RESUMEN
Therapeutic angiogenesis with gene encoding vascular endothelial growth factor (VEGF) is a new potential treatment in cardiovascular disease. However, unregulated VEGF-mediated angiogenesis has the potential to promote tumor growth, accelerate diabetic proliferative retinopathy, and promote rupture of atherosclerotic plaque. To be safe and effective, gene therapy with VEGF must be regulated. To limit the risk of pathological angiogenesis, we developed a hypoxia-inducible VEGF gene therapy system using the erythropoietin (Epo) enhancer and water-soluble lipopolymer (WSLP). pEpo-SV-VEGF or pSV-VEGF-Epo was constructed by insertion of the Epo enhancer upstream of the Simian Virus 40 (SV40) promoter or downstream of the poly(A) signal of pSV-VEGF. In vitro transfection showed that pEpo-SV-VEGF, not pSV-VEGF-Epo, induced the VEGF expression in hypoxic cells. In addition, the VEGF protein, which was produced from the Epo-SV-VEGF-transfected and hypoxia-incubated cells, was able to enhance the proliferation of the endothelial cells. Injection of the pEpo-SV-VEGF/WSLP complex showed that the expression of VEGF was induced in ischemic myocardium, compared to normal myo-cardium. Therefore, with the localized induction of VEGF and the low cytotoxicity of WSLP, the pEpo-SV-VEGF/WSLP system may be helpful to eventually treat ischemic heart disease.
Asunto(s)
Factores de Crecimiento Endotelial/genética , Terapia Genética/métodos , Hipoxia , Péptidos y Proteínas de Señalización Intercelular/genética , Linfocinas/genética , Isquemia Miocárdica/terapia , Neovascularización Fisiológica , Células Madre/metabolismo , Animales , División Celular , Endotelio Vascular/citología , Elementos de Facilitación Genéticos , Ensayo de Inmunoadsorción Enzimática/métodos , Eritropoyetina , Expresión Génica , Ingeniería Genética , Humanos , Liposomas , Luciferasas/genética , Modelos Animales , Isquemia Miocárdica/fisiopatología , Polímeros , Conejos , Transfección/métodos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Water-soluble lipopolymer (WSLP), which consisted of polyethylenimine (PEI, 1800 Da) and cholesterol, was characterized as a gene carrier to smooth muscle cells and myocardium. Acid-base titration showed that WSLP had a proton-buffering effect. The size of WSLP/plasmid DNA (pDNA) complex was around 70 nm. WSLP/pDNA complex was transfected to A7R5 cells, a smooth muscle cell line. WSLP showed the highest transfection at a 40/1 N/P ratio. WSLP has higher transfection efficiency than PEI (1800 and 25 000 Da), SuperFect, and lipofectamine. In addition, WSLP has less cytotoxicity than PEI (25 000 Da), SuperFect, and lipofectamine. Since WSLP has cholesterol moiety, it may utilize cellular cholesterol uptake pathway, in which low-density lipoprotein (LDL) is involved. An inhibition study with free cholesterol or low-density lipoprotein (LDL) showed that transfection was inhibited by cholesterol or LDL, suggesting that WSLP/pDNA complex is transfected to the cells through the cholesterol uptake pathway. To evaluate the transfection efficiency to myocardium, WSLP/pDNA complex was injected into the rabbit myocardium. WSLP showed higher transfection than PEI and naked pDNA. WSLP expressed the transgene for more than 2 weeks. In conclusion, WSLP is an efficient carrier for local gene transfection to myocardium, and useful in in vivo gene therapy.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/genética , Miocardio/metabolismo , Transfección/métodos , Animales , Colesterol/genética , Expresión Génica , Liposomas , Luciferasas/genética , Músculo Liso/metabolismo , Polietileneimina , ConejosRESUMEN
A modification of the subtalar extraarticular arthrodesis is described in which local bone graft from the calcaneus and talus is used with internal fixation to obtain fusion. Twenty-eight patients with cerebral palsy underwent 52 local bone-graft subtalar arthrodeses to correct valgus deformity of the hindfoot at an average age of 7 years 5 months. The preoperative lateral talocalcaneal angle averaged 44.5 degrees, with the intraoperative and postoperative angles being 35 and 33.5 degrees, respectively. These postoperative figures were significantly different from the preoperative films and remained stable over the average 41-month follow-up period. Six feet did not have radiographic union, yet only two lost correction requiring repeated surgery. Thus 88% of the feet had radiographic union, and 96% had a stable talocalcaneal angle at follow-up. This procedure is quick without the necessity of a distant bone graft and the inherent morbidity associated with it. It is as successful in stabilizing the subtalar joint as are other techniques reported in the literature.
Asunto(s)
Articulación del Tobillo/cirugía , Artrodesis/métodos , Trasplante Óseo , Parálisis Cerebral/cirugía , Deformidades Adquiridas del Pie/cirugía , Calcáneo , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Masculino , Astrágalo , Resultado del TratamientoRESUMEN
A new technique is presented for obtaining gas concentration by measuring the slope of the anomalous dispersion at a resonance. We describe the equations that govern this process using a Lorentz model and show that the slope of the anomalous dispersion is directly related to the absorption coefficient. The slope is obtained from an interferometric setup and a frequency modulation spectroscopy technique. Experimental data are presented that illustrate this technique for two different sample cells containing water vapor.
RESUMEN
BACKGROUND: Hypoxia and warm ischemia produce severe injury to cardiac grafts harvested from non-heartbeating donors. To potentially improve recovery of such grafts, we studied the effects of intravenous phenylephrine preconditioning. METHODS: Thirty-seven blood-perfused rabbit hearts were studied. Three groups of non-heart-beating donors underwent intravenous treatment with phenylephrine at 12.5 (n = 8), 25 (n = 7), or 50 microg/kg (n = 7) before initiation of apnea. Non-heart-beating controls (n = 8) received saline vehicle. Hypoxic cardiac arrest occurred after 6 to 12 minutes of apnea, followed by 20 minutes of warm in vivo ischemia. A 45-minute period of ex vivo reperfusion ensued. Nonischemic controls (n = 7) were perfused without antecedent hypoxia or ischemia. RESULTS: Phenylephrine 25 microg/kg significantly delayed the onset of hypoxic cardiac arrest compared with saline controls (9.6 +/- 0.5 versus 7.7 +/- 0.4 minutes; p = 0.00001), yet improved recovery of left ventricular developed pressure compared with saline controls (57.1 +/- 5.3 versus 41.0 +/- 3.4 mm Hg; p = 0.04). Phenylephrine 25 microg/kg also yielded a trend toward less myocardial edema than saline vehicle (p = 0.09). CONCLUSIONS: Functional recovery of nonbeating cardiac grafts is improved by preconditioning. We provide evidence that the myocardium can be preconditioned with phenylephrine against hypoxic cardiac arrest.
Asunto(s)
Paro Cardíaco/cirugía , Trasplante de Corazón/métodos , Precondicionamiento Isquémico Miocárdico/métodos , Fenilefrina/administración & dosificación , Análisis de Varianza , Animales , Agua Corporal/metabolismo , Infusiones Intravenosas , Isquemia Miocárdica/cirugía , Reperfusión Miocárdica , Miocardio/metabolismo , Consumo de Oxígeno , Conejos , Función Ventricular IzquierdaRESUMEN
Gastric acid and pepsin secreted in 3 hr and antral gastrin released in response to vagal excitation induced by 2-deoxy-D-glucose (2DG), 625 mumol/kg i.v., were studied in six conscious trained gastric fistula dogs. During a 2-hr infusion, Met-enkephalin (96 nmol/kg/hr; delta receptor) reduced the 2DG response by 50%; when the enkephalin was stopped there was a rapid rebound to peak values. Met-enkephalin also blocked the release of gastrin in the first 15 min. By itself, Met-enkephalin did not stimulate secretion and slightly depressed gastrin. By contrast, morphine (96 nmol/kg/hr; mu receptor) augmented and sustained the 2DG gastric acid secretory response. This effect was blocked by naloxone. Morphine alone caused a small rise in serum gastrin after 90 min, followed by a delayed gastric acid secretion of about 30% of the peak 2DG response. Naloxone, a mu opiate antagonist (mu/delta, 27:1), also inhibited the 2DG gastric secretory response by about 50% and augmented the Met-enkephalin inhibition of secretion without blocking either the secretory rebound or the effect on gastrin release. None of the three opiates changed the direct cholinergic gastric secretory or gastrin-releasing effects of bethanechol. Thus, vagal stimulation of the stomach involves pathways which can be influenced by both mu and delta opiates, with apparently opposite effects, proximal to the level of acetylcholine action on the gastric mucosa. The central and peripheral control points in the activation of the stomach via the vagus which are sensitive to opiates have yet to be located and explained.
Asunto(s)
Desoxiazúcares/farmacología , Desoxiglucosa/farmacología , Endorfinas/farmacología , Encefalinas/farmacología , Ácido Gástrico/metabolismo , Gastrinas/metabolismo , Morfina/farmacología , Naloxona/farmacología , Nervio Vago/fisiología , Animales , Compuestos de Betanecol/farmacología , Perros , Encefalina Metionina , Femenino , Masculino , Pepsina A/metabolismo , Estimulación Química , Nervio Vago/efectos de los fármacosRESUMEN
Although low doses of histamine (less than 150 nM/kg.hr) stimulate pepsin secretion, higher doses inhibit pepsin secretion in a dose-dependent manner. To better histamine stimulation of pepsin, histamine was used at doses at the lower end of the dose-response scale in five dogs with gastric fistula. Five doses of histamine below the ED50 for acid, viz, 9, 22.5, 67.5, 90 and 112 nM/kg.hr in 45-min steps, provided values for pepsin secretion from which Ed50 = 11.4 nmol/kg.hr (i.e., about 1/12 the ED50 for acid) and calculated maximum 22,600 peptic U/30 min were calculated. To document the inhibition, pepsin secretion was first stimulated by an infusion of bethanechol (0.4 mumol/kg.hr). A super-added injection of the histamine H-2 agonist 4-methylhistamine (0.4 or 0.8 mumol/kg) produced strong additional acid stimulation and immediate 40% suppression of pepsin secretion. The ratio pepsin/acid was reduced to one-third of control for the 90 min after 4-methylhistamine. The most specific H-2 agonist impromidine had the same effects, whereas pentagastrin (1.95 nmol/kg) inhibited both acid and pepsin secretion stimulated by bethanechol. The specificity of H-2 effect of impromidine was confirmed by simultaneous tachycardia and hypotension; pentagastrin did not produce cardiovascular effects. These studies confirm the unique effect of histamine on the peptic cell of the dog in which both stimulation and inhibition are H-2 receptor-mediated effects.
Asunto(s)
Pepsina A/metabolismo , Receptores Histamínicos H2/efectos de los fármacos , Receptores Histamínicos/efectos de los fármacos , Animales , Perros , Jugo Gástrico/metabolismo , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Guanidinas/farmacología , Histamina/farmacología , Imidazoles/farmacología , Impromidina , Metilhistaminas/farmacologíaRESUMEN
Histamine H-2 receptor-mediated effects of the very selective H-2 agonist (H-1:H-2 < 1:1000) impromidine on gastric acid, chloride and pepsin secretion and on heart rate (HR) and systolic blood pressure were compared to those of histamine in five conscious gastric fistula dogs. In each dog, impromidine in a step-dose response (0.46 to 46 nmol/kg.hr) in 45-min steps was given without and with a background infusion of cimetidine (2 mumol/kg.hr). Histamine acid phosphate was given in a seven step-dose response (18 to 1350 nmol/kg. hr). Impromidine produced the same maximum stimulation of gastric HCI output, increase of HR and fall in systolic blood pressure as histamine. Impromidine was some 38 times more potent than histamine in stimulation of acid (ED50 3.8 vs. 145 nmol/kg.hr) and 30 times more potent in raising HR (ED50 5.6 vs. 172 nmol/kg.hr). Cimetidine competitively inhibited the effects of impromidine with similar pA2 values for each effect (acid, 5.99; chloride, 6.03; change in HR, 6.03; and change in systolic blood pressure, 6.32). The effects of impromidine on pepsin secretion were qualitatively and quantitatively similar to those of histamine and other H-2 agonists with weak stimulation at low doses and progressive inhibition with increasing doses of impromidine. Coupling the results with the known high specificity of impromidine, gastric acid secretion, chronotropism and hypotension all seem to be purely H-2-mediated effects of histamine in the intact conscious dog.
Asunto(s)
Presión Sanguínea , Jugo Gástrico/metabolismo , Guanidinas , Frecuencia Cardíaca , Imidazoles , Receptores Histamínicos H2/fisiología , Receptores Histamínicos/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Cimetidina , Perros , Relación Dosis-Respuesta a Droga , Frecuencia Cardíaca/efectos de los fármacos , Impromidina , Pepsina A/metabolismo , Receptores Histamínicos H2/efectos de los fármacos , Factores de TiempoRESUMEN
Two new H-2 agonists 5,N'-dimethyl histamine (DMH), an imidazole analog, and dimaprit, a non-imidazole compound, and histamine (H), a mixed H-1, H-2 agonist, were given to four conscious gastric fistula dogs. Gastric acid was stimulated dose responsively. Dimaprit stimulated a 20% greater maximum output of acid, and more pepsin than the other agents, but inhibited pepsin secretion as doses of greater than 0.63 mumol/kg-h. Heart rate was increased dose responsively by all drugs to approximately 200 beats/min with ED50 of 0.17:0.42:0.80 mumol/kg-h (H:DMH:dimaprit). Normalized dose responses showed that histamine was equipotent on acid and heart rate (ED50 0.15 vs. 0.14 respectively) while DMH (ED50 0.21 vs. 0.40) and dimaprit (ED50 0.27 vs. 0.85) stimulated heart rate less effectively than acid. Histamine was more effective at reducing blood pressure, with approximate ED50 of 0.28:0.60:0.96 for H, DMH and dimaprit respectively. The results indicate considerable heterogeneity of histamine responses for different actions mediated by the H-2 receptors, as well as differences for any one action between H-2 agonists.