In contrast, farnesyl pyrophosphate and geranylgeranyl pyrophosphate repletion negated the statin effect. When the prenyltransferase inhibitors were studied we

found that the geranylgeranyl transferase inhibitor selectively blocked hypoxic proliferation, while the farnesyl transferase inhibitor had no effect. This indicated that FPP was negating the statin effect via its action as a substrate for GGPP synthesis, rather than by direct restoration of a farnesylated protein. These results lead us to conclude that a geranylgeranylated signaling protein plays an obligate role in proliferative hypoxic signaling in PAFs, acting upstream of p38 MAP kinase. Post-translational modification with the addition of a geranylgeranyl pyrophosphate moiety has been shown to be essential for the normal function of various cellular proteins (40). Of these, the best characterized are the Rho-GTPases, of which RhoA, Rac1, and Cdc42 have been most widely studied. Inhibition of the geranylgeranylation modification step leads to failure of association of these proteins with the plasma membrane, disrupting the signaling pathway. Of the characterized Rho-GTPase signaling systems, the RhoA–Rho kinase pathway and the Rac1-NADPH oxidase system seemed the most likely mediators of hypoxic-p38 MAPK-proliferative signaling in

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b Figure 7. The effect of Rho kinase and Rac1 inhibition on acute hypoxia-induced DNA synthesis and p38 MAP kinase phosphorylation in rat pulmonary artery fibroblast cells. Growth-arrested RPAFs were stimulated for 24 hours, in normoxia or hypoxia (35 mm Hg), in the presence of 1% serum 6 hydroxyfasudil (HF, 20 mM) or the Rac1 GTPase inhibitor NSC23766 (NSC, 100 mM). (A) DNA synthesis was assessed by [3H]thymidine uptake. Scintillation counts are expressed as a ratio versus the control count from the individual experiment. Normoxic serum–induced proliferation was unaffected by inhibitors. NSC23766 inhibited hypoxic-induced proliferation, but hydroxyfasudil had no effect. Values shown are mean 6 SD from four replicate experiments on cells from the same animal; experiments were repeated on cells from more than four animals, and representative graphs are shown. (* value significantly greater than normoxia-1%serum, P , 0.05). (B) Cells grown to 90% confluence and quiesced in 6-well plates were stimulated for 16 hours. Levels of total (tp38) and phosphorylated (pp38) p38 MAP kinase in the cell lysates of normoxic (N) and hypoxic (H) cells were determined by Western blot analysis as described (see MATERIALS AND METHODS). Hypoxia-induced p38 phosphorylation was completely inhibited by NSC23766 but was unaffected by hydroxyfasudil. Results shown are typical of experiments conducted on RPAF cells from four different animals.

pulmonary adventitial fibroblasts (41–43). To assess if either of these were involved, we compared the effect of the Rho kinase inhibitor hydroxyfasudil and the selective Rac1 inhibitor NSC23766. The positive results (i.e., inhibition of hypoxia-induced proliferation and p38 MAP kinase phosphorylation) with the Rac1 inhibitor suggest that this system is activated by acute hypoxia in RPAFs. These findings also suggest that disruption of Rac1 signaling is the mechanism of the identified effects of low-dose statin on these cells. We have not directly shown that 1 mM statin causes failure of membrane association of Rac1, or that Rac1 (and/or other GTPases) are activated in acute hypoxia: these are important directions for future research and, currently, inferences made about the involvement of Rac1 in hypoxic-p38proliferative signaling in RPAFs are indirect. However, it is considered striking that NSC23766 inhibits the hypoxic processes here completely, but selectively. Rac1 has been shown to couple extracellular signals via association with NADPH oxidases and subsequent generation

of intracellular reactive oxygen species (ROS). Whether intracellular ROS levels go up or down in the pulmonary vasculature in acute hypoxia and whether they have any role in mediating cellular responses to hypoxia is contentious (44). One study, however, has documented increased intracellular ROS levels in fibroblasts after acute hypoxia and shown that this increase leads to p38 MAP kinase activation and proliferation (45). GTPases such as Rac1 were not assessed in this study, but the complementary results of our own data suggest an acute hypoxic signaling pathway comprising Rac1-NADPH oxidase activation, leading to ROS generation, which in turn leads sequentially to p38 MAP kinase phosphorylation and HIF-1a stabilization and proliferation (18). Intriguingly, Rac1 has also separately been linked the PI3-kinase–ribosomal protein p70 S6 kinase pathway in other cells types (46). Activation of this P13-kinase–p70S6K pathway has been shown to be essential for acute hypoxiainduced proliferation of bovine pulmonary artery fibroblasts (47) and for serotonin-induced growth of pulmonary artery smooth muscle cells (48). It seems relevant to speculate that there may be cross talk between the PI3-K-Akt-mTOR proliferative pathway and the Rac1-p38-HIF-1a pathway that we propose here and in our previous work (18). It may also be that we are simply looking at the same pathway through different windows, though the observation that statins activate rather than inhibit Akt (albeit in a different model) is confounding (49). Further study of these pathways, in particular to fully clarify the nature of the geranylgeranylated protein pathway upstream of p38 MAP kinase in PAFs, would seem to be important future work. When considering the potential application of these results, we have identified statins as agents that can selectively inhibit acute hypoxia-induced pulmonary adventitial fibroblast proliferation

TABLE 1. RELEVANT PROPERTIES OF STATINS IN CLINICAL USE Parameter Cmax, mM/L Solubility Metabolism

Atorvastatin

Fluvastatin

Simvastatin

Lovastatin

Pravastatin

Rosuvastatin

0.02–0.05 Lipophilic CYP3A4

1.035 Lipophilic CYP2C9



0.1–0.123 Hydrophilic Sulfation

0.045 Hydrophilic CYP2C9/C19

Maximum serum concentrations are following a 40-mg oral dose of statin. Lipophilic statins have effects on nonhepatic cells, whereas hydrophilic statins (rosuvastatin and pravastatin) typically do not. Statins metabolized via CYP3A4 have an increased potential for drug interactions (adapted from Ref. 25).


in the pulmonary circulation. Unwanted, nonspecific effects on systemic fibroblasts are not seen, with appropriate dosage. Pharmacokinetic data would suggest that an antifibroproliferative dose of fluvastatin could be achieved in the pulmonary circulation, with standard oral doses. The safety and tolerability of statins at these doses is established. Proof of concept clinical trials of simvastatin for pulmonary arterial hypertension are in progress. There is an evolving body of evidence supporting a large clinical trial of statins for human pulmonary hypertension. The results of the study we present here would suggest that fluvastatin may have specific potential for the treatment of selected forms of pulmonary hypertension, particularly those that are driven by hypoxia and adventitial fibroblast proliferation. Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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