Wu W, Platoshyn O, Firth AL, Yuan JX. Hypoxia divergently regulates production of reactive oxygen species in human pulmonary and coronary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 293: L952-L959, 2007. First published August 10, 2007; doi:10.1152/ajplung.00203.2007.-Acute hypoxia causes pulmonary vasoconstriction and coronary vasodilation. The divergent effects of hypoxia on pulmonary and coronary vascular smooth muscle cells suggest that the mechanisms involved in oxygen sensing and downstream effectors are different in these two types of cells. Since production of reactive oxygen species (ROS) is regulated by oxygen tension, ROS have been hypothesized to be a signaling mechanism in hypoxia-induced pulmonary vasoconstriction and vascular remodeling. Furthermore, an increased ROS production is also implicated in arteriosclerosis. In this study, we determined and compared the effects of hypoxia on ROS levels in human pulmonary arterial smooth muscle cells (PASMC) and coronary arterial smooth muscle cells (CASMC). Our results indicated that acute exposure to hypoxia (PO 2 ϭ 25-30 mmHg for 5-10 min) significantly and rapidly decreased ROS levels in both PASMC and CASMC. However, chronic exposure to hypoxia (PO 2 ϭ 30 mmHg for 48 h) markedly increased ROS levels in PASMC, but decreased ROS production in CASMC. Furthermore, chronic treatment with endothelin-1, a potent vasoconstrictor and mitogen, caused a significant increase in ROS production in both PASMC and CASMC. The inhibitory effect of acute hypoxia on ROS production in PASMC was also accelerated in cells chronically treated with endothelin-1. While the decreased ROS in PASMC and CASMC after acute exposure to hypoxia may reflect the lower level of oxygen substrate available for ROS production, the increased ROS production in PASMC during chronic hypoxia may reflect a pathophysiological response unique to the pulmonary vasculature that contributes to the development of pulmonary vascular remodeling in patients with hypoxia-associated pulmonary hypertension. 2 •Ϫ ) and hydrogen superoxide (HO Ϫ ) formed from sequential transfer of electrons from molecular oxygen, in vascular smooth muscle cells plays an important role in the physiological regulation of vascular tone and vascular remodeling (20,22). These reactive intermediates act as progenitors for other forms of ROS such as hydrogen peroxide (H 2 O 2 ), peroxynitrite (ONOO Ϫ ), and the hydroxyl radical. There are currently debates as to the nature of the dysregulation of ROS production that may contribute to pathophysiological conditions, such as pulmonary hypertension and atherosclerosis, and the disturbance of normal vascular function. Not surprisingly, the mechanisms by which hypoxia causes pulmonary vessels to constrict and coronary vessels to relax are therefore an active area of research.It is now known that hypoxic pulmonary vasoconstriction (HPV) is an intrinsic property of the pulmonary vasculature and specifically resides in pulmonary arterial smooth muscle cells (PASMC) (31,...

Growing evidence from both prokaryotes and eukaryotes indicates that pyrimidine 5-methyl groups can have profound biological consequences that are mediated by the affinity of DNA-protein interactions. The presence of the 5-methyl group could potentially create a steric block preventing the binding of some proteins whereas the affinity of many other proteins is substantially increased by pyrimidine methylation. In this paper, we have constructed a series of oligonucleotides containing cytosine and a series of 5-substituted cytosine analogues including all halogens. This set of oligonucleotides has been used to probe the relationship between the size of the substituent and its capacity to modulate cleavage by the methylation-sensitive restriction endonucleases MspI and HpaII. Additionally, we have examined the impact of the halogen substitution on the corresponding bacterial methyltransferase (M.HpaII). We observed that MspI cleavage is only subtly affected by substituted cytosine analogues at the inner position of the CCGG recognition site. In contrast, HpaII cleaves cytosine-containing oligonucleotides completely whereas 5-fluorocytosine-containing oligonucleotides are cleaved at a reduced rate. The presence of the larger halogens Cl, Br, or I as well as a methyl group completely prevents cleavage by HpaII. These data suggest that the steric wall is encountered by HpaII slightly beyond the fluorine substituent, at about 2.65 A from the pyrimidine C5-position. It is known that 5-fluorocytosine in an oligonucleotide can form a covalent irreversible suicide complex with either prokaryotic or eukaryotic methyltransferases. Kinetic data reported here suggest that the 5-fluorocytosine-containing oligonucleotide can also inhibit M.HpaII by formation of a reversible, noncovalent complex. Our results indicate that although a 5-Cl substituent has electronic properties similar to 5-F, 5-chlorocytosine duplexes neither form a complex with M.HpaII nor inhibit enzymatic methylation. Emerging data suggest that halogenation of cytosine can occur in DNA in vivo from inflammation-mediated reactive molecules. The results reported here suggest that the inadvertent halogenation of cytosine residues in DNA could alter the affinity of sequence-specific DNA-binding proteins.

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Uterine carcinosarcoma is an uncommon and aggressive histologic variant of endometrial carcinoma with a poor prognosis.Inactivating DNA polymerase ɛ () mutations have been associated with high tumor mutational burden (TMB) and response to immune checkpoint inhibition.To the authors' knowledge, this is the first report of response to immune checkpoint inhibitor therapy in a patient with uterine carcinosarcoma.This case further supports expanding genomic profiling to include assessment of tumor mutational burden across tumor types, given the potential for immune checkpoint inhibitor therapy in TMB-high tumors.