Xichen Wu

Long-Term Treatment With Oral Sildenafil Is Safe and Improves Functional Capacity and Hemodynamics in Patients With Pulmonary Arterial Hypertension

Background—The prognosis and functional capacity of patients with pulmonary arterial hypertension (PAH) is poor, and there is a need for safe, effective, inexpensive oral treatments. A single dose of sildenafil, an oral phosphodiesterase type-5 (PD-5) inhibitor, is an effective and selective pulmonary vasodilator in PAH. However, the long-term effects of PD-5 inhibition and its mechanism of action in human pulmonary arteries (PAs) are unknown. Methods and Results—We hypothesized that 3 months of sildenafil (50 mg orally every 8 hours) added to standard treatment would be safe and improve functional capacity and hemodynamics in PAH patients. We studied 5 consecutive patients (4 with primary pulmonary hypertension, 1 with Eisenmenger’s syndrome; New York Heart Association class II to III). Functional class improved by ≥1 class in all patients. Pretreatment versus posttreatment values (mean±SEM) were as follows: 6-minute walk, 376±30 versus 504±27 m, P <0.0001; mean PA pressure, 70±3 versus 52±3 mm Hg, P <0.007; pulmonary vascular resistance index 1702±151 versus 996±92 dyne · s · cm−5 · m−2, P <0.006. The systemic arterial pressure was unchanged, and no adverse effects occurred. Sildenafil also reduced right ventricular mass measured by MRI. In 7 human PAs (6 cardiac transplant donors and 1 patient with PAH on autopsy), we showed that PD-5 is present in PA smooth muscle cells and that sildenafil causes relaxation by activating large-conductance, calcium-activated potassium channels. Conclusion—This small pilot study suggests that long-term sildenafil therapy might be a safe and effective treatment for PAH. At a monthly cost of

Dichloroacetate Prevents and Reverses Pulmonary Hypertension by Inducing Pulmonary Artery Smooth Muscle Cell Apoptosis

492 Canadian, sildenafil is more affordable than most approved PAH therapies. A large multicenter trial is indicated to directly compare sildenafil with existing PAH treatments.

Endothelium-Derived Hyperpolarizing Factor in Human Internal Mammary Artery Is 11,12-Epoxyeicosatrienoic Acid and Causes Relaxation by Activating Smooth Muscle BKCa Channels

The pulmonary arteries (PA) in pulmonary arterial hypertension (PAH) are constricted and remodeled;. They have suppressed apoptosis, partly attributable to suppression of the bone morphogenetic protein axis and selective downregulation of PA smooth muscle cell (PASMC) voltage-gated K+ channels, including Kv1.5. The Kv downregulation-induced increase in [K+]i, tonically inhibits caspases, further suppressing apoptosis. Mitochondria control apoptosis and produce activated oxygen species like H2O2, which regulate vascular tone by activating K+ channels, but their role in PAH is unknown. We show that dichloroacetate (DCA), a metabolic modulator that increases mitochondrial oxidative phosphorylation, prevents and reverses established monocrotaline-induced PAH (MCT-PAH), significantly improving mortality. Compared with MCT-PAH, DCA-treated rats (80 mg/kg per day in drinking water on day 14 after MCT, studied on day 21) have decreased pulmonary, but not systemic, vascular resistance (63% decrease, P<0.002), PA medial thickness (28% decrease, P<0.0001), and right ventricular hypertrophy (34% decrease, P<0.001). DCA is similarly effective when given at day 1 or day 21 after MCT (studied day 28) but has no effect on normal rats. DCA depolarizes MCT-PAH PASMC mitochondria and causes release of H2O2 and cytochrome c, inducing a 10-fold increase in apoptosis within the PA media (TUNEL and caspase 3 activity) and decreasing proliferation (proliferating-cell nuclear antigen and BrdU assays). Immunoblots, immunohistochemistry, laser-captured microdissection-quantitative reverse-transcription polymerase chain reaction and patch-clamping show that DCA reverses the Kv1.5 downregulation in resistance PAs. In summary, DCA reverses PA remodeling by increasing the mitochondria-dependent apoptosis/proliferation ratio and upregulating Kv1.5 in the media. We identify mitochondria-dependent apoptosis as a potential target for therapy and DCA as an effective and selective treatment for PAH.

In Vivo Gene Transfer of the O2-Sensitive Potassium Channel Kv1.5 Reduces Pulmonary Hypertension and Restores Hypoxic Pulmonary Vasoconstriction in Chronically Hypoxic Rats

Background—Left internal mammary arteries (LIMAs) synthesize endothelium-derived hyperpolarizing factor (EDHF), a short-lived K+ channel activator that persists after inhibition of nitric oxide (NO) and prostaglandin synthesis. EDHF hyperpolarizes and relaxes smooth muscle cells (SMCs). The identity of EDHF in humans is unknown. We hypothesized that EDHF (1) is 11,12-epoxyeicosatrienoic acid (11,12-EET); (2) is generated by cytochrome P450-2C, CYP450-2C; and (3) causes relaxation by opening SMC large-conductance Ca2+-activated K+ channels (BKCa). Methods and Results—The identity of EDHF and its mechanism of action were assessed in 120 distal human LIMAs and 20 saphenous veins (SVs) obtained during CABG. The predominant EET synthesized by LIMAs is 11,12-EET. Relaxations to exogenous 11,12-EET and endogenous EDHF are of similar magnitudes. Inhibition of EET synthesis by chemically distinct CYP450 inhibitors (17-octadecynoic acid, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide), or a selective EET antagonist (4,15-epoxyeicosa-5(Z)-enoic acid) impairs EDHF relaxation. 11,12-EET activates a BKCa current and hyperpolarizes LIMA SMCs. Inhibitors of BKCa but not inward-rectifier or small-conductance KCa channels abolish relaxation to endogenous EDHF and exogenous 11,12-EET. BKCa and CYP450-2C mRNA and proteins are more abundant in LIMAs than in SVs, perhaps explaining the lack of EDHF activity of the SV. Laser capture microdissection and quantitative RT-PCR demonstrate that BKCa channels are primarily in vascular SMCs, whereas the CYP450-2C enzyme is present in both the endothelium and SMCs. Conclusions—In human LIMAs, EDHF is 11,12-EET produced by an EDHF synthase CYP450-2C and accounting for ≈40% of net endothelial relaxation. 11,12-EET causes relaxation by activating SMC BKCa channels.

Dynamin-Related Protein 1–Mediated Mitochondrial Mitotic Fission Permits Hyperproliferation of Vascular Smooth Muscle Cells and Offers a Novel Therapeutic Target in Pulmonary Hypertension

Background—Alveolar hypoxia acutely elicits pulmonary vasoconstriction (HPV). Chronic hypoxia (CH), despite attenuating HPV, causes pulmonary hypertension (CH-PHT). HPV results, in part, from inhibition of O2-sensitive, voltage-gated potassium channels (Kv) in pulmonary artery smooth muscle cells (PASMCs). CH decreases Kv channel current/expression and depolarizes and causes Ca2+ overload in PASMCs. We hypothesize that Kv gene transfer would normalize the pulmonary circulation (restore HPV and reduce CH-PHT), despite ongoing hypoxia. Methods and Results—Adult male Sprague-Dawley rats were exposed to normoxia or CH for 3 to 4 weeks and then nebulized orotracheally with saline or adenovirus (Ad5) carrying genes for the reporter, green fluorescent protein reporter±human Kv1.5 (cloned from normal PA). HPV was assessed in isolated lungs. Hemodynamics, including Fick and thermodilution cardiac output, were measured in vivo 3 and 14 days after gene therapy by use of micromanometer-tipped catheters. Transgene expression, measured by quantitative RT-PCR, was confined to the lung, persisted for 2 to 3 weeks, and did not alter endogenous Kv1.5 levels. Ad5-Kv1.5 caused no mortality or morbidity, except for sporadic, mild elevation of liver transaminases. Ad5-Kv1.5 restored the O2-sensitive K+ current of PASMCs, normalized HPV, and reduced pulmonary vascular resistance. Pulmonary vascular resistance decreased at day 2 because of increased cardiac output, and remained reduced at day 14, at which time there was concomitant regression of right ventricular hypertrophy and PA medial hypertrophy. Conclusions—Kv1.5 is an important O2-sensitive channel and potential therapeutic target in PHT. Kv1.5 gene therapy restores HPV and improves PHT. This is, to the best of our knowledge, the first example of K+ channel gene therapy for a vascular disease.

Preferential Expression and Function of Voltage-Gated, O2-Sensitive K+ Channels in Resistance Pulmonary Arteries Explains Regional Heterogeneity in Hypoxic Pulmonary Vasoconstriction: Ionic Diversity in Smooth Muscle Cells

Rationale: Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction caused, in part, by pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Mitochondrial fragmentation and normoxic activation of hypoxia-inducible factor-1&agr; (HIF-1&agr;) have been observed in PAH PASMCs; however, their relationship and relevance to the development of PAH are unknown. Dynamin-related protein-1 (DRP1) is a GTPase that, when activated by kinases that phosphorylate serine 616, causes mitochondrial fission. It is, however, unknown whether mitochondrial fission is a prerequisite for proliferation. Objective: We hypothesize that DRP1 activation is responsible for increased mitochondrial fission in PAH PASMCs and that DRP1 inhibition may slow proliferation and have therapeutic potential. Methods and Results: Experiments were conducted using human control and PAH lungs (n=5) and PASMCs in culture. Parallel experiments were performed in rat lung sections and PASMCs and in rodent PAH models induced by the HIF-1&agr; activator, cobalt, chronic hypoxia, and monocrotaline. HIF-1&agr; activation in human PAH leads to mitochondrial fission by cyclin B1/CDK1–dependent phosphorylation of DRP1 at serine 616. In normal PASMCs, HIF-1&agr; activation by CoCl2 or desferrioxamine causes DRP1-mediated fission. HIF-1&agr; inhibition reduces DRP1 activation, prevents fission, and reduces PASMC proliferation. Both the DRP1 inhibitor Mdivi-1 and siDRP1 prevent mitotic fission and arrest PAH PASMCs at the G2/M interphase. Mdivi-1 is antiproliferative in human PAH PASMCs and in rodent models. Mdivi-1 improves exercise capacity, right ventricular function, and hemodynamics in experimental PAH. Conclusions: DRP-1–mediated mitotic fission is a cell-cycle checkpoint that can be therapeutically targeted in hyperproliferative disorders such as PAH.

O2 Sensing in the Human Ductus Arteriosus: Regulation of Voltage-Gated K+ Channels in Smooth Muscle Cells by a Mitochondrial Redox Sensor

Hypoxic pulmonary vasoconstriction (HPV) is initiated by inhibition of O2-sensitive, voltage-gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs). Kv inhibition depolarizes membrane potential (EM), thereby activating Ca2+ influx via voltage-gated Ca2+ channels. HPV is weak in extrapulmonary, conduit pulmonary arteries (PA) and strong in precapillary resistance arteries. We hypothesized that regional heterogeneity in HPV reflects a longitudinal gradient in the function/expression of PASMC O2-sensitive Kv channels. In adult male Sprague Dawley rats, constrictions to hypoxia, the Kv blocker 4-aminopyridine (4-AP), and correolide, a Kv1.x channel inhibitor, were endothelium-independent and greater in resistance versus conduit PAs. Moreover, HPV was dependent on Kv-inhibition, being completely inhibited by pretreatment with 4-AP. Kv1.2, 1.5, Kv2.1, Kv3.1b, Kv4.3, and Kv9.3. mRNA increased as arterial caliber decreased; however, only Kv1.5 protein expression was greater in resistance PAs. Resistance PASMCs had greater K+ current (IK) and a more hyperpolarized EM and were uniquely O2− and correolide-sensitive. The O2-sensitive current (active at −65 mV) was resistant to iberiotoxin, with minimal tityustoxin sensitivity. In resistance PASMCs, 4-AP and hypoxia inhibited IK 57% and 49%, respectively, versus 34% for correolide. Intracellular administration of anti-Kv1.5 antibodies inhibited correolide’s effects. The hypoxia-sensitive, correolide-insensitive IK (15%) was conducted by Kv2.1. Anti-Kv1.5 and anti-Kv2.1 caused additive depolarization in resistance PASMCs (Kv1.5>Kv2.1) and inhibited hypoxic depolarization. Heterologously expressed human PASMC Kv1.5 generated an O2− and correolide-sensitive IK like that in resistance PASMCs. In conclusion, Kv1.5 and Kv2.1 account for virtually all the O2-sensitive current. HPV occurs in a Kv-enriched resistance zone because resistance PASMCs preferentially express O2-sensitive Kv-channels.

Impairment of hypoxic pulmonary vasoconstriction in mice lacking the voltage-gated potassium channel Kv1.5

Abstract— Functional closure of the human ductus arteriosus (DA) is initiated within minutes of birth by O2 constriction. It occurs by an incompletely understood mechanism that is intrinsic to the DA smooth muscle cell (DASMC). We hypothesized that O2 alters the function of an O2 sensor (the mitochondrial electron transport chain, ETC) thereby increasing production of a diffusible redox-mediator (H2O2), thus triggering an effector mechanism (inhibition of DASMC voltage-gated K+ channels, Kv). O2 constriction was evaluated in 26 human DAs (12 female, aged 9±2 days) studied in their normal hypoxic state or after normoxic tissue culture. In fresh, hypoxic DAs, 4-aminopyridine (4-AP), a Kv inhibitor, and O2 cause similar constriction and K+ current inhibition (IK). Tissue culture for 72 hours, particularly in normoxia, causes ionic remodeling, characterized by decreased O2 and 4-AP constriction in DA rings and reduced O2- and 4-AP–sensitive IK in DASMCs. Remodeled DAMSCs are depolarized and express less O2-sensitive channels (including Kv2.1, Kv1.5, Kv9.3, Kv4.3, and BKCa). Kv2.1 adenoviral gene-transfer significantly reverses ionic remodeling, partially restoring both the electrophysiological and tone responses to 4-AP and O2. In fresh DASMCs, ETC inhibitors (rotenone and antimycin) mimic hypoxia, increasing IK and reversing constriction to O2, but not phenylephrine. O2 increases, whereas hypoxia and ETC inhibitors decrease H2O2 production by altering mitochondrial membrane potential (&Dgr;&PSgr;m). H2O2, like O2, inhibits IK and depolarizes DASMCs. We conclude that O2 controls human DA tone by modulating the function of the mitochondrial ETC thereby varying &Dgr;&PSgr;m and the production of H2O2, which regulates DASMC Kv channel activity and DA tone.

The neurovascular mechanism of clitoral erection: nitric oxide and cGMP-stimulated activation of BKCa channels

Hypoxic pulmonary vasoconstriction (HPV) is initiated by the inhibition of several 4‐aminopyridine (4‐AP)‐sensitive, voltage‐gated, K+ channels (Kv). Several O2‐sensitive candidate channels (Kv1.2, Kv1.5, Kv2.1, and Kv3.1b) have been proposed, based on similarities between their characteristics in expression systems and the properties of the O2‐sensitive K+ current (IK) in pulmonary artery smooth muscle cells (PASMCs). We used gene targeting to delete Kv1.5 in mice by creating a SWAP mouse that is functionally a Kv1.5 knockout. We hypothesized that SWAP mice would display impaired HPV. The Kv1.5 α‐subunits present in the endothelium and PASMCs of wild‐type mice were absent in the lungs of SWAP mice, whereas expression of other channels Kv (1.1, 1.2, 2.1, 3.1, 4.3), Kir 3.1, Kir 6.1, and BKCa was unaltered. In isolated lungs and resistance PA rings, HPV was reduced significantly in SWAP versus wild‐type mice. Consistent with this finding, PASMCs from SWAP PAs were slightly depolarized and lacked IKv1.5, a 4‐AP and hypoxia‐sensitive component of IK that activated between ‐50 mV and ‐30 mV. We conclude that a K+ channel containing Kv1.5 α‐subunits is an important effector of HPV in mice.

Sildenafil Reverses O2 Constriction of the Rabbit Ductus Arteriosus by Inhibiting Type 5 Phosphodiesterase and Activating BKCa Channels

Female sexual function is under‐studied, and mechanisms of clitoral engorgement‐relaxation are incompletely understood. Penile erection results from nitric oxide (NO) ‐induced cyclic guanosine monophosphate (cGMP) accumulation. cGMP‐dependent protein kinase (PKG) activates large‐conductance, calcium‐activated potassium channels (BKCa), thereby hyperpolarizing and relaxing vascular and trabecular smooth muscle cells, allowing engorgement. We hypothesize rat clitorises relax by a similar mechanism. Rat clitorises express components of the proposed pathway: neuronal and endothelial NO synthases, soluble guanylyl cyclase (sGC), type 5 phosphodiesterase (PDE‐5), and BKCa channels. The NO donor diethylamine NONOate (DEANO), the PKG activator 8‐pCPT‐cGMP, and the PDE‐5 inhibitor sildenafil, cause dose‐dependent clitoral relaxation that is inhibited by antagonists of PKG (Rp‐8‐Br‐cGMPS) or BKCa channels (iberiotoxin). Electrical field stimulation induces tetrodotoxin‐sensitive NO release and relaxation that is inhibited by the Na+ channel blocker tetrodotoxin or sGC inhibitor 1H‐(1,2,4)oxadiozolo(4,3‐a)quinoxalin‐1‐one. Human BKCa channels, transferred to Chinese hamster ovary cells via an adenoviral vector, and endogenous rat clitoral smooth muscle K+ current are activated by this PKG‐dependent mechanism. Laser confocal microscopy reveals protein expression of BKCa channels on clitoral smooth muscle cells; these cells exhibit BKCa channel activity that is activated by both DEANO and sildenafil. We conclude that neurovascular derived NO causes clitoral relaxation via a PKG‐dependent activation of BKCa channels. The BKCa channel is an appealing target for drug therapy of female erectile dysfunction.— Gragasin, F. S., Michelakis, E. D., Hogan, A., Moudgil, R., Hashimoto, K., Wu, X., Bonnet, S., Haromy, A., Archer, S. L. The neurovascular mechanism of clitoral erection: nitric oxide and cGMP‐stimulated activation of BKCa channels. FASEB J. 18, 1382‐1391 (2004)