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Cyclosporin A Inhibits Hypoxia-induced Pulmonary Hypertension and Right Ventricle Hypertrophy

Centre 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

	ABSTRACT

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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 delays development of polycythemia, right ventricular hypertrophy, pulmonary hypertension, and hypoxia-induced pulmonary artery smooth muscle cell depolarization and increased calcium levels (11, 15).

HIF-1 is a heterodimeric transcription factor consisting of a regulated subunit and a constitutive subunit (16). Under normoxic conditions, HIF-1 is hydroxylated and acetylated, resulting in its recognition by the von Hippel-Lindau protein, leading to its ubiquitinylation and subsequent degradation through the proteasomal pathway (17). Hypoxia blocks both HIF-1 hydroxylation and acetylation, resulting in its stabilization. Moreover, growth factors or cytokines can induce HIF-1 expression in a hypoxia-independent manner (18). In addition, biochemical factors have been shown to affect HIF-1 stabilization. Cyclosporin A (CsA), a well-known inhibitor of the calcium/calmodulin-dependent protein phosphatase calcineurin, can block hypoxia-induced HIF-1 accumulation by activating Pro-564 hydroxylation in cultured cells (19). However, whether repeated CsA administration is able to prevent the transcriptional activity of HIF-1 in vivo, and to modulate the known responses to hypoxia, has not been examined.

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.

	METHODS

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This investigation was performed in accordance with the Helsinki Accords for Humane Treatment of Animals during Experimentation.

Experimental Design
Early responses to hypoxia.
Twenty-four rats received intraperitoneally either CsA (Sandimmune, 25 mg · kg^–1 · d^–1; Novartis, Basel, Switzerland; T group) or an equivalent dose of sodium chloride solution (U group). Groups were divided into subgroups of n = 6 rats, exposed or not for 6 h to hypoxia in a chamber (PO₂ = 10%).

Long-term responses to hypoxia.
Forty-three rats were exposed for 3 wk to either hypoxia (H group) or normoxia (N group). Groups were divided in subgroups, in which rats were injected intraperitoneally with either CsA at 25 mg · kg^–1 · d^–1 (N-CsA and H-CsA, n = 11 and n = 10, respectively), or the equivalent dose of sodium chloride solution (N-C and H-C, n = 11 each). This dose of CsA was chosen according to previous studies using CsA to prevent cardiac hypertrophy in rodents (26, 27).

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
Fourteen animals (5 from the H-CsA group and 3 each from the N-C, N-CsA, and H-C groups) were subjected to hemodynamic measurements after 3 wk of conditioning. Right functional parameters were measured in closed-chest anesthetized rats with a ultraminiature catheter pressure transducer inserted into the right jugular vein and advanced downstream to the right ventricle (catheter PR-249 attached to control unit model TC100 [Millar Instruments, Inc., Houston, TX] and connected to a recorder [2000 series; Gould Instrument Systems/Data Sciences International, Arden Hills, MN]).

Tissue Processing
All other animals were decapitated at the end of the study. Blood was sampled for hematocrit measurement. The middle lobe of the right lung and kidney cortex were immediately sampled, and frozen in liquid nitrogen. Thereafter, the heart was removed and dissected, with the right ventricle weighed and frozen in liquid nitrogen.

mRNA Analysis
Total RNA was extracted from 5 mg of right ventricle and from 15 mg of kidney and lung, using the RNeasy mini kit procedure (Qiagen, Courtaboeuf, France) in accordance with the company protocol. The RNA concentration was determined by determining optical density at 260 nm. Reverse transcription was performed with a reverse transcription core kit (Eurogentec, Saraing, Belgium) with 2.5 µM oligo(dT)₁₅ primer, according to the manufacturer's instructions.

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.

Statistical Analysis
Data are presented as means ± SEM. CsA treatment and hypoxia effects were determined by two-way analysis of variance. Differences between groups were tested with a Newman-Keuls post hoc test. Statistical significance was accepted at p < 0.05.

	RESULTS

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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.

View larger version (7K): [in this window] [in a new window]   Figure 1. Transcript levels of erythropoietin (EPO) in kidney were measured by real-time polymerase chain reaction, and normalized by geometric averaging of two internal control genes, cyclophilin A and hypoxanthine–guanine phosphoribosyltransferase. EPO mRNA levels was significantly decreased in normoxic cyclosporin A (CsA)–treated rats. The dramatic increase observed in rats subjected to 6 h of hypoxia was completely blocked in hypoxic CsA-treated rats. Results are expressed as a relative percentage of normoxic value. *** Different from normoxic values (p < 0.001); , different from untreated animals (p < 0.05 and p < 0.001, respectively).

View larger version (17K): [in this window] [in a new window]   Figure 2. Transcript levels of hypoxia-inducible factor-1 (HIF-1) target genes in lung, namely endothelin-1 (ET-1; A), glucose transporter type 1 (Glut-1; B), lactate dehydrogenase type A (LDH-A; C), and serotonin transporter (5-HTT; D), were increased by hypoxia and CsA treatment nearly abolished the effects of hypoxia. Legend is as described in Figure 1. *, *** Different from normoxic values (p < 0.05 and p < 0.001, respectively); different from untreated animals (p < 0.05).

View larger version (9K): [in this window] [in a new window]   Figure 3. Transcript levels of modulatory calcineurin-interacting protein-1 (MCIP-1) in lung were decreased by CsA treatment in normoxic animals. Six hours of hypoxia induced a large increase in MCIP-1 expression that was completely blocked by CsA treatment. * Different from normoxic values (p < 0.05); different from untreated animals (p < 0.05).

View larger version (103K): [in this window] [in a new window]   Figure 4. Right ventricle pressure (Prv) recording from normoxic and hypoxic rats treated (N-CsA, H-CsA) or not treated (N-C, H-C) with CsA. Right ventricular pressure was not altered by CsA in control animals but was significantly increased by hypoxia. CsA treatment abolished the effects of hypoxia.

Hypoxia increases and CsA blocks calcineurin activation in the right ventricle.
We then investigated whether cardiac calcineurin was activated in response to hypoxia and whether CsA was able to block this effect. Both hypoxia (p < 0.001) and CsA treatment (p < 0.001) affected MCIP-1 mRNA levels in the right ventricle, with a strong interaction (p < 0.001) between these two factors (Figure 5). MCIP-1 mRNA levels markedly increased after hypoxia exposure in nontreated rats (+437%; p < 0.001). This hypoxia-induced increase in MCIP-1 mRNA levels was fully prevented by CsA treatment, so that there was no difference between N-C, N-CsA, and H-CsA groups, showing that calcineurin activation is involved in right ventricle hypertrophy resulting from the hypoxia-mediated pressure overload.

View larger version (9K): [in this window] [in a new window]   Figure 5. Transcript levels of MCIP-1 in the right ventricle of normoxic and hypoxic rats treated with CsA (N-CsA, H-CsA), or not treated with CsA (N-C, H-C). MCIP-1 was up-regulated by hypoxia, and CsA treatment also abolished this effect. Results are expressed as a relative percentage of N-C values. *** Different from normoxic values (p < 0.001); different from untreated animals (p < 0.001).

	DISCUSSION

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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 hydroxylation, leading to its ubiquitinylation and subsequent degradation, although a possible involvement of calcineurin has not been ruled out in this study (19). Conversely, because HIF-1 subunit can be up-regulated in a hypoxia-independent manner but also in a Ca²⁺/calcineurin-dependent manner (38), it is possible that calcineurin may directly up-regulate HIF-1 subunit expression, thus participating in the sustained induction of HIF-1–dependent gene expression. These findings thus suggest that calcineurin may act as a downstream effector of hypoxia in the lung, leading to the proliferation of pulmonary artery smooth muscle cells and adventitial fibroblasts. CsA treatment could prevent pulmonary hypertension both by canceling hypoxia-induced 5-HTT upregulation and inhibiting local ET-1 release, and by inhibiting calcium-dependent calcineurin activation within pulmonary vascular smooth muscle cells and/or adventitial fibroblasts (Figure 6).

View larger version (24K): [in this window] [in a new window]   Figure 6. Scheme depicting the possible role of calcineurin and HIF-1 in hypoxia-induced pulmonary hypertension. HIF-1 induces the release and activates the binding of serotonin (5-HT) and/or ET-1 on their membrane receptor. Receptor stimulation and hypoxia-induced activation of calcium channels lead to Ca2+ release from the sarcoplasmic reticulum (SR) and sustained elevation of intracellular calcium. This increase in cytosolic Ca2+ concentration activates calcineurin, leading to possibly HIF-1 gene transcription and to an increase in the transcription of proliferative genes that stimulates pulmonary artery smooth muscle cells and/or adventitial fibroblast proliferation. CsA administration canceled both the calcineurin activation and the hypoxia-induced increase in 5-HTT and ET-1. G = G protein; IP3 = inositol triphosphate; NFAT = nuclear factor–activated T-cell transcription factor.

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.

	FOOTNOTES

 
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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