Published ahead of print on June 23, 2006, doi:10.1164/rccm.200512-1976OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200512-1976OC
Cyclosporin A Inhibits Hypoxia-induced Pulmonary Hypertension and Right Ventricle HypertrophyCentre de Recherches du Service de Santé des Armées, La Tronche; E0221 INSERM, Université Joseph Fourier, Grenoble; and U-769 INSERM, Université Paris-Sud, Châtenay-Malabry, France Correspondence and requests for reprints should be addressed to Nathalie Koulmann, M.D., Ph.D., Department of Human Factors, Centre de Recherches du Service de Santé des Armées, BP 87-38702 La Tronche Cedex, France. E-mail: nkoulmann{at}crssa.net
Rationale: Hypoxia-induced pulmonary hypertension involves hypoxia-inducible factor-1  (HIF-1) activation as well as elevated resting calcium levels. Cyclosporin A (CsA) inhibits calcium-induced calcineurin activation and blocks the stabilization of HIF-1 in cultured cells. Objectives: We hypothesized that treatment of rats with CsA would prevent HIF-1–dependent gene transcription, lower specific responses to acute hypoxia, and prevent pulmonary hypertension and right ventricle hypertrophy resulting from prolonged exposure to hypoxia. Methods: Acute and chronic responses to hypoxia were studied in rats treated or not treated with CsA (25 mg · kg–1 · d–1). Measurements: Transcript levels of genes encoding the serotonin transporter or four HIF-1 target genes, in rats exposed for 6 h to ambient hypoxia, treated or not by CsA, were measured. In vivo hemodynamics, hematocrit, and heart morphologic characteristics were assessed in rats subjected to hypoxia for 3 wk, treated or not treated with CsA. Changes in mRNA levels of the modulatory calcineurin-interacting protein-1 (MCIP-1) were used as a sensitive indicator of calcineurin activity in lung and heart. Main results: Acute exposure to hypoxia led to a marked increase in mRNA levels of serotonin transporter, modulatory calcineurin-interacting protein-1, and HIF-1 target genes, which was blunted by CsA treatment. Prolonged exposure to hypoxia raised right ventricle pressure, induced right ventricle hypertrophy, and activated cardiac calcineurin, effects that were fully prevented by CsA treatment. Conclusions: These results suggest that CsA prevents hypoxia-induced pulmonary hypertension and right ventricle hypertrophy, either by inhibiting HIF-1 transcriptional activity in lung, by decreasing calcineurin activity in lung and heart, by direct effects of CsA, or by a combination of these factors.
Key Words: pulmonary hypertension • right ventricle hypertrophy • signal transduction Exposure to chronic hypoxia leads to specific responses, including erythrocytosis, pulmonary hypertension, and right ventricular hypertrophy. Sixty years after the first report of hypoxia-induced pulmonary vasoconstriction, the fundamental mechanisms are still to be elucidated (1). The pathogenesis of pulmonary hypertension is highly complex and involves several intracellular pathways (2). Hypoxia-induced remodeling of the pulmonary circulation and increased pulmonary vascular tone involve the release of vasoactive agents, among which serotonin (5-hydroxytryptamine [5-HT]) plays a critical role (3) through the action of the serotonin transporter (5-HTT) (4–7) and various 5-HT receptors (8–10). It is also associated with elevated intracellular calcium resting levels (11), suggesting the possible involvement of calcium in cellular remodeling.
The transcription factor hypoxia-inducible factor-1 (HIF-1) is a critical regulator of the physiological responses to hypoxia, such as increased glycolysis, angiogenesis, and erythropoiesis (12, 13). HIF-1 was originally identified as an activator of erythropoietin (EPO) gene expression (14). HIF-1 plays a pivotal role in the pathogenesis of hypoxia-induced pulmonary hypertension (11, 15). Partial deficiency of HIF-1
HIF-1 is a heterodimeric transcription factor consisting of a regulated Hypoxia-induced pulmonary hypertension leads to increase right ventricular pressure and to right ventricle hypertrophy. Cardiac hypertrophy is a complex process in which calcineurin clearly plays a critical role (20). Calcineurin is an effective inducer of cardiac growth in response to numerous pathologic stimuli (21). Inhibition of calcineurin activity with CsA can prevent cardiac hypertrophy in response to various stimuli (22). Whether enhanced calcineurin activity is involved in the development of pulmonary hypertension and right ventricle hypertrophy in response to chronic hypoxia has not been examined. Thus, the aim of this experiment was to test the hypothesis that CsA administration in rats would prevent HIF-1–dependent gene transcription, pulmonary hypertension, and right ventricle hypertrophy resulting from chronic exposure to hypoxia. Some of the results of these studies have been previously reported in the form of abstracts (23–25). This article reports our complete study findings.
This investigation was performed in accordance with the Helsinki Accords for Humane Treatment of Animals during Experimentation.
Experimental Design
Long-term responses to hypoxia. Because both hypoxia and CsA treatment decreased the food intake of animals, with an additive effect in the H-CsA group, two additional groups were fed as CsA-treated hypoxic animals maintained in normoxia and treated (PF-CsA) or not treated (PF-C) with CsA, to rule out a possible toxic effect of CsA.
Hemodynamic Measurements
Tissue Processing
mRNA Analysis Oligonucleotide primers used in this study were designed with Mac Vector software (Accelrys, San Diego, CA) and synthesized at Eurogentec (Table 1). Considering probable disparities due to variations in input RNA or in reverse transcription efficiencies, cyclophilin A and hypoxanthine–guanine phosphoribosyltransferase mRNA were also quantified as endogenous housekeeping genes.
Quantitative PCR was performed with a LightCycler FastStart DNA Master SYBR Green I kit with a LightCycler system (Roche Applied Science, Meylan, France). The threshold cycle value was calculated from LightCycler software. Quantification was achieved with a pool of cDNA samples as calibrator according to the comparative threshold cycle method (28). Accurate normalization was performed by geometric averaging of the two endogenous housekeeping genes (29). Relative mRNA values were calculated with RealQuant software (Roche Applied Science, Mannheim, Germany).
Statistical Analysis
Early Responses to Hypoxia HIF-1–responsive genes are up-regulated by acute exposure to ambient hypoxia and blunted by CsA treatment in both kidney and lung. In preliminary experiments we examined the induction of hypoxia responsive genes after 2 and 6 h of exposure to hypoxia. Because results obtained after 6 h gave more robust results, a 6-h exposure time was chosen. EPO was the first hypoxia-sensitive gene described, and is expressed mainly in kidney. CsA treatment lowered EPO mRNA levels in kidney of normoxic rats (p < 0.001; Figure 1). Moreover, EPO mRNA levels were markedly affected by hypoxia (p < 0.001). In untreated animals EPO mRNA levels were increased after 6 h of hypoxia (p < 0.001). CsA administration completely abolished the hypoxia-induced increase in EPO mRNA levels (p < 0.001). An interaction between hypoxia and CsA was observed (p < 0.001), suggesting that the hypoxia effects differed in untreated and in CsA-treated rats.
Endothelin-1 (ET-1), glucose transporter type 1 (Glut-1), lactate dehydrogenase type A (LDH-A), and 5-HTT are also hypoxia-sensitive genes that are commonly expressed in the lung. In the present study, no global effect of CsA treatment was observed for those genes (Figure 2). A global effect of hypoxia was observed for the four hypoxia-sensitive genes ET-1, Glut-1, LDH-A, and 5-HTT (p < 0.001, p < 0.01, p < 0.05, and p < 0.05, respectively). An interaction between CsA treatment and hypoxia was found, indicating that the acute responses to hypoxia differed between treated and untreated animals. In untreated rats, all mRNA levels were markedly induced by acute hypoxia (p < 0.001 for ET-1, and p < 0.05 for Glut-1, 5-HTT, and LDH-A). CsA administration completely abolished the hypoxia-induced expression of HIF-1 target genes. These changes cannot be attributed to a generalized effect of either hypoxia or CsA on gene expression because no significant changes in mRNA levels of the two endogenous housekeeping genes (cyclophilin A and hypoxanthine–guanine phosphoribosyltransferase) used for normalization were observed.
Calcineurin activation is involved in the acute response to hypoxia in the lung. The specific effects of hypoxia and/or CsA administration on calcineurin activity were assessed by measuring mRNA levels of modulatory calcineurin-interacting protein-1 (MCIP-1) in lung (30). As expected, CsA lowered MCIP-1 mRNA levels in normoxic rats (p < 0.001). In untreated animals the MCIP-1 mRNA level paralleled the expression of hypoxia-responsive genes (Figure 3); it was increased 2.5-fold 6 h after hypoxia exposure (p < 0.001), and CsA treatment completely blocked the hypoxia-induced increase in MCIP-1 mRNA expression.
Long-term Responses to Hypoxia Hematocrit. The increase in red blood cell production is an early general response to chronic hypoxia. Hematocrit was affected both by hypoxia (p < 0.001) and CsA treatment (p < 0.001), without interaction (Table 2). Hypoxia induced an increase in hematocrit both in nontreated rats (p < 0.001) and in CsA-treated rats (p < 0.001), but hematocrit values observed in hypoxic rats were lower in CsA-treated rats (p < 0.001).
Hypoxia-induced right ventricle pressure increase is blunted by CsA treatment. CsA treatment alone did not affect mean right ventricle pressure in normoxic rats (26.5 ± 3.5 vs. 31.3 ± 2.4 mm Hg in normoxia; Figure 4). A clear consequence of chronic hypoxia is an increased load to the right ventricle. This is evidenced in Figure 4, where right ventricle pressure increased more than twofold in hypoxic untreated animals (72.7 ± 4.7 mm Hg, p < 0.001). CsA administration fully prevented the hypoxia-induced increase in right ventricle pressure (22.0 ± 2.3 mm Hg).
CsA treatment reduces the hypoxia-induced absolute and relative increase in right ventricle weight. Both hypoxia (p < 0.001) and CsA treatment (p < 0.001) globally affected right ventricle weight, when expressed either as absolute values or relative to body weight (Table 2). Hypoxia exposure increased absolute and relative right ventricle weight in nontreated rats (+126 and +152%, respectively; p < 0.001). CsA treatment strongly reduced the hypoxia-induced increase in absolute and relative right ventricle weight (p < 0.001). The absolute right ventricle weight was lower in the N-CsA group than in the N-C group (p < 0.05); however, when expressed relative to body weight, relative right ventricle weight did not differ between these two groups. Whereas absolute right ventricle weight was not different between N-CsA and H-CsA rats, hypoxia induced an increase in relative right ventricle weight in CsA-treated rats (+45%; p < 0.01). Because CsA and hypoxia induce a decrease in food intake, changes in heart weight were compared also in pair-fed animals. Absolute and relative right ventricle weights were slightly higher in the H-CsA group than in the PF-C group (+21 and +24% for absolute and relative right ventricle weight, respectively) and the PF-CsA group (+27 and +22% for relative right ventricle weight, respectively), which shows that CsA treatment reduces the hypoxia-induced absolute and relative increase in right ventricle weight independently of toxic effects.
Hypoxia increases and CsA blocks calcineurin activation in the right ventricle.
The main findings of this study are that, in vivo, (1) acute hypoxia increased calcineurin activity in lung, as assessed by MCIP-1 mRNA levels; (2) CsA administration abolished the hypoxia-induced increase in calcineurin activity and in the transcription of numerous HIF-1 target genes; (3) the hypoxia-induced increase in right ventricle pressure was fully abolished by CsA treatment; and (4) the subsequent right ventricle hypertrophy, which was associated with an enhancement in calcineurin activity, was also fully prevented by CsA treatment. Taken together, these results show that CsA prevents hypoxia-induced pulmonary hypertension and its associated right ventricle hypertrophy, either by inhibiting HIF-1 transcriptional activity in lung, decreasing calcineurin activity in lung and heart, by direct effects of CsA, or by a combination of these factors. This is the first evidence that calcineurin activation might be involved in hypoxic signaling leading to pulmonary hypertension.
Acute hypoxia induced the expression of early HIF-1–responsive gene expression. In the present study, calcineurin activity assessed through the level of MCIP-1 gene transcription was dramatically increased in lung after 6 h of acute hypoxia. Administration of CsA, a well-known inhibitor of calcineurin activity (31), led to a 50% decrease in calcineurin activation in the lungs of normoxic CsA-treated animals, and CsA completely abolished the hypoxia-induced increase in MCIP-1 transcription, suggesting that hypoxia activates calcineurin. ET-1 and 5-HT have been shown to play a central role in pulmonary hypertension, acting both as potent pulmonary vasoconstrictors and cellular mitogens on both pulmonary artery smooth muscle cells and adventitial fibroblasts (2), through increased intracellular calcium levels (32, 33). A link between 5-HT signaling, calcineurin activation, and hypertrophy has been established in cardiac muscle cells (34). Moreover, in pulmonary arterial smooth muscle cells, acute hypoxia causes depolarization and increased intracellular calcium concentration through store-operated and voltage-operated calcium channels (35). Chronic elevation of calcium activates calcineurin, which plays a critical role in smooth muscle cell remodeling through dephosphorylation of nuclear factor–activated T-cell transcription factors (36). Thus, the hypoxia-induced increase in calcium in pulmonary arterial smooth muscle cells may be responsible for calcineurin activation and MCIP-1 expression in lung after 6 h of acute hypoxia. Interestingly, CsA treatment also inhibited the hypoxia-dependent gene transcription of ET-1, Glut-1, and LDH-A in the lung and of EPO in the kidney (12). Transcript levels of these hypoxia-responsive genes rose markedly, in response to acute hypoxia in untreated animals, and this increase was fully prevented in CsA-treated animals, in parallel with calcineurin activity. It has been previously suggested that 5-HTT could also be an oxygen-sensitive gene (6, 37). Again, 5-HTT transcription was up-regulated by hypoxia in association with increased calcineurin activity, and this response was also completely blunted by CsA treatment. These results show for the first time that CsA administration in rats prevents the hypoxia-induced transcriptional activation of 5-HTT and of four well-known HIF-1 target genes. However, whether this results from calcineurin activation or from direct effects of CsA remains to be established. Indeed, it has been shown in cultured cells that CsA may interfere with the hypoxic signaling cascade by activating HIF-1
Consistent with the prevention of hypoxia-responsive gene activation, CsA treatment was able to prevent some of the specific responses to hypoxia, including erythrocytosis, pulmonary hypertension, and its associated right ventricle hypertrophy. As expected, hematocrit values increased in response to hypoxia and in parallel to increased transcription of the EPO gene in kidney, in a CsA-sensitive manner. Moreover, CsA treatment also prevented both the increase in right ventricular pressure and cardiac hypertrophy usually observed in response to chronic hypoxia (39). Interestingly, these effects appear similar to those induced by partial deficiency in HIF-1 (35), suggesting a close link between HIF-1 and calcineurin. Calcineurin activation is known to participate in cardiac hypertrophy induced by diverse stimuli (40). To examine its possible involvement in hypoxia-induced cardiac hypertrophy, we measured the level of MCIP-1 transcript expression. Chronic hypoxia indeed led to increased levels of MCIP-1 mRNA in the right ventricle of untreated animals, an effect that was completely blocked by CsA treatment. The present study suggests for the first time that the calcineurin/nuclear factor–activated T-cell pathway is also involved in hypoxia-mediated right ventricle hypertrophy. However, because CsA blocks pulmonary hypertension, inhibition of hypoxia-induced right ventricle hypertrophy by CsA is due to its antihypertensive rather than antihypertrophic effects. In conclusion, the present study shows that, in an animal model, CsA administration fully prevented the hypoxia-induced increase in the expression of numerous hypoxia-responsive genes in parallel with changes in calcineurin activity. These early alterations may have long-term consequences on the prevention of hypoxia-induced pulmonary hypertension and its associated right ventricle hypertrophy. Hence, our results suggest a role for calcineurin activity in hypoxia-induced pulmonary hypertension. However, CsA also blocks the adaptive increase in hematocrit in response to chronic hypoxia. Further experiments are needed to define whether calcineurin inhibitors, alone or in conjunction with inhibitors of other signaling pathways (41–43), might be useful to prevent and/or reverse hypoxia-induced pulmonary hypertension. Clearly, the present findings have important clinical consequences in providing calcineurin as a new therapeutic target to prevent pulmonary hypertension.
Originally Published in Press as DOI: 10.1164/rccm.200512-1976OC on June 23, 2006 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. Received in original form December 29, 2005; accepted in final form June 19, 2006
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