The role of hypoxia and p53 in the regulation of angiogenesis in bladder cancer.

PURPOSE: Our previous studies defined thrombospondin-1 (TSP-1) and vascular endothelial growth factor (VEGF) as the primary mediators of angiogenesis in the bladder and the loss of inhibitory TSP-1 as a key event in the transition to an angiogenic phenotype during bladder cancer development. We evaluated the role of p53, which is commonly inactivated in bladder cancer, and hypoxia in the regulation of angiogenesis in the bladder. MATERIALS AND METHODS: The p53 status was modulated in normal urothelial and bladder cancer cells, and conditioned media was collected under normal oxygen or hypoxic (0.5% O2) conditions. Angiogenic activity was evaluated with the endothelial cell migration assay, and the levels of secreted TSP-1 and VEGF were determined by Western blot analysis and enzyme-linked immunosorbent assay, respectively. RESULTS: Retroviral mediated expression of the E6 oncoprotein reduced wild-type p53 levels in normal urothelial cells by greater than 90% but did not significantly alter TSP-1 or VEGF levels, while total inductive and inhibitory activities remained unchanged. Adenoviral mediated expression of wild-type p53 was confirmed in 4 bladder cancer cell lines by Western blot analysis for p53 and its downstream effector protein p21 (2.5 to 5.0-fold increase). TSP-1 levels remained unchanged but the levels of secreted VEGF in the high grade UMUC-3 and 253J cell lines were significantly decreased 5 to 50-fold and a corresponding decrease in net angiogenic activity was observed. However, (increased expression) of p53 had no effect on the angiogenic activity of the low grade RT4 or high grade HT1376 bladder cancer cells. Hypoxia converted normal urothelial cell derived conditioned media from anti-angiogenic to angiogenic and increased the angiogenic activity of bladder cancer cell derived conditioned media. This change was due to 2.5 to 6-fold hypoxic up-regulation of VEGF because the expression of inhibitory TSP-1 was not significantly altered. CONCLUSIONS: Our results suggest that p53 does not regulate angiogenesis in the bladder in the setting of an otherwise normal genome and gene therapy with wild-type p53, which is currently being studied for this cancer, may have only limited effects on angiogenesis. In contrast, hypoxia regulates angiogenesis in this system, primarily through its effects on VEGF.

Molecular mediators of angiogenesis in bladder cancer.

Bladder tumors are characterized by markedly increased angiogenesis when compared to the normal urothelium (NU) from which they are derived. Here, we use both cultured cells and immunohistochemistry to demonstrate a primary regulatory role for thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis, in the development of bladder tumor angiogenesis. Secretions from bladder cancer (CA) cells stimulated endothelial cell migration and corneal neovascularization, whereas those from NU cells were inhibitory. The antiangiogenic activity of NU cells was primarily due to secreted TSP-1 because neutralizing antibodies completely relieved the inhibition. Neutralizing antibodies to several putative angiogenesis inducers identified vascular endothelial growth factor (VEGF) and, to a lesser extent, basic fibroblast growth factor as the primary inducers secreted by bladder cancer cells. The secretion of TSP-1 by low- and high-grade cancer cells was reduced >94% when compared to NU cells, and this loss of inhibitory TSP-1 accounted for the development of an angiogenic phenotype because both NU cells and cancer cells secreted similar levels of total stimulatory activity and VEGF. Immunohistochemistry showed that TSP-1 was significantly reduced in all grades of bladder cancer when compared to NU, whereas VEGF staining remained relatively constant. Taken together, these data suggest that down-regulation of TSP-1 secretion is a key event in the switch from an antiangiogenic to an angiogenic phenotype, which occurs early in the development of bladder cancer.

Respiratory dyskinesia presenting as acute respiratory distress.

Incapacitating respiratory distress was the presenting manifestation of a choreiform movement disorder. Because the patient also had asthma, respiratory distress was at first mistakenly attributed to this condition. Despite vigorous asthma management, there was no improvement. However, once the neurologic condition was recognized, use of specific therapy (haloperidol and reserpine) resulted in rapid and sustained remission of respiratory symptoms.

Lung clearance of inhaled 99mTc-DTPA in the dog.

We studied the reproducibility of measuring an index of permeability of respiratory epithelium in dogs using aerosolized 99mTc-DTPA (diethylenetriaminepentaacetate). The method uses a gamma camera to measure the rate of clearance of soluble radioactive aerosol deposited in the lung. A solution of 99mTc-DTPA in normal saline was aerosolized by an ultrasonic nebulizer. Eleven anesthetized dogs breathing spontaneously inhaled the resulting droplets for 2 min. Mass median aerodynamic diameter of the droplets was 4.4 micron with a geometric standard deviation of 2.1. Clearance from the lung was monitored by quantitative gamma camera imaging for up to 2 h. For a 60-min observation period, the biological half-life for clearance of 99mTc-DTPA from both lungs was 66 +/- 11 (SD) min. Apical regions cleared significantly slower than basal regions, probably because of a larger portion of bronchial tissue in the apical region of the dog's lung. The best reproducibility of absorption of 99mTc-DTPA in the dog was obtained from basal regions and peripheral zones of the lung within 30 min after inhalation of the radioaerosol.

Evidence of two temperate episodes in late Pleistocene deposits at Marsworth, UK.

In the British Quaternary, two post-Cromerian interglacials, the Hoxnian and the Ipswichian, are recognized. Evidence of additional interglacials in this interval is widely accepted in the oceanic record of Quaternary events, and the possibility that at least one additional interglacial of this age is represented in Britain has been discussed. However, in the absence of datable interglacial deposits which are seen to overlie one another, the issue has remained controversial. We describe here deposits at Marsworth, UK (Fig. 1) where there is evidence of two temperate episodes, and of intervening periglacial conditions. Stratigraphical superposition is established beyond any reasonable doubt. The later deposit relates to the temperate woodland stage of the Ipswichian Interglacial. Dating of the earlier temperate material by the 230Th/234U disequilibrium method indicates an interglacial episode not previously established in the British Quaternary.