Citations

Background— Caveolin-1 is a regulator of signaling events originating from plasma membrane microdomains termed caveolae. This study was performed to determine the regulatory role of caveolin-1 on the proliferative events induced by platelet-derived growth factor (PDGF) in vascular smooth muscle cells (VSMCs). Methods and Results— Treatment of VSMCs with PDGF for 24 hours resulted in a loss of caveolin-1 protein expression and plasma membrane–associated caveolae, despite a 3-fold increase in caveolin-1 mRNA. Pretreatment of VSMCs with chloroquine, an inhibitor of lysosomal function, inhibited the PDGF-induced loss of caveolin-1. These studies demonstrated that caveolin-1 was a target of PDGF signaling events. Adenoviral overexpression of caveolin-1 was associated with a switch in PDGF-induced signaling events from a proliferative response to an apoptotic response. This overexpression inhibited PDGF-induced expression of cyclin D1 in the presence of unaffected mitogen-activated protein kinase activation. Conclusions— Taken together, these studies suggest that caveolin-1 is an inhibitor of PDGF proliferative responses and might be capable of transforming PDGF-induced proliferative signals into death signals.

Background and Purpose— Hypercholesterolemia is associated with endothelial dysfunction. Inducible nitric oxide synthase (iNOS) is upregulated in atherosclerotic vessels. However, its role in the regulation of vascular function is not completely understood. We examined the effect of adenovirus-mediated gene transfer of iNOS to the hypercholesterolemic rabbit carotid artery in vivo. Methods— Rabbits were fed a high-cholesterol or chow diet for 10 weeks. Two doses (1×10 8 and 1×10 9 plaque-forming units [pfu]/mL) of adenoviral vectors encoding iNOS (AdiNOS) or β-galactosidase (Adβgal) were luminally delivered to the carotid arteries from chow- and cholesterol-fed animals. Vascular reactivity and superoxide levels were assessed in Adβgal- and AdiNOS-transduced vessels from chow- and cholesterol-fed animals after 3 days. Results— Endothelium-dependent vasorelaxation was impaired in the carotid artery from cholesterol-fed animals. In AdiNOS-transduced arteries, transgene expression was demonstrated by positive immunostaining in the endothelium. Transduction with low-dose (1×10 8 pfu/mL) AdiNOS did not affect vascular reactivity in arteries from chow- and cholesterol-fed animals. In contrast, high-dose (1×10 9 pfu/mL) AdiNOS significantly reduced endothelium-dependent relaxation in vessels from cholesterol- but not chow-fed rabbits. After both low- and high-dose iNOS gene transfer, levels of O 2 · − were significantly ( P <0.05) elevated in carotid arteries from cholesterol-fed animals. Incubation with an O 2 · − scavenger did not reverse vascular dysfunction in these arteries. Conclusions— Adenoviral-mediated overexpression of iNOS results in increased production of O 2 · − in carotid arteries from cholesterol- but not chow-fed animals. High-dose AdiNOS gene transfer is associated with reduced endothelium-dependent and -independent relaxation in vessels from cholesterol-fed animals.

Purpose: A phase III, randomized, double-blind study was conducted by the North Central Cancer Treatment Group to determine the efficacy and toxicity of oral glutamine for the prevention of acute diarrhea in patients receiving pelvic radiation therapy (RT). Patients and Methods: All 129 patients enrolled from 14 institutions between February 1998 and October 1999 were eligible. Patients received 4 g of glutamine or placebo orally, twice a day, beginning with the first or second day of RT and continuing for 2 weeks after RT. During treatment, patients were assessed weekly for toxicity, and a bowel function questionnaire was administered. The primary measures of treatment efficacy were diarrhea levels measured by maximum grade of diarrhea, incidence of diarrhea, and average diarrhea score. After completion of RT, the bowel function questionnaire was administered weekly for 4 weeks and at 12 and 24 months. Toxicity was measured by National Cancer Institute common toxicity criteria. Results: The median age of patients was 69 years (range, 34 to 86 years). The two treatment arms were balanced with respect to all baseline factors. There were no significant differences in toxicity by treatment. Quality-of-life scores and the mean number of problems reported on the bowel function questionnaire were virtually identical for both treatment groups. The incidence of grade 3 or higher diarrhea was 20% for the glutamine arm and 19% for the placebo arm (P = .99). The maximum number of stools per day was 5.1 for the glutamine arm and 5.2 for the placebo arm (P = .99). Conclusion: There is no evidence of a beneficial effect of glutamine during pelvic RT.

Renal artery stenosis (RAS) may cause hypertension, azotemia, episodes of flash pulmonary edema and congestive heart failure. Renal artery angioplasty and stenting was performed in 207 patients from 1991 to 1997. Thirty-nine of these patients (19%) underwent renal artery stenting for the control of recurrent episodes of congestive heart failure and flash pulmonary edema. All patients had angiographic evidence of severe (>70%) bilateral RAS (n = 18) or severe RAS to a solitary functioning kidney (n = 21). Sixteen patients (41%) were male and 23 (59%) were female, mean age 69.9 years (range 50- 85 years). Of the 18 patients with bilateral RAS, 12 (66.6%) underwent bilateral stenting. Mean blood pressure decreased from 174/85 6 32/23 mmHg to 148/72 6 24/14 mmHg (p < 0.001). Mean number of blood pressure medications decreased from 3 6 1 to 2.5 6 1 (p = 0.006). Twenty-eight patients (71.8%) had improvement in blood pressure control. The mean serum creatinine decreased from 3.16 6 1.61 to 2.65 6 1.87 (p = 0.06). Six of 39 patients (15.4%) used angiotensin converting enzyme (ACE) inhibitors prior to stenting whereas 19 of 39 patients (48.7%) used ACE inhibitors poststenting (p = 0.004). Twenty of 39 patients (51.4%) demonstrated improvement in serum creatinine, 10 of 39 patients (25.6%) had stabilization of serum creatinine and nine of 39 patients (23%) demonstrated worsening. The number of hospitalizations due to congestive heart failure in the year preceding renal artery stenting was 2.4 6 1.4 and poststenting was 0.3 6 0.7 (p < 0.001). The New York Heart Association Functional Class decreased from 2.9 6 0.9 prestenting to 1.6 6 0.9 poststenting (p < 0.001). Thirty of 39 patients (77%) had no hospitalizations for congestive heart failure during a mean follow-up period of 21.3 months. Nine patients expired during the course of follow up; eight of the nine patients died within the first year after renal artery stenting. Renal artery stenting decreased the frequency of congestive heart failure, flash pulmonary edema, and the need for hospitalization in most patients. Blood pressure was markedly improved in the majority of patients with improved or stabilized renal function. Evaluation for RAS is important in hypertensive patients who present with recurrent congestive heart failure or flash pulmonary edema.

Acute vascular or humoral rejection, a vexing outcome of organ transplantation, has been attributed by some to activation and by others to apoptosis of endothelial cells in the graft. We asked which of these processes causes acute vascular rejection by tracing the processes during the development of acute vascular rejection in porcine cardiac xenografts performed in baboons. Apoptosis, assayed by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL), expression of activated caspase-3, and proapoptotic genes Bax and Bcl-x L , was not detected until acute vascular rejection was well advanced, and even then, apoptosis was largely confined to myocytes. Activation of the endothelium, as evidenced by expansion of rough endoplasmic reticulum and increased ribosomal antigen and phospho-p70 S6 kinase, occurred early in the course of acute vascular rejection and progressed through the disease process. These findings suggest that acute vascular rejection is caused by an active metabolic process and not by apoptosis in the endothelium.

PURPOSE: To evaluate quality of life (QOL) and tumor response after administration of an oral chemotherapy regimen in patients with previously untreated metastatic colorectal cancer. PATIENTS AND METHODS: Seventy-eight patients received a mean number of 5.8 cycles of therapy. QOL data were analyzed at baseline, after every two cycles of therapy, and at the time of treatment discontinuation. The Uniscale and the European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire C30 were both utilized. RESULTS: The confirmed response rate was 26% (95% confidence interval [CI], 17% to 37%). Median survival was 11.3 months (95% CI, 9.6 to 15.1 months). Global QOL scores were unchanged over the course of therapy by either tool. Only the physical function subscale score had worsened at the end of therapy. In an analysis of responding patients, significant and durable improvements in both global QOL measures as well as select subscale scores were observed. Diarrhea and physical function QOL scores had declined at the time of treatment discontinuation. Patients who did not respond to therapy had preserved QOL scores when they were evaluated after two cycles of therapy. CONCLUSION: This oral treatment strategy preserved QOL in treated patients. Global QOL measures as well as several QOL subscale scores significantly improved in patients with a documented response to therapy. The profile of improved QOL components indicated that patient well-being was related to tumor response in specific and perceivable ways. Nonresponding patients reported preserved QOL during the first two cycles of therapy. QOL analysis was feasible and informative in this moderately sized multicenter phase II trial.

In a phase 2 study, 23 patients with myelofibrosis with myeloid metaplasia were treated with imatinib mesylate at a constant dose of 400 mg/d. Treatment was held in 16 patients (70%), after 1 to 12 weeks, because of side effects (neutropenia, 6 patients; musculoskeletal pain, 5 patients; thrombocytosis, 4 patients; edema, 3 patients; diarrhea and hyperbilirubinemia, 1 patient). Including patients in whom retreatment at a reduced dose was possible, 11 patients (48%) were able to continue treatment beyond 3 months. None of the patients experienced a response in anemia, and only 2 had partial responses in splenomegaly. A greater than 50% increase in platelet count was documented in 11 (48%) patients, but not in those with baseline platelet counts of less than 100 × 109/L. In vitro, imatinib mesylate caused variable degrees of growth suppression of myeloid and erythroid progenitors that unfortunately did not translate into clinical benefit.

ABSTRACT Caveolin-1 (Cav-1) is a major protein constituent of caveolae, a type of plasma membrane raft. We observed that coexpression of human Cav-1 with human immunodeficiency virus type 1 (HIV-1) blocked virion production from cells that are ordinarily highly permissive. Further investigation showed that this effect is specific, occurs at low ratios of Cav-1 to HIV-1 DNA, depends on expression of Cav-1 protein, and involves severely impaired expression of HIV-1 proteins. Cav-1 also blocked HIV-2 expression. In contrast, Cav-1 did not inhibit protein expression by a paramyxovirus and did not induce apoptosis or affect cellular morphology, cell viability, or cell cycle progression. Although only small amounts of HIV-1 virions were released from Cav-1-transfected cells, these were fully infectious. Deletion mutagenesis showed that the C-terminal 78 residues were as active as the full-length (178-amino-acid) protein in producing the block. In contrast, the 100 most N-terminal amino acids of Cav-1, which include the previously identified oligomerization and scaffolding domains, were shown to be dispensable. Study of single-amino-acid-exchange mutants of Cav-1 established that palmitoylation was not required. Additional deletion mutants then identified the hydrophobic, membrane-associated domain (residues 101 to 135) as the main determinant. Cellular distribution of wild-type and mutant proteins correlated with ability to block HIV-1 expression. Finally, Cav-2 also blocked HIV-1 expression. These data show that coexpression of caveolins can markedly inhibit expression of HIV proviral DNA and establish that the inhibition is mediated by the hydrophobic, membrane-associated domain.

Background and Purpose — Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179DeNOS) increases nitric oxide (NO) production constitutively in the absence of agonist stimulus. The present study was designed to determine the effect of mutant S1179DeNOS gene expression on vasomotor function of canine cerebral arteries. Methods — Isolated basilar and middle cerebral arteries were exposed ex vivo (30 minutes at 37°C) to an adenoviral vector (10 10 plaque-forming units per milliliter) encoding the S1179DeNOS gene (AdCMVS1179DeNOS), the wild-type eNOS gene (AdCMVeNOS), or the green fluorescent protein (GFP) reporter gene (AdCMVGFP). Twenty-four hours after transduction, arteries were suspended in an organ chamber for isometric force recording, and levels of cGMP were measured by radioimmunoassay. Results — Transgene protein expression was detected mainly in the vascular adventitia. In AdCMVS1179DeNOS-transduced arteries, basal levels of cGMP were significantly elevated compared with those in control (nontransduced), AdCMVGFP-, or AdCMVeNOS-transduced vessels (n=8; P <0.01). The elevation of cGMP was abolished by a NOS inhibitor, N G -nitro- l -arginine methyl ester (L-NAME), or by incubation in the calcium-free medium in the presence of calcium chelators. In AdCMVS1179DeNOS-transduced arteries, contractions to endothelin-1 (10 −10 to 10 −8 mol/L) were significantly reduced compared with those in control and AdCMVGFP-transduced arteries (n=7; P <0.05). The vasoconstrictor effect of endothelin-1 was restored in the presence of the NOS inhibitor L-NAME. Conclusions — Our results suggest that in cerebral arteries, expression of recombinant S1179DeNOS increases basal production of NO and inhibits the vasoconstrictor effect of endothelin-1. This effect may have therapeutic application in prevention and treatment of cerebrovascular diseases.

Background and Purpose — Post-subarachnoid hemorrhage (SAH) cerebral vasospasm is a potentially devastating condition whose pathogenesis involves impaired nitric oxide (NO) bioavailability. We aimed to determine whether recombinant endothelial NO synthase (eNOS) gene expression may protect vasomotor function and prevent vasospasm in a canine experimental SAH model. Methods — Recombinant adenoviral vectors (5×10 9 plaque-forming units/animal) encoding genes for eNOS (AdeNOS) and β-galactosidase (AdLacZ) or vehicle were injected into the cerebrospinal fluid (CSF) of dogs on day −1 (ie, 24 hours before the first intra-CSF injection of blood on day 0). Cerebral angiography was performed at day 0 (baseline) and day 7 (immediately before death), and tissues were harvested for additional studies. Results — Western analysis and immunohistochemistry detected recombinant eNOS exclusively in cerebral arteries isolated from AdeNOS-transduced dogs, and in this group of animals CSF NO concentrations were significantly elevated by day 2. Analysis of day 7 versus day 0 cerebral angiograms for each group revealed significant spasm at the basilar artery midpoint in AdLacZ-transduced and nontransduced dogs but not in AdeNOS-transduced dogs. Isometric force recording of basilar arteries isolated from AdeNOS-transduced dogs showed significantly augmented relaxations to bradykinin and reduced contractions to endothelin-1. Conclusions — Our results suggest that expression of recombinant eNOS in the adventitia of cerebral arteries may contribute toward protection against post-SAH vasospasm.