Citations

AbstractBackgroundThe addition of bevacizumab to chemotherapy improved outcomes for patients with metastatic colon cancer. E5204 was designed to test whether the addition of bevacizumab to mFOLFOX6, following neoadjuvant chemoradiation and definitive surgery, could improve overall survival (OS) in patients with stage II/III adenocarcinoma of the rectum.Subjects, Materials, and MethodsPatients with stage II/III rectal cancer who had completed neoadjuvant 5-fluorouracil-based chemoradiation and had undergone complete resection were enrolled. Patients were randomized to mFOLFOX6 (Arm A) or mFOLFOX6 with bevacizumab (Arm B) administered every 2 weeks for 12 cycles.ResultsE5204 registered only 355 patients (17% of planned accrual goal) as it was terminated prematurely owing to poor accrual. At a median follow-up of 72 months, there was no difference in 5-year overall survival (88.3% vs. 83.7%) or 5-year disease-free survival (71.2% vs. 76.5%) between the two arms. The rate of treatment-related grade ≥ 3 adverse events (AEs) was 68.8% on Arm A and 70.7% on Arm B. Arm B had a higher proportion of patients who discontinued therapy early as a result of AEs and patient withdrawal than did Arm A (32.4% vs. 21.5%, p = .029).The most common grade 3–4 treatment-related AEs were neutropenia, leukopenia, neuropathy, diarrhea (without prior colostomy), and fatigue.ConclusionAt 17% of its planned accrual, E5204 did not meet its primary endpoint. The addition of bevacizumab to FOLFOX6 in the adjuvant setting did not significantly improve OS in patients with stage II/III rectal cancer.Implications for PracticeAt 17% of its planned accrual, E5204 was terminated early owing to poor accrual. At a median follow-up of 72 months, there was no significant difference in 5-year overall survival (88.3% vs. 83.7%) or in 5-year disease-free survival (71.2% vs. 76.5%) between the two arms. Despite significant advances in the treatment of rectal cancer, especially in improving local control rates, the risk of distant metastases and the need to further improve quality of life remain a challenge. Strategies combining novel agents with chemoradiation to improve both distant and local control are needed.

PURPOSE Observation is the current standard of care for smoldering multiple myeloma. We hypothesized that early intervention with lenalidomide could delay progression to symptomatic multiple myeloma. METHODS We conducted a randomized trial that assessed the efficacy of single-agent lenalidomide compared with observation in patients with intermediate- or high-risk smoldering multiple myeloma. Lenalidomide was administered orally at a dose of 25 mg on days 1 to 21 of a 28-day cycle. The primary end point was progression-free survival, with disease progression requiring the development of end-organ damage attributable to multiple myeloma and biochemical progression. RESULTS One hundred eighty-two patients were randomly assigned—92 patients to the lenalidomide arm and 90 to the observation arm. Median follow-up is 35 months. Response to therapy was observed in 50% (95% CI, 39% to 61%) of patients in the lenalidomide arm, with no responses in the observation arm. Progression-free survival was significantly longer with lenalidomide compared with observation (hazard ratio, 0.28; 95% CI, 0.12 to 0.62; P = .002). One-, 2-, and 3-year progression-free survival was 98%, 93%, and 91% for the lenalidomide arm versus 89%, 76%, and 66% for the observation arm, respectively. Only six deaths have been reported, two in the lenalidomide arm versus four in the observation arm (hazard ratio for death, 0.46; 95% CI, 0.08 to 2.53). Grade 3 or 4 nonhematologic adverse events occurred in 25 patients (28%) on lenalidomide. CONCLUSION Early intervention with lenalidomide in smoldering multiple myeloma significantly delays progression to symptomatic multiple myeloma and the development of end-organ damage.

Increased production of oxygen free radicals is an important mechanism of endothelial dysfunction in diabetes mellitus. Our goal was to test whether adenovirus (Ad)-mediated gene transfer of copper/zinc (CuZn) or manganese superoxide dismutase (Mn SOD) improves relaxation of diabetic vessels. The aortas from 9 alloxan-induced diabetic mellitus (DM) and 16 control rabbits were used. Control and DM rings were transduced ex vivo with Ad vectors encoding Mn SOD (AdMn SOD), CuZn SOD (AdCuZn SOD), β-galactosidase (Adβgal), or diluents. In the absence of gene transfer, SOD activity was significantly increased in DM aortas. Transgene expression in DM AdCuZn SOD and DM AdMn SOD-transduced vessels was confirmed by Western blot analysis and by increased SOD activity (DM AdCuZn SOD, 76.2 ± 9.3; DM AdMn SOD, 65.2 ± 4.8; P < 0.05 vs. DM Adβgal; 50.9 ± 4.4 U/mg protein). Superoxide production was increased in DM Adβgal-transduced aorta and relaxations to acetylcholine were impaired in these vessels. Gene transfer of CuZn SOD and Mn SOD corrected both of these defects. Thus Ad-mediated gene transfer CuZn and Mn SOD to the diabetic aorta improves endothelium-dependent relaxation.

We recently reported that expression of recombinant endothelial nitric oxide (NO) synthase (eNOS) gene in adventitial fibroblasts restores NO formation in canine cerebral arteries without endothelium in response to bradykinin ex vivo and in vivo. The present study was designed to further characterize the stimuli that can activate recombinant eNOS enzyme expressed in the adventitia of cerebral arteries. To stimulate recombinant eNOS, we used serum (0.1–10%), substance P (10−11–3 × 10−9M), and ANG II (10−7–10−5M) because they increase intracellular calcium concentrations in fibroblasts. Endothelium-denuded segments of canine basilar arteries were incubated with an adenoviral vector encoding β-galactosidase gene or eNOS gene for 30 min at 37°C. After 24 h, vasomotor activity and cGMP formation in eNOS or β-galactosidase arteries were examined by isometric force recording and by radioimmunoassay, respectively. In control arteries and β-galactosidase gene-transduced arteries, serum caused concentration-dependent contractions, whereas in recombinant eNOS gene-transduced arteries, serum produced concentration-dependent relaxations. Substance P and ANG II had no effect on vascular tone in control and β-galactosidase arteries but caused concentration-dependent relaxations as well as a significant increase in cGMP levels in eNOS arteries. These relaxations were blocked by the NOS inhibitor NG-nitro-l-arginine methyl ester. Chemical treatment or mechanical inactivation of adventitial function significantly attenuated substance P-induced relaxations and ANG II-induced relaxations. These findings demonstrate that serum, substance P, and ANG II cause adventitia-dependent relaxations in cerebral arteries expressing the recombinant eNOS gene. This mechanism of vasodilatation may have beneficial effects in the prevention and treatment of vascular disorders characterized by the diminished bioavailability of NO, such as cerebral vasospasm.

Resistance arteries are an important target for vascular gene therapy because they play a key role in the regulation of tissue blood flow. The present study was designed to determine the effects of recombinant endothelial (e) nitric oxide synthase (NOS) gene expression on vasomotor reactivity of small brain stem arteries (internal diameter, 253 ± 2.5 μm). Arterial rings were exposed ex vivo to an adenoviral vector (109and 1010plaque-forming units/ml) encoding eNOS gene or β-galactosidase gene. Twenty-four hours after transduction, vascular function was examined by isometric force studies. Transgene expression was evident mainly in adventitia. In arteries with endothelium transduced with eNOS gene but not with control β-galactosidase gene, relaxations to bradykinin and substance P were significantly augmented. Removal of endothelium abolished relaxations to bradykinin and substance P in control and β-galactosidase arteries. However, in endothelium-denuded arteries transduced with recombinant eNOS, bradykinin and substance P caused relaxations that were abolished in the presence of the NOS inhibitor NG-nitro-l-arginine methyl ester. In control arteries, endothelium removal augmented relaxations to the nitric oxide donors sodium nitroprusside and diethylamine NONOate. This augmentation was absent in eNOS gene-transduced arteries without endothelium. Our results suggest that, in small brain stem arteries, expression of recombinant eNOS increases biosynthesis of nitric oxide. Adventitia of small arteries is a good target for expression of recombinant eNOS. Genetically engineered adventitial cells may serve as a substitute source of nitric oxide in cerebral arteries with dysfunctional endothelium.

Our previous ex vivo and in vivo studies reported that expression of the recombinant endothelial nitric oxide (NO) synthase (eNOS) gene in adventitial fibroblasts recovers NO production in arteries without endothelium in response to bradykinin. The present study was designed to characterize subtypes of bradykinin receptors on adventitial fibroblasts coupled to the activation of recombinant eNOS. Endothelium-denuded segments of canine basilar arteries were transduced with β-galactosidase (β-Gal) gene or eNOS gene ex vivo, using a replication-defective adenoviral vector (1010plaque-forming units/ml) for 30 min at 37°C. Twenty-four hours later, isometric force recording or cGMP measurement was carried out. B1bradykinin receptor agonist (des-Arg9-bradykinin, 10−10-10−8mol/l) did not significantly affect vascular tone in control or β-Gal gene-transduced canine basilar arteries without endothelium. In contrast, this agonist caused concentration-dependent relaxations in recombinant eNOS gene-transduced arteries without endothelium. Relaxations to B1receptor agonist in the eNOS arteries were abolished by B1receptor antagonist (des-Arg9-[Leu8]bradykinin, 6 × 10−9mol/l) but not by B2receptor antagonist (Hoe-140, 5 × 10−8mol/l). Bradykinin did not significantly alter vascular tone in control or β-gal arteries without endothelium, whereas this peptide (10−11-10−8mol/l) induced concentration-dependent relaxations, as well as an increase in cGMP formation in endothelium-denuded eNOS-transduced arteries. Stimulatory effects of bradykinin were prevented in the presence of a B2receptor antagonist but not in the presence of a B1receptor antagonist. B1and B2receptor antagonists had no effect on relaxations to substance P, confirming the selectivity of the compounds. Our results suggest that B1and B2bradykinin receptors are coupled to activation of recombinant eNOS expressed in adventitial fibroblasts.

The present study was designed to determine the effect of recombinant inducible nitric oxide (NO) synthase (iNOS) gene expression on vasomotor function in cerebral arteries. Isolated canine basilar arteries were exposed ex vivo (30 min at 37°C) to an adenoviral vector [107, 108, or 109plaque-forming units (pfu)/ml] encoding either the iNOS gene or the β-galactosidase reporter gene. Twenty-four hours after transduction, Western blot analysis demonstrated expression of iNOS protein only in iNOS (109pfu/ml)-transduced arteries. Immunohistochemical analysis localized iNOS expression predominantly in adventitia. Vascular reactivity of isolated basilar arteries was studied by isometric force recording. Concentration-response curves to UTP (10−9–10−3M) and diethylaminodiazen-1-ium-1,2-dioate (10−10–10−5M) were significantly shifted to the right in iNOS gene (109pfu/ml)-transduced rings compared with control and β-galactosidase-transduced rings ( P < 0.05, n = 5–6). Endothelium-dependent relaxation to bradykinin was significantly attenuated in iNOS-transduced rings ( P < 0.001, n = 8). The basal level of cGMP and superoxide anion (O[Formula: see text]·) production were elevated in iNOS-transduced rings ( P < 0.05, n = 7 for cGMP; P < 0.01, n = 6–9 for O[Formula: see text]· production). Our results suggest that expression of recombinant iNOS in cerebral arteries reduces vasomotor reactivity to both vasoconstrictor and vasodilator agonists. Attenuation of contractions is most likely due to functional antagonism between UTP and cGMP. Reduction of endothelium-dependent relaxation to bradykinin appears to be mediated in part by reduced reactivity of smooth muscle cells to NO.

Although hydrogen peroxide (H2O2) induces proliferation of vascular smooth muscle cells, its role in endothelial cell proliferation is unclear. Our aim was to study the role of hydrogen peroxide in endothelial cell proliferation by overexpressing catalase. Human aortic endothelial cells were transduced with adenoviral vectors encoding β-galactosidase (Adβgal) or catalase (AdCat) or were exposed to diluent alone (control). Transgene expression was demonstrated by β-galactosidase staining, Western analysis, and significantly increased enzyme activity in AdCat-transduced cells. Overexpression of catalase decreased DNA synthesis in AdCat compared with control and Adβgal-transduced cells (536.8 ± 31 vs. 1,875.1 ± 132.9 vs. 1,347.5 ± 93.7 dpm/well, respectively; P < 0.05 vs. control and Adβgal). Six days after transduction with AdCat (multiplicity of infection = 50), cell numbers were significantly reduced (AdCat: 38 ± 1.8% of cell counts in control, P < 0.05; and 45 ± 2% of cell count in Adβgal, P < 0.05). Incubation with aminotriazole 10 mmol/l, an inhibitor of catalase, prevented this effect. The number of apoptotic cells was increased one- and threefold 2 and 4 days, respectively, after transduction with AdCat. Exogenous administration of low concentrations of H2O2(50 μM) significantly increased cell proliferation, whereas it was inhibited by higher concentrations. These results suggest that H2O2is an important modulator of endothelial cell proliferation.