Dov Gal

Use of the rabbit ear artery to serially assess foreign protein secretion after site-specific arterial gene transfer in vivo. Evidence that anatomic identification of successful gene transfer may underestimate the potential magnitude of transgene expression.

BackgroundThe development of molecular strategies for the treatment of restenosis has been hindered by low efficiencies of in vivo arterial transfection. Expression of intracellular marker proteins is generally evident in <1% of vascular smooth muscle cells after in vivo arterial transfection. Efforts to improve the efficiency of in vivo gene transfer have been further impeded by the use of transgenes encoding for intracellular marker proteins, necessitating tissue removal and limiting survey for expression to one point in time. Methods and ResultsTo study gene expression on a serial basis in vivo and determine the relation between a secreted gene product and transfection efficiency after in vivo arterial gene transfer, a method for performing and serially monitoring gene expression in vivo was developed using the central artery of the rabbit ear. Liposome-mediated transfection of plasmid DNA containing the gene for human growth hormone (hGH) was successfully performed in 18 of 23 arteries. Serum hGH levels measured 5 days after transfection ranged from 0.1 to 3.8 ng/mL (mean, 0.97 ng/mL); in contrast, serum drawn from the control arteries demonstrated no evidence of hGH proene duction. Serial measurement of hGH from transfected arteries demonstrated maximum hGH secretion 5 days after transfection and no detectable hormone after 20 days. Despite these levels of secreted gene product documented in vivo, immunohistochemical staining of sections taken from the rabbit ear artery at necropsy disclosed only rare cells in which there was evidence of successful transfection. ConclusionsThese experiments demonstrate a useful method of performing serial in vivo analyses of gene expression after vascular transfection and that anatomic analyses of transfection efficiency may underestimate the potential magnitude of expression in the case of a secreted gene product. These findings have implications for the clinical application of somatic gene therapy because low-efficiency transfection with a gene encoding for a secreted protein may achieve therapeutic effects not realized by transfection with genes encoding for proteins that remain intracellular.

Pulsed ultraviolet laser irradiation produces endothelium-independent relaxation of vascular smooth muscle.

Recent studies have shown that continuous wave laser irradiation induces contraction of vascular smooth muscle, except at powers far below the threshold for tissue ablation. To determine the corresponding effects of pulsed laser irradiation on vascular smooth muscle tone, vascular rings of rabbit thoracic aorta were mounted isometrically with 1 g tension in Krebs-bicarbonate buffer and irradiated with 308 or 351 nm from an excimer laser through a 400-microns optical fiber. A total of 250 exposures were performed with 1-6.5 mJ/pulse (fluence = 0.8-5.5 J/cm2), 10-50 Hz, and cumulative exposures of 10-120 seconds. Excimer laser irradiation in combinations of pulse energy (PE), repetition rate (RR), and cumulative exposure below, at, or above threshold for tissue ablation consistently produced relaxation unassociated with contraction in each of the 250 exposures. For the total 250 exposures, the magnitude of relaxation (reduction in recorded tension, Rmax) was 55 +/- 4% (mean +/- SEM) of maximum vasomotor reactivity recorded in the specimen in response to administration of serotonin. Rmax varied directly with both PE and RR. When PE was increased from 1 to 5 mJ/pulse (n = 13), Rmax increased from 57 +/- 19% to 80 +/- 19% (p less than 0.0001); when RR was increased from 10 to 50 Hz (n = 10), Rmax increased from 27 +/- 8 to 46 +/- 8 (p less than 0.0001). Rmax varied independently of endothelial integrity (assessed anatomically and pharmacologically) and wavelength (308 vs. 351 nm). Simultaneously recorded tissue-temperature profiles disclosed that during pulsed laser irradiation, tissue temperature rise did not exceed 5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Percutaneous delivery of low-level laser energy reverses histamine-induced spasm in atherosclerotic Yucatan microswine.

BackgroundPrevious in vitro experiments performed in our laboratory have shown that low-level laser energy may produce prompt reduction in isometric tension of vascular smooth muscle. The present study was designed to extend these previous in vitro findings to an in vivo model and thereby investigate the hypothesis that laser light delivered percutaneously in vivo could successfully reverse arterial spasm. Methods and ResultsSpasm defined as >50% reversible reduction in luminal diameter persisting for ≥5 minutes was successfully provoked by injection of histamine (100–400 μg/kg) in 13 arteries among 10 atherosclerotic Yucatan microswine; the magnitude of histamineinduced vasoconstriction was then documented angiographically by repeated injections of contrast media for as long as 30 minutes (controls). After return of angiographic luminal diameter to baseline, spasm was reproduced with a second injection of histamine into the same artery. Representative wavelengths generated by ultraviolet (UV), visible, and infrared lasers were then delivered percutaneously via conventional fiberoptics to the site of spasm, and angiographic assessment was repeated for as long as 30 minutes (treatment trial). In three arteries treated with UV (351 nm) light from an excimer laser, angiographic luminal diameter narrowing decreased from 100% to 23.9%, 50.0% to 9.3%, and 76.0% to 42.3%, respectively. The magnitude of laser-induced increase in luminal diameter was 50.2±22.7%, which was significantly greater than the magnitude of relaxation observed spontaneously during the control trials (10.9±9.8%, p=0.02). Visible light from a helium-neon (632 nm) laser accomplished complete reversal of histamine-induced spasm in two of four arteries; in the remaining two arteries, luminal diameter narrowing percentages were reduced from 57.0% to 20.0% and from 76.5% to 30.8%, respectively. The magnitude of helium-neon laser-induced relaxation (55.8±17.9%) was again significantly greater than that observed during the control trials (0.9±1.9%, p=0.0l). Finally, infrared irradiation from a diode-pumped neodymium:yttrium aluminum garnet (1,064 nm) laser decreased histamine-induced luminal diameter narrowing in three arteries from 100%lv to 21.4%, 56.0% to 8.7%, and 68.3% to 35.3%, respectively. The magnitude of infrared laser-induced improvement in luminal diameter narrowing was 53.0±23.3%, which was significantly greater than that observed during the control trials (12.9±10.7%, p=0.01). In three additional arteries, fiberoptic sham trials (without laser irradiation) failed to produce relaxation of histamine-induced spasm. ConclusionsThese findings document for the first time that light-induced relaxation of vascular smooth muscle, previously documented in vitro, may be reproduced in vivo.

Vascular spasm complicates continuous wave but not pulsed laser irradiation

Preliminary clinical experience with laser angioplasty has suggested that arterial spasm may complicate attempts to employ laser light to accomplish vascular recanalization. The present study was designed to investigate the role of energy profile on the development of arterial spasm during laser angioplasty. Laser irradiation was delivered percutaneously in vivo to New Zealand white rabbits and to Yucatan microswine with or without atherosclerotic lesions induced by a combination of balloon endothelial denudation and atherogenic diet. Continuous wave (CW) laser irradiation from an argon ion gas laser (wavelength 488 to 514 nm) was applied to 23 arteries, while 16 arteries were irradiated using a pulsed xenon chloride (308 nm) or xenon fluoride (351 nm) excimer laser. Arterial spasm, defined as greater than 50% reduction in luminal diameter narrowing, complicated delivery of laser light to 17 (74%) of the 23 arteries irradiated with the CW argon laser. Spasm was consistently observed at powers greater than 2 W, at cumulative exposures greater than 200 seconds, and at total energy greater than 200 joules. Spasm was typically diffuse (including the length of the vessel) and protracted (lasting up to 120 minutes). Intra-arterial nitroglycerin (up to 300 micrograms) produced only temporary and incomplete resolution of laser-induced spasm. In contrast, spasm was never observed in any of the 16 arteries in which laser angioplasty was performed using a pulsed laser (0.95 to 6.37 joules/cm2, 10 to 50 Hz, 48 to 370 seconds). Thus CW but not pulsed laser angioplasty may be complicated by arterial spasm.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure of angiography to diagnose thermal perforation complicating laser angioplasty in a rabbit

Abstract Perforation of the vascular wall underlying the site of intended recanalization constitutes the principal risk of laser angioplasty. 1–3 In clinical trials of percutaneous laser angioplasty, absence of contrast extravasation on angiographic examination after laser treatment is usually interpreted to indicate that laser angioplasty was uncomplicated by vessel wall perforation. 1–3 We describe failure of angiography to diagnose necropsy-proved thermal perforation complicating laser recanalization of experimentally induced atherosclerotic narrowing in a rabbit.

Percutaneous Laser Myoplasty for Treatment of Hypertrophic Cardiomyopathy

The intraoperative application of argon-laser irradiation for the treatment of hypertrophic cardiomyopathy (HC) represents the first successful use of laser light for the treatment of cardiovascular disease in a live patient [1]. Conventional surgical treatment for patients with HC who are refractory to medical therapy involves a left ventricular septal myotomy and myectomy (Morrow procedure) [2] or myotomy alone [3, 4]. Both operations are performed via a complete median sternotomy. Following institution of cardiopulmonary bypass, an aortotomy is created and the left ventricular outflow tract is visualized below the aortic valve. Retracting the valve cusps, an incision (myotomy) is then made in the thickened basal portion of the ventricular septum. The procedure as designed by Morrow includes removal of a relatively small piece (0.5–2.0 gm) of septal myocardium (myectomy).