Oxidative stress induced pulmonary endothelial cell proliferation is not mediated by superoxide

Cellular hyper-proliferation, endothelial dysfunction and oxidative stress are hallmarks of the pathobiology of pulmonary hypertension. Indeed, pulmonary endothelial cells proliferation is susceptible to redox state modulation. Some studies suggest that superoxide stimulates endothelial cell proliferation while others have linked the proliferative response to an up-regulation of peroxynitrite in lungs under oxidative stress. Given the divergence of opinion on the subject, it is important to establish the agents mediating cellular hyper-proliferation under oxidative stress. Using the combination of xanthine and xanthine oxidase, the current study demonstrates that neither superoxide nor hydrogen peroxide stimulated pulmonary endothelial cell proliferation. Alone, low level superoxide (100 RLU/s) did not alter DNA synthesis in endothelial cell and high concentration (500 RLU/s) superoxide decreased DNA synthesis to 31.8\ub13%, 30.4\ub12%, and 53.8\ub15% control at 0.1, 0.5 and 2.5% basal growth stimulation, respectively. Nonetheless, the formation of peroxynitrite under this condition stimulated proliferation to 49.2\ub19%, 51.1\ub18% and 71.2\ub12%, respectively. Taken together, pulmonary endothelial cell proliferation occurred only under conditions producing nitric oxide and superoxide in concert

Genotoxic Damage Activates the AMPK-α1 Isoform in the Nucleus via Ca2+/CaMKK2 Signaling to Enhance Tumor Cell Survival

2017 American Association for Cancer Research. Many genotoxic cancer treatments activate AMP-activated protein kinase (AMPK), but the mechanisms of AMPK activation in response to DNA damage, and its downstream consequences, have been unclear. In this study, etoposide activates the a1 but not the a2 isoform of AMPK, primarily within the nucleus. AMPK activation is independent of ataxia-telangiectasia mutated (ATM), a DNA damage-activated kinase, and the principal upstream kinase for AMPK, LKB1, but correlates with increased nuclear Ca2þ and requires the Ca2þ/calmodulin-dependent kinase, CaMKK2. Intriguingly, Ca2þ-dependent activation of AMPK in two different LKB1-null cancer cell lines caused G1-phase cell-cycle arrest, and enhanced cell viability/ survival after etoposide treatment, with both effects being abolished by knockout of AMPK-a1 and a2. The CDK4/6 inhibitor palbociclib also caused G1 arrest in G361 but not HeLa cells and, consistent with this, enhanced cell survival after etoposide treatment only in G361 cells. These results suggest that AMPK activation protects cells against etoposide by limiting entry into S-phase, where cells would be more vulnerable to genotoxic stress. Implications: These results reveal that the a1 isoform of AMPK promotes tumorigenesis by protecting cells against genotoxic stress, which may explain findings that the gene encoding AMPK-a1 (but not -a2) is amplified in some human cancers. Furthermore, a1-selective inhibitors might enhance the anticancer effects of genotoxic-based therapies

Carbonic Anhydrase Inhibitors Suppress Platelet Procoagulant Responses and In Vivo Thrombosis

Carbonic anhydrase (CA) inhibitors have a long history of safe clinical use as mild diuretics, in the treatment of glaucoma and for altitude sickness prevention. In this study, we aimed to determine if CA inhibition may be an alternative approach to control thrombosis. We utilized a high-resolution dynamic imaging approach to provide mechanistic evidence that CA inhibitors may be potent anti-procoagulant agents in vitro and effective anti-thrombotics in vivo. Acetazolamide and methazolamide, while sparing platelet secretion, attenuated intracellular chloride ion entry and suppressed the procoagulant response of activated platelets in vitro and thrombosis in vivo. The chemically similar N-methyl acetazolamide, which lacks CA inhibitory activity, did not affect platelet procoagulant response in vitro. Outputs from rotational thromboelastometry did not reflect changes in procoagulant activity and reveal the need for a suitable clinical test for procoagulant activity. Drugs specifically targeting procoagulant remodeling of activated platelets, by blockade of carbonic anhydrases, may provide a new way to control platelet-driven thrombosis without blocking essential platelet secretion responses

The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin α IIb β 3 Outside-in Signaling

The class I PI3K family of lipid kinases plays an important role in integrin αIIbβ3 function, thereby supporting thrombus growth and consolidation. Here, we identify Ras/Rap1GAP Rasa3 (GAP1IP4BP) as a major phosphatidylinositol 3,4,5-trisphosphate-binding protein in human platelets and a key regulator of integrin αIIbβ3 outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI3K-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin αIIbβ3-expressing CHO cells blocked Rap1 activity and integrin αIIbβ3-mediated spreading on fibrinogen. In contrast, Rap1GAP-deficient (P489V) and Ras/Rap1GAP-deficient (R371Q) Rasa3 had no effect. We furthermore show that two Rasa3 mutants (H794L and G125V), which are expressed in different mouse models of thrombocytopenia, lack both Ras and Rap1GAP activity and do not affect integrin αIIbβ3-mediated spreading of CHO cells on fibrinogen. Platelets from thrombocytopenic mice expressing GAP-deficient Rasa3 (H794L) show increased spreading on fibrinogen, which in contrast to wild-type platelets is insensitive to PI3K inhibitors. Together, these results support an important role for Rasa3 in PI3K-dependent integrin αIIbβ3-mediated outside-in signaling and cell spreading

Oxidative stress induced pulmonary endothelial cell proliferation is not mediated by superoxide

Cellular hyper-proliferation, endothelial dysfunction and oxidative stress are hallmarks of the pathobiology of pulmonary hypertension. Indeed, pulmonary endothelial cells proliferation is susceptible to redox state modulation. Some studies suggest that superoxide stimulates endothelial cell proliferation while others have linked the proliferative response to an up-regulation of peroxynitrite in lungs under oxidative stress. Given the divergence of opinion on the subject, it is important to establish the agents mediating cellular hyper-proliferation under oxidative stress. Using the combination of xanthine and xanthine oxidase, the current study demonstrates that neither superoxide nor hydrogen peroxide stimulated pulmonary endothelial cell proliferation. Alone, low level superoxide (100 RLU/s) did not alter DNA synthesis in endothelial cell and high concentration (500 RLU/s) superoxide decreased DNA synthesis to 31.8±3%, 30.4±2%, and 53.8±5% control at 0.1, 0.5 and 2.5% basal growth stimulation, respectively. Nonetheless, the formation of peroxynitrite under this condition stimulated proliferation to 49.2±9%, 51.1±8% and 71.2±2%, respectively. Taken together, pulmonary endothelial cell proliferation occurred only under conditions producing nitric oxide and superoxide in concert

Aquaporins in platelet function

Structurally, aquaporins (AQPs) are small channel proteins with monomers of ~ 30 kDa that are assembled as tetramers to form pores on cell membranes. Aquaporins mediate the conduction of water but at times also small solutes including glycerol across cell membranes and along osmotic gradients. Thirteen isoforms of AQPs have been reported in mammalian cells, and several of these are likely expressed in platelets. Osmotic swelling mediated by AQP1 sustains the calcium entry required for platelet phosphatidylserine exposure and microvesiculation, through calcium permeable stretch-activated or mechanosensitive cation channels. Notably, deletion of AQP1 diminishes platelet procoagulant membrane dynamics in vitro and arterial thrombosis in vivo, independent of platelet granule secretion and without affecting hemostasis. Water entry into platelets promotes procoagulant activity, and AQPs may also be critical for the initiation and progression of venous thrombosis. Platelet AQPs may therefore represent valuable targets for future development of a new class of antithrombotics, namely, anti-procoagulant antithrombotics, that are mechanistically distinct from current antithrombotics. However, the structure of AQPs does not make for easy targeting of these channels, hence they remain elusive drug targets. Nevertheless, thrombosis data in animal models provide compelling reasons to continue the pursuit of AQP-targeted antithrombotics. In this review, we discuss the role of aquaporins in platelet secretion, aggregation and procoagulation, the challenge of drugging AQPs, and the prospects of targeting AQPs for arterial and venous antithrombosis

Acute hypoxia stimulates intracellular peroxynitrite formation associated with pulmonary artery smooth muscle cell proliferation

There is separate evidence for peroxynitrite formation and hypoxia-induced cell proliferation in several models of hypoxic pulmonary hypertension. We therefore hypothesized that the stimulation of pulmonary artery smooth muscle cells (PASMCs) proliferation by hypoxia is due to peroxynitrite formation. The effect of hypoxia alone and in combination with <0.2 mu M peroxynitrite on PASMCs was investigated in explants from bovine lungs grown in 1%, 5%, or 10% oxygen for 24 hours with or without peroxynitrite. At 0.1% fetal bovine serum, DNA synthesis of PASMCs (assessed by H-3 thymidine incorporation) was increased by transient exposure to 0.2 mu M peroxynitrite (by 158% +/- 14%, P < 0.01) or to 24 hours of hypoxia (5% oxygen) (by 221% +/- 17%, P < 0.01). Results were similar at 2.5% fetal bovine serum. Treatment of PASMCs with 0.2 mu M peroxynitrite or 5% O-2 hypoxia caused a significant increase in nitrotyrosine formation to a similar extent and intensity. The proliferative response to 0.2 mu M peroxynitrite or to the combination of peroxynitrite plus 5% O-2 was similar to the effect of 5% O-2 alone and was abolished by simultaneous treatment with peroxynitrite scavenger-ebselen (5 mu M). Our present data indicate that hypoxia can initiate peroxynitrite-induced proliferative events and suggest a mechanism for the vascular hypertrophy associated with pulmonary hypertension