Pulmonary hypertension (PHT) is associated with increased vascular resistance due to sustained contraction and enhanced proliferation of pulmonary arterial smooth muscle cells (PASMC); the abnormal tone and remodeling in the pulmonary vasculature may relate, at least in part, to decreased cyclic nucleotide levels. Cyclic nucleotide phosphodiesterases (PDEs), of which 11 families have been identified, catalyze the hydrolysis of cAMP and cGMP. We tested the hypothesis that PASMC isolated from patients with PHT, either idiopathic pulmonary arterial hypertension (IPAH) or secondary pulmonary hypertension (SPH), have increased expression and activity of PDE isoforms that reduce the responsiveness of agents that raise cellular cAMP. Real-time PCR and immunoblotting demonstrated that the expression of PDE1A, PDE1C, PDE3B, and PDE5A was enhanced in PASMC from both IPAH and SPH patients compared with control PASMC. Consistent with this enhanced expression of PDEs, agonist-stimulated cAMP levels were significantly reduced in IPAH and SPH PASMC unless a PDE inhibitor was present. The use of specific PDE inhibitors revealed that an increase in PDE1 and PDE3 activity largely accounted for reduced agonist-induced cAMP levels and increased proliferation in IPAH and SPH PASMC. Treatment with PDE1C-targeted small interference RNA enhanced cAMP accumulation and inhibited cellular proliferation to a greater extent in PHT PASMC than controls. The results imply that an increase in PDE isoforms, in particular PDE1C, contributes to decreased cAMP and increased proliferation of PASMC in patients with PHT. PDE1 isoforms may provide novel targets for the treatment of both primary and secondary forms of the disease.
- pulmonary circulation and disease
- signal transduction
pulmonary hypertension (PHT) is associated with increased pulmonary vascular resistance (PVR) due to sustained pulmonary vasoconstriction and vascular wall remodeling (6, 34). PHT occurs as a primary illness [idiopathic pulmonary arterial hypertension (IPAH)], the result of a sporadic/familial mutation (i.e., in bone morphogenetic protein receptor II), or secondary (SPH) to disorders such as chronic obstructive pulmonary disease, congenital heart disease, left ventricular failure, or sustained exposure to hypoxia. Despite different initiating mechanisms of PHT, the prognosis is poor in both primary and secondary forms of the disease (6, 34).
The intracellular second messenger adenosine 3′,5′-cyclic monophosphate (cAMP) helps regulate vascular tone, cellular proliferation, and hypertrophy that remodel the pulmonary artery (PA). For example, PAs from rats with chronic hypoxia-induced PHT have decreased cAMP levels (10), and certain drugs currently used for the treatment of PHT, such as prostacyclin and its analogs, iloprost or beraprost, increase cellular content of cAMP. The balance between formation by adenylyl cyclases (ACs) and degradation by cyclic nucleotide phosphodiesterases (PDEs) determines cellular cAMP levels and activation of downstream mediators of cAMP action, i.e., protein kinase A, Epac (exchange protein activated by cAMP), and cyclic nucleotide-gated channels.
The 11 families of cyclic nucleotide PDEs (which encompass over 30 different isoforms) have distinctive substrate specificities (cAMP and/or cGMP), regulatory characteristics, tissue distribution, and susceptibility to inhibitors (2, 12, 26). In PAs from rats with chronic hypoxia-induced PHT, total PDE activity is increased, primarily because of upregulated mRNA and protein expression and increased activity of PDE3A/B (a cGMP-inhibited/cAMP-stimulated cAMP-hydrolyzing isoform) and PDE5A (a cGMP-specific isoform) (9, 14). PDE3 and PDE5 inhibitors both decrease pulmonary arterial pressure (PAP) and PVR in animal models of PHT (7, 11); PDE3A, PDE3B, and PDE5 are expressed in pulmonary arterial smooth muscle cells (PASMC) (14, 19). Inhibition of PDE5 decreases proliferation of human PASMC and is more effective at raising cGMP than NO donors or a soluble guanylyl cyclase inhibitor (30). Sildenafil, a selective PDE5 inhibitor, promotes pulmonary vasodilation in patients with PHT (13). Although PDE5 inhibition has been used to treat PHT, other PDE isoforms may provide unique targets for treatment of the disease.
By contrast with results obtained in cells from experimental animals, previous studies of PDEs in human PASMCs, in particular with respect to PHT, have largely ignored the role of individual PDE isoforms (19, 24). We therefore sought to 1) define the expression of PDEs in human PASMC from normal subjects and patients with IPAH and SPH and 2) investigate whether altered PDE expression has a functional effect on cAMP accumulation and proliferation of PASMC. The results define a role for specific PDE isoforms, particularly isoforms that hydrolyze cAMP, in regulating the function of PASMC and blunting abnormal pathophysiology in PHT.
MATERIALS AND METHODS
All chemicals were purchased from Sigma-Aldrich with the exception of those outlined in the methods below and the following radioisotopes, which were obtained from Amersham Biosciences: [3H]cGMP, [3H]cAMP, [3H]thymidine, and 125I-cAMP.
PASMC isolation and culture.
PASMC were isolated from patients after lung/heart transplantation, as described previously (33), after University of California San Diego Institutional Review Board approval and informed consent by each subject. The mean PAP was 51 and 53 mmHg for the IPAH patients (a 57-yr-old woman and a 31-yr-old man, IPAH-1 and IPAH-2, respectively) and 26 and 33 mmHg for the SPH patients (a 69-yr-old man with idiopathic pulmonary fibrosis and a 58-yr-old woman with emphysema, SPH-1 and SPH-2, respectively). PASMC from a normal subject (a 49-yr-old man with normal PAP) were isolated as outlined above or obtained commercially from Cambrex. PASMC were grown in 5% CO2-95% air at 37°C in smooth muscle growth medium (Cambrex), composed of smooth muscle basal medium (SMBM), 5% fetal bovine serum (FBS), 0.5 ng/ml human recombinant epidermal growth factor, 2 ng/ml human recombinant fibroblast growth factor, 5 μg/ml insulin, and 50 μg/ml each of gentamicin and amphotericin-B. PASMC were used in experiments between the fourth and sixth passages, throughout which time no changes in cell morphology were noted. In each experiment the passages of the control and patient cells were matched. Cell viability was measured by Trypan blue staining.
RT-PCR and real-time PCR.
RNA was isolated from PASMC (RNeasy mini kit; Qiagen) from which cDNA was synthesized (Superscript first-strand synthesis system for RT-PCR; Invitrogen). Both PCR (MJ Research PTC-100 using Platinum Taq DNA polymerase) and real-time PCR (MJ Research Opticon 2 using Eurogentec QPCR Mastermix Plus SYBR green kit) were performed using 100 ng of cDNA and 0.05 μM sense/antisense primers; two primer sets were designed for each PDE isoform (Table 1). Cycle conditions were 95°C for 10 min (1 cycle), 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s (40 cycles). PCR products were separated by gel electrophoresis and sequenced (Eton Bioscience). Real-time data were normalized to RNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Western blot analysis.
Protein samples from PASMC (10 μg) were separated by gel electrophoresis and immunoblotted using the NuPage gel system (Invitrogen) following the manufacturer's instructions and visualized using horseradish peroxidase and enhanced chemiluminescence. Antibodies used included PDE1A, PDE1C, PDE4C (Fabgennix), PDE3A, PDE3B (Santa Cruz Biotechnology), and PDE5A (Cell Signaling). GAPDH (Abcam) was used to normalize loading.
PDE activity was determined using 1 μM cyclic nucleotide as substrate via a two-step radioassay procedure adapted from Thompson and Appleman (29). Briefly, substrate and protein sample were incubated over a period of time that PDE activity was linear, after which they were boiled for 2 min to terminate the reaction. Results are expressed relative to the protein concentration. To identify the contribution of activity of specific PDEs, we performed assays in the presence of specific PDE inhibitors [PDE1, 30 μM vinpocetine, 30 μM 8-methoxy-methyl-3-isobutyl-1-methylxanthine (8-MM-IBMX); PDE2, 10 μM erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) in the presence of excess cGMP; PDE3, 10 μM milrinone; and PDE4, 10 μM rolipram] and with or without calcium in the presence of EGTA.
cAMP accumulation was measured as previously described (16). PASMC were equilibrated in serum and NaHCO3-free Dulbecco's modified Eagle's medium (pH 7.4) supplemented with 20 mM HEPES for 30 min at 37°C and then incubated with stimulatory agonists for 10 min in the absence and presence of PDE inhibitors (added 20 min before the addition of agonists). Reactions were terminated by aspiration of the medium and addition of 250 μl of cold 7.5% (wt/vol) trichloroacetic acid (TCA). cAMP content in TCA extracts was determined by radioimmunoassay and normalized to the amount of protein per well.
Cell proliferation assay.
[3H]Thymidine incorporation was used to assess DNA synthesis. PASMC were seeded in six-well plates, grown to 70% confluence, and then serum-starved with SMBM for 24 h. SMBM was replaced with SMBM containing 5% FBS or 10 ng/ml platelet-derived growth factor-BB (PDGF-BB) in addition to 1 μCi [3H]thymidine for 48 h with or without PDE inhibitors. Wells were washed with cold PBS and twice with cold 7.5% TCA, and then the content of each well was dissolved in 0.5 M NaOH before liquid scintillation counting.
Measurement of cytosolic Ca2+ concentration.
Calcium was measured using fura-2, as described previously (28). Cytosolic Ca2+ concentration ([Ca2+]cyt) in single human PASMC was measured using the Ca2+-sensitive fluorescent indicator fura-2 AM. Cells on 25-mm coverslips were loaded with fura-2 AM (3 μM) for 30 min in the dark at room temperature (22–24°C) under an atmosphere of 5% CO2-95% air. The fura-2 AM-loaded cells were then transferred to a perfusion chamber on the microscope stage and superfused with modified Krebs solution (MKS) for 30 min to remove extracellular dye and allow intracellular esterases to cleave cytosolic fura-2 AM into active fura-2. The MKS contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, pH 7.4. In Ca2+-free MKS, CaCl2 was replaced by equimolar MgCl2, and 0.1 mM EGTA was added to chelate residual Ca2+. Fura-2 fluorescence (510-nm light emission excited by 340- and 380-nm illuminations) from the cells, as well as background fluorescence, was collected at room temperature (22°C) using a ×40 Nikon UV-Fluor objective and a charge-coupled device camera. The fluorescence signals emitted from the cells were monitored using an Intracellular Imaging fluorescence microscopy system and recorded on a computer for later analysis. Multiple cells were imaged in a single field.
Three siRNA sequences designed to target PDE1C and a negative scrambled small interference RNA (siRNA) sequence were purchased from Ambion (2 of the siRNA sequences significantly reduced PDE1C protein expression; the 3rd was not used in further experiments). PASMC were grown to ∼70% confluence in six-well plates, after which 1.5 μg of siRNA was transfected using a GenePORTER 2 transfection kit (Gene Therapy Systems) according to the manufacturer's instructions. In brief, after the siRNA was incubated in diluent B for 5 min at room temperature, GenePORTER was added and incubated for a further 10 min. The GenePORTER 2/DNA complex was then added directly to the well to give a final volume of 1 ml with culture medium. Cells were collected 48 h after siRNA treatment. siRNA transfection efficiency was determined using fluorescently labeled siRNA (Ambion).
Data are expressed as means ± SE. Statistical comparisons between two populations were performed using paired and unpaired Student's t-tests where appropriate or using one-way ANOVA in combination with Tukey's post hoc test to compare differences between multiple groups, with a probability value of P < 0.05 considered to be significant. All experiments were preformed at least in triplicate with each sample from each control, IPAH, or SPH donor.
PDEs in PASMC: altered expression in patients with IPAH or SPH.
We found that PASMC express mRNA for multiple PDE isoforms: 1A, 1C, 2A, 3A, 3B, 4A, 4B, 4C, 4D, 5A, 7A, 7B, 8A, 9A, 9B, 10A, and 11A (data not shown). Neither PDE1B (absence confirmed by immunoblot, data not shown) nor PDE8B was detected in PASMC, but the primers were validated using rat brain mRNA. To ensure that our findings were not the result of differing primer efficiency, we designed a second set of primers for each PDE and obtained similar results.
Real-time PCR and immunoblotting demonstrated an increase in expression of four PDE isoforms in PASMC from patients with IPAH (Figs. 1A and 2) and SPH (Figs. 1B and 2) compared with control: PDE1A, PDE1C, PDE3B, and PDE5A with PDE1C showing the most prominent increase in mRNA. Total cAMP and cGMP PDE activity increased in PASMC from patients with IPAH (∼3-fold and ∼2-fold, respectively) and SPH (∼2.5-fold and ∼1.5-fold, respectively) compared with control (Fig. 3, A and B). Although mRNA for PDE2A and PDE7A was increased in PASMC from patients with IPAH and SPH, we did not observe increased protein levels for those isoforms (data not shown). Increases in PDE3A protein levels were inconsistent, and none of the PDE4 isoforms was significantly enhanced in patients with IPAH or SPH (Figs. 1 and 2). Using cAMP-PDE inhibitors [PDE1, 30 μM vinpocetine and 30 μM 8-MM-IBMX; PDE2, 10 μM EHNA in the presence of excess cGMP; PDE3, 10 μM milrinone; and PDE4, 10 μM rolipram, and the addition of excess calcium in the presence of EGTA (PDE1)], we calculated the relative contribution of each isoform to total cAMP PDE activity (Fig. 3C) and found that PDE1 and PDE3 activities were increased in PASMC from patients with IPAH and SPH compared with control and other cAMP-PDE isoforms.
Increased PDE expression and activity with IPAH and SPH decreases agonist-induced cAMP and contributes to an increase in proliferation and capacitative Ca2+ entry in PASMC.
To investigate the functional impact of increased PDE expression and activity, we added PDE inhibitors to assays of cAMP accumulation, cell proliferation, and capacitative Ca2+ entry [CCE; induced by passive store depletion by the sarco(endo)plasmic Ca2+-ATPase inhibitor cyclopiazonic acid (CPA)]. In the absence of a PDE inhibitor, forskolin (a direct AC activator) and beraprost (a PGI2 receptor agonist) stimulated substantially less accumulation of cAMP in both IPAH and SPH PASMC than in control cells (Fig. 4, A and C). Addition of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 200 μM) prominently enhanced cAMP accumulation in PASMC from IPAH and SPH such that inclusion of IBMX yielded cAMP generation in response to forskolin and beraprost that was not statistically different in the PHT PASMC compared with controls (Figs. 4, B and D, and 5; Table 2).
As shown in Fig. 5, both forskolin and beraprost (10 μM, 10 min) stimulated cAMP generation in control PASMC in both the presence and absence of IBMX compared with basal, but unless IBMX was present, neither agonist was able to elevate cAMP levels in PASMC from IPAH and SPH patients, thus indicating that IBMX increased both the efficacy and potency of the agonists in the stimulation of cAMP formation. Consistent with the patterns observed for increased expression of the PDE isoforms (Figs. 1 and 2), PDE1 and PDE3 inhibitors were more effective than PDE2 and PDE4 inhibitors in enhancing cAMP in response to forskolin in IPAH and SPH PASMC compared with controls (Fig. 5E). Because 8-MM-IBMX can also inhibit PDE5 at a concentration similar to that used in the above experiment (1), we tested 10 μM zaprinast (a PDE5 inhibitor) and found that it did not significantly increase cAMP in PASMC, implying that the effect of 8-MM-IBMX to enhance agonist-induced cAMP levels is due to inhibition of PDE1 and not PDE5. Vinpocetine, another PDE1 inhibitor, also increased the cAMP response in IPAH and SPH. Although vinpocetine also has been shown to inhibit PDE7B in addition to PDE1 (23), we concluded a role for PDE1 because of the effectiveness of both PDE1 inhibitors and because of the absence of an increase of PDE7B in IPAH PASMC (Fig. 1A). We conclude that IPAH and SPH PASMC have increased PDE1 and PDE3 activities that appear to be responsible for the blunted ability of these cells to increase cAMP levels.
Serum (5% FBS)- and PDGF (10 ng/ml)-stimulated [3H]thymidine incorporation was increased 3-fold and 1.5-fold in IPAH and SPH PASMC, respectively, and was consistent with the effect of IBMX on cAMP accumulation; this PDE inhibitor reduced [3H]thymidine incorporation to a greater extent in PASMC from patients than in controls (Fig. 6, A and B). Among several PDE subtype-selective inhibitors tested, those that blocked PDE1 and PDE3 were more effective in reducing serum-stimulated proliferation of IPAH and SPH PASMC compared with control PASMC (Fig. 6C).
We also examined the effect of increased PDE activity on store depletion-mediated Ca2+ influx or CCE in PASMC. In these experiments, CCE was induced by passive depletion of intracellular stores using 5 μM CPA. When cells were superfused with Ca2+-free solution, extracellular application of CPA transiently increased [Ca2+]cyt due to Ca2+ leakage from the sarcoplasmic reticulum. Approximately 15 min later (i.e., when the intracellular stores were depleted with CPA), restoration of extracellular Ca2+ caused an additional increase in [Ca2+]cyt due to Ca2+ influx through store-operated Ca2+ channels or CCE. As shown in Fig. 7A, the amplitude of CCE in PASMC from IPAH patients was significantly greater than in control PASMC from normal subjects (P < 0.05). Consistent with the cell proliferation results shown in Fig. 6, we found that CCE, which is higher in IPAH PASMC (28), could only be significantly reduced by forskolin if it was added in the presence of PDE inhibitor: 200 μM IBMX, 30 μM 8-MM-IBMX (PDE1), 10 μM milrinone (PDE3), or 10 μM rolipram (PDE4, Fig. 7).
Together these data suggest that PDE inhibitors, in addition to their contribution to lowering cAMP levels, are important in regulating cell proliferation and CCE in PASMC from IPAH and SPH patients.
Role of PDE1C: studies with siRNA.
On the basis of the prominent increase in expression of PDE1C mRNA in IPAH and SPH PASMC (Fig. 1) and the absence of a specific pharmacological inhibitor for this PDE isoform, we assessed the role of PDE1C by using siRNA to knockdown its expression. Figure 8A shows that treatment of PASMC with two different siRNAs designed to target PDE1C reduced its expression by 60–70%. Scrambled siRNA did not significantly reduce PDE1C expression; moreover, siRNA targeted to PDE1C did not reduce PDE1A expression. Incubation of cells with PDE1C siRNA helped restore forskolin-induced cAMP levels in IPAH and SPH PASMC (having a greater effect than in control PASMC) and decreased serum-stimulated proliferation to a greater extent than did the siRNA negative control (Fig. 8, B and C). Viability of PASMC was decreased ∼10% following transfection with either scrambled or PDE1C siRNA (Fig. 8D), thus demonstrating that the decrease in proliferation was not the result of increased cell death.
The data shown demonstrate that multiple PDE isoforms are expressed in human PASMC and that expression of certain of these (PDE1A, PDE1C, PDE3B and PDE5A) are increased in PASMC from patients with IPAH and SPH. In addition, incubation with PDE subtype-selective inhibitors reduced proliferation, CCE, and increased cAMP generation more effectively in PASMC from IPAH and SPH patients than from controls. Based on the consistent increase in PDE isoform expression that we observed in PASMC from patients with either IPAH or SPH, settings that have different initiating mechanisms, increased PDE isoform expression, although presumably a secondary consequence of PHT, likely contributes to pathophysiology.
RT-PCR and immunoblotting revealed that mRNA and protein for many PDE isoforms are expressed in human PASMC. Phillips et al. (19) demonstrated the profile of PDEs in PASMC isolated from rat proximal and distal pulmonary arteries and found, similar to our results, that PDE3 and PDE4 are principal PDE isoforms that contribute to cAMP PDE degradation. Our results indicate that changes in the relative contribution of PDE isoforms to total PDE activity accompany both IPAH and SPH, with increased contribution of PDE1 and PDE3 and a decreased role of PDE4 relative to control PASMC. Thus we found that PDE1 and PDE3 inhibitors were more effective in increasing cAMP generation, reducing CCE, and inhibiting proliferation in PASMC from patients with PHT than in PASMC from controls.
Previous studies with animal models of PHT and PASMC from patients with IPAH have suggested that PDE5 and PDE3 are elevated and involved in the hyperproliferation of PASMC (7, 9, 14, 18, 19, 24, 30). Our results support the idea that specific PDE subtypes, e.g., PDE3B and PDE5A, contribute to IPAH and SPH because their expression, particularly at the protein level, is substantially increased compared with that in control PASMC. PDE5 inhibitors can reduce proliferation in a manner that is thought to partially depend on cross talk with the cAMP signaling pathway via PDE3 inhibition (15, 30). Wharton et al. (30) demonstrated that inhibition of PDE5, through increasing cGMP, reduced DNA synthesis and stimulated apoptosis of human PASMC. Sildenafil, a selective PDE5 inhibitor, has been shown to reduce resting [Ca2+] in PASMCs isolated from chronic hypoxic rats (18). Our findings showing that use of zaprinast at concentrations specific for PDE5 inhibition did not significantly raise cAMP imply that the beneficial effects of PDE5 inhibition are primarily via an increase in cGMP and, thus, PDE inhibitors that raise cAMP may provide an alternative means to improve abnormal pathophysiology in IPAH and SPH.
We found that the most striking increase in both IPAH and SPH PASMC occurred in the mRNA of PDE1 family members. PDE1 is encoded by three separate genes, PDE1A, PDE1B (which appears to be absent in human PASMC), and PDE1C, which hydrolyzes cAMP and cGMP with different affinities and mediates, at least in part, the decrease in cAMP accumulation in response to an increase in intracellular [Ca2+] ([Ca2+]i) (3, 31, 32). Both PDE1A and PDE1B preferentially hydrolyze cGMP, whereas PDE1C hydrolyzes cAMP and cGMP with equal affinity (31). An increase in PDE1A expression has been shown to contribute to reduced response (“tolerance”) to drugs that increase NO; the PDE1 inhibitor vinpocetine is able to partially restore sensitivity of the vasculature to nitroglycerin (8, 27). Increased CCE and a generalized rise in [Ca2+]i also can lead to activation of PDE1A in other cell types, such as human astrocytoma cells (5). PDE1C is present in proliferating aortic smooth muscle cells but absent in intact human thoracic aorta, findings that suggest PDE1C contributes to the proliferative phenotype of vascular smooth muscle cells (21, 22). Such ideas are consistent with our data showing that treatment with PDE1C siRNA inhibits proliferation of PASMC, especially in cells from PHT patients. Overall, our results imply that PDE1, and in particular PDE1C, could be a useful therapeutic target for IPAH and SPH and for other disorders with increased PASMC proliferation. Increased PDE1C expression has not been implicated in the proliferation of smooth muscle cells in species other than human (20, 21), thus making it difficult to study the importance of this PDE isoform in PHT using animal models. PDE1 family members may provide cross talk between increased [Ca2+]i and decreased levels of cyclic nucleotides with PHT; accordingly, PDE1 inhibition, perhaps in particular that of PDE1C, might be a therapeutic approach to inhibit the enhanced smooth muscle proliferation of PHT.
Because PDE1A, PDE1C, and PDE3B are not only found in the pulmonary circulation, inhibitors of these PDEs have the potential to lead to side effects such as systemic hypotension. PDE3 has long been thought to play a role in PHT; in fact, PDE3 inhibitors have been shown effective in both preventing and reversing the development of PHT in experimental models (7, 14, 19, 24). Even though PDE3 inhibitors were effective in decreasing PVR, fatal cardiovascular side effects occurred and led to their withdrawal from clinical use (17, 20). Would it be possible to use low concentrations of specific PDE1 and PDE3 inhibitors, perhaps in combination with other approved therapies for PHT (even a PDE5 inhibitor), as a means to decrease blood pressure in the pulmonary circulation without producing systemic effects? This possibility is consistent with evidence from animal models of PHT in which subthreshold doses of PDE1 and PDE3 inhibitors can increase cAMP-dependent vasodilation in response to inhaled prostanoids without affecting systemic blood pressure (4, 24, 25). Our findings that identify increases in PDE1A and PDE1C mRNA and protein and total PDE1 activity in both IPAH and SPH PASMC, as well as efficacy of PDE1C siRNA in reducing PASMC proliferation, suggest a potentially important novel role for PDE1 in PHT. Thus development of more selective pharmacological inhibitors or appropriately targeted PDE1 isoform-specific genetic antagonists might be unique therapeutic approaches for the rational, pathophysiologically targeted treatment of PHT.
In conclusion, the current study provides new evidence for a biochemically and physiologically important role of several PDE isoforms in PASMC from patients with IPAH and SPH. Based on its high level of expression (Fig. 1), PDE1C appears to be a particularly useful marker of remodeling in the pulmonary circulation. The use of inhibitors targeted to cAMP-hydrolyzing PDEs whose expression is increased in PHT has the potential to provide a means to regulate the magnitude and duration of cAMP levels and response, and thereby to produce beneficial effects in PASMC. PDE1-selective inhibitors appear to be particularly attractive as novel therapeutics to attenuate pathophysiological abnormalities that occur in PHT.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-66941 and HL-64045 and American Lung Association Grant RT-9094-N.
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