Observations by Kozieł et al. reported in this issue of the Biochemical Journal suggest the existence of novel regulatory processes associated with new evidence for increased Nox4 (NAPDH oxidase 4) regulation of mitochondrial function in a cultured endothelial cell aging-induced senescence model. Cellular aging appears to promote a Nox4 interaction with mitochondria that disrupts complex I in the electron transport chain and increases the detection of mitochondrial H2O2. Nox4 appears to maintain a highly interconnected mitochondrial network, which may influence mitochondrial fission and/or fusion mechanisms in a manner that could be a contributing factor in the loss of replicative lifespan seen in senescence.
A study reported in this issue of the Biochemical Journal by Kozieł et al.  defines several novel roles for increased Nox4 (NADPH oxidase 4) regulation of mitochondrial function in a cultured endothelial cell aging-induced senescence model. It is shown that Nox4 depletion by shRNA (short hairpin RNA) in this endothelial senescence model induced loss of the highly interconnected mitochondrial network seen in the presence of Nox4. However, with aging, Nox4 depletion stabilized mitochondrial membrane potential, and it decreased the detection of mitochondrial H2O2. Data from high-resolution respirometry in permeabilized cells from this model indicate that Nox4 appears to cause an inhibition of mitochondrial electron transport in complex I, and this was associated with decreased expression of complex I subunits. Previous studies by this group demonstrated that shRNA depletion of Nox4, the major NADPH oxidase in endothelium, caused a shift from the senescent phenotype to one with a significant extension of the replicative lifespan . Whereas the development of senescence did not appear to be associated with increased Nox4 expression, it was associated with oxidative DNA and protein damage . Thus the new information from the study of Kozieł et al.  provides evidence for the development with aging of novel interactions between Nox4 and mitochondria that appear to promote increased mitochondrial peroxide generation and senescence in a manner which supports both free radical and mitochondrial dysfunction theories on aging .
There is substantial evidence for increased Nox4 expression in multiple cardiovascular diseases . Nox4 overexpression and mitochondrial dysfunction have been observed in some aging-associated disease processes such as pressure overload-induced cardiac hypertrophy . It was demonstrated in a cardiac overexpressing Nox4 mouse model, that Nox4 promoted increased mitochondrial superoxide associated with cardiac hypertrophy . In addition, it has been shown that Nox4 has a mitochondrial localization signal, and deletion of this localization signal has been shown to prevent Nox4 from co-localizing with mitochondria in the perinuclear region of cardiac myocytes . Thus, although evidence exists for multiple cellular localizations for Nox4 [3,4], there is rather strong evidence that it can have a mitochondrial localization [4,5].
Nox4 activity is controlled primarily through changes in expression of Nox4, and the primary reactive oxygen species generated by Nox4 appears to be H2O2 . This oxidase is linked to regulating processes in the vasculature that extend from oxygen sensing under basal conditions, which can be associated with the regulation of vascular function , to processes contributing to growth and remodelling as Nox4 activity increases . For example, Nox4 regulates endothelial progenitor cell function and related stem cell-associated remodelling and repair processes. Signalling systems in close proximity to Nox4 are likely to be regulated by this oxidase under baseline physiological conditions. Under pathophysiological conditions, Nox4 can contribute to senescence and apoptotic cell death . Although it is logical to suggest that a more generalized level of cellular oxidant stress participates in these processes, the study of Kozieł et al.  provides evidence that a localized generation of H2O2, which appears to be related to co-localization of Nox4 with mitochondria, may be a major factor in promoting mitochondrial dysfunction associated with endothelial cell senescence.
The observed inhibition of the function and expression of complex I in the mitochondrial electron transport chain by Nox4 seems to be a disruptive metabolic process that would be consistent with endothelial cell senescence. However, observations suggesting that maintaining Nox4 activity in the cultured endothelial cell aging model studied appear to function through promoting a highly interconnected mitochondrial network raises interesting questions about relationships between the status of this network and mitochondrial function. Although mitochondrial fragmentation was initially associated with pathophysiological processes, recent studies with mice deficient in proteins controlling mitochondrial fission such as mitofusins suggest that the efficiency of mitochondrial energy metabolism to support physiological function may not be closely associated with the extent of mitochondrial fission or fusion . Since evidence is emerging that mitochondrial fission is needed for the distribution of mitochondria and the progression of cell division associated with cardiovascular cell proliferation , perhaps the loss of this process is a factor in the senescence associated with increased Nox4. It is likely that interesting and important signalling mechanisms controlled by Nox4 interactions with mitochondria influence the regulation of mitochondrial fusion or fission-associated fragmentation of mitochondria.
Observations in the study by Kozieł et al.  raise additional questions, including the possibility of processes controlling an increased Nox4 interaction with or localization to mitochondria, which could be important in other pathophysiological aspects of aging. On the basis of the effects of depletion of Nox4, increased Nox4 activity is a key regulator of the disruption of complex I and the detection of increased mitochondria-derived H2O2. As discussed by Kozieł et al. , Nox4 is found in multiple cellular localizations in addition to mitochondria, such as the endoplasmic reticulum and nuclear regions. One could hypothesize that there might be processes associated with cellular aging that promote increased mitochondrial Nox4. Increased mitochondrial generation of reactive oxygen species has been viewed as a fundamental process associated with aging and many diseases related to aging. Although there is evidence for important roles for increased mitochondrial Nox4 in cardiac myocyte hypertrophy , the scientific literature has very little information regarding how important Nox4 is in the many pathophysiological conditions that are known to be associated with oxidant-related mitochondrial dysfunction. For example, pulmonary hypertension is associated with increased pulmonary arterial smooth muscle Nox4 expression , oxidant-associated mitochondrial dysfunction and changes in the mitochondrial network structure . Interestingly, increased endothelial cell Nox4 expression and superoxide production appear to contribute to the impaired angiogenesis seen in fetal pulmonary arteries with in utero pulmonary hypertension . Relationships between mitochondria and Nox4 may also exist in the oxygen-sensing field, where there is much controversy related to the importance of both of these systems . Thus observations reported in the study of Kozieł et al.  on the influence of Nox4 in an endothelial cell aging model of senescence appear to raise possibilities of new regulatory mechanisms that remain to be defined.
My laboratory is funded by the National Institutes of Health [grant number HL043023].