RESUMEN
We have previously shown that hypoxic proliferation of human pulmonary microvascular endothelial cells (hPMVECs) depends on epidermal growth factor receptor (EGFR) activation. To determine downstream signaling leading to proliferation, we tested the hypothesis that hypoxia-induced proliferation in hPMVECs would require EGFR-mediated activation of extracellular signal-regulated kinase (ERK) leading to arginase II induction. To test this hypothesis, hPMVECs were incubated in either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) and Western blotting was performed for EGFR, arginase II, phosphorylated-ERK (pERK), and total ERK (ERK). Hypoxia led to greater EGFR, pERK, and arginase II protein levels than did normoxia in hPMVECs. To examine the role of EGFR in these hypoxia-induced changes, hPMVECs were transfected with siRNA against EGFR or a scrambled siRNA and placed in hypoxia. Inhibition of EGFR using siRNA attenuated hypoxia-induced pERK and arginase II expression as well as the hypoxia-induced increase in viable cell numbers. hPMVECs were then treated with vehicle, an EGFR inhibitor (AG1478), or an ERK pathway inhibitor (U0126) and placed in hypoxia. Pharmacologic inhibition of EGFR significantly attenuated the hypoxia-induced increase in pERK level. Both AG1478 and U0126 also significantly attenuated the hypoxia-induced increase in viable hPMVECs numbers. hPMVECs were transfected with an adenoviral vector containing arginase II (AdArg2) and overexpression of arginase II rescued the U0126-mediated decrease in viable cell numbers in hypoxic hPMVECs. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic induction of arginase II and cellular proliferation.
Asunto(s)
Células Endoteliales/enzimología , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Pulmón/irrigación sanguínea , Microvasos/patología , Arginasa/metabolismo , Butadienos/farmacología , Recuento de Células , Hipoxia de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Silenciador del Gen/efectos de los fármacos , Humanos , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Quinazolinas/farmacología , ARN Interferente Pequeño/metabolismo , Tirfostinos/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
AIM: Pulmonary hypertension (PH) develops in 25-40% of bronchopulmonary dysplasia (BPD) patients, substantially increasing mortality. We have previously found that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) production, is elevated in patients with BPD-associated PH. ADMA is metabolised by N(á´³) ,N(á´³) -dimethylarginine dimethylaminohydrolase (DDAH). Presently, we test the hypothesis that there are single nucleotide polymorphisms (SNPs) in DDAH1 and/or DDAH2 associated with the development of PH in BPD patients. METHODS: BPD patients were enrolled (n = 98) at Nationwide Children's Hospital. Clinical characteristics and 36 SNPs in DDAH1 and DDAH2 were compared between BPD-associated PH patients (cases) and BPD-alone patients (controls). RESULTS: In BPD patients, 25 (26%) had echocardiographic evidence of PH (cases). In this cohort, DDAH1 wild-type rs480414 was 92% sensitive and 53% specific for PH in BPD, and the DDAH1 SNP rs480414 decreased the risk of PH in an additive model of inheritance (OR = 0.39; 95% CI [0.18-0.88], p = 0.01). CONCLUSION: The rs480414 SNP in DDAH1 may be protective against the development of PH in patients with BPD. Furthermore, the DDAH1 rs480414 may be a useful biomarker in developing predictive models for PH in patients with BPD.
Asunto(s)
Amidohidrolasas/genética , Displasia Broncopulmonar/complicaciones , Hipertensión Pulmonar/genética , Estudios de Cohortes , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Polimorfismo de Nucleótido SimpleRESUMEN
Exposure of newborn mice to hyperoxia arrests lung development, with resultant pathological characteristics similar to bronchopulmonary dysplasia in infants born prematurely. We tested the hypothesis that aberrations in lung development caused by 14 days of sublethal hyperoxia would be reversed during 14 days of recovery to room air (RA) when the concentration of oxygen exposure was weaned gradually. Newborn FVB mice were exposed to 85% oxygen or RA for 14 days. Weaning from hyperoxia was by either transfer directly into RA or a decrease in the concentration of oxygen by 10% per days. At 28 days, pups were euthanized, and the lungs were inflation fixed and assessed. At postnatal day 28, lungs of mice weaned abruptly from hyperoxia had fewer (6 ± 0.6 versus 10 ± 0.7; P < 0.001) alveoli per high-powered field and larger alveoli (4050 ± 207 versus 2305 ± 182 µm(2)) than animals weaned gradually; both hyperoxia-exposed groups were different from lungs obtained from air-breathing controls (20 ± 0.5 alveoli per high-powered field; P < 0.001). The results are consistent with the absence of catch-up alveolarization in this model and indicate that the long-term consequences of early exposures to hyperoxia merit closer examination. The effects of abrupt weaning to RA observed further suggest that weaning should be considered in experimental models of newborn exposure to hyperoxia.
Asunto(s)
Displasia Broncopulmonar/etiología , Displasia Broncopulmonar/patología , Hiperoxia/complicaciones , Pulmón/patología , Respiración Artificial/métodos , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , RatonesRESUMEN
Overexpression of GALGT2 in skeletal muscle can stimulate the glycosylation of α dystroglycan and the upregulation of normally synaptic dystroglycan-binding proteins, some of which are dystrophin and laminin α2 surrogates known to be therapeutic for several forms of muscular dystrophy. This article describes the vascular delivery of GALGT2 gene therapy in a large animal model, the rhesus macaque. Recombinant adeno-associated virus, rhesus serotype 74 (rAAVrh74), was used to deliver GALGT2 via the femoral artery to the gastrocnemius muscle using an isolated focal limb perfusion method. GALGT2 expression averaged 44 ± 4% of myofibers after treatment in macaques with low preexisting anti-rAAVrh74 serum antibodies, and expression was reduced to 9 ± 4% of myofibers in macaques with high preexisting rAAVrh74 immunity (P < 0.001; n = 12 per group). This was the case regardless of the addition of immunosuppressants, including prednisolone, tacrolimus, and mycophenolate mofetil. GALGT2-treated macaque muscles showed increased glycosylation of α dystroglycan and increased expression of dystrophin and laminin α2 surrogate proteins, including utrophin, plectin1, agrin, and laminin α5. These experiments demonstrate successful transduction of rhesus macaque muscle with rAAVrh74.MCK.GALGT2 after vascular delivery and induction of molecular changes thought to be therapeutic in several forms of muscular dystrophy.
Asunto(s)
Distrofina/biosíntesis , Técnicas de Transferencia de Gen , Terapia Genética , Laminina/biosíntesis , Distrofias Musculares/genética , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Distroglicanos/genética , Distroglicanos/metabolismo , Distrofina/genética , Regulación de la Expresión Génica , Glicosiltransferasas/genética , Laminina/genética , Macaca mulatta/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofias Musculares/patología , Distrofias Musculares/terapiaRESUMEN
Pulmonary artery smooth muscle cell (PASMC) proliferation plays a fundamental role in the vascular remodeling seen in pulmonary hypertensive diseases associated with hypoxia. Arginase II, an enzyme regulating the first step in polyamine and proline synthesis, has been shown to play a critical role in hypoxia-induced proliferation of human PASMC (hPASMC). In addition, there is evidence that patients with pulmonary hypertension have elevated levels of arginase in the vascular wall. Resveratrol, a natural polyphenol found in red wine and grape skins, has diverse biochemical and physiological actions including antiproliferative properties. Furthermore, resveratrol has been shown to attenuate right ventricular and pulmonary artery remodeling, both pathological components of pulmonary hypertension. The present studies tested the hypothesis that resveratrol would prevent hypoxia-induced pulmonary artery smooth muscle cell proliferation by inhibiting hypoxia-induced arginase II expression. Our data indicate that hypoxia-induced hPASMC proliferation is abrogated following treatment with resveratrol. In addition, the hypoxic induction of arginase II was directly attenuated by resveratrol treatment. Furthermore, we found that the inhibitory effect of resveratrol on arginase II in hPASMC was mediated through the PI3K-Akt signaling pathway. Supporting these in vitro findings, resveratrol normalized right ventricular hypertrophy in an in vivo neonatal rat model of chronic hypoxia-induced pulmonary hypertension. These novel data support the notion that resveratrol may be a potential therapeutic agent in pulmonary hypertension by preventing PASMC arginase II induction and proliferation.
Asunto(s)
Arginasa/biosíntesis , Proliferación Celular/efectos de los fármacos , Hipoxia/fisiopatología , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/fisiología , Estilbenos/farmacología , Animales , Arginasa/antagonistas & inhibidores , Células Cultivadas , Humanos , Hipertensión Pulmonar/fisiopatología , Hipertrofia Ventricular Derecha/tratamiento farmacológico , Hipertrofia Ventricular Derecha/etiología , Hipoxia/complicaciones , Miocitos del Músculo Liso/metabolismo , Ratas , ResveratrolRESUMEN
Asymmetric dimethylarginine (ADMA) is an endogenously produced nitric oxide synthase (NOS) inhibitor. L-Arginine can be metabolised by NOS and arginase, and arginase is the first step in polyamine production necessary for cellular proliferation. We tested the hypothesis that ADMA would inhibit NOS but not arginase activity and that this pattern of inhibition would result in greater L-arginine bioavailability to arginase, thereby increasing viable cell number. Bovine arginase was used in in vitro activity assays with various concentrations of substrate (L-arginine, ADMA, N(G) -monomethyl-L-arginine (L-NMMA) and N(G) -nitro-L-arginine methyl ester (L-NAME)). Only L-arginine resulted in measurable urea production (Km = 6.9 ± 0.8 mmol/L; Vmax = 6.6 ± 0.3 µmol/mg protein per min). We then incubated bovine arginase with increasing concentrations of ADMA, L-NMMA and L-NAME in the presence of 1 mmol/L l-arginine and found no effect of any of the tested compounds on arginase activity. Using bovine pulmonary arterial endothelial cells (bPAEC) we determined the effects of ADMA on nitric oxide (NO) and urea production and found significantly lower NO production and greater urea production (P < 0.003) with ADMA, without changes in arginase protein levels. In addition, ADMA treatment resulted in an approximately 30% greater number of viable cells after 48 h than in control bPAEC. These results demonstrate that ADMA is neither a substrate nor an inhibitor of arginase activity and that in bPAEC ADMA inhibits NO production and enhances urea production, leading to more viable cells. These results may have pathophysiological implications in disorders associated with higher ADMA levels, such as pulmonary hypertension.
Asunto(s)
Arginasa/antagonistas & inhibidores , Arginina/análogos & derivados , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Pulmón/citología , Animales , Arginina/farmacología , Bovinos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Especificidad por Sustrato , Urea/metabolismo , omega-N-Metilarginina/farmacologíaRESUMEN
Our objective was to determine the best measure of heparin anticoagulation in neonatal patients on extracorporeal membrane oxygenation. Activated clotting time (ACT), activated partial thromboplastin time (aPTT), and antifactor Xa levels, along with corresponding heparin infusion rates and heparin bolus volumes, were collected from neonates receiving ECMO at our institution from 2008 to 2013. After natural log transformation of antifactor Xa, ACT, and aPTT, overall correlations between antifactor Xa levels and either ACT or aPTT and correlations between these tests and heparin infusion rates were evaluated using linear mixed models that accounted for both within- and between-patient correlations. Twenty-six neonates with an average weight of 3.4 kg (standard deviation .7) had a total of 27 separate ECMO runs during the study period. Within each patient, ACT (r = .40, p < .0001) and aPTT (r = .48, p < .0001) were both directly correlated with antifactor Xa levels. In contrast, between patients, only aPTT maintained a direct correlation with antifactor Xa (r = .61, p = .07), whereas ACT showed a statistically significant inverse correlation with antifactor Xa (r = -.48, p = .04). Compared with ACT, aPTT is more consistently reflective of the anticoagulation status both within each patient on ECMO and between patients treated with ECMO. Future efforts to develop standardized heparin infusion algorithms for patients on ECMO should consider using aPTT levels to monitor anticoagulation.
Asunto(s)
Pruebas de Coagulación Sanguínea/métodos , Monitoreo de Drogas/métodos , Oxigenación por Membrana Extracorpórea/efectos adversos , Heparina/administración & dosificación , Trombosis/sangre , Trombosis/etiología , Trombosis/prevención & control , Anticoagulantes/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Recién Nacido , Masculino , Reproducibilidad de los Resultados , Estudios Retrospectivos , Sensibilidad y Especificidad , Resultado del TratamientoRESUMEN
Chronic hypoxia (CH)-induced pulmonary hypertension is characterized by vasoconstriction and vascular remodeling, leading to right ventricular dysfunction. Given the role of arterial compliance (C(a)) in right ventricular work, a decrease in C(a) would add to right ventricular work. Nitric oxide (NO) is a potent vasodilator made by NO synthases from L-arginine (L-Arg). However, little is known of the effect of L-Arg on vascular compliance (C(v)) in the lung. We hypothesized that exposure to CH would decrease C(a) and that this effect would be reversed by exogenous L-Arg. Sprague-Dawley rats were exposed to either normoxia or CH for 14 days; the lungs were then isolated and perfused. Vascular occlusions were performed and modeled using a three-compliance, two-resistor model. Pressure-flow curves were generated, and a distensible vessel model was used to estimate distensibility and a vascular resistance parameter (R(0)). Hypoxia resulted in the expected increase in arterial resistance (R(a)) as well as a decrease in both C(a) and C(v). L-Arg had little effect on R(a), C(a), or C(v) in isolated lungs from normoxic animals. L-Arg decreased R(a) in lungs from CH rats and redistributed compliance to approximately that found in normoxic lungs. CH increased R(0), and L-Arg reversed this increase in R(0). L-Arg increased exhaled NO, and inhibition of L-Arg uptake attenuated the L-Arg-induced increase in exhaled NO. These data demonstrate that the CH-induced decrease in C(a) was reversed by L-Arg, suggesting that L-Arg may improve CH-induced right ventricular dysfunction.
Asunto(s)
Arginina/farmacología , Hemodinámica/efectos de los fármacos , Hipertensión Pulmonar/tratamiento farmacológico , Hipoxia/tratamiento farmacológico , Pulmón/irrigación sanguínea , Arteria Pulmonar/efectos de los fármacos , Venas Pulmonares/efectos de los fármacos , Disfunción Ventricular Derecha/tratamiento farmacológico , Animales , Arginina/metabolismo , Presión Sanguínea/efectos de los fármacos , Enfermedad Crónica , Citrulina/farmacología , Adaptabilidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/fisiopatología , Hipoxia/complicaciones , Hipoxia/metabolismo , Hipoxia/fisiopatología , Pulmón/metabolismo , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Perfusión , Arteria Pulmonar/metabolismo , Arteria Pulmonar/fisiopatología , Venas Pulmonares/metabolismo , Venas Pulmonares/fisiopatología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Resistencia Vascular/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Disfunción Ventricular Derecha/etiología , Disfunción Ventricular Derecha/metabolismo , Disfunción Ventricular Derecha/fisiopatologíaRESUMEN
Adeno-associated virus type 9 (AAV9) is a powerful tool for delivering genes throughout the central nervous system (CNS) following intravenous injection. Preclinical results in pediatric models of spinal muscular atrophy (SMA) and lysosomal storage disorders provide a compelling case for advancing AAV9 to the clinic. An important translational step is to demonstrate efficient CNS targeting in large animals at various ages. In the present study, we tested systemically injected AAV9 in cynomolgus macaques, administered at birth through 3 years of age for targeting CNS and peripheral tissues. We show that AAV9 was efficient at crossing the blood-brain barrier (BBB) at all time points investigated. Transgene expression was detected primarily in glial cells throughout the brain, dorsal root ganglia neurons and motor neurons within the spinal cord, providing confidence for translation to SMA patients. Systemic injection also efficiently targeted skeletal muscle and peripheral organs. To specifically target the CNS, we explored AAV9 delivery to cerebrospinal fluid (CSF). CSF injection efficiently targeted motor neurons, and restricted gene expression to the CNS, providing an alternate delivery route and potentially lower manufacturing requirements for older, larger patients. Our findings support the use of AAV9 for gene transfer to the CNS for disorders in pediatric populations.
Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética , Atrofia Muscular Espinal/terapia , Animales , Encéfalo/metabolismo , Dependovirus/genética , Regulación de la Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Células HEK293 , Humanos , Inyecciones Epidurales , Inyecciones Intraarteriales , Macaca , Masculino , Neuronas Motoras/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular Espinal/genética , Neuroglía/metabolismo , Médula Espinal/metabolismo , Porcinos , Factores de Tiempo , Transducción Genética , Transgenes/genéticaRESUMEN
In a phase 1/2, open-label dose escalation trial, we delivered rAAVrh74.MCK.GALGT2 (also B4GALNT2) bilaterally to the legs of two boys with Duchenne muscular dystrophy using intravascular limb infusion. Subject 1 (age 8.9 years at dosing) received 2.5 × 1013 vector genome (vg)/kg per leg (5 × 1013 vg/kg total) and subject 2 (age 6.9 years at dosing) received 5 × 1013 vg/kg per leg (1 × 1014 vg/kg total). No serious adverse events were observed. Muscle biopsy evaluated 3 or 4 months post treatment versus baseline showed evidence of GALGT2 gene expression and GALGT2-induced muscle cell glycosylation. Functionally, subject 1 showed a decline in 6-min walk test (6MWT) distance; an increase in time to run 100 m, and a decline in North Star Ambulatory Assessment (NSAA) score until ambulation was lost at 24 months. Subject 2, treated at a younger age and at a higher dose, demonstrated an improvement over 24 months in NSAA score (from 20 to 23 points), an increase in 6MWT distance (from 405 to 478 m), and only a minimal increase in 100 m time (45.6-48.4 s). These data suggest preliminary safety at a dose of 1 × 1014 vg/kg and functional stabilization in one patient.
RESUMEN
Inducible nitric oxide (NO) synthase (iNOS) is a stress response protein upregulated in inflammatory conditions, and NO may suppress cellular proliferation. We hypothesized that preventing L-arginine (L-arg) uptake in endothelial cells would prevent lipopolysaccharide/tumor necrosis factor-α (LPS/TNF)-induced, NO-mediated suppression of cellular proliferation. Bovine pulmonary arterial endothelial cells (bPAEC) were treated with LPS/TNF or vehicle (control), and either 10 mM L-leucine [L-leu; a competitive inhibitor of L-arg uptake by the cationic amino acid transporter (CAT)] or its vehicle. In parallel experiments, iNOS or arginase II were overexpressed in bPAEC using an adenoviral vector (AdiNOS or AdArgII, respectively). LPS/TNF treatment increased the expression of iNOS, arginase II, CAT-1, and CAT-2 mRNA in bPAEC, resulting in greater NO and urea production than in control bPAEC, which was prevented by L-leu. LPS/TNF treatment resulted in fewer viable cells than in controls, and LPS/TNF-stimulated bPAEC treated with L-leu had more viable cells than LPS/TNF treatment alone. LPS/TNF treatment resulted in cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase expression, which was attenuated by L-leu. AdiNOS reduced viable cell number, and treatment of AdiNOS transfected bPAEC with L-leu preserved cell number. AdArgII increased viable cell number, and treatment of AdArgII transfected bPAEC with L-leu prevented the increase in cell number. These data demonstrate that iNOS expression in pulmonary endothelial cells leads to decreased cellular proliferation, which can be attenuated by preventing cellular L-arg uptake. We speculate that CAT activity may represent a novel therapeutic target in inflammatory lung diseases characterized by NO overproduction.
Asunto(s)
Transportador de Aminoácidos Catiónicos 1/metabolismo , Transportador de Aminoácidos Catiônicos 2/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Pulmón/citología , Óxido Nítrico/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Adenoviridae/genética , Animales , Arginasa/metabolismo , Arginina/metabolismo , Transportador de Aminoácidos Catiónicos 1/genética , Transportador de Aminoácidos Catiônicos 2/genética , Bovinos , Recuento de Células , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transfección , Urea/metabolismoRESUMEN
Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50-79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials.
Asunto(s)
Distrofina/metabolismo , Inyecciones Intraarteriales/métodos , Inyecciones Intramusculares/métodos , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/terapia , Péptidos/metabolismo , Animales , Western Blotting , Dependovirus/genética , Distrofina/genética , Ensayo de Inmunoadsorción Enzimática , Terapia Genética , Vectores Genéticos/genética , Humanos , Macaca mulatta , Ratones , Ratones Endogámicos C57BL , Oligopéptidos , Péptidos/genéticaRESUMEN
Down syndrome (DS) patients have an increased risk of developing pulmonary hypertension (PH). Increased plasma levels of asymmetric dimethylarginine (ADMA) may contribute to vascular dysfunction in adults with idiopathic pulmonary hypertension. Our goal was to test the hypothesis that DS patients with PH have higher plasma levels of ADMA than DS patients without PH. DS patients with definitive PH (n = 6) and DS patients with no evidence of PH (n = 12) were studied. Plasma levels of arginine, ADMA, and nitrite/nitrate (NOx; stable metabolites of nitric oxide (NO)) were measured. Plasma arginine concentration was lower (p < 0.05) in PH patients (23 ± 11 µM) versus non-PH patients (46 ± 24 µM). Plasma ADMA concentration was higher (p < 0.005) in PH patients (18.0 ± 4.2 µM) versus non-PH patients (8.6 ± 5.9 µM). Plasma NOx was lower (p < 0.05) in PH patients (4.5 ± 1.7 µM) versus non-PH patients (8.5 ± 7.3 µM). These results are consistent with ADMA contributing to lower NO production in DS patients with PH and suggest that ADMA levels may be a potential biomarker for PH in DS patients.
Asunto(s)
Arginina/análogos & derivados , Síndrome de Down/sangre , Hipertensión Pulmonar/sangre , Arginina/sangre , Biomarcadores/sangre , Síndrome de Down/complicaciones , Femenino , Humanos , Hipertensión Pulmonar/complicaciones , Lactante , Masculino , Nitratos/sangre , Nitritos/sangreRESUMEN
1. The endogenous production of and/or the bioavailability of nitric oxide (NO) is decreased in pulmonary hypertensive diseases. L-arginine (L-arg) is the substrate for NO synthase (NOS). L-arg is transported into cells via the cationic amino acid transporters (CAT), of which there are two isoforms in endothelial cells, CAT-1 and CAT-2. 2. To test the hypothesis that hypoxia will decrease CAT expression and L-arg uptake resulting in decreased NO production in human pulmonary microvascular endothelial cells (hPMVEC), cells were incubated in either normoxia (21% O(2), 5% CO(2), balance N(2)) or hypoxia (1% O(2), 5% CO(2), balance N(2)). 3. The hPMVEC incubated in hypoxia had 80% less NO production than cells incubated in normoxia (P < 0.01). The hPMVEC incubated in hypoxia had significantly lower CAT-2 mRNA levels than normoxic hPMVEC (P < 0.005), and the transport of L-arg was 40% lower in hypoxic than in normoxic hPMVEC (P < 0.01). In hypoxic cells, overexpression of CAT-1 resulted in significantly greater L-arg transport and NO production (P < 0.05). 4. These results demonstrate that in hPMVEC, hypoxia decreased CAT-2 expression, L-arg uptake and NO production. Furthermore, the hypoxia-induced decrease in NO production in hPMVEC can be attenuated by overexpressing CAT in these cells. We speculate that the CAT may represent a novel therapeutic target for treating pulmonary hypertensive disorders.
Asunto(s)
Transportador de Aminoácidos Catiónicos 1/biosíntesis , Endotelio Vascular/metabolismo , Pulmón/irrigación sanguínea , Óxido Nítrico/biosíntesis , Sistemas de Transporte de Aminoácidos Básicos/biosíntesis , Arginina/metabolismo , Línea Celular , Endotelio Vascular/citología , Humanos , Hipoxia/metabolismo , Microvasos/citología , Microvasos/metabolismoRESUMEN
We hypothesized that hypoxia would activate epidermal growth factor receptor (EGFR) tyrosine kinase, leading to increased arginase expression and resulting in proliferation of human pulmonary microvascular endothelial cell (hPMVEC). To test this hypothesis, hPMVEC were incubated in normoxia (20% O(2), 5% CO(2)) or hypoxia (1% O(2), 5% CO(2)). Immunoblotting for EGFR and proliferating cell nuclear antigen was done, and protein levels of both total EGFR and proliferating cell nuclear antigen were greater in hypoxic hPMVEC than in normoxic hPMVEC. Furthermore, hypoxic hPMVEC had greater levels of EGFR activity than did normoxic hPMVEC. Hypoxic hPMVEC had a twofold greater level of proliferation compared with normoxic controls, and this increase in proliferation was prevented by the addition of AG-1478 (a pharmacological inhibitor of EGFR). Immunoblotting for arginase I and arginase II demonstrated a threefold induction in arginase II protein levels in hypoxia, with little change in arginase I protein levels. The hypoxic induction of arginase II protein was prevented by treatment with AG-1478. Proliferation assays were performed in the presence of arginase inhibitors, and hypoxia-induced proliferation was also prevented by arginase inhibition. Finally, treatment with an EGFR small interfering RNA prevented hypoxia-induced proliferation and urea production. These findings demonstrate that hypoxia activates EGFR tyrosine kinase, leading to arginase expression and thereby promoting proliferation in hPMVEC.
Asunto(s)
Células Endoteliales/citología , Células Endoteliales/enzimología , Receptores ErbB/metabolismo , Pulmón/irrigación sanguínea , Microvasos/citología , Arginasa/antagonistas & inhibidores , Arginasa/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Biológicos , Quinazolinas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Factores de Tiempo , Tirfostinos/farmacología , Urea/metabolismoRESUMEN
The mitogen-activated protein (MAP) kinases are involved in cellular responses to many stimuli, including hypoxia. MAP kinase signaling is regulated by a family of phosphatases that include MAP kinase phosphatase-1 (MKP-1). We hypothesized that mice lacking the Mkp-1 gene would have exaggerated chronic hypoxia-induced pulmonary hypertension. Wild-type (WT) and Mkp-1(-/-) mice were exposed to either 4 wk of normoxia or hypobaric hypoxia. Following chronic hypoxia, both genotypes demonstrated elevated right ventricular pressures, right ventricular hypertrophy as demonstrated by the ratio of the right ventricle to the left ventricle plus septum weights [RV(LV + S)], and greater vascular remodeling. However, the right ventricular systolic pressures, the RV/(LV + S), and the medial wall thickness of 100- to 300-microm vessels was significantly greater in the Mkp-1(-/-) mice than in the WT mice following 4 wk of hypobaric hypoxia. Chronic hypoxic exposure caused no detectable change in eNOS protein levels in the lungs in either genotype; however, Mkp-1(-/-) mice had lower levels of eNOS protein and lower lung NO production than did WT mice. No iNOS protein was detected in the lungs by Western blotting in any condition in either genotype. Both arginase I and arginase II protein levels were greater in the lungs of hypoxic Mkp-1(-/-) mice than those in hypoxic WT mice. Lung levels of proliferating cell nuclear antigen were greater in hypoxic Mkp-1(-/-) than in hypoxic WT mice. These data are consistent with the concept that MKP-1 acts to restrain hypoxia-induced arginase expression and thereby reduces vascular remodeling and the severity of pulmonary hypertension.
Asunto(s)
Arginasa/metabolismo , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/fisiología , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Hipoxia/genética , Hipoxia/metabolismo , Pulmón/metabolismo , Proteínas/metabolismo , Animales , Western Blotting , Peso Corporal/fisiología , Enfermedad Crónica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo I/biosíntesis , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Óxido Nítrico Sintasa de Tipo III/genética , Nitritos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Función Ventricular Derecha/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Inhaled nitric oxide is being evaluated as a preventative therapy for patients at risk for bronchopulmonary dysplasia (BPD). Nitric oxide (NO), in the presence of superoxide, forms peroxynitrite, which reacts with tyrosine residues on proteins to form 3-nitrotyrosine (3-NT). However, NO can also act as an antioxidant and was recently found to improve the oxidative balance in preterm infants. Thus, we tested the hypothesis that the addition of a therapeutically relevant concentration (10 ppm) of NO to a hyperoxic exposure would lead to decreased 3-NT formation in the lung. FVB mouse pups were exposed to either room air (21% O(2)) or >95% O(2) with or without 10 ppm NO within 24 h of birth. In the first set of studies, body weights and survival were monitored for 7 days, and exposure to >95% O(2) resulted in impaired weight gain and near 100% mortality by 7 days. However, the mortality occurred earlier in pups exposed to >95% O(2) + NO than in pups exposed to >95% O(2) alone. In a second set of studies, lungs were harvested at 72 h. Immunohistochemistry of the lungs at 72 h revealed that the addition of NO decreased alveolar, bronchial, and vascular 3-NT staining in pups exposed to both room air and hyperoxia. The lung nitrite levels were higher in animals exposed to >95% oxygen + NO than in animals exposed to >95% oxygen alone. The protein levels of myeloperoxidase, monocyte chemotactic protein-1, and intracellular adhesion molecule-1 were assessed after 72 h of exposure and found to be greatest in the lungs of pups exposed to >95% O(2). This hyperoxia-induced protein expression was significantly attenuated by the addition of 10 ppm NO. We propose that in the presence of >95% O(2), peroxynitrite formation results in protein nitration; however, adding excess NO to the >95% O(2) exposure prevents 3-NT formation by NO reacting with peroxynitrite to produce nitrite and NO(2). We speculate that the decreased protein nitration observed with the addition of NO may be a potential mechanism limiting hyperoxic lung injury.
Asunto(s)
Lesión Pulmonar/prevención & control , Óxido Nítrico/administración & dosificación , Tirosina/análogos & derivados , Animales , Quimiocina CCL2/análisis , Hiperoxia/metabolismo , Hiperoxia/patología , Molécula 1 de Adhesión Intercelular/análisis , Pulmón/efectos de los fármacos , Pulmón/patología , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/patología , Ratones , Oxígeno/efectos adversos , Peroxidasa/análisis , Tirosina/antagonistas & inhibidores , Tirosina/biosíntesisRESUMEN
A recently completed study quantified the percent of fentanyl or morphine sulfate lost to uncoated polyvinylchloride (PVC) tubing or to one of two hollow fiber oxygenators within the extracorporeal life support (ECLS) circuit. The results demonstrated the majority of drug loss was due to adsorption by the PVC tubing. The purpose of this study was to determine if a tubing coating process affects fentanyl or morphine Sulfate adsorption. The goal was to quantify fentanyl or morphine sulfate lost due to adhesion within surface modified tubing. The following surface modifications were studied: 1) Maquet Safeline (synthetic immobilized albumin); 2) Maquet Softline (a heparin free biopassive polymer); 3) Maquet Bioline (recombinant human albumin + heparin) (Maquet Cardiopulmonary AG, Hirrlingen, Germany); 4) Terumo X Coating (poly2methoxylacrylate)) (Terumo Cardiovascular Systems Corporation, Ann Arbor, MI); 5) Medtronic Carmeda (covalently bonded heparin); and 6) Medtronic Trillium (covalently bonded heparin) (Medtronic, Minneapolis, MN). A total of 36 individual circuits were built from the above six available modified surface coatings, for a total of six individual circuits of each coating type. Blood samples were drawn at 5 minutes, 120 minutes, and 360 minutes followed by High-Performance Liquid Chromatography to determine available circulating levels of either fentanyl or morphine sulfate. Fentanyl concentrations decreased to an average final available concentration of 35% (+/- 5%) within the 18 circuits. Morphine sulfate however, decreased to a final available concentration of 57% (+ 1%) in all Maquet tubing and the Medtronic Trillium tubing, while it decreased to a final concentration of 35% (+ 1%) in the Medtronic Carmeda coated tubing and in the Terumo X Coating tubing. Biocompatible ECLS circuit surface coatings affected drug-adsorption and availability. Further evaluation is necessary to understand the adsorptive loss of other drugs administered to our patients while on modified surface coated ECLS circuits.
Asunto(s)
Materiales Biocompatibles Revestidos , Circulación Extracorporea/instrumentación , Fentanilo/química , Morfina/química , Adsorción , Analgésicos/administración & dosificación , Analgésicos/farmacocinética , Fentanilo/farmacocinética , Humanos , Hipnóticos y Sedantes/química , Hipnóticos y Sedantes/farmacocinética , Técnicas In Vitro , Morfina/farmacocinética , Cloruro de Polivinilo , Propiedades de SuperficieRESUMEN
Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development program to regional limb perfusion, comparing two methods previously established for gene therapy, peripheral venous limb perfusion (VLP) and an intra-arterial push and dwell (IAPD) using rAAV1 and rAAV8 in a non-human primate (rhesus macaque) study. The rhesus AAT transgene was used with a c-myc tag to enable quantification of transgene expression. 5 cohorts of animals were treated with rAAV1-IM, rAAV1-VLP, rAAV1-IAPD, rAAV8-VLP, and rAAV8-IAPD (n = 2-3), with a dose of 6 × 1012 vg/kg. All methods were well tolerated clinically. Potency, as determined by serum levels of AAT, of rAAV1 by the VLP method was twice that observed with direct IM injection; 90 µg/mL with VLP versus 38 µg/mL with direct IM injection. There was an approximately 25-fold advantage in estimated vector genomes retained within the muscle tissue with VLP and a 5-fold improvement in the ratio of total vector genomes retained within muscle as compared with liver. The other methods were intermediate in the potency and retention of vector genomes. Examination of muscle enzyme (CK) levels indicated rAAV1-VLP to be equally safe as compared with IM injection, while the IAPD method showed significant CK elevation. Overall, rAAV1-VLP demonstrates higher potency per vector genome injected and a greater total vector retention within the muscle, as compared to IM injection, while enabling a much greater total dose to be delivered, with equivalent safety. These data provide the basis for continuation of the dose escalation of the rAAV1-AAT program in patients and bode well for rAAV-VLP as a platform for replacement of secreted proteins.
RESUMEN
In a previous limb-girdle muscular dystrophy type 2D (LGMD2D) clinical trial, robust alpha-sarcoglycan gene expression was confirmed following intramuscular gene (SGCA) transfer. This paved the way for first-in-human isolated limb infusion (ILI) gene transfer trial to the lower limbs. Delivery of scAAVrh74.tMCK.hSGCA via an intravascular route through the femoral artery predicted improved ambulation. This method was initially chosen to avoid safety concerns required for large systemic vascular delivery viral loads. ILI methods were adopted from the extensive chemotherapy experience for treatment of malignancies confined to the extremities. Six LGMD2D subjects were enrolled in a dose-ascending open-label clinical trial. Safety of the procedure was initially assessed in the single limb of a non-ambulant affected adult at a dose of 1 × 1012 vg/kg. Subsequently, ambulatory children (aged 8-13 years) were enrolled and dosed bilaterally with either 1 × 1012 vg/kg/limb or 3 × 1012 vg/kg/limb. The six-minute walk test (6MWT) served as the primary clinical outcome; secondary outcomes included muscle strength (maximum voluntary isometric force testing) and SGCA expression at 6 months. All ambulatory participants except one had pre- and post-treatment muscle biopsies. All four subjects biopsied had confirmed SGCA gene delivery by immunofluorescence, Western blot analysis (14-25% of normal), and vector genome copies (5.4 × 103-7.7 × 104 vg/µg). Muscle strength in the knee extensors (assessed by force generation in kilograms) showed improvement in two subjects that correlated with an increase in fiber diameter post gene delivery. Six-minute walk times decreased or remained the same. Vascular delivery of AAVrh74.tMCK.hSGCA was effective at producing SGCA protein at low doses that correlated with vector copies and local functional improvement restricted to targeted muscles. Future trials will focus on systemic administration to enable targeting of proximal muscles to maximize clinical benefit.