Biomedical subjects

T O'Brien

Publications and source records attributed to T O'Brien.

At least 91 records · Page 5Linked to original sources

PubMed1998

Transfer and expression of recombinant nitric oxide synthase genes in the cardiovascular system.

Gene therapy involves the transfer of a functional gene into host cells to correct the malfunction of a specific gene or to alleviate the symptoms of a disease. For gene transfer to the cardiovascular system, adenoviral vectors are the most efficient means of transfer. Recently, transfer and functional expression of recombinant nitrio oxide synthase (NOS) genes to cerebral and cardiovascular beds have been demonstrated both ex vivo and in vivo. Here, Alex Chen and colleagues review current progress in the field of vascular NOS gene transfer and the potential use of NOS gene therapy for a number of cardiovascular diseases. Although the feasibility of the NOS gene transfer approach has been demonstrated in animal models, currently available vectors have a number of technical and safety limitations that have to be solved before human NOS gene therapy for cardiovascular disease can be attempted.

Animals↗

PubMed1998

Influence of temperature on adenovirus-mediated gene transfer.

OBJECTIVE: The transfer of recombinant genes to donor organs may allow for novel therapeutic approaches to the challenges of acute and chronic rejection. Adenoviral vectors are capable of efficient gene transfer, but use of these vectors during donor organ preservation may be less efficient due to the low temperature. This study was designed to examine the effect of temperature on the efficiency of adenovirus-mediated gene transfer. METHODS: Gene transfer to human endothelial cells, porcine vascular smooth muscle cells and cultured rat thoracic aortas was examined. Incubation with an adenoviral vector encoding for E. coli beta-galactosidase was performed for 1 h at three different temperatures: 4 degrees C, 10 degrees C and 37 degrees C. Transgene expression was assessed by histochemical staining for beta-galactosidase in transduced cells and by evaluation of beta-galactosidase activity in organ cultures. RESULTS: Both in human endothelial cells and vascular smooth muscle cells the percentage of positively staining cells following transduction at 37 degrees C was significantly greater than at 4 degrees C and at 10 degrees C (30.55 +/- 7.26% vs. 14.29 +/- 3.79% and 12.43 +/- 2.47%, respectively for endothelial cells, P < 0.01 vs. 4 degrees C and 10 degrees C; and 28.25 +/- 4.52% vs. 17.91 +/- 3.76% and 16.63 +/- 3.92%, respectively for smooth muscle cells, P < 0.05 vs. 4 degrees C, P < 0.01 vs. 10 degrees C). Beta-galactosidase activity was significantly greater in aortas transduced at 37 degrees C than in vessels transduced at 4 degrees C and 10 degrees C (289,700 +/- 113,300 vs. 149,600 +/- 54,390 and 108,800 +/- 23,140 relative chemiluminesce units/mg of total protein, respectively; P < 0.05 vs. 4 degrees C, P < 0.001 vs. 10 degrees C). CONCLUSIONS: The present study demonstrates that the efficiency of adenovirus-mediated gene transfer is significantly reduced at lower temperatures. The need for cold preservation of donor organs may render efficient adenovirus-mediated gene transfer more difficult in the transplantation setting.

Adenoviridae↗

PubMed1998

Functional analysis of the human TAFII250 N-terminal kinase domain.

The largest subunit of the human transcription factor TFIID, TAFII250, was previously reported to contain serine/threonine kinase domains that can autophosphorylate and transphosphorylate the large subunit of the basal factor TFIIF. Here, we identify the regions of the N-terminal kinase domain (amino acids 1-414) necessary for kinase activity and examine its function in vivo. Point mutations within two patches of amino acids in the kinase domain decrease both autophosphorylation and transphosphorylation activities. Importantly, we find that TAFII250-bearing mutations within the N-terminal kinase domain exhibit a significantly reduced ability to rescue ts13 cells that express a temperature-sensitive TAFII250. Moreover, transcription from the cyclin A and cdc2 promoters becomes impaired when cotransfected with hTAFII250 containing inactive forms of the N-terminal kinase domain. Our results suggest that the TAFII250 kinase activity is required to direct transcription of at least some genes in vivo.

Cell Line↗

PubMed1998

High expression of Wnt7b in human superficial bladder cancer vs invasive bladder cancer.

Aberrant Wnt gene expression is involved in the development of breast cancer, but its role in other tumours is unknown. Wnts regulate cadherin function, previously shown to be more commonly deregulated in invasive bladder cancer. This study investigated whether factors upstream of cadherins were aberrantly expressed in superficial bladder cancer. The expression of one transforming (Wnt7b) and one non-transforming (Wnt5a) Wnt gene in four human bladder carcinoma cell lines, and in normal human bladder tissues (n = 8) and bladder cancers (n = 48) were analysed by ribonuclease protection analysis. All cell lines expressed an approximately equal level of Wnt7b mRNA. Wnt5a and Wnt7b mRNAs were both expressed in normal bladder tissues and bladder tumours. The median expression of Wnt7b was fourfold higher in superficial tumours (n = 29) than in normal tissues (n = 8, P = 0.002) and five fold higher than in invasive tumours (n = 17, P = 0.003). There was no significant difference between normal tissues and invasive tumours (P = 0.3). The expression of Wnt5a did not vary significantly between normal tissues and superficial tumours (P = 0.4), normal tissues and invasive tumours (P = 0.3) or superficial tumours and invasive tumours (P = 0.2). The differential expression of Wnt7b suggests a role in the early events of superficial bladder tumorigenesis involving cell adhesion and provides further evidence of different pathways of evolution of superficial and invasive cancer.

Carcinoma↗

PubMed1998

Medical-resource use for suspected tuberculosis in a New York City hospital.

OBJECTIVE: To compare resource use by diagnostic outcome among hospital admissions during which tuberculosis (TB) was suspected. DESIGN: Retrospective study based on chart review and microbiology laboratory data. SETTING: The department of medicine in a municipal hospital serving central Brooklyn, New York. PARTICIPANTS: We identified all adult admissions in 1993 during which TB was suspected. We assigned each admission to one of four mutually exclusive groups defined by the results of microbiological tests (acid-fast bacilli [AFB] smear and culture): culture-positive and smear-positive (C+S+); culture-positive and smear-negative (C+S-); culture-negative and smear-positive (C-S+); or culture-negative and smear-negative (C-S-). Each admission was divided into two separate periods to which the utilization of medical resources was assigned: the diagnostic and the postdiagnostic periods, which were separated by the date of receipt of the first definitive culture report. RESULTS: Data on 519 admissions (93 C+S+; 57 C+S-; 30 C-S+; and 339 C-S-) were analyzed. Although C+S+ were more likely than other groups to have an admitting diagnosis of TB, approximately one quarter of the admissions without TB (C-S+, C-S-) were admitted with the principal diagnosis of TB. For the four groups, C+S+, C+S-, C-S+, and C-S-, the respective rates of TB isolation and anti-TB treatment, and median lengths of isolation were 98%, 87%, and 34 days; 74%, 74%, and 7 days; 83%, 83%, and 15 days; and 44%, 29%, and 0 days. During the diagnostic period, the rate and length of isolation were similar in the AFB-smear-positive groups (C+S+ and C-S+). We estimated that admissions without culture-proven TB (C-S+ and C-S-) accounted for 3,174 (36%) of the 8,712 days of TB isolation expended and for 65% of the 16,671 days of anti-TB treatment. The vast majority of this resource consumption (2,737 [86%] of 3,174 days of isolation) occurred during the diagnostic period before a definitive culture result was known. CONCLUSIONS: Our results suggest that prolonged diagnostic uncertainty and misclassification of cases due to false-positive and false-negative smears are associated with substantial medical-resource consumption. New diagnostic modalities that reduce the period of diagnostic uncertainty could reduce the utilization of resources later found to be unnecessary.

Adult↗

PubMed1998

In vitro evaluation of a new lithium dilution method of measuring cardiac output and shunt fraction in patients undergoing venovenous extracorporeal membrane oxygenation.

OBJECTIVE: To evaluate, in vitro, a method of measuring cardiac output and shunt fraction during venovenous extracorporeal membrane oxygenation (ECMO). DESIGN: Experimental study using an in vitro model. SETTING: A teaching hospital. MODEL: An ECMO circuit was set up in parallel with a patient circuit consisting of tubing through which saline was circulated from a 50-L reservoir by a pump which was set at 3 L/min to represent cardiac output. A second pump in the ECMO circuit drew saline from the patient circuit and passed it through a membrane oxygenator. The flow from the membrane oxygenator either returned directly to the patient circuit or was diverted, via a third pump, back into the ECMO circuit, thereby producing a shunt. INTERVENTIONS: By adjusting the flow rates of the second (ECMO) and third (shunt) pumps, three shunt fractions of 12%, 25%, and 50% were produced at three different ECMO flow rates. Lithium chloride (0.15 mmol) was injected just downstream of the membrane oxygenator; the lithium ion concentration-time curves were recorded simultaneously in the flow returning to the saline reservoir and in the flow just upstream of the membrane oxygenator using lithium selective electrodes. MEASUREMENTS AND MAIN RESULTS: Nine pairs of curves were recorded, one pair for each combination of ECMO and shunt flow rates. Analysis of these curves allowed shunt flow and "cardiac output" to be calculated and compared with the flow rates delivered by the pumps. Mean "cardiac output" derived from the lithium dilution curves was 2.98 +/- 0.18 (SD) L/min, compared with a delivered pump flow of 3 L/min. Measured shunt flow = 0.008 + 1.09 x actual shunt flow (R = 0.997). CONCLUSIONS: This method would allow cardiac output and shunt flow to be measured in patients undergoing venovenous ECMO. It could result in better patient management and improved cannula design.

Blood Flow Velocity↗

PubMed1998

Adenovirus-mediated gene transfer of macrophage colony stimulating factor to the arterial wall in vivo.

Macrophage colony stimulating factor (MCSF) is believed to play a key role in one of the earliest events in atherosclerosis, ie, monocyte to macrophage differentiation in the arterial intima. The aim of this study was to examine the biological effects of vascular wall expression of MCSF. A recombinant adenovirus vector encoding human MCSF (AdMCSF) was generated by standard techniques of homologous recombination in 293 cells. The rabbit carotid artery was transduced with AdMCSF. As negative controls, carotid arteries were transduced with either an adenoviral vector encoding beta-galactosidase, an adenoviral vector encoding apolipoprotein E, or diluent alone. Intima-media thickness ratio was calculated 5 and 21 days after transduction. The cell type present in intimal infiltrates was analyzed by immunohistochemistry. MCSF expression was demonstrated in the vessel wall of AdMCSF-transduced vessels by reverse transcription-polymerase chain reaction and immunofluorescence. In contrast to control vessels, adenovirus-mediated MCSF expression was associated with an intimal cellular infiltrate consisting of smooth muscle cells and small numbers of macrophages. Whereas the intima-media thickness ratio was greater in AdMCSF-transduced vessels at 5 days, this difference was no longer statistically significant at 21 days. These results suggest that MCSF may play a role in recruitment of monocytes and macrophages to the vessel wall and may contribute to smooth muscle cell proliferation and migration.

Adenoviridae↗

PubMed1998

Adventitial expression of recombinant eNOS gene restores NO production in arteries without endothelium.

The current study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on endothelium-dependent relaxations to bradykinin in isolated canine basilar, coronary, or femoral arteries. Arterial rings were exposed ex vivo (30 minutes at 37 degrees C) to an adenoviral vector encoding either the eNOS gene (AdCMVeNOS) or the beta-galactosidase reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the adventitia. Expression of recombinant proteins was much higher in basilar arteries than in coronary or femoral arteries. Rings of control, AdCMVbeta-Gal, and AdCMVeNOS arteries with and without endothelium were suspended for isometric tension recording. Levels of cGMP were measured by radioimmunoassay. In AdCMVeNOS basilar arteries with endothelium, relaxations to low concentrations of bradykinin (3 x 10(-11) to 10(-9) mol/L) were significantly augmented. In contrast, in coronary and femoral arteries with endothelium, AdCMVeNOS transduction did not affect relaxations to bradykinin. Removal of the endothelium abolished bradykinin-induced relaxations in control and AdCMVbeta-Gal basilar arteries. However, in basilar arteries transduced with AdCMVeNOS even when the endothelium was removed, stimulation with bradykinin (3 x 10(-11) to 10(-9) mol/L) caused relaxations as well as increases in cGMP production. The relaxations to bradykinin were completely blocked by an NOS inhibitor, NG-nitro-L-arginine methyl ester. Electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of the basilar artery adventitia. These results suggest that genetically modified adventitial fibroblasts may restore production of NO in cerebral arteries without endothelium. Our findings support a novel concept in vascular biology that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.

Adenoviridae↗

PubMed1998

Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery after experimental subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Gene transfer with recombinant viral vectors encoding vasodilator proteins may be useful in therapy of cerebral vasospasm after subarachnoid hemorrhage (SAH). Relaxations mediated by nitric oxide are impaired in cerebral arteries affected by SAH. The present study was designed to determine the effect of SAH on the efficiency of ex vivo adenovirus-mediated gene transfer to canine basilar arteries and to examine whether expression of recombinant endothelial nitric oxide synthase (eNOS) gene may have functional effects on vasomotor reactivity of spastic arteries affected by SAH. METHODS: Replication-deficient recombinant adenovirus vectors encoding bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVbeta-Gal) genes were used for ex vivo gene transfer. Rings of basilar arteries obtained from control dogs and dogs exposed to SAH were incubated with the vectors in minimum essential medium. Twenty-four hours after gene transfer, expression and function of the recombinant genes were evaluated by (1) histochemical or immunohistochemical staining, (2) beta-galactosidase protein measurement, and (3) isometric tension recording. RESULTS: Transduction with AdCMVbeta-Gal and AdCMVeNOS resulted in the expression of recombinant beta-galactosidase and eNOS proteins mostly in the vascular adventitia. The expression of beta-galactosidase protein was approximately 2-fold higher in SAH arteries than in normal arteries. Endothelium-dependent relaxations caused by bradykinin and substance P were suppressed in SAH arteries. The relaxations to bradykinin were significantly augmented in both normal and SAH arteries after AdCMVeNOS transduction but not after AdCMVbeta-Gal transduction. The relaxations to substance P were augmented by AdCMVeNOS transduction only in normal arteries. Bradykinin and substance P caused relaxations even in endothelium-denuded arteries, when the vessels were transduced with AdCMVeNOS. These endothelium-independent (adventitia-dependent) relaxations to bradykinin observed after AdCMVeNOS transduction were similar between normal and SAH arteries, whereas those to substance P were significantly reduced in SAH arteries compared with normal arteries. CONCLUSIONS: These results suggest that expression of recombinant proteins after adenovirus-mediated gene transfer may be enhanced in cerebral arteries affected by SAH and that successful eNOS gene transfer to spastic arteries can at least partly restore the impaired nitric oxide-mediated relaxations through local (adventitial) production of nitric oxide.

Adenoviridae↗

PubMed1998

Primary and secondary reinforcers in performance of a 1.0-mile walk/jog by adolescents with moderate mental retardation.

Based on visual inspection of data, reinforcement procedures, namely, use of verbal praise and verbal praise plus token exchange, were at least mildly influential in improving performance of 5 youth with moderate mental retardation compared to their initial performance without reinforcers. These findings suggest specific reinforcement may improve the time for aerobic activity by adolescents with moderate mental retardation.

Adolescent↗

PubMed1998

Adenoviral-mediated gene transfer to human adipocytes in vitro, and human adipose tissue ex vivo and rabbit femoral adipose tissue in vivo.

Adenoviral-mediated gene transfer has proven useful in several organ systems to understand gene action and to provide a potential therapeutic modality for localized, organ-specific gene overexpression. However, the application of adenoviral-mediated gene transfer to adipocytes and adipose tissue has not been evaluated. We evaluated the feasibility of in vitro and ex vivo transfer of the beta-galactosidase gene to human adipocytes and adipose tissue by means of adenoviral vectors. The efficiency (percentage of cells transduced) of adenoviral-mediated gene transfer of the beta-galactosidase gene to human adipocytes in vitro and to human adipose tissue ex vivo was 21 +/- 3% and 14 +/- 3%, respectively. Adenoviral-mediated gene transfer in a rabbit femoral adipose tissue was also demonstrated in vivo. Adenoviral-mediated gene transfer may facilitate studies on understanding the biology of adipocytes and provide a potential tool for the modulation of adipocyte function in vivo and thereby for the treatment of obesity.

Adenoviridae↗

PubMed1998

Hyperlipidemia and diabetes mellitus.

The increased risk of coronary artery disease in subjects with diabetes mellitus can be partially explained by the lipoprotein abnormalities associated with diabetes mellitus. Hypertriglyceridemia and low levels of high-density lipoprotein are the most common lipid abnormalities. In type 1 diabetes mellitus, these abnormalities can usually be reversed with glycemic control. In contrast, in type 2 diabetes mellitus, although lipid values improve, abnormalities commonly persist even after optimal glycemic control has been achieved. Screening for dyslipidemia is recommended in subjects with diabetes mellitus. A goal of low-density lipoprotein cholesterol of less than 130 mg/dL and triglycerides lower than 200 mg/dL should be sought. Several secondary prevention trials, which included subjects with diabetes, have demonstrated the effectiveness of lowering low-density lipoprotein cholesterol in preventing death from coronary artery disease. The benefit of lowering triglycerides is less clear. Initial approaches to lowering the levels of lipids in subjects with diabetes mellitus should include glycemic control, diet, weight loss, and exercise. When goals are not met, the most common drugs used are hydroxymethylglutaryl coenzyme A reductase inhibitors or fibrates.

Coronary Disease↗

PubMed1997

Vascular endothelial growth factor is a predictor of relapse and stage progression in superficial bladder cancer.

Tumor development is angiogenesis dependent, and vascular endothelial growth factor (VEGF) is a key growth factor in this process. We demonstrate that high expression of VEGF mRNA in 55 superficial bladder cancers was associated with earlier recurrence (P = 0.001; hazard ratio, 3.09) and progression to a more invasive phenotype (P = 0.02; hazard ratio, 5.33). VEGF mRNA expression correlated with protein levels in superficial tumors (r = 0.59, P = 0.003) and normal bladder (r = 0.65, P < 0.05), although the ratio of VEGF protein to mRNA was elevated in tumors compared to normal bladder (P = 0.004), suggesting posttranscriptional regulation. In this study, VEGF is implicated as a major downstream mediator of the effects of the p53 tumor suppressor gene by the association between high p53 protein (determined immunochemically) and high VEGF protein and mRNA expression (P < 0.02), although in cases without high p53 protein expression, high VEGF mRNA also predicts a poor prognosis. The relationship between VEGF and early tumor recurrence suggests that seeding via angiogenesis may be a major mechanism in the pathogenesis of recurrence. These studies indicate that VEGF can predict the behavior of superficial bladder tumors and is a therapeutic target for intravesical therapy.

Carcinoma, Transitional Cell↗

PubMed1997

Effects of in vivo adventitial expression of recombinant endothelial nitric oxide synthase gene in cerebral arteries.

Nitric oxide (NO), synthesized from L-arginine by NO synthases (NOS), plays an essential role in the regulation of cerebrovascular tone. Adenoviral vectors have been widely used to transfer recombinant genes to different vascular beds. To determine whether the recombinant endothelial NOS (eNOS) gene can be delivered in vivo to the adventitia of cerebral arteries and functionally expressed, a replication-incompetent adenoviral vector encoding eNOS gene (AdCMVNOS) or beta-galactosidase reporter gene (AdCMVLacZ) was injected into canine cerebrospinal fluid (CSF) via the cisterna magna (final viral titer in CSF, 10(9) pfu/ml). Adventitial transgene expression was demonstrated 24 h later by beta-galactosidase histochemistry and quantification, eNOS immunohistochemistry, and Western blot analysis of recombinant eNOS. Electron microscopy immunogold labeling indicated that recombinant eNOS protein was expressed in adventitial fibroblasts. In AdCMVNOS-transduced arteries, basal cGMP production and bradykinin-induced relaxations were significantly augmented when compared with AdCMVLacZ-transduced vessels (P < 0.05). The increased receptor-mediated relaxations and cGMP production were inhibited by eNOS inhibitors. In addition, the increase in cGMP production was reversed in the absence of calcium, suggesting that the increased NO production did not result from inducible NOS expression. The present study demonstrates the successful in vivo transfer and functional expression of recombinant eNOS gene in large cerebral arteries. It also suggests that perivascular eNOS gene delivery via the CSF is a feasible approach that does not require interruption of cerebral blood flow.

Adenoviridae↗

PubMed1997

Recombinant endothelial nitric oxide synthase-transduced human saphenous veins: gene therapy to augment nitric oxide production in bypass conduits.

BACKGROUND: Nitric oxide is a potent vasodilator that also inhibits platelet aggregation and smooth muscle cell proliferation, properties that may prevent early and late occlusion of saphenous vein coronary bypass conduits. We determined whether human saphenous veins can be transduced with adenovirus vector-encoding bovine endothelial nitric oxide synthase (Ad.CMVeNOS), resulting in functional expression of recombinant nitric oxide synthase. METHODS AND RESULTS: Harvested segments of human saphenous vein were exposed for 1 hour at 37 degrees C to replication-deficient Ad.CMVeNOS (5 x 10(9) PFU/mL) or control adenovirus-encoding Escherichia coli beta-galactosidase (Ad.CMVLacZ; 5 x 10(9) PFU/mL). The vein segments were analyzed for recombinant endothelial nitric oxide synthase expression and activity 48 hours later. Histochemical staining for recombinant beta-galactosidase activity was localized to the luminal endothelium and adventitia of vein segments transduced with Ad.CMVLacZ. Similarly, immunohistochemical staining with a monoclonal antibody for nitric oxide synthase localized recombinant gene expression to endothelial and adventitial cells in Ad.CMVeNOS veins; only endogenous nitric oxide synthase was identified in the endothelium of Ad.CMVLacZ veins. Nitrite generation after stimulation with calcium ionophore increased in Ad.CMVeNOS veins (1420.0+/-298.2 nM/mg versus 130.3+/-19.9 nM/mg; n=3; P<.05). Isometric tension recording demonstrated augmented maximal relaxation to calcium ionophore (32+/-4.5% versus 17.4+/-7.4%; n=6; P<.05) after precontraction with norepinephrine. Bioassay superfusion demonstrated a twofold augmentation of the biodetector ring relaxation during calcium ionophore stimulation of Ad.CMVeNOS veins. CONCLUSIONS: Adenovirus-mediated gene transfer to human saphenous veins resulted in functional transgene expression with increased nitric oxide release. These or similar molecular techniques to increase nitric oxide production may reduce the risk of early thrombosis in saphenous vein grafts.

Adenoviridae↗

PubMed1997

Adventitial gene transfer of recombinant endothelial nitric oxide synthase to rabbit carotid arteries alters vascular reactivity.

BACKGROUND: Adventitial gene transfer may serve as a tool to study vascular biology and may have therapeutic potential. We investigated the hypothesis that adenovirus-mediated transfer of the gene for endothelial nitric oxide synthase (eNOS) to the adventitia would alter vascular reactivity. METHODS AND RESULTS: Rabbit carotid arteries were surgically isolated and adenoviral vectors encoding eNOS (AdeNOS) or beta-galactosidase instilled into the periarterial sheath at a concentration of 1 x 10(10) pfu/mL. Arteries were harvested 4 days later for immunostaining, NOS enzymatic assay, measurement of cGMP, and vasomotor studies. Transgene expression in the adventitia was confirmed by histochemistry for beta-galactosidase and immunostaining for eNOS with a monoclonal antibody. Calcium-dependent NOS enzymatic activity and cGMP levels were significantly greater in the AdeNOS-transduced arteries. Maximal contractions to phenylephrine (10(-5) mol/L) were diminished in the AdeNOS-transduced arteries (4.6+/-0.2 versus 5.6+/-0.2 g; P<.05), but in the presence of the eNOS inhibitor N(G)-monomethyl-L-argininc (3x10(-4) mol/L) there was no difference between the two groups (7.1+/-0.2 versus 7.5+/-0.3 g; P=NS). Relaxations to calcium ionophore obtained during submaximal contractions to phenylephrine were significantly enhanced in the AdeNOS-transduced arteries (-log EC50, 7.77+/-0.08 versus 7.45+/-0.07; P<.02). CONCLUSIONS: We conclude that eNOS gene transfer to the adventitia alters vascular reactivity, as demonstrated by diminished contractile responses to phenylephrine and enhanced relaxations to calcium ionophore. This may represent a therapeutic strategy for vascular diseases characterized by decreased bioavailability of NO.

1-Methyl-3-isobutylxanthine↗

PubMed1997

Two mechanisms of basic fibroblast growth factor-induced angiogenesis in bladder cancer.

In the urine of patients with bladder cancer, levels of the angiogenio peptide basic fibroblast growth factor (bFGF) may be elevated 100-fold. To date, levels of expression of bFGF in bladder tumor tissue have not been determined, nor has the cellular source of the urinary bFGF been identified. bFGF mRNA expression was quantified using RNase protection analysis in 32 primary bladder tumors and 8 normal bladder specimens. In addition, bFGF protein expression in the tumor cytosol was determined using a Quantikine ELISA, and bFGF protein expression was localized with immunohistochemistry. bFGF mRNA expression was absent in 28 of 32 (87%) bladder cancers despite detectable expression in 7 of 8 (87%) normal bladder specimens (P = 0.0001). In only one tumor was bFGF mRNA expression higher than in normal bladder tissue. Median bFGF protein expression was also higher in the normal bladder specimens than in the superficial tumors (3800 pg/g protein versus 1140 pg/g protein; P < 0.02), but there was no statistically significant difference between protein expression in normal bladder and invasive cancers (3800 pg/g versus 3600 pg/g). Median bFGF protein expression was higher in invasive cancers than in superficial tumors (P < 0.05). Intense bFGF immunoreactivity was seen in the basal lamina of normal transitional epithelium, in normal human detrusor muscle, and in vessels within tumors. Tumor cell immunoreactivity was rare and was usually weak. Only in the tumor which strongly overexpressed bFGF mRNA and protein was cytoplasmic staining detectable in the neoplastic cells. There are two mechanisms of bFGF-induced angiogenesis in bladder cancer. Rarely, neoplastic cells synthesize bFGF but more commonly bFGF is released by degradation of epithelial basement membranes and detrusor muscle, from where it can diffuse into the tumor microenvironment and bind to blood vessels. Mechanisms of extracellular matrix degradation may be important in bladder cancer angiogenesis and progression and as such are potential therapeutic targets.

Fibroblast Growth Factor 2↗

PubMed1997

Expression and function of a recombinant endothelial nitric oxide synthase gene in porcine coronary arteries.

OBJECTIVES: Direct gene transfer of exogenous nitric oxide synthase, with the subsequent increase in nitric oxide production, could represent a potential therapeutic strategy in the treatment of vascular proliferative disorders. The goal of the present study was to determine if porcine coronary arteries could be transduced with an adenoviral vector encoding endothelial nitric oxide synthase (Ad.CMVeNOS) resulting in functional expression. METHODS AND RESULTS: Segments of porcine right coronary artery were exposed for 1 h at 37 degrees C to either replication-deficient adenovirus encoding bovine endothelial nitric oxide synthase (Ad.CMVeNOS, 5 x 10(9) pfu/ml) or control adenovirus encoding Escherichia coli beta-galactosidase (Ad.CMVLacZ, 5 x 10(9) pfu/ml). Immunohistochemistry with a monoclonal antibody specific for nitric oxide synthase (NOS) demonstrated recombinant gene expression in both the endothelial and adventitial layers of Ad.CMVeNOS transduced coronaries with only endogenous NOS confirmed in the endothelium of Ad.CMVLacZ arteries. Coronary arteries transduced with Ad.CMVeNOS yielded 517 +/- 110 (mean +/- S.E.M.) nM/ng nitrite while vessels transduced with Ad.CMVLacZ yielded 126 +/- 84 nM/ng (P < 0.05, n = 6). Isometric tension recording, following prostaglandin F2 alpha constriction, documented a reduced tension in Ad.CMVeNOS transduced coronaries, compared to matched Ad.CMVLacZ coronaries (6.10 +/- 1.08 g vs. 8.45 +/- 1.19 g, respectively, P = 0.05, n = 8). This tension differential was eliminated with prior incubation in NG-monomethyl-L-arginine (L-NMMA, 10(-4) M). The EC50 for calcium ionophore relaxation of Ad.CMVeNOS coronary arteries was reduced compared to Ad.CMVLacZ (-7.90 +/- 0.03 logM vs. -7.26 +/- 0.11 logM, respectively, P < 0.05, n = 8). CONCLUSIONS: These studies demonstrate successful transfer of endothelial nitric oxide synthase into porcine coronary arteries as verified by histochemical localization of recombinant protein with an increase of nitric oxide release as demonstrated by enhanced nitrite production and an alteration in vasomotor function.

Adenoviridae↗