The cysteine protease dipeptidyl aminopeptidase 3 does not contribute to egress of Plasmodium falciparum from host red blood cells.

The ability of Plasmodium parasites to egress from their host red blood cell is critical for the amplification of these parasites in the blood. Previous forward chemical genetic approaches have implicated the subtilisin-like protease (SUB1) and the cysteine protease dipeptidyl aminopeptidase 3 (DPAP3) as key players in egress, with the final step of SUB1 maturation thought to be due to the activity of DPAP3. In this study, we have utilized a reverse genetics approach to engineer transgenic Plasmodium falciparum parasites in which dpap3 expression can be conditionally regulated using the glmS ribozyme based RNA-degrading system. We show that DPAP3, which is expressed in schizont stages and merozoites and localizes to organelles distinct from the micronemes, rhoptries and dense granules, is not required for the trafficking of apical proteins or processing of SUB1 substrates, nor for parasite maturation and egress from red blood cells. Thus, our findings argue against a role for DPAP3 in parasite egress and indicate that the phenotypes observed with DPAP3 inhibitors are due to off-target effects.

NOX4-derived reactive oxygen species limit fibrosis and inhibit proliferation of vascular smooth muscle cells in diabetic atherosclerosis.

Smooth muscle cell (SMC) proliferation and fibrosis contribute to the development of advanced atherosclerotic lesions. Oxidative stress caused by increased production or unphysiological location of reactive oxygen species (ROS) is a known major pathomechanism. However, in atherosclerosis, in particular under hyperglycaemic/diabetic conditions, the hydrogen peroxide-producing NADPH oxidase type 4 (NOX4) is protective. Here we aim to elucidate the mechanisms underlying this paradoxical atheroprotection of vascular smooth muscle NOX4 under conditions of normo- and hyperglycaemia both in vivo and ex vivo. Following 20-weeks of streptozotocin-induced diabetes, Apoe(-/-) mice showed a reduction in SM-alpha-actin and calponin gene expression with concomitant increases in platelet-derived growth factor (PDGF), osteopontin (OPN) and the extracellular matrix (ECM) protein fibronectin when compared to non-diabetic controls. Genetic deletion of Nox4 (Nox4(-/)(-)Apoe(-/-)) exacerbated diabetes-induced expression of PDGF, OPN, collagen I, and proliferation marker Ki67. Aortic SMCs isolated from NOX4-deficient mice exhibited a dedifferentiated phenotype including loss of contractile gene expression, increased proliferation and ECM production as well as elevated levels of NOX1-associated ROS. Mechanistic studies revealed that elevated PDGF signalling in NOX4-deficient SMCs mediated the loss of calponin and increase in fibronectin, while the upregulation of NOX1 was associated with the increased expression of OPN and markers of proliferation. These findings demonstrate that NOX4 actively regulates SMC pathophysiological responses in diabetic Apoe(-/-) mice and in primary mouse SMCs through the activities of PDGF and NOX1.

Differential effects of NOX4 and NOX1 on immune cell-mediated inflammation in the aortic sinus of diabetic ApoE-/- mice.

Oxidative stress and inflammation are central mediators of atherosclerosis particularly in the context of diabetes. The potential interactions between the major producers of vascular reactive oxygen species (ROS), NADPH oxidase (NOX) enzymes and immune-inflammatory processes remain to be fully elucidated. In the present study we investigated the roles of the NADPH oxidase subunit isoforms, NOX4 and NOX1, in immune cell activation and recruitment to the aortic sinus atherosclerotic plaque in diabetic ApoE(-/-) mice. Plaque area analysis showed that NOX4- and NOX1-derived ROS contribute to atherosclerosis in the aortic sinus following 10 weeks of diabetes. Immunohistochemical staining of the plaques revealed that NOX4-derived ROS regulate T-cell recruitment. In addition, NOX4-deficient mice showed a reduction in activated CD4(+) T-cells in the draining lymph nodes of the aortic sinus coupled with reduced pro-inflammatory gene expression in the aortic sinus. Conversely, NOX1-derived ROS appeared to play a more important role in macrophage accumulation. These findings demonstrate distinct roles for NOX4 and NOX1 in immune-inflammatory responses that drive atherosclerosis in the aortic sinus of diabetic mice.

Reactive Oxygen Species Can Provide Atheroprotection via NOX4-Dependent Inhibition of Inflammation and Vascular Remodeling.

OBJECTIVE: Oxidative stress is considered a hallmark of atherosclerosis. In particular, the superoxide-generating type 1 NADPH oxidase (NOX1) has been shown to be induced and play a pivotal role in early phases of mouse models of atherosclerosis and in the context of diabetes mellitus. Here, we investigated the role of the most abundant type 4 isoform (NOX4) in human and mouse advanced atherosclerosis. APPROACH AND RESULTS: Plaques of patients with cardiovascular events or established diabetes mellitus showed a surprising reduction in expression of the most abundant but hydrogen peroxide (H2O2)-generating type 4 isoform (Nox4), whereas Nox1 mRNA was elevated, when compared with respective controls. As these data suggested that NOX4-derived reactive oxygen species may convey a surprisingly protective effect during plaque progression, we examined a mouse model of accelerated and advanced diabetic atherosclerosis, the streptozotocin-treated ApoE(-/-) mouse, with (NOX4(-/-)) and without genetic deletion of Nox4. Similar to the human data, advanced versus early plaques of wild-type mice showed reduced Nox4 mRNA expression. Consistent with a rather protective role of NOX4-derived reactive oxygen species, NOX4(-/-) mice showed increased atherosclerosis when compared with wild-type mice. Deleting NOX4 was associated with reduced H2O2 forming activity and attenuation of the proinflammatory markers, monocyte chemotratic protein-1, interleukin-1ß, and tumor necrosis factor-α, as well as vascular macrophage accumulation. Furthermore, there was a greater accumulation of fibrillar collagen fibres within the vascular wall and plaque in diabetic Nox4(-/-)ApoE(-/-) mice, indicative of plaque remodeling. These data could be replicated in human aortic endothelial cells in vitro, where Nox4 overexpression increased H2O2 and reduced the expression of pro-oxidants and profibrotic markers. Interestingly, Nox4 levels inversely correlated with Nox2 gene and protein levels. Although NOX2 is not constitutively active unlike NOX4 and forms rather superoxide, this opens up the possibility that at least some effects of NOX4 deletion are mediated by NOX2 activation. CONCLUSIONS: Thus, the appearance of reactive oxygen species in atherosclerosis is apparently not always a nondesirable oxidative stress, but can also have protective effects. Both in humans and in mouse, the H2O2-forming NOX4, unlike the superoxide-forming NOX1, can act as a negative modulator of inflammation and remodeling and convey atheroprotection. These results have implications on how to judge reactive oxygen species formation in cardiovascular disease and need to be considered in the development of NOX inhibitory drugs.