Rapid and Efficient Production of Coronary Artery Ligation and Myocardial Infarction in Mice Using Surgical Clips

Aims

The coronary artery ligation model in rodents mimics human myocardial infarction (MI). Normally mechanical ventilation and prolonged anesthesia period are needed. Recently, a method has been developed to create MI by popping-out the heart (without ventilation) followed by immediate suture ligation. Mortality is high due to the time-consuming suture ligation process while the heart is exposed. We sought to improve this method and reduce mortality by rapid coronary ligation using a surgical clip instead of a suture.

Methods and Results

Mice were randomized into 3 groups: clip MI (CMI), suture MI (SMI), or sham (SHAM). In all groups, heart was manually exposed without intubation through a small incision on the chest wall. Unlike the conventional SMI method, mice in the CMI group received a metal clip on left anterior descending artery (LAD), quickly dispensed by an AutoSuture Surgiclip^™. The CMI method took only 1/3 of ligation time of the standard SMI method and improved post-MI survival rate. TTC staining and Masson’s trichrome staining revealed a similar degree of infarct size in the SMI and CMI groups. Echocardiograph confirmed that both SMI and CMI groups had a similar reduction of ejection fraction and fraction shortening over the time. Histological analysis showed that the numbers of CD68⁺ macrophages and apoptotic cells (TUNEL-positive) are indistinguishable between the two groups.

Conclusion

This new method, taking only less than 3 minutes to complete, represents an efficient myocardial infarction model in rodents.     

Sildenafil promotes adipogenesis through a PKG pathway

Sildenafil is the first oral PDE5 inhibitor for the treatment of erectile dysfunction and pulmonary arterial hypertension. In the present study, we investigated the effect of sildenafil on adipogenesis in 3T3L1 preadipocytes. Treatment with sildenafil for 8 days significantly promoted adipogenesis characterized by increased lipid droplet and triglyceride content in 3T3L1 cells. Meanwhile, sildenafil induced a pronounced up-regulation of the expression of adipocyte-specific genes, such as aP2 and GLUT4. The results by RT-PCR and Western blotting further showed that sildenafil increased the sequential expression of C/EBPβ, PPARγ and C/EBPα. Additionally, we found that the other two PDE5 inhibitors (vardenafil and tadalafil) and the cGMP analog 8-pCPT-cGMP also increased adipogenesis. Likewise, 8-pCPT-cGMP could up-regulate the expression of adipogenic and adipocyte-specific genes. Importantly, the PKG inhibitor Rp-8-pCPT-cGMP was able to inhibit both sildenafil and 8-pCPT-cGMP-induced adipogenesis. Furthermore, sildenafil promoted basal and insulin-mediated glucose uptake in 3T3L1 cells, which was counteracted by Rp-8-pCPT-cGMP. These results indicate that sildenafil could promote adipogenesis accompanied by increased glucose uptake through a PKG pathway at least partly.     

The effects of PDE5 inhibitory drugs on renal ischemia/reperfusion injury in rats

The aim of the present study was to evaluate the effects of phosphodiesterase type 5 (PDE5) inhibitory drugs, Tadalafil and Sildenafil, on inducible NOS (iNOS), endothelial NOS (eNOS) and p53 genes expressions and apoptosis in ischemia/reperfusion (I/R) induced oxidative injury in rat renal tissue. Eighty Sprague-Dawley rats (300-350?g) were divided into four groups. In ischemia/reperfusion group, rats were subjected to renal ischemia by clamping the left pedicle for 60?min, and then reperfused for 90?min. On the other hand, in other two groups the rats were individually pretreated with Tadalafil and Sildenafil 1?h before the induction of ischemia. Malondialdehyde (MDA) is determined in renal tissue homogenates by high-performance liquid chromatography, the number of apoptotic cell were calculated by TUNEL method and p53 and eNOS expression were detected with immunohistochemistry. On the other hand, myeloperoxidase (MPO) levels were measured by spectrophotometric method and the mRNA level of iNOS in renal tissue was determined by Real-time PCR (RT-PCR). Our results indicate that MDA and MPO levels were increased in the I/R group than those in the control group. Both Tadalafil and Sildenafil treatment decreased the MDA levels in ischemia/reperfusion group, whereas this effect was more potent with Sildenafil. RT-PCR results showed that, iNOS gen expression increased in the I/R group, but decreased in the PDE5 inhibitory drugs treated group. Apoptotic cells, eNOS levels and p53 positive cells were also decreased in PDE5 inhibitory drugs treated group. We suggest that Tadalafil and Sildenafil have beneficial effects against I/R related renal tissue injury and this protective effect is clearer for Sildenafil than Tadalafil.     

Effect of sildenafil on renin secretion in human subjects

Sildenafil is a potent and selective inhibitor of the cyclic GMP-specific phosphodiesterase (PDE5) that is very effective in the treatment of male impotence. It inhibits breakdown of cyclic guanosine monophosphate (cGMP) formed in penile smooth muscle cells in response to stimulation by nitric oxide resulting in muscle relaxation. PDE5 is widely distributed in the body, being present in the vasculature, platelets, and kidneys. In the kidney, PDE5 is involved in the regulation of sodium excretion and renin secretion. The aim of the present investigation was to investigate the effect of sildenafil, in doses used clinically, on renin secretion in human subjects. The studies were performed in two groups of healthy normotensive subjects: one in which sodium intake was unrestricted, and one in which sodium intake was restricted to 600 mg/day. Blood pressure and heart rate were monitored throughout the study, and blood samples for the measurement of plasma cGMP and cAMP concentrations and plasma renin activity (PRA) were collected. After control measurements, the subjects ingested a capsule containing sildenafil or placebo. Cardiovascular measurements and blood sampling continued for the next 120 min. Sildenafil had only minor cardiovascular effects. Diastolic pressure tended to be lower and heart rate was generally higher after sildenafil than after placebo, but the differences were small. Sildenafil caused a prompt and sustained increase in plasma cGMP concentration and a more gradual increase in plasma cAMP concentration. After the subjects received placebo, there was a progressive decrease in PRA during the 2-hr observation period, presumably reflecting the circadian rhythm in renin secretion. In contrast, PRA failed to decrease after the subjects received sildenafil, thus indicating that sildenafil exerts a stimulatory action on renin secretion. This action on renin secretion may help explain why sildenafil only has minor effect on blood pressure despite the widespread distribution of PDE5 in vascular tissues.     

Sildenafil citrate induces migration of mouse aortic endothelial cells and proteinase secretion

Vascular endothelial cells release proteinases that degrade the extracellular matrix (ECM), thus enabling cell migration during angiogenesis and vasculogenesis. Sildenafil citrate stimulates the nitric oxide-cyclic guanosine monophosphate pathway through inhibition of phosphodiesterase type V (PDE5). In this report, we examined the mechanisms underlying sildenafil citrate-induced cell migration using cultured mouse aortic endothelial cells (MAECs). Sildenafil citrate induced migration and proteinase secretion by murine endothelial cells. Sildenafil citrate induced the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9, which is inhibited by NF-κB inhibitors. Sildenafil citrate also induced the secretion of plasmin, which is inhibited by Pl 3′-kinase inhibitors. It is suggested that sildenafil citrate-induced migrating activity in endothelial cells may be accomplished by increased secretion of proteinases.     

Sildenafil-mediated acute cardioprotection is independent of the NO/cGMP pathway

Sildenafil, a potent inhibitor of phosphodiesterase type 5, has recently been investigated in animal models of myocardial ischemia-reperfusion (MI/R) injury. Previous studies have suggested that the protective effects of sildenafil are mediated via activation of endothelial nitric oxide (NO) synthesis (eNOS) and inducible NOS (iNOS). To further investigate the protective mechanism of sildenafil, we subjected wild-type, eNOS, and iNOS null animals to 30 min of myocardial ischemia and 24 h of reperfusion. Treatment with 0.06 mg/kg sildenafil 5 min before reperfusion significantly reduced myocardial infarct size in wild-type, eNOS null mice (eNOS(-/-)), and iNOS(-/-) animals. Additionally, the low dose utilized in this study did not alter myocardial cGMP. These results suggest that acute low-dose sildenafil-mediated cardioprotection is independent of eNOS, iNOS, and cGMP. In a second series of experiments, we investigated sildenafil in db/db diabetic mice subjected to MI/R. We found that sildenafil failed to protect diabetic mice against MI/R. However, NO(.) donor therapy was found to significantly protect against MI/R injury in both nondiabetic and diabetic mice, suggesting that protection could be conferred in diabetic mice and that the upstream modulator of soluble guanylyl cyclase, NO(.), may mediate protection independent of cGMP signaling. The present study suggests that further research is needed to delineate the precise mechanisms by which sildenafil exerts cardioprotection.     

A cDNA microarray assessment of gene expression in the liver of rainbow trout (Oncorhynchus mykiss) in response to a handling and confinement stressor

A purpose-designed microarray platform (Stressgenes, Phase 1) was utilised to investigate the changes in gene expression within the liver of rainbow trout during exposure to a prolonged period of confinement. Tissue and blood samples were collected from trout at intervals up to 648 h after transfer to a standardised confinement stressor, together with matched samples from undisturbed control fish. Plasma ACTH, cortisol, glucose and lactate were analysed to confirm that the neuroendocrine response to confinement was consistent with previous findings and to provide a phenotypic context to assist interpretation of gene expression data. Liver samples for suppression subtractive hybridisation (SSH) library construction were selected from within the experimental groups comprising “early” stress (2–48 h) and “late” stress (96–504 h). In order to reduce redundancy within the four SSH libraries and yield a higher number of unique clones an additional subtraction was carried out. After printing of the arrays a series of 55 hybridisations were executed to cover 6 time points. At 2 h, 6 h, 24 h, 168 h and 504 h 5 individual confined fish and 5 individual control fish were used with control fish only at 0 h. A preliminary list of 314 clones considered differentially regulated over the complete time course was generated by a combination of data analysis approaches and the most significant gene expression changes were found to occur during the 24 h to 168 h time period with a general approach to control levels by 504 h. Few changes in expression were apparent over the first 6 h. The list of genes whose expression was significantly altered comprised predominantly genes belonging to the biological process category (response to stimulus) and one cellular component category (extracellular region) and were dominated by so-called acute phase proteins. Analysis of the gene expression profile in liver tissue during confinement revealed a number of significant clusters. The major patterns comprised genes that were up-regulated at 24 h and beyond, the primary examples being haptoglobin, β-fibrinogen and EST10729. Two representative genes from each of the six k-means clusters were validated by qPCR. Correlations between microarray and qPCR expression patterns were significant for most of the genes tested. qPCR analysis revealed that haptoglobin expression was up-regulated approximately 8-fold at 24 h and over 13-fold by 168 h.     

Effect of PDE5 inhibition on coronary hemodynamics in pacing-induced heart failure

Inhibition of phosphodiesterase type 5 (PDE5) can relax systemic and coronary vessels by causing accumulation of cGMP. Both the endothelial dysfunction with decreased nitric oxide production and increased natriuretic peptide levels in congestive heart failure (CHF) have the potential to alter cGMP production, thereby influencing the response to PDE5 inhibition. Consequently, this study examined the effects of PDE5 inhibition with sildenafil in dogs with CHF produced by rapid ventricular pacing. CHF resulted in decreases of left ventricular (LV) systolic pressure, coronary blood flow, and the maximal first time derivative of LV pressure (LV dP/dt(max)) at rest and during treadmill exercise compared with normal, whereas resting LV end-diastolic pressure increased from 10 +/- 1.4 to 23 +/- 1.4 mmHg. Sildenafil (2 and 10 mg/kg per os) caused a 5- to 6-mmHg decrease of aortic pressure (P < 0.05), with no change of heart rate, LV systolic pressure, or LV dP/dt(max). Sildenafil caused no change in coronary flow or myocardial oxygen consumption in animals with CHF at rest or during exercise. In contrast to findings in normal animals, sildenafil did not augment endothelium-dependent coronary vasodilation in response to acetylcholine in animals with CHF. Furthermore, Western blotting showed decreased PDE5 protein expression in myocardium from failing hearts. These findings demonstrate that PDE5 contributes little to regulation of coronary hemodynamics in CHF.     

Protein kinase G-dependent cardioprotective mechanism of phosphodiesterase-5 inhibition involves phosphorylation of ERK and GSK3beta

Sildenafil, a potent inhibitor of phosphodiesterase-5 (PDE-5) induces powerful protection against myocardial ischemia-reperfusion injury. PDE-5 inhibition increases cGMP levels that activate cGMP-dependent protein kinase (PKG). However, the cause and effect relationship of PKG in sildenafil-induced cardioprotection and the downstream targets of PKG remain unclear. Adult ventricular myocytes were treated with sildenafil and subjected to simulated ischemia and reoxygenation. Sildenafil treatment significantly decreased cardiomyocyte necrosis and apoptosis. The PKG inhibitors, KT5823, guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chloro-phenylthio) (R(p)-8-pCPT-cGMPs), or DT-2 blocked the anti-necrotic and anti-apoptotic effect of sildenafil. Selective knockdown of PKG in cardiomyocytes with adenoviral vector containing short hairpin RNA of PKG also abolished sildenafil-induced protection. Furthermore, intra-coronary infusion of sildenafil in Langendorff-isolated mouse hearts prior to ischemia-reperfusion significantly reduced myocardial infarct size after 20 min ischemia and 30 min reperfusion, which was abrogated by KT5823. Sildenafil significantly increased PKG activity in intact hearts and cardiomyocytes. Sildenafil also enhanced the Bcl-2/Bax ratio, phosphorylation of Akt, ERK1/2, and glycogen synthase kinase 3beta. All these changes (except Akt phosphorylation) were significantly blocked by KT5823 and short hairpin RNA of PKG. These studies provide the first evidence for an essential role of PKG in sildenafil-induced cardioprotection. Moreover, our results demonstrate that sildenafil activates a PKG-dependent novel signaling cascade that involves activation of ERK and inhibition of glycogen synthase kinase 3beta leading to cytoprotection.     

Sildenafil potentiates nitric oxide mediated inhibition of human platelet aggregation

Nitric oxide (NO) inhibits platelet aggregation primarily via a cyclic 3'5'-guanosine monophosphate (cGMP)-dependent process. Sildenafil is a phosphodiesterase type 5 (PDE5) inhibitor that potentiates NO action by reducing cGMP breakdown. We hypothesised that sildenafil would augment the inhibitory effects of NO on in vitro platelet aggregation. After incubation with sildenafil or the soluble guanylate cyclase inhibitor H-(1,2,4)oxadiazolo(4,3-a)quinoxallin-1-one (ODQ), collagen-mediated human platelet aggregation was assessed in the presence of two NO donors, the cGMP-dependent sodium nitroprusside (SNP) and the cGMP-independent diethylamine diazeniumdiolate (DEA/NO). SNP and DEA/NO caused a concentration-dependent inhibition of platelet aggregation. ODQ inhibited and sildenafil augmented the effect of SNP, and to a lesser extent the effect of DEA/NO. We conclude that sildenafil potentiates NO-mediated inhibition of platelet aggregation through blockade of cGMP metabolism and that PDE5 inhibitors may have important antiplatelet actions relevant to the prevention of cardiovascular disease.     

3D-QSAR studies on sildenafil analogues, selective phosphodiesterase 5 inhibitors

Sildenafil, one of selective phosphodiesterase 5 (PDE5) inhibitors, is a widely used oral agent for the treatment of erectile dysfunction. To develop new PDE5 inhibitors with improved therapeutic efficacy, a series of sildenafil analogues have been prepared and their in vitro PDE5 inhibitory activities were evaluated. Their IC(50) values ranged from 423 to 0.05 nM. Herein, the results of 3D-QSAR (CoMFA and CoMSIA) analyses on these inhibitors are reported. Both CoMFA and CoMSIA gave reliable models with q(2) values >0.75 and r(2) values >0.99. The resulting CoMFA and CoMSIA models reveal a good correlation between the contour maps and the active site residues critical for the interaction with inhibitor, and nicely predict the key structural features of new analogues with improved activity and selectivity.     

The effect of selective phosphodiesterase inhibitors,alone and in combination,on a murine model of allergic asthma

BackgroundThe anti-inflammatory effects of the selective phosphodiesterase (PDE) inhibitors cilostazol (PDE 3), RO 20-1724 (PDE 4) and sildenafil (PDE 5) were examined in a murine model of allergic asthma. These compounds were used alone and in combination to determine any potential synergism, with dexamethasone included as a positive control.MethodsControl and ovalbumin sensitised Balb/C mice were administered orally with each of the possible combinations of drugs at a dose of 3 mg/Kg for 10 days.ResultsWhen used alone, RO 20-1724 significantly reduced eosinophil influx into lungs and lowered tumour necrosis factor-α, interleukin-4 and interleukin-5 levels in the bronchoalveolar lavage fluid when compared to untreated mice. Treatment with cilostazol or sildenafil did not significantly inhibit any markers of inflammation measured. Combining any of these PDE inhibitors produced no additive or synergistic effects. Indeed, the anti-inflammatory effects of RO 20-1724 were attenuated by co-administration of either cilostazol or sildenafil.ConclusionsThese results suggest that concurrent treatment with a PDE 3 and/or PDE 5 inhibitor will reduce the anti-inflammatory effectiveness of a PDE 4 inhibitor.     

Sildenafil alters calcium signaling and vascular tone in pulmonary arteries from chronically hypoxic rats

Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been recently proposed as a therapeutic tool to treat or prevent pulmonary artery hypertension (PAHT). We thus studied the effect of sildenafil on both the calcium signaling of isolated pulmonary artery smooth muscle cells (PASMCs) and the reactivity of pulmonary artery (PA) obtained from chronic hypoxia (CH)-induced pulmonary hypertensive rats compared with control (normoxic) rats. CH rats were maintained in an hypobaric chamber (50.5 kPa) for 3 wk leading to full development of PAHT. Intracellular calcium concentration ([Ca2+]i) was measured in PASMCs loaded with the calcium fluorophore indo 1. Unlike in control rats, sildenafil (10-100 nM) decreased the resting [Ca2+]i value in PASMCs obtained from CH rats. In PASMCs from both control and CH rats, sildenafil concentration dependently inhibited the [Ca2+]i response induced by G-coupled membrane receptor agonists such as angiotensin II and phenylephrine but had no effect on the amplitude of the [Ca2+]i response induced by caffeine. Sildenafil (0.1 nM-1 microM) concentration dependently reduced basal PA tone that is present in CH rats and relaxed PA rings precontracted with phenylephrine in both control and CH rats. These data show that sildenafil is a potent pulmonary artery relaxant in CH rats and that it normalizes CH-induced increases in resting [Ca2+]i and basal tone. Consequently, pharmacological inhibition of sildenafil-sensitive PDE5 downregulates the Ca2+ signaling pathway involved in this model of pulmonary hypertension.     

Sildenafil is a strong activator of mammalian carbonic anhydrase isoforms I–XIV

Sildenafil citrate, a phosphodiesterase-5 (PDE5) inhibitor widely used for the treatment of erectile dysfunction was investigated for its interaction with the zinc-enzyme carbonic anhydrase (CA, EC 4.2.1.1), as it has in its molecule a piperazine moiety also found in some CA activators (CAAs). Sildenafil was a potent, low micromolar activator of several CA isozymes, such as CA I, VA and VI (K_As in the range of 1.08–6.54 μM), and activated slightly less the isoforms CA III, IV and VA (K_As of 13.4–16.8 μM). CA isozymes II, IX, XIII and XIV showed activation constants in the range of 27.5–34.0 μM, whereas the least activated isoforms were CA VII and XII (K_As of 72.9–73.0 μM). Sildenafil citrate was also given orally to Sprague-Dawley rats at 1 mg/kg body weight. Red blood cell CA activity was inhibited in the treated animals at 3–5 h post-administration (in the range of 60–85%), probably due to NO/nitrite formed by PDE5 inhibition or by another, unknown mechanism. Whether CA activation by sildenafil has clinical consequences in humans is beyond the scope of the present work and warrants further studies.     

Effects of Sildenafil on the Gastrocnemius and Cardiac Muscles of Rats in a Model of Prolonged Moderate Exercise Training

Moderate exercise training improves energetic metabolism, tissue perfusion and induces cardiac and skeletal muscle remodeling. Sildenafil, a potent phosphodiesterase-5 inhibitor used to treat erectile dysfunction, reduces infarct size and increases tissue oxygenation in experimental models of cardiovascular disease. We have evaluated the effects of prolonged moderate exercise training and a repeat administration of sildenafil on the rat gastrocnemius and cardiac muscles. Animals were divided into two groups: sedentary and trained. Each group was subdivided into animals treated with vehicle or with two doses of sildenafil (10 or 15 mg/kg/day) during the last week of training. Physical exercise did not induce cardiac hypertrophy, whereas it increased mRNA levels of the PGC-1α, HIF-1α and VEGF genes, which are involved in mitochondrial biogenesis and angiogenesis, and reduced mRNA levels of FoxO3a, MuRF-1 and Atrogin-1. Sildenafil dose-dependently promoted both angiogenesis, as shown by increased capillary density, and muscle atrophy, as shown by muscle fibre size. These effects were more pronounced in trained animals. Our data confirm the beneficial effects of a moderate and prolonged training on cardiovascular and skeletal systems and document the positive and negative effects of sildenafil on these tissues at doses higher than those used in clinical practice. This report may impact on the use of sildenafil as a substance able to influence sports performance.     

Conformational conversion of PDE5 by incubation with sildenafil or metal ion is accompanied by stimulation of allosteric cGMP binding

Phosphodiesterase-5 (PDE5) is a dimer containing a cGMP-specific catalytic domain and an allosteric cGMP-binding subdomain (GAF A) on each subunit. PDE5 exhibits three conformational forms that can be separated by Native PAGE and are denoted as Bands 1, 2, and 3 in decreasing order of mobility. A preparation comprised mainly of Band 2 PDE5 was partially converted to Band 3 PDE5 by 1 h incubation with cGMP or the PDE5-specific inhibitors sildenafil, vardenafil, or tadalafil, but not with cAMP, milrinone (PDE3-specific), or rolipram (PDE4-specific). Band 2 PDE5 was converted almost entirely to Band 3 PDE5 by overnight incubation with sildenafil at 30 °C. This time-dependent conversion was accompanied by a 7-fold increase in allosteric cGMP-binding activity, suggesting that Band 3 PDE5 is a much more active form than Band 2 PDE5 for allosteric cGMP binding. Conversion of Band 2 PDE5 to Band 3 PDE5 occurred faster by pre-incubation with cGMP, which binds to both the allosteric and catalytic sites of PDE5, than with catalytic site-specific sildenafil. Overnight incubation of a Band 2/Band 3 PDE5 mixture with EDTA caused time-dependent conversion to Band 1 PDE5 (apoenzyme), and this conversion was accompanied by a 50% loss in cGMP-binding activity. After incubation with EDTA, addition of Mn⁺⁺ or Mg⁺⁺ caused reversion of Band 1 to a Band 2/Band 3 PDE5 mixture in which Band 3 PDE5 predominated. This reversion was accompanied by a 3-fold increase in allosteric cGMP-binding activity. The combination of results implied that physiological conversion of Band 2 to Band 3 PDE5 by cGMP and/or divalent metal ion occupancy of the catalytic domain would increase allosteric cGMP binding to the enzyme. This conversion would produce a greater negative feedback effect on cGMP action by increasing sequestration of cGMP at the allosteric cGMP-binding site of PDE5 and by increasing cGMP degradation at the catalytic site of the enzyme. This conversion would also increase PDE5 inhibitor binding to the enzyme.     

Lung vasodilatory response to inhaled iloprost in experimental pulmonary hypertension: amplification by different type phosphodiesterase inhibitors

Inhaled prostanoids and phosphodiesterase (PDE) inhibitors have been suggested for treatment of severe pulmonary hypertension. In catheterized rabbits with acute pulmonary hypertension induced by continuous infusion of the stable thromboxane analogue U46619, we asked whether sildenafil (PDE1/5/6 inhibitor), motapizone (PDE3 inhibitor) or 8-Methoxymethyl-IBMX (PDE1 inhibitor) synergize with inhaled iloprost. Inhalation of iloprost caused a transient pulmonary artery pressure decline, levelling off within <20 min, without significant changes in blood gases or systemic hemodynamics. Infusion of 8-Methoxymethyl-IBMX, motapizone and sildenafil caused each a dose-dependent decrease in pulmonary artery pressure, with sildenafil possessing the highest efficacy and at the same time selectivity for the pulmonary circulation. When combining a per se ineffective dose of each PDE inhibitor (200 μg/kg × min 8-Methoxymethyl-IBMX, 1 μg/kg × min sildenafil, 5 μg/kg × min motapizone) with subsequent iloprost nebulization, marked amplification of the prostanoid induced pulmonary vasodilatory response was noted and the area under the curve of P_PA reduction was nearly threefold increased with all approaches, as compared to sole iloprost administration. Further amplification was achieved with the combination of inhaled iloprost with sildenafil plus motapizone, but not with sildenafil plus 8MM-IBMX. Systemic hemodynamics and gas exchange were not altered for all combinations. We conclude that co-administration of minute systemic doses of selective PDE inhibitors with inhaled iloprost markedly enhances and prolongs the pulmonary vasodilatory response to inhaled iloprost, with maintenance of pulmonary selectivity and ventilation perfusion matching. The prominent effect of sildenafil may be operative via both PDE1 and PDE5, and is further enhanced by co-application of a PDE3 inhibitor.     

Mitigation of the progression of heart failure with sildenafil involves inhibition of RhoA/Rho-kinase pathway

Chronic inhibition of phosphodiesterase-5 with sildenafil immediately after permanent occlusion of the left anterior descending coronary artery was shown to limit ischemic heart failure (HF) in mice. To mimic a more clinical scenario, we postulated that treatment with sildenafil beginning at 3 days post-myocardial infarction (MI) would also reduce HF progression through the inhibition of the RhoA/Rho-kinase pathway. Adult male ICR mice with fractional shortening < 25% at day 3 following permanent left anterior descending coronary artery ligation were continuously treated with either saline (volume matched, ip, 2 times/day) or sildenafil (21 mg/kg, ip, 2 times/day) for 25 days. Echocardiography showed fractional shortening preservation and less left ventricular end-diastolic dilatation with sildenafil treatment compared with saline treatment at 7 and 28 days post-MI (P < 0.05). Both fibrosis and apoptosis, determined by Masson's trichrome and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), respectively, were attenuated in the sildenafil-treated mice (P < 0.05 vs. saline). Western blot analysis showed enchanced Bcl-2-to-Bax ratio with sildenafil treatment (P < 0.05 vs. saline). Activity assay showed sildenafil-mediated PKG activation 1 day after treatment (P < 0.05 vs. sham and saline). PKG activation was associated with sildenafil-mediated inhibition of Rho kinase (P < 0.05) compared with saline treatment, whereas PKG inhibition with KT-5823 abolished this inhibitory effect of sildenafil. In conclusion, for the first time, our findings show that chronic sildenafil treatment, initiated at 3 days post-MI, attenuates left ventricular dysfunction independent of its infarct-sparing effect, and this cardioprotection involves the inhibition of the RhoA/Rho-kinase pathway. Sildenafil may be a promising therapeutic tool for advanced HF in patients.