SU-E-I-69: Magnetic Resonance Metal Artifact Evaluation with Routine Clinical Cardiac Sequences.

PURPOSE: To examine schemes to grade the severity of metal susceptibility artifacts on image quality using cardiac MRI pulse sequences. METHODS: A post-thoracotomy patient was simulated with a stainless steel sternal wire (Syneture,MA; size=6, diameter=48mm), placed securely on an ACR MRI phantom. Phantom was scanned on a 1.5-T Siemens using cardiac MRI sequences:1)TrueFISP, 2)Gradient-Recalled-Echo (GRE), 3)Turbo-Spin- Echo (TSE), 4)Turbo-Inversion-Recovery-Magnitude (TIRM), 5)Dark- blood-IR-FS (DBFS) with and without the wire (FOV=30×30cm, slice- thickness/slice-gap=7.0/1.5mm, matrix size=192×192, slices=17). Image quality degradation was assessed in terms of signal loss and spatial deformation; signal loss by a) measuring the largest diameter of signal drop and b) number of slices with a signal drop and spatial deformation in VelocityAI (Atlanta,GA) by computing the rigid transformation indices between the phantom's internal grid with and without the metal. RESULTS: Image quality was evaluated in terms of signal loss, spatial deformation and ring artifacts. Signal loss: TruFISP and GRE showed the largest signal drop diameter (13 and 16cm respectively). GRE sequence showed a signal drop in -12 slices where as signal drop occurred in only ∼4-5 slices with other sequences. Spatial deformation: GRE sequence showed the maximum with a ∼9mm grid deflection followed by TSE and DBFS (∼8mm). An average deflection of 5.4mm was observed on most of the sequences except T rueFISP and TIRM (Omm). Rigid body transformation showed a maximum x,y,z-translation of -4.7, 0.3 and 1.69 mm and x,y,z-angular rotation of 0.2, - 1.5 and 0.5° for GRE sequence followed by TSE and DBFS confirming the spatial distortion results. Concentric ring artifacts with signal loss were also observed on TrueFISP and DBFS images. CONCLUSIONS: Quantification of cardiac MR sequences to metal tolerance and the impact on image quality has shown that GRE and TrueFISP are the most metal-susceptible and TIRM is the most metal-tolerant sequence in terms of both signal loss and spatial deformation. This study helped in creating a separate cardiac metal protocol comprising of mainly metal-tolerant sequences thus reducing scan time and patient discomfort.

Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF(121) (AdVEGF121) versus maximum medical treatment.

The demonstration that angiogenic growth factors can stimulate new blood vessel growth and restore perfusion in animal models of myocardial ischemia has led to the development of strategies designed for the local production of angiogenic growth factors in patients who are not candidates for conventional revascularization. The results of recent clinical trials of proangiogenesis gene therapy have been disappointing; however, significant limitations in experimental design, in particular in gene transfer strategies, preclude drawing definitive conclusions. In the REVASC study cardiac gene transfer was optimized by direct intramyocardial delivery of a replication-deficient adenovirus-containing vascular endothelial growth factor (AdVEGF121, 4 x 10(10) particle units (p.u.)). Sixty-seven patients with severe angina due to coronary artery disease and no conventional options for revascularization were randomized to AdVEGF121 gene transfer via mini-thoracotomy or continuation of maximal medical treatment. Exercise time to 1 mm ST-segment depression, the predefined primary end-point analysis, was significantly increased in the AdVEGF121 group compared to control at 26 weeks (P=0.026), but not at 12 weeks. As well, total exercise duration and time to moderate angina at weeks 12 and 26, and in angina symptoms as measured by the Canadian Cardiovascular Society Angina Class and Seattle Angina Questionnaire were all improved by VEGF gene transfer (all P-values at 12 and 26 weeks < or =0.001). However, if anything the results of nuclear perfusion imaging favored the control group, although the AdVEGF121 group achieved higher workloads. Overall there was no significant difference in adverse events between the two groups, despite the fact that procedure-related events were seen only in the thoracotomy group. Therefore, administration of AdVEGF121 by direct intramyocardial injections resulted in objective improvement in exercise-induced ischemia in patients with refractory ischemic heart disease.

Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin.

Small-conductance Ca2+-activated K+ channels (SK channels) are independent of voltage and gated solely by intracellular Ca2+. These membrane channels are heteromeric complexes that comprise pore-forming alpha-subunits and the Ca2+-binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM-binding domain (CaMBD), which is located in an intracellular region of the alpha-subunit immediately carboxy-terminal to the pore. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM. Here we report the 1.60 A crystal structure of the SK channel CaMBD/Ca2+/CaM complex. The CaMBD forms an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three alpha-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium-dependent and -independent CaM/protein interactions. Together with biochemical data, the structure suggests a possible gating mechanism for the SK channel.

Control of electrical activity in central neurons by modulating the gating of small conductance Ca2+-activated K+ channels.

In most central neurons, action potentials are followed by an afterhyperpolarization (AHP) that controls firing pattern and excitability. The medium and slow components of the AHP have been ascribed to the activation of small conductance Ca(2+)-activated potassium (SK) channels. Cloned SK channels are heteromeric complexes of SK alpha-subunits and calmodulin. The channels are activated by Ca(2+) binding to calmodulin that induces conformational changes resulting in channel opening, and channel deactivation is the reverse process brought about by dissociation of Ca(2+) from calmodulin. Here we show that SK channel gating is effectively modulated by 1-ethyl-2-benzimidazolinone (EBIO). Application of EBIO to cloned SK channels shifts the Ca(2+) concentration-response relation into the lower nanomolar range and slows channel deactivation by almost 10-fold. In hippocampal CA1 neurons, EBIO increased both the medium and slow AHP, strongly reducing electrical activity. Moreover, EBIO suppressed the hyperexcitability induced by low Mg(2+) in cultured cortical neurons. These results underscore the importance of SK channels for shaping the electrical response patterns of central neurons and suggest that modulating SK channel gating is a potent mechanism for controlling excitability in the central nervous system.

Effect of percutaneous adenovirus-mediated Gax gene delivery to the arterial wall in double-injured atheromatous stented rabbit iliac arteries.

Though the efficacy of intravascular gene transfer has been demonstrated in native vessels following acute injury, this methodology has not been validated in complex models of vascular injury that more closely mimic clinical angioplasty procedures. Previous studies have shown that Gax gene overexpression modulates the injury-induced remodeling of the vessel in rat carotid and normal rabbit iliac arteries. Here, we evaluated the effect of the Gax gene delivery in atheromatous stented vessels. Rabbits were fed 120 g daily of 1% cholesterol diet for 3 weeks. At 1 week they underwent initial injury on the external iliac artery, then balloon angioplasty was performed at 3 weeks at the same site with a 2.5 mm diameter channel balloon catheter (three times 1 min at 6 atm). Either saline (n = 4) or the control viral construct Ad-CMVluc (5 x 109 p.f.u.) (n = 5) or Ad-CMVGax (5 x 10(9) p.f.u.) (n = 4) was delivered with a poloxamer mixture via a channel balloon (6 atm, 30 min), and a 15 mm long Palmaz-Schatz stent (PS154) was then deployed at the site (1 min, 8 atm). Arteries were analyzed 1 month later. At 1 month, the Ad-CMVGax treated arteries exhibited a lower maximal intimal area (1. 15+/-0.1 mm2) than saline (1.87+/-0.15 mm2, P = 0.007) or Ad-CMVluc-treated vessels (1.98+/-0.31 mm2, P = 0.04). Likewise Ad-CMVGax-treated vessels displayed a lower maximal percentage cross-sectional area narrowing (35.1+/-3.5%) than saline (65.3+/-9.4%, P = 0.01) or Ad-CMVluc-treated vessels (62.7+/-6.7%, P = 0.02). Angiographic analysis revealed larger minimal lumen diameter in Ad-CMVGax treated arteries (2.0+/-0.1 mm) than saline (1.14+/-0.36 mm, P = 0.06) or Ad-CMVluc-treated vessels (1.23+/-0.25 mm, P = 0.02). Overexpression of the Gax gene inhibits neointimal hyperplasia and lumen loss in atheromatous stented rabbit iliac arteries.

Age-dependent defect in vascular endothelial growth factor expression is associated with reduced hypoxia-inducible factor 1 activity.

Previous studies have indicated that advanced age is associated with impaired angiogenesis in part because of reduced levels of vascular endothelial growth factor (VEGF) expression. To investigate potential mechanisms responsible for this age-dependent defect in VEGF expression, aortic smooth muscle cells isolated from young rabbits (ages 6-8 months) or old rabbits (ages 4-5 years) were exposed to normoxic (21% oxygen) or hypoxic (0.1% oxygen) conditions. Hypoxia-induced VEGF expression was significantly lower in old versus young cells. VEGF mRNA stability in hypoxic conditions was similar in both young and old cells. However, transient transfection with a luciferase reporter gene that was transcriptionally regulated by the VEGF promoter revealed a significant defect in VEGF up-regulation following hypoxia in old versus young cells (a 43 versus 117% increase in luciferase activity, p < 0.05); this difference was not seen when a deletion construct lacking the hypoxia-inducible 1 (HIF-1) binding site was used. Moreover, although HIF-1 alpha-mRNA expression was shown to be similar in young and old smooth muscle cells, HIF-1 alpha protein and DNA binding activity were significantly reduced in old versus young smooth muscle cells that were exposed to hypoxia. We propose that age-dependent reduction in hypoxia-induced VEGF expression results from reduced HIF-1 activity and may explain the previously described age-dependent impairment of angiogenesis in response to ischemia.

Vascular smooth muscle cell proliferation in the pathogenesis of atherosclerotic cardiovascular diseases.

Atherosclerosis is the principal cause of myocardial infarction, stroke, and peripheral vascular disease, accounting for nearly half of all mortality in developed countries. For example, it has been estimated that atherosclerosis leads to approximately 500,000 deaths from coronary artery disease and 150,000 deaths from stroke every year in the United States (American Heart Association, 1996). Percutaneous transluminal angioplasty has become a well-established technique for revascularization of occluded arteries. However, the long-term efficacy of the procedure remains limited by progressive vessel renarrowing (restenosis) within the following few months after angioplasty. Abnormal vascular smooth muscle cell (VSMC) proliferation is thought to play an important role in the pathogenesis of both atherosclerosis and restenosis. Accordingly, considerable effort has been devoted to elucidate the mechanisms that regulate cell cycle progression in VSMCs. In the present article, we will review the different factors that are involved in the control of VSMC proliferation, especially in the context of cardiovascular disease. Ultimately, a thorough understanding of these regulatory networks may lead to the development of novel drug and gene therapies for the treatment of cardiovascular diseases. Therapeutic approaches that targeted specific cell-cycle control genes or growth regulatory molecules which effectively inhibited neointimal lesion formation will be also discussed.

Age-dependent increase in c-fos activity and cyclin A expression in vascular smooth muscle cells. A potential link between aging, smooth muscle cell proliferation and atherosclerosis.

OBJECTIVE: Aging can be defined as a progressive deterioration of biological functions after the organism has attained its maximal reproductive competence, which is usually associated with a decrease in proliferative ability in most cell types. However, in certain pathological situations such as atherosclerosis and restenosis, aging has been shown to be associated with a higher level of vascular smooth muscle cell (VSMC) proliferation and neointimal lesion formation after angioplasty. In the present study, we investigated potential mechanisms involved in the age-dependent increase in VSMC proliferation. METHODS AND RESULTS: Primary cultures of VSMCs were isolated from young (6-8-month-old) and old (4-5-year-old) New Zealand rabbits. Results from cell counting assays and FACS analysis were consistent with a shortening of the cell cycle in old VSMCs. Western blot analysis in serum stimulated cells showed a significant increase in the level of cyclin A and cyclin-dependent kinase 2 proteins in the old vs. young VSMCs. In marked contrast, expression of cyclin E in VSMCs was not influenced by aging. Transient transfection assays showed an age-dependent increase in transcription from the human cyclin A promoter. Parallel studies demonstrated that the expression of the AP1 transcription factor c-fos, which interacts with the cyclin A promoter and stimulates VSMC proliferation, was also increased in old VSMCs. Consistent with this notion, electrophoretic mobility shift assays demonstrated an increase in AP1 DNA-binding activity in old VSMCs. CONCLUSIONS: These studies suggest that age-associated increase in c-fos activity contributes to augmented cyclin A expression and VSMC proliferation in old animals. These mechanisms might contribute to the higher prevalence and severity of atherosclerosis in the elderly.

Domains responsible for constitutive and Ca(2+)-dependent interactions between calmodulin and small conductance Ca(2+)-activated potassium channels.

Small conductance Ca(2+)-activated potassium channels (SK channels) are coassembled complexes of pore-forming SK alpha subunits and calmodulin. We proposed a model for channel activation in which Ca2+ binding to calmodulin induces conformational rearrangements in calmodulin and the alpha subunits that result in channel gating. We now report fluorescence measurements that indicate conformational changes in the alpha subunit after calmodulin binding and Ca2+ binding to the alpha subunit-calmodulin complex. Two-hybrid experiments showed that the Ca(2+)-independent interaction of calmodulin with the alpha subunits requires only the C-terminal domain of calmodulin and is mediated by two noncontiguous subregions; the ability of the E-F hands to bind Ca2+ is not required. Although SK alpha subunits lack a consensus calmodulin-binding motif, mutagenesis experiments identified two positively charged residues required for Ca(2+)-independent interactions with calmodulin. Electrophysiological recordings of SK2 channels in membrane patches from oocytes coexpressing mutant calmodulins revealed that channel gating is mediated by Ca2+ binding to the first and second E-F hand motifs in the N-terminal domain of calmodulin. Taken together, the results support a calmodulin- and Ca(2+)-calmodulin-dependent conformational change in the channel alpha subunits, in which different domains of calmodulin are responsible for Ca(2+)-dependent and Ca(2+)-independent interactions. In addition, calmodulin is associated with each alpha subunit and must bind at least one Ca2+ ion for channel gating. Based on these results, a state model for Ca2+ gating was developed that simulates alterations in SK channel Ca2+ sensitivity and cooperativity associated with mutations in CaM.

Tissue inhibition of angiotensin-converting enzyme activity stimulates angiogenesis in vivo.

BACKGROUND: Endothelial cells (ECs) represent the critical cellular element responsible for postnatal angiogenesis. Because ACE inhibitors may favorably affect endothelial function, we investigated the hypothesis that administration of the ACE inhibitor quinaprilat could enhance angiogenesis in vivo. METHODS AND RESULTS: Ten days after resection of 1 femoral artery, New Zealand White (NZW) rabbits were randomly assigned to receive recombinant human vascular endothelial growth factor (rhVEGF) administered as a single intra-arterial injection (n=6), quinaprilat (n=8) or captopril (n=7) administered as a daily subcutaneous injection, or no treatment (controls, n=6). Angiogenesis was monitored in vivo by measurement of blood pressure, vasoreactivity, and resistance in ischemic versus normal limbs at day 10 (D10) and D40; angiographic studies to identify sites of neovascularization were performed at D10 and D40, and morphometric analysis of capillary density in the ischemic limb was performed at necropsy (D40). Both functional and morphological outcomes documented augmented angiogenesis in quinaprilat-treated rabbits similar to that observed for rhVEGF and superior to that observed with either captopril or no drug (controls). Residual ACE activity was equivalent for the captopril and quinaprilat groups in plasma (42.54+/-0.03% versus 41.53+/-0.02%, P=NS) but not in tissue, where quinaprilat lowered ACE activity significantly (P<0.01) compared with captopril (13% versus 61%). CONCLUSIONS: ACE inhibition with quinaprilat promotes angiogenesis in a rabbit model of hindlimb ischemia. Thus, nonsulfhydryl ACE inhibitors with high tissue affinity may be potentially useful for therapeutic angiogenesis in ischemic tissues. Moreover, previous evidence that ACE inhibition benefits patients with myocardial ischemia may be due in part to augmented collateral development.

Early cell loss after angioplasty results in a disproportionate decrease in percutaneous gene transfer to the vessel wall.

Acute cell loss has been documented following angioplasty of normal rat and rabbit arteries. Here we analyzed the effects of balloon injury intensity on early cellular loss in single- and double-injury models and how it influences the efficiency of percutaneous gene delivery to the vessel wall. Rabbits underwent bilateral iliac angioplasties (n = 52) with 2.5-mm (balloon-to-artery [B/A] ratio, 1.08 to 1.13) and 3.0-mm (B/A ratio, 1.29 to 1.34) balloons. In the single-injury model, the 3.0-mm balloon induced a 61% reduction in medial cellularity at 3 days postinjury (p < 0.001) while the 2.5-mm balloon did not produce significant cell loss. In the double-injury model, the effects were more pronounced, with 35% (p < 0.01) and 91% (p < 0.001) reductions in medial cellularity at 3 days with the 2.5- and 3.0-mm balloons, respectively, but neointimal cellularity was decreased only with the 3.0-mm balloon (37% reduction, p = 0.025). Adenovirus-mediated beta-galactosidase gene delivery with a channel balloon (n = 24) revealed that larger balloon-to-artery ratios decreased both absolute levels and relative frequencies of transgene expression in the vessel wall. In the single-injury model, gene transfer efficiency was 4.2+/-1.1 and 1.3+/-0.25% (p < 0.05) for the small and large balloons, respectively. In the double-injury model, gene transfer efficiency was 6.6+/-1.6 and 2.3+/-0.8% (p < 0.05) in the neointima and 4.1+/-1.2 and 2.6+/-1.2% (p = NS) in the media for the small and large balloon, respectively. We conclude that early cell loss is dependent on the intensity of the injury in both single- and double-injury models of balloon angioplasty, with greater frequencies of cell loss occurring in the media than in the neointima. In both models, larger balloon-to-artery ratios result in disproportionate reductions in percutaneous adenovirus-mediated gene delivery.

Rescue of diabetes-related impairment of angiogenesis by intramuscular gene therapy with adeno-VEGF.

Diabetes is a major risk factor for coronary and peripheral artery diseases. Although diabetic patients often present with advanced forms of these diseases, it is not known whether the compensatory mechanisms to vascular ischemia are affected in this condition. Accordingly, we sought to determine whether diabetes could: 1) impair the development of new collateral vessel formation in response to tissue ischemia and 2) inhibit cytokine-induced therapeutic neovascularization. Hindlimb ischemia was created by femoral artery ligation in nonobese diabetic mice (NOD mice, n = 20) and in control C57 mice (n = 20). Hindlimb perfusion was evaluated by serial laser Doppler studies after the surgery. In NOD mice, measurement of the Doppler flow ratio between the ischemic and the normal limb indicated that restoration of perfusion in the ischemic hindlimb was significantly impaired. At day 14 after surgery, Doppler flow ratio in the NOD mice was 0.49+/-0.04 versus 0.73+/-0.06 for the C57 mice (P< or =0.005). This impairment in blood flow recovery persisted throughout the duration of the study with Doppler flow ratio values at day 35 of 0.50+/-0.05 versus 0.90+/-0.07 in the NOD and C57 mice, respectively (P< or =0.001). CD31 immunostaining confirmed the laser Doppler data by showing a significant reduction in capillary density in the NOD mice at 35 days after surgery (302+/-4 capillaries/mm2 versus 782+/-78 in C57 mice (P< or =0.005). The reduction in neovascularization in the NOD mice was the result of a lower level of vascular endothelial growth factor (VEGF) in the ischemic tissues, as assessed by Northern blot, Western blot and immunohistochemistry. The central role of VEGF was confirmed by showing that normal levels of neovascularization (compared with C57) could be achieved in NOD mice that had been supplemented for this growth factor via intramuscular injection of an adenoviral vector encoding for VEGF. We conclude that 1) diabetes impairs endogenous neovascularization of ischemic tissues; 2) the impairment in new blood vessel formation results from reduced expression of VEGF; and 3) cytokine supplementation achieved by intramuscular adeno-VEGF gene transfer restores neovascularization in a mouse model of diabetes.

Age-dependent impairment of angiogenesis.

BACKGROUND: The effect of aging on angiogenesis in ischemic vascular disease has not been studied. Accordingly, we investigated the hypothesis that angiogenesis is impaired as a function of age. METHODS AND RESULTS: Forty days after the resection of 1 femoral artery, collateral vessel development was significantly impaired in old (aged 4 to 5 years; n=7) versus young (aged 6 to 8 months; n=6) New Zealand White (NZW) rabbits on the basis of reduced hindlimb perfusion (ischemic: normal blood pressure ratio=0.58+/-0.05 versus 0.77+/-0.06; P<0.005), reduced number of angiographically visible vessels (angiographic score=0.48+/-0.05 versus 0.70+/-0.05; P<0.01), and lower capillary density in the ischemic limb (130.3+/-5.8/mm2 versus 171.4+/-9.5/mm2; P<0.001). Angiogenesis was also impaired in old (aged 2 years) versus young (aged 12 weeks) mice as shown by reduced hindlimb perfusion (measured by laser Doppler imaging) and lower capillary density (353.0+/-14.3/mm2 versus 713.3+/-63.4/mm2; P<0.01). Impaired angiogenesis in old animals was the result of impaired endothelial function (lower basal NO release and decreased vasodilation in response to acetylcholine) and a lower expression of vascular endothelial growth factor (VEGF) in ischemic tissues (by Northern blot, Western blot, and immunohistochemistry). When recombinant VEGF protein was administered to young and old rabbits, both groups exhibited a significant and similar increase in blood pressure ratio, angiographic score, and capillary density. CONCLUSIONS: Angiogenesis responsible for collateral development in limb ischemia is impaired with aging; responsible mechanisms include age-related endothelial dysfunction and reduced VEGF expression. Advanced age, however, does not preclude augmentation of collateral vessel development in response to exogenous angiogenic cytokines.

Mechanism of calcium gating in small-conductance calcium-activated potassium channels.

The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike-frequency adaptation) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. Here we study the mechanism of calcium gating and find that SK channels are not gated by calcium binding directly to the channel alpha-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the alpha-subunits in a calcium-independent manner. Our data support a model in which calcium gating of SK channels is mediated by binding of calcium to calmodulin and subsequent conformational alterations in the channel protein.

Effects of poloxamer 407 on transfection time and percutaneous adenovirus-mediated gene transfer in native and stented vessels.

UNLABELLED: Reduction in transfection time and the ability to perform gene transfer in conjunction with endovascular stent implantation constitute two important challenges for percutaneous adenovirus-mediated gene transfer to vessel walls. Studies have suggested that the use of biocompatible polyol poloxamer 407 could be useful. We first evaluated the use of poloxamer 407 for percutaneous gene transfer in nonstented rabbit iliac arteries. A 200-microl mixture of Ad-RSVbetagal or Ad-CMVLuc in either phosphate-buffered saline (PBS) or 20% poloxamer was delivered. After 3 days, gene transfection was evaluated by X-Gal staining or measurement of luciferase activity. Poloxamer use resulted in a 3- to 15-fold increase in the percentage of transfected cells (X-Gal, p = 0.001) and a 16-fold increase in protein product (luciferase activity, p = 0.03), and allowed a decrease in transfection time from 30 to 5 min with minimal reduction in transfection efficiency. We then evaluated the feasibility of percutaneous gene transfer, using Ad-RSVbetagal diluted in pure PBS or 20% poloxamer, in conjunction with stent implantation. Gene delivery was performed either immediately before (pre-) or after (post-) stent implantation. When adenoviruses were diluted in PBS, gene transfer had a low efficiency (prestent, 0.3%; poststent, 0.2%; NS). With poloxamer, the efficacy was much higher (p = 0.0001) and similar "pre" (2.2%) or "post" (1.7%) stent delivery (NS). CONCLUSIONS: (1) The use of poloxamer, rather than PBS, as a vehicle increases the efficacy of percutaneous adenovirus-mediated gene transfer and reduces transfection time; (2) gene transfer performed during stent implantation with poloxamer is feasible and achieves a significant level of gene expression. Thus percutaneous gene delivery is applicable to conventional stents and could present an attractive method by which to achieve local biological effects in a stent environment.

Aromatic residues affecting permeation and gating in dSlo BK channels.

Structural determinants of permeation in large unit conductance calcium-activated potassium channels (BK channels) were investigated. Y293 and F294 in the P-region of dSlo were substituted by tryptophans. Compared to wild-type channels, Y293W channels displayed reduced inward unitary currents while F294W channels exhibited normal inward current amplitudes but flickery kinetics. Both mutations produced changes in current/voltage relations under bi-ionic conditions. Sensitivity to block by external tetraethylammonium (TEA) was affected in both channels, and the voltage dependence of TEA block was increased in F294W channels. Both mutations also affected gating by shifting the half-maximal activation voltage of macroscopic conductance/voltage relations to more positive potentials, and eliminating a slow component of deactivation. The double mutant did not produce ionic currents. These data are consistent with a model in which Y293 contributes to a potassium-binding site close to the outer mouth of the dSlo pore, while F294 contributes to an energy barrier near this site.

Vascular endothelial growth factor/vascular permeability factor produces nitric oxide-dependent hypotension. Evidence for a maintenance role in quiescent adult endothelium.

In vitro studies suggest that vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) may stimulate release of nitric oxide (NO) from endothelial cells. To investigate the hemodynamic consequences of recombinant VEGF/VPF administered in vivo, recombinant human VEGF/VPF was administered as a bolus dose of 500 micrograms to anesthetized (n = 6) or conscious (n = 5) New Zealand White rabbits, as well as anesthetized rabbits with diet-induced hypercholesterolemia (HC; n = 7). Anesthetized Yorkshire farm pigs (no specific dietary pretreatment) were studied before and after receiving 500 micrograms intravenous (IV; n = 5) or intracoronary (IC; n = 5) VEGF/VPF. In anesthetized, normal rabbits, mean arterial pressure (MAP) fell by 20.5 +/- 1.4% (P < .05 versus baseline) within 3 minutes after IV VEGF/VPF. Pretreatment with N omega-nitro-L-arginine caused a significant inhibition of VEGF/VPF-induced hypotension. In conscious, normal rabbits, VEGF/VPF produced a consistent though lesser reduction in MAP. The fall in MAP induced by VEGF/VPF in anesthetized, HC rabbits (21.5 +/- 2.5% from baseline) was no different from that observed in normal anesthetized rabbits. In pigs, both IV and IC administration of VEGF/VPF produced a prompt reduction in MAP. Heart rate increased, while cardiac output, stroke volume, left atrial pressure, and total peripheral resistance all declined to a similar, statistically significant degree in both IV and IC groups. Epicardial echocardiography disclosed neither global nor segmental wall motion abnormalities in response to VEGF/VPF. We conclude that (1) VEGF/VPF-stimulated release of NO, previously suggested in vitro, occurs in vivo; (2) this finding suggests that functional VEGF/VPF receptors are present on quiescent adult endothelium, consistent with a maintenance function for VEGF/VPF, which may include regulation of NO; and (3) the preserved response of HC rabbits suggests that endothelial cell receptors for VEGF/VPF are spared in the setting of hypercholesterolemia.