Dichloroacetate treatment partially regresses established pulmonary hypertension in mice with SM22alpha-targeted overexpression of the serotonin transporter.

Voltage-gated potassium (Kv)1.5 is decreased in pulmonary arteries (PAs) of patients with idiopathic pulmonary arterial hypertension (IPAH) and in experimental models including mice with SM22alpha-targeted overexpression of the serotonin transporter (5-HTT). The mechanisms underlying these abnormalities, however, remain unknown. Dichloroacetate (DCA) inhibits chronic hypoxia- or monocrotaline-induced PAH by inhibiting nuclear factor of activated T-cells (NFAT)c2 and increasing Kv1.5. Therefore, we hypothesized that DCA could regress established PAH in SM22-5-HTT+ mice. We evaluated pulmonary hemodynamics, vascular remodeling, NFATc2, and Kv1.5 protein in 20-wk-old SM22-5-HTT+ or wild-type mice treated for 1, 7, and 21 d with DCA, cyclosporine-A (NFAT inhibitor), or vehicle. DCA partially reversed PAH in SM22-5-HTT+ mice by decreasing proliferation and increasing apoptosis in muscularized PAs. Furthermore, serotonin (10(-8)-10(-6) M) dose-dependently increased PA-smooth muscle cell (PA-SMC) proliferation in culture (EC(50)=0.97 x 10(-7) M) and DCA (5 x 10(-4) M) vs. PBS markedly reduced the growth of PA-SMC from IPAH and control patients treated with the highest dose of serotonin by 50 and 30%, respectively. Finally, although serotonin induces NFATc2 activation in PA-SMCs, inhibition of NFATc2 alone with cyclosporine-A was not sufficient for reversing PAH in this model. Our results support the possibility that DCA may be an interesting agent for investigation in patients with PAH.

RhoA and Rho kinase activation in human pulmonary hypertension: role of 5-HT signaling.

RATIONALE: The complex and multifactorial pathogenesis of pulmonary hypertension (PH) involves constriction, remodeling, and in situ thrombosis of pulmonary vessels. Both serotonin (5-HT) and Rho kinase signaling may contribute to these alterations. OBJECTIVES: To investigate possible links between the 5-HT transporter (5-HTT) and RhoA/Rho kinase pathways, as well as their involvement in the progression of human and experimental PH. METHODS: Biochemical and functional analyses of lungs, platelets, and pulmonary artery smooth muscle cells (PA-SMCs) from patients with idiopathic PH (iPH) and 5-HTT overexpressing mice. MEASUREMENTS AND MAIN RESULTS: Lungs, platelets, and PA-SMCs from patients with iPH were characterized by marked elevation in RhoA and Rho kinase activities and a strong increase in 5-HT binding to RhoA indicating RhoA serotonylation. The 5-HTT inhibitor fluoxetine and the type 2 transglutaminase inhibitor monodansylcadaverin prevented 5-HT-induced RhoA serotonylation and RhoA/Rho kinase activation, as well as 5-HT-induced proliferation of PA-SMCs from iPH patients that was also inhibited by the Rho kinase inhibitor fasudil. Increased Rho kinase activity, RhoA activation, and RhoA serotonylation were also observed in lungs from SM22-5-HTT(+)mice, which overexpress 5-HTT in smooth muscle and spontaneously develop PH. Treatment of SM22-5-HTT(+) mice with either fasudil or fluoxetine limited PH progression and RhoA/Rho kinase activation. CONCLUSIONS: RhoA and Rho kinase activities are increased in iPH, in association with enhanced RhoA serotonylation. Direct involvement of the 5-HTT/RhoA/Rho kinase signaling pathway in 5-HT-mediated PA-SMC proliferation and platelet activation during PH progression identify RhoA/Rho kinase signaling as a promising target for new treatments against PH.

Effects of roflumilast, a phosphodiesterase-4 inhibitor, on hypoxia- and monocrotaline-induced pulmonary hypertension in rats.

Phosphodiesterase type 4 (PDE4) is involved in the hydrolysis of cAMP in pulmonary vascular smooth muscle (PA-SMC) and immune inflammatory cells. Given that intracellular cAMP accumulation inhibits contraction and growth of PA-SMCs as well as inflammatory cell functions, we investigated the effects of the PDE4 inhibitor 3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide (roflumilast), on pulmonary hypertension (PH) in rats. Treatment with roflumilast (0.5 or 1.5 mg x kg(-1) day(-1)) from day 1 to day 21 after monocrotaline (MCT) injection (60 mg x kg(-1) s.c.) attenuated PH development: pulmonary artery pressure, right ventricular hypertrophy, and muscularization of distal vessels on day 21 were decreased compared to control MCT-treated rats. Roflumilast (1.5 mg x kg(-1) day(-1)) also reduced the increases in interleukin-6 and monocyte chemotactic protein-1 mRNAs observed in lung tissue on day 21 without affecting the rise in interleukin-1beta mRNA on days 1 and 21. Roflumilast (1.5 mg x kg(-1) day(-1)) from day 21 to day 42 after MCT reversed established PH, almost normalizing pulmonary artery pressure and structure, and suppressing proliferating cell nuclear antigen-positive cells in pulmonary vascular walls. Treatment with roflumilast similarly attenuated PH development due to chronic hypoxia. Treatment of human PA-SMCs with roflumilast N-oxide, the active metabolite of roflumilast, at concentrations up to 10(-6) M, potentiated PA-SMC growth inhibition induced by prostacyclin (10(-6) M) or interleukin-1beta (10 ng x ml(-1)) but was inactive on its own. In conclusion, the PDE4 inhibitor roflumilast significantly attenuates pulmonary vascular remodeling and hypertension induced by chronic hypoxia or MCT and reverses established PH after MCT administration.

Transgenic mice overexpressing the 5-hydroxytryptamine transporter gene in smooth muscle develop pulmonary hypertension.

One intrinsic abnormality of pulmonary artery smooth muscle cells (PA-SMCs) in human idiopathic pulmonary hypertension (iPH) is an exaggerated proliferative response to internalized serotonin (5-HT) caused by increased expression of the 5-HT transporter (5-HTT). To investigate whether 5-HTT overexpression in PA-SMCs is sufficient to produce PH, we generated transgenic mice overexpressing 5-HTT under the control of the SM22 promoter. Studies in SM22-LacZ(+) mice showed that the transgene was expressed predominantly in SMCs of pulmonary and systemic vessels. Compared with wild-type mice, SM22-5-HTT(+) mice exhibited a 3- to 4-fold increase in lung 5-HTT mRNA and protein, together with increased lung 5-HT uptake activity, but no changes in platelet 5-HTT activity or blood 5-HT levels. At 8 weeks of age, SM22-5-HTT(+) mice exhibited PH, with marked increases in right ventricular systolic pressure (RVSP), right ventricle/left ventricle+septum ratio, and muscularization of distal pulmonary vessels, but no changes in systemic arterial pressure. PH worsened with age. Except a marked decrease in Kv channels, no changes in the lung expression of mediators of pulmonary vascular remodeling were observed in SM22-5-HTT(+) mice. Compared with wild-type mice, SM22-5-HTT(+) mice showed depressed hypoxic pulmonary vasoconstriction contrasting with greater severity of hypoxia- or monocrotaline-induced PH. These results show that increased 5-HTT expression in PA-SMCs, to a level close to that found in human iPH, lead to PH in mice. They further support a central role for 5-HTT in the pathogenesis of PH, making 5-HTT a potential therapeutic target.

Bax and heart mitochondria: uncoupling and inhibition of respiration without permeability transition.

The effects of Bax (full-length, FL, and C-terminal truncated, DeltaC) on respiration rate, membrane potential, MgATPase activity and kinetics of regulation of respiration were studied in isolated rat heart mitochondria and permeabilized cardiomyocytes. The results showed that while both Bax-FL and Bax-DeltaC permeabilized the outer mitochondrial membrane, released cytochrome c and reduced the respiration rate, the latter could be fully restored by exogenous cytochrome c only in the case of Bax-DeltaC, but not in presence of Bax-FL. In addition, Bax-FL but not Bax-DeltaC increased the MgATPase activity, and their effects on the mitochondrial membrane potential were quantitatively different. None of these effects was sensitive to cyclosporin A (CsA). It is concluded that Bax-FL affects both the outer and the inner mitochondrial membranes by: (1) opening large pores in the outer membrane; (2) inhibiting some segments of the respiratory chain in the inner membrane; and (3) uncoupling the inner mitochondrial membrane by increasing proton leak without opening the permeability transition pore (PTP).

Nebivolol for improving endothelial dysfunction, pulmonary vascular remodeling, and right heart function in pulmonary hypertension.

BACKGROUND: Endothelial cell (EC) dysfunction plays a central role in the pathogenesis of pulmonary arterial hypertension (PAH), promoting vasoconstriction, smooth muscle proliferation, and inflammation. OBJECTIVES: This study sought to test the hypothesis that nebivolol, a ß1-antagonist and ß2,3-agonist, may improve PAH and reverse the PAH-related phenotype of pulmonary ECs (P-EC). METHODS: We compared the effects of nebivolol with metoprolol, a first-generation ß1-selective ß-blocker, on human cultured PAH and control P-EC proliferation, vasoactive and proinflammatory factor production, and crosstalk with PA smooth muscle cells. We assessed the effects of both ß-blockers in precontracted PA rings. We also compared the effects of both ß-blockers in experimental PAH. RESULTS: PAH P-ECs overexpressed the proinflammatory mediators interleukin-6 and monocyte chemoattractant protein-1, fibroblast growth factor-2, and the potent vasoconstrictive agent endothelin-1 as compared with control cells. This pathological phenotype was corrected by nebivolol but not metoprolol in a dose-dependent fashion. We confirmed that PAH P-EC proliferate more than control cells and stimulate more PA smooth muscle cell mitosis, a growth abnormality that was normalized by nebivolol but not by metoprolol. Nebivolol but not metoprolol induced endothelium-dependent and nitric oxide-dependent relaxation of PA. Nebivolol was more potent than metoprolol in improving cardiac function, pulmonary vascular remodeling, and inflammation of rats with monocrotaline-induced pulmonary hypertension. CONCLUSIONS: Nebivolol could be a promising option for the management of PAH, improving endothelial dysfunction, pulmonary vascular remodeling, and right heart function. Until clinical studies are undertaken, however, routine use of ß-blockers in PAH cannot be recommended.

Key role of the endothelial TGF-ß/ALK1/endoglin signaling pathway in humans and rodents pulmonary hypertension.

Mutations affecting transforming growth factor-beta (TGF-ß) superfamily receptors, activin receptor-like kinase (ALK)-1, and endoglin (ENG) occur in patients with pulmonary arterial hypertension (PAH). To determine whether the TGF-ß/ALK1/ENG pathway was involved in PAH, we investigated pulmonary TGF-ß, ALK1, ALK5, and ENG expressions in human lung tissue and cultured pulmonary-artery smooth-muscle-cells (PA-SMCs) and pulmonary endothelial cells (PECs) from 14 patients with idiopathic PAH (iPAH) and 15 controls. Seeing that ENG was highly expressed in PEC, we assessed the effects of TGF-ß on Smad1/5/8 and Smad2/3 activation and on growth factor production by the cells. Finally, we studied the consequence of ENG deficiency on the chronic hypoxic-PH development by measuring right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary arteriolar remodeling in ENG-deficient (Eng+/-) and wild-type (Eng+/+) mice. We also evaluated the pulmonary blood vessel density, macrophage infiltration, and cytokine expression in the lungs of the animals. Compared to controls, iPAH patients had higher serum and pulmonary TGF-ß levels and increased ALK1 and ENG expressions in lung tissue, predominantly in PECs. Incubation of the cells with TGF-ß led to Smad1/5/8 phosphorylation and to a production of FGF2, PDGFb and endothelin-inducing PA-SMC growth. Endoglin deficiency protected mice from hypoxic PH. As compared to wild-type, Eng+/- mice had a lower pulmonary vessel density, and no change in macrophage infiltration after exposure to chronic hypoxia despite the higher pulmonary expressions of interleukin-6 and monocyte chemoattractant protein-1. The TGF-ß/ALK1/ENG signaling pathway plays a key role in iPAH and experimental hypoxic PH via a direct effect on PECs leading to production of growth factors and inflammatory cytokines involved in the pathogenesis of PAH.

The beneficial effect of suramin on monocrotaline-induced pulmonary hypertension in rats.

BACKGROUND: Pulmonary hypertension (PH) is a progressive disorder characterized by an increase in pulmonary artery pressure and structural changes in the pulmonary vasculature. Several observations indicate that growth factors play a key role in PH by modulating pulmonary artery smooth muscle cell (PA-SMC) function. In rats, established monocrotaline-induced PH (MCT-PH) can be reversed by blocking platelet-derived growth factor receptors (PDGF-R), epidermal growth factor receptors (EGF-R), or fibroblast growth factor receptors (FGF-R). All these receptors belong to the receptor tyrosine kinase (RTK) family. METHODS AND RESULTS: We evaluated whether RTK blockade by the nonspecific growth factor inhibitor, suramin, reversed advanced MCT-PH in rats via its effects on growth-factor signaling pathways. We found that suramin inhibited RTK and ERK1/2 phosphorylation in cultured human PA-SMCs. Suramin inhibited PA-SMC proliferation induced by serum, PDGF, FGF2, or EGF in vitro and ex vivo. Treatment with suramin from day 1 to day 21 after monocrotaline injection attenuated PH development, as shown by lower values for pulmonary artery pressure, right ventricular hypertrophy, and distal vessel muscularization on day 21 compared to control rats. Treatment with suramin from day 21 to day 42 after monocrotaline injection reversed established PH, thereby normalizing the pulmonary artery pressure values and vessel structure. Suramin treatment suppressed PA-SMC proliferation and attenuated both the inflammatory response and the deposition of collagen. CONCLUSIONS: RTK blockade by suramin can prevent MCT-PH and reverse established MCT-PH in rats. This study suggests that an anti-RTK strategy that targets multiple RTKs could be useful in the treatment of pulmonary hypertension.

Apoptosis and effects of intracavernous bone marrow cell injection in a rat model of postprostatectomy erectile dysfunction.

OBJECTIVES: To investigate the pathophysiology of postprostatectomy erectile dysfunction (pPED) in a rat model of bilateral cavernous nerve ablation (BCNA) and to assess the effects of local bone marrow mononuclear cell (BMMNC) injection on erectile dysfunction (ED) and cavernosal cellular abnormalities caused by BCNA. DESIGN, SETTING, AND PARTICIPANTS: This was an experimental study in Fisher rats with BCNA. INTERVENTION: Intervention included BNCA, electrical stimulation of the pelvic ganglion, and local BMMNC injection. MEASUREMENTS: Erectile responses to electric pelvic ganglion stimulation were studied. Cavernous tissue was examined to determine the cell types undergoing apoptosis and to detect changes in protein and gene expression of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) using real-time quantitative polymerase chain reaction (RTQ-PCR) and Western blotting. The effects of local BMMNC injection on these parameters were studied. RESULTS AND LIMITATIONS: Diffuse apoptosis was noted in the connective tissue mesenchymal cells and vascular smooth muscle and endothelial cells. Compared with sham-operated controls, nNOS and eNOS levels were decreased after 3 wk and were normal (eNOS) or increased (nNOS) after 5 wk, suggesting spontaneous nerve regeneration. Despite nNOS recovery, erectile responses to electrical stimulation remained impaired after 5 wk, when mesenchymal cell apoptosis was the main persistent biologic abnormality. BMMNC injection decreased apoptotic cell numbers, accelerated the normalisation of nNOS and eNOS, and partially restored erectile responses at week 5. CONCLUSIONS: Massive cell apoptosis may play a key role in the pathophysiology of pPED. In this animal model, apoptosis persisted despite spontaneous nerve regeneration, suggesting that the course of BCNA-induced cell dysfunction was independent of reinnervation. BMMNC improved erectile function by inhibiting apoptosis and may hold promise for repairing penile cell damage caused by radical prostatectomy (RP).

Endothelial-derived FGF2 contributes to the progression of pulmonary hypertension in humans and rodents.

Pulmonary hypertension (PH) is a progressive, lethal lung disease characterized by pulmonary artery SMC (PA-SMC) hyperplasia leading to right-sided heart failure. Molecular events originating in pulmonary ECs (P-ECs) may contribute to the PA-SMC hyperplasia in PH. Thus, we exposed cultured human PA-SMC to medium conditioned by P-EC from patients with idiopathic PH (IPH) or controls and found that IPH P-EC-conditioned medium increased PA-SMC proliferation more than control P-EC medium. Levels of FGF2 were increased in the medium of IPH P-ECs over controls, while there was no detectable difference in TGF-beta1, PDGF-BB, or EGF levels. No difference in FGF2-induced proliferation or FGF receptor type 1 (FGFR1) mRNA levels was detected between IPH and control PA-SMCs. Knockdown of FGF2 in P-EC using siRNA reduced the PA-SMC growth-stimulating effects of IPH P-EC medium by 60% and control P-EC medium by 10%. In situ hybridization showed FGF2 overproduction predominantly in the remodeled vascular endothelium of lungs from patients with IPH. Repeated intravenous FGF2-siRNA administration abolished lung FGF2 production, both preventing and nearly reversing a rat model of PH. Similarly, pharmacological FGFR1 inhibition with SU5402 reversed established PH in the same model. Thus, endothelial FGF2 is overproduced in IPH and contributes to SMC hyperplasia in IPH, identifying FGF2 as a promising target for new treatments against PH.