Rationale Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction due in part to pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Mitochondrial fragmentation and normoxic activation of hypoxia-inducible factor-1α (HIF-1α) have been observed in PAH PASMCs, however their relationship and relevance to the development of PAH is 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-1α activator, cobalt, chronic hypoxia, and monocrotaline. HIF-1α activation in human PAH leads to mitochondrial fission by cyclin B1/CDK1-dependent phosphorylation of DRP1 at Serine-616. In normal PASMC, HIF-1α activation by CoCl2 or desferrioxamine causes DRP1-mediated fission. HIF-1α 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 PASMC and in rodent models. Mdivi-1 improves exercise capacity, right ventricular function and hemodynamics in experimental PAH. Conclusion DRP-1-mediated mitotic fission is a cell cycle checkpoint that can be therapeutically targeted in hyperproliferative disorders such as PAH.

Abstract-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 ϩ channels, including Kv1.5. The Kv downregulation-induced increase in [K ϩ ] i , tonically inhibits caspases, further suppressing apoptosis. Mitochondria control apoptosis and produce activated oxygen species like H 2 O 2 , which regulate vascular tone by activating K ϩ 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, PϽ0.002), PA medial thickness (28% decrease, PϽ0.0001), and right ventricular hypertrophy (34% decrease, PϽ0.001). 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 H 2 O 2 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 mitochondriadependent apoptosis as a potential target for therapy and DCA as an effective and selective treatment for PAH. Key Words: apoptosis Ⅲ proliferation Ⅲ smooth muscle Ⅲ vascular remodeling P ulmonary arterial hypertension (PAH) is defined by an elevated pulmonary vascular resistance (PVR), which leads to right heart failure and premature death. The cause remains unknown and available treatments are limited, expensive, and often associated with significant side effects. 1,2 The pulmonary arteries (PAs) are affected by varying degrees of vasoconstriction and vascular remodeling, including cellular proliferation in both the intima and media and distal PA muscularization. 1,2 Vascular medial remodeling results from an imbalance between smooth muscle cell (SMC) proliferation and apoptosis, favoring proliferation. Gene microarray studies show that lungs from patients with PAH have a decrease in the proapoptotic/antiapoptotic gene expression ratio. 3 Furthermore, several loss-of-function germline or acquired mutations have been described in receptors of the transforming growth factor-␤) superfamily, such as bone morphogenetic protein receptor-2 (BM...

Background-Left internal mammary arteries (LIMAs) synthesize endothelium-derived hyperpolarizing factor (EDHF), a short-lived K ϩ 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)

Background-Alveolar hypoxia acutely elicits pulmonary vasoconstriction (HPV). Chronic hypoxia (CH), despiteattenuating HPV, causes pulmonary hypertension (CH-PHT). HPV results, in part, from inhibition of O 2 -sensitive, voltage-gated potassium channels (Kv) in pulmonary artery smooth muscle cells (PASMCs). CH decreases Kv channel current/expression and depolarizes and causes Ca 2ϩ 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 reporterϮhuman 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 O 2 -sensitive K ϩ 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 O 2 -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 ϩ channel gene therapy for a vascular disease.

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 Ն1 class in all patients. Pretreatment versus posttreatment values (meanϮSEM) were as follows: 6-minute walk, 376Ϯ30 versus 504Ϯ27 m, PϽ0.0001; mean PA pressure, 70Ϯ3 versus 52Ϯ3 mm Hg, PϽ0.007; pulmonary vascular resistance index 1702Ϯ151 versus 996Ϯ92 dyne · s · cm Ϫ5 · m Ϫ2, PϽ0.006. 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. Figure 1. Short-term inhaled nitric oxide (iNO) reduces pulmonary vascular resistance in PAH, 4,5 but ambulatory delivery in humans is cumbersome. Another strategy is to prolong the survival of cGMP in PASMCs by inhibiting type-5 phosphodiesterase (PD-5), an isoform that is primarily located in the penis and lungs, which rapidly degrades cGMP. Because of PD-5's tissue distribution (pulmonaryϾsystemic vasculature), PD-5 inhibitors are attractive candidate pulmonary vasodilators that minimally decrease systemic blood pressure. (75 mg) is an effective and relatively selective pulmonary vasodilator. 4,5 We hypothesized that PD-5 inhibition acutely causes human PA dilatation, in part by opening of BK Ca channels, and that it chronically improves hemodynamics and functional capacity in moderately severe PAH. Conclusion-This MethodsWe studied 5 consecutive patients with PAH (nϭ4 New York Heart Association [NYHA] class III; patient 3 class II). All subjects provided informed consent. Patients with class IV PAH were excluded because they often require epoprostenol, which could confound the assessment of sildenafil's effects. All the patients had been stable for Ͼ3 months, and their standard therapy was not altered before initiation of sildenafil. All were on diuretics and coumadin, and patients 2 and 4 were on Ca 2ϩ channel blockers because they had been shown to respond to iNO with Ͼ20% decrease in pulmonary vascular resistance (Figure 2). No patient was taking nitrates. All patients had primary pulmonary hypertension (PPH) except patient 2, w...

Abstract-Hypoxic pulmonary vasoconstriction (HPV) is initiated by inhibition of O 2 -sensitive, voltage-gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs). Kv inhibition depolarizes membrane potential (E M ), thereby activating Ca 2ϩ influx via voltage-gated Ca 2ϩ 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 O 2 -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 Kvinhibition, 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 ϩ current (I K ) and a more hyperpolarized E M and were uniquely O 2 Ϫ and correolide-sensitive. The O 2 -sensitive current (active at Ϫ65 mV) was resistant to iberiotoxin, with minimal tityustoxin sensitivity. In resistance PASMCs, 4-AP and hypoxia inhibited I K 57% and 49%, respectively, versus 34% for correolide. Intracellular administration of anti-Kv1.5 antibodies inhibited correolide's effects. The hypoxia-sensitive, correolide-insensitive I K (15%) was conducted by Kv2.1. Anti-Kv1.5 and anti-Kv2.1 caused additive depolarization in resistance PASMCs (Kv1.5ϾKv2.1) and inhibited hypoxic depolarization. Heterologously expressed human PASMC Kv1.5 generated an O 2 Ϫ and correolide-sensitive I K like that in resistance PASMCs. In conclusion, Kv1.5 and Kv2.1 account for virtually all the O 2 -sensitive current. HPV occurs in a Kv-enriched resistance zone because resistance PASMCs preferentially express O 2 -sensitive Kv-channels. Key Words: immunoelectropharmacology Ⅲ laser capture microdissection Ⅲ voltage-gated channels Ⅲ pulmonary circulation Ⅲ adenoviral gene transfer T he adult pulmonary circulation is a low-resistance circuit designed for gas exchange that is perfused by a thinwalled, afterload-intolerant right ventricle. The pulmonary vasculature consists of large, elastic, extraparenchymal "conduit" arteries and small, muscular intrapulmonary arteries, which control regional distribution of blood flow and largely determine pulmonary vascular resistance (PVR). Hypoxic pulmonary vasoconstriction (HPV) is a widely conserved mechanism for ventilation-perfusion matching. 1 With segmental hypoxia (eg, atelectasis), resistance pulmonary arteries (PAs) serving the hypoxic lobes constrict, diverting blood to better-oxygenated segments, thereby optimizing systemic PO 2 , without increasing PVR. 2,3 Microangiography 4 and micropuncture 5 reveal that HPV primarily occurs in resistance PAs (Ͻ200 mol/L diameter, division 4, and distal), with lesser contributions from larger i...

SummaryIncretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation underlies the stimulatory effect of incretins and that glutamate uptake into insulin granules mediated by cAMP/PKA signaling amplifies insulin release. Glutamate production is diminished in an incretin-unresponsive, insulin-secreting β cell line and pancreatic islets of animal models of human diabetes and obesity. Conversely, a membrane-permeable glutamate precursor restores amplification of insulin secretion in these models. Thus, cytosolic glutamate represents the elusive link between glucose metabolism and cAMP action in incretin-induced insulin secretion.

Fetal to maternal blood flow matching in the placenta, necessary for optimal fetal blood oxygenation, may occur via hypoxic fetoplacental vasoconstriction (HFPV). We hypothesized that HFPV is mediated by K+ channel inhibition in fetoplacental vascular smooth muscle, as occurs in several other O2-sensitive tissues. With the use of an isolated human placental cotyledon perfused at a constant flow rate, we found that hypoxia reversibly increased perfusion pressure by >20%. HFPV was unaffected by cyclooxygenase or nitric oxide synthase inhibition. HFPV and 4-aminopyridine, an inhibitor of voltage-dependent K+(Kv) channels, increased pressure in a nonadditive manner, suggesting they act via a common mechanism. Iberiotoxin, a large conductance Ca2+-sensitive K+(BKCa) channel inhibitor, had little effect on normoxic pressure. Immunoblotting and RT-PCR showed expression of several putative O2-sensitive K+ channels in peripheral fetoplacental vessels. In patch-clamp experiments with smooth muscle cells isolated from peripheral fetoplacental arteries, hypoxia reversibly inhibited Kv but not BKCa or ATP-dependent currents. We conclude that human fetoplacental vessels constrict in response to hypoxia. This response is largely mediated by hypoxic inhibition of Kv channels in the smooth muscle of small fetoplacental arteries.