ABSTRACT

Significant numbers of myocytes die by apoptosis during myocardial infarction. The molecular mechanism of this process, however, remains largely unexplored. To facilitate a molecular genetic analysis, we have developed a model of ischemia-induced cardiac myocyte apoptosis in the mouse. Surgical occlusion of the left coronary artery results in apoptosis, as indicated by the presence of nucleosome ladders and in situ DNA strand breaks. Apoptosis occurs mainly in cardiac myocytes, and is shown for the first time to be limited to hypoxic regions during acute infarction. Since hypoxia-induced apoptosis in other cell types is dependent on p53, and p53 is induced by hypoxia in cardiac myocytes, we investigated the necessity of p53 for myocyte apoptosis during myocardial infarction. Myocyte apoptosis occurs as readily, however, in the hearts of mice nullizygous for p53 as in wild-type littermates. These data demonstrate the existence of a p53-independent pathway that mediates myocyte apoptosis during myocardial infarction. ( J.

No abstract

Permanent closure of the ductus arteriosus requires loss of cells from the muscle media and development of neointimal mounds, composed in part of proliferating endothelial cells. We hypothesized that postnatal ductus constriction produces hypoxia of the inner vessel wall; we also hypothesized that hypoxia might lead to cell death and the production of vascular endothelial cell growth factor (VEGF), a hypoxia-inducible growth factor that stimulates endothelial proliferation. We mapped the distribution of hypoxia in newborn baboons and correlated it with the appearance of cell death (TUNEL technique), VEGF expression, and endothelial proliferation (proliferating cell nuclear antigen expression). In the full-term baboon (n=10), the ductus was functionally closed on Doppler examination by 24 h after delivery. Regions of the ductus where the lumen was most constricted were associated with moderate/intense hypoxia; VEGF expression was increased in the hypoxic muscle media, and luminal endothelial cells, adjacent to the hypoxic media, were proliferating. Cells in the most hypoxic regions of the ductus wall were undergoing DNA fragmentation. In contrast, regions of the ductus with mild degrees of hypoxia had no evidence of cell death, VEGF expression, or endothelial proliferation. Cell death and endothelial proliferation seemed to be limited to regions of the full-term ductus experiencing moderate/intense hypoxia. In the premature baboon (67% gestation) (n=24), only 29% closed their ductus by Doppler examination before d 6. None of the premature baboons, including those with a closed ductus by Doppler, had evidence of moderate/intense hypoxia; also, there was no evidence of cell death, VEGF expression, endothelial proliferation, or neointima formation by d 6. Therefore, the premature ductus is resistant to developing hypoxia, even when its lumen is constricted; this may make it susceptible to later reopening.

Hypoxia is known to be an important prognostic marker in many human cancers. We report the use of two oxygen measurement techniques in human brain tumors and compare these data with semiquantitative histological end points. Oxygenation was measured using the Eppendorf needle electrode and/or EF5 binding in 28 brain tumors. These data were compared with necrosis, mitosis, and endothelial proliferation. In some tumors, absolute EF5 binding was converted to tissue pO 2 based on in vitro calibrations. Eppendorf electrode readings could not be used to identify WHO grade 1/2 versus WHO grade 3/4 tumors, they could not differentiate grade 3 versus grade 4 glial-derived neoplasms, nor did they correlate with necrosis or endothelial proliferation scores. EF5 binding increased as the tumor grade increased and was significantly associated with necrosis and endothelial proliferation. There was no statistically significant correlation between the two hypoxia detection techniques, although both methods indicated similar absolute ranges of tissue pO 2 . There was substantial inter-and intratumoral heterogeneity of EF5 binding in WHO grade 4 glial neoplasms. The majority of cells in glial-derived tumor had levels of hypoxia that were mild to moderate (defined herein as 10% to 0.5% pO 2 ) rather than severe (defined as approximately 0.1% pO 2 ). Immunohistochemical detection of EF5 binding tracks histological parameters in adult brain tumors, with increased binding associated with increasing necrosis and endothelial proliferation. The proportion of moderately to severely hypoxic cells is relatively low, even in the high-grade tumors. Human brain tumors are dominated by oxic to moderately hypoxic cells.

No abstract

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway can increase vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1A (HIF-1A) expression. We examined the effect of nelfinavir, an HIV protease inhibitor that inhibits Akt signaling, on VEGF and HIF-1A expression and on angiogenesis, tumor oxygenation, and radiosensitization. Nelfinavir decreases VEGF expression under normoxia via the transcription factor Sp1, which regulates the proximal core VEGF promoter. Nelfinavir decreased Sp1 phosphorylation and decreased Sp1 binding to a probe corresponding to the proximal VEGF promoter in a gel shift assay. Nelfinavir also decreased the hypoxic induction of HIF-1A, which also regulates the VEGF promoter, most likely by decreasing its translation. The effect of nelfinavir on VEGF expression had the functional consequence of decreasing angiogenesis in an in vivo Matrigel plug assay. To determine the effect this might have on tumor radiosensitization, we did tumor regrowth assays with xenografts in nude mice. The combination of nelfinavir and radiation increased time to regrowth compared with radiation alone whereas nelfinavir alone had little effect on tumor regrowth. This radiosensitizing effect was greater than suggested by in vitro clonogenic survival assays. One possible explanation for the discordance is that nelfinavir has an effect on tumor oxygenation. Therefore, we examined this with the hypoxia marker EF5 and found that nelfinavir leads to increased oxygenation within tumor xenografts. Our results suggest that nelfinavir decreases HIF-1A/VEGF expression and tumor hypoxia, which could play a role in its in vivo radiosensitizing effect. These data support the use of nelfinavir in combination with radiation in future clinical trials.

The aim of this study was to evaluate 2-(2-nitro-1 H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5) labeled with 18 F-fluorine to image hypoxia in patients with squamous cell carcinoma of the head and neck (HNSCC). Methods: Fifteen patients with HNSCC were studied. Measurement of tumor blood flow was followed by an 18 F-EF5 PET/CT scan. On a separate day, 18 F-FDG PET/CT was performed to determine the metabolically active tumor volume. In 6 patients, dynamic 18 F-EF5 images of the head and neck area were acquired, followed by static images acquired at 1, 2, and 3 h after injection. In the remaining 9 patients, only static images were obtained. 18 F-EF5 uptake in tumors was compared with that in neck muscle, and the 18 F-EF5 findings were correlated with the 18 F-FDG PET/CT studies. Results: A total of 13 primary tumors and 5 lymph node metastases were evaluated for their uptake of 18 F-EF5. The median tumor-to-muscle 18 F-EF5 uptake ratio (T/M) increased over time and was 1.38 (range, 1.1-3.2) 3 h after tracer injection. The median blood flow in tumors was 36.7 mL/100 g/min (range, 23.3-78.6 mL/100 g/min). Voxel-by-voxel analysis of coregistered blood flow and 18 F-EF5 images revealed a distinct pattern, resulting in a T/M of 1.5 at 3 h to be chosen as a cutoff for clinically significant hypoxia. Fourteen of 18 tumors (78%) had subvolumes within the metabolically active tumor volumes with T/M greater than or equal to 1.5. Conclusion: On the basis of these data, the potential of 18 F-EF5 to detect hypoxia in HNSCC is encouraging. Further development of 18 F-EF5 for eventual targeting of antihypoxia therapies is warranted.