Forestalling age-impaired angiogenesis and blood flow by targeting NOX: Interplay of NOX1, IL-6, and SASP in propagating cell senescence.

In an aging population, intense interest has shifted toward prolonging health span. Mounting evidence suggests that cellular reactive species are propagators of cell damage, inflammation, and cellular senescence. Thus, such species have emerged as putative provocateurs and targets for senolysis, and a clearer understanding of their molecular origin and regulation is of paramount importance. In an inquiry into signaling triggered by aging and proxy instigator, hyperglycemia, we show that NADPH Oxidase (NOX) drives cell DNA damage and alters nuclear envelope integrity, inflammation, tissue dysfunction, and cellular senescence in mice and humans with similar causality. Most notably, selective NOX1 inhibition rescues age-impaired blood flow and angiogenesis, vasodilation, and the endothelial cell wound response. Indeed, NOX1i delivery in vivo completely reversed age-impaired hind-limb blood flow and angiogenesis while disrupting a NOX1-IL-6 senescence-associated secretory phenotype (SASP) proinflammatory signaling loop. Relevant to its comorbidity with age, clinical samples from diabetic versus nondiabetic subjects reveal as operant this NOX1-mediated vascular senescence and inflammation in humans. On a mechanistic level, our findings support a previously unidentified role for IL-6 in this feedforward inflammatory loop and peroxisome proliferator-activated receptor gamma (PPARγ) down-regulation as inversely modulating p65-mediated NOX1 transcription. Targeting this previously unidentified NOX1-SASP signaling axis in aging is predicted to be an effective strategy for mitigating senescence in the vasculature and other organ systems.

Notch2 suppression mimicking changes in human pulmonary hypertension modulates Notch1 and promotes endothelial cell proliferation.

Pulmonary arterial hypertension (PAH) is a fatal cardiopulmonary disease characterized by increased vascular cell proliferation with apoptosis resistance and occlusive remodeling of the small pulmonary arteries. The Notch family of proteins subserves proximal signaling of an evolutionarily conserved pathway that effects cell proliferation, fate determination, and development. In endothelial cells (ECs), Notch receptor 2 (Notch2) was shown to promote endothelial apoptosis. However, a pro- or antiproliferative role for Notch2 in pulmonary endothelial proliferation and ensuing PAH is unknown. We postulated that suppressed Notch2 signaling drives pulmonary endothelial proliferation in the context of PAH. We observed that levels of Notch2 are ablated in lungs from PAH subjects compared with non-PAH controls. Notch2 expression was attenuated in human pulmonary artery endothelial cells (hPAECs) exposed to vasoactive stimuli including hypoxia, TGF-ß, ET-1, and IGF-1. Notch2-deficient hPAECs activated Akt, Erk1/2, and antiapoptotic protein Bcl-2 and reduced levels of p21cip and Bax associated with increased EC proliferation and reduced apoptosis. In addition, Notch2 suppression elicited a paradoxical activation of Notch1 and canonical Notch target gene Hes1, Hey1, and Hey2 transcription. Furthermore, reduction in Rb and increased E2F1 binding to the Notch1 promoter appear to explain the Notch1 upregulation. Yet, when Notch1 was decreased in Notch2-suppressed cells, the wound injury response was augmented. In aggregate, our results demonstrate that loss of Notch2 in hPAECs derepresses Notch1 and elicits EC hallmarks of PAH. Augmented EC proliferation upon Notch1 knockdown points to a context-dependent role for Notch1 and 2 in endothelial cell homeostasis.NEW & NOTEWORTHY This study demonstrates a previously unidentified role for Notch2 in the maintenance of lung vascular endothelial cell quiescence and pulmonary artery hypertension (PAH). A key novel finding is that Notch2 suppression activates Notch1 via Rb-E2F1-mediated signaling and induces proliferation and apoptosis resistance in human pulmonary artery endothelial cells. Notably, PAH patients show reduced levels of endothelial Notch2 in their pulmonary arteries, supporting Notch2 as a fundamental driver of PAH pathogenesis.

The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

Senescent cells withdraw from the cell cycle and do not proliferate. The prevalence of senescent compared to normally functioning parenchymal cells increases with age, impairing tissue and organ homeostasis. A contentious principle governing this process has been the redox theory of aging. We linked matricellular protein thrombospondin 1 (TSP1) and its receptor CD47 to the activation of NADPH oxidase 1 (Nox1), but not of the other closely related Nox isoforms, and associated oxidative stress, and to senescence in human cells and aged tissue. In human endothelial cells, TSP1 promoted senescence and attenuated cell cycle progression and proliferation. At the molecular level, TSP1 increased Nox1-dependent generation of reactive oxygen species (ROS), leading to the increased abundance of the transcription factor p53. p53 mediated a DNA damage response that led to senescence through Rb and p21cip, both of which inhibit cell cycle progression. Nox1 inhibition blocked the ability of TSP1 to increase p53 nuclear localization and p21cip abundance and its ability to promote senescence. Mice lacking TSP1 showed decreases in ROS production, p21cip expression, p53 activity, and aging-induced senescence. Conversely, lung tissue from aging humans displayed increases in the abundance of vascular TSP1, Nox1, p53, and p21cip Finally, genetic ablation or pharmacological blockade of Nox1 in human endothelial cells attenuated TSP1-mediated ROS generation, restored cell cycle progression, and protected against senescence. Together, our results provide insights into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence and suggest potential targets for controlling the aging process at the molecular level.

Selective recapitulation of conserved and nonconserved regions of putative NOXA1 protein activation domain confers isoform-specific inhibition of Nox1 oxidase and attenuation of endothelial cell migration.

Excessive vascular and colon epithelial reactive oxygen species production by NADPH oxidase isoform 1 (Nox1) has been implicated in a number of disease states, including hypertension, atherosclerosis, and neoplasia. A peptide that mimics a putative activation domain of the Nox1 activator subunit NOXA1 (NOXA1 docking sequence, also known as NoxA1ds) potently inhibited Nox1-derived superoxide anion (O2·-) production in a reconstituted Nox1 cell-free system, with no effect on Nox2-, Nox4-, Nox5-, or xanthine oxidase-derived reactive oxygen species production as measured by cytochrome c reduction, Amplex Red fluorescence, and electron paramagnetic resonance. The ability of NoxA1ds to cross the plasma membrane was tested by confocal microscopy in a human colon cancer cell line exclusively expressing Nox1 (HT-29) using FITC-labeled NoxA1ds. NoxA1ds significantly inhibited whole HT-29 carcinoma cell-derived O2·- generation. ELISA and fluorescence recovery after photobleaching experiments indicate that NoxA1ds, but not its scrambled control, binds Nox1. FRET experiments conducted using Nox1-YFP and NOXA1-CFP illustrate that NoxA1ds disrupts the binding interaction between Nox1 and NOXA1, whereas a control peptide did not. Moreover, hypoxia-induced human pulmonary artery endothelial cell O2·- production was completely inhibited by NoxA1ds. Human pulmonary artery endothelial cell migration under hypoxic conditions was also reduced by pretreatment with NoxA1ds. Our data indicate that a peptide recapitulating a putative activation subdomain of NOXA1 (NoxA1ds) is a highly efficacious and selective inhibitor of Nox1 activity and establishes a critical interaction site for Nox1-NOXA1 binding required for enzyme activation.

Endothelial cell protein C receptor opposes mesothelioma growth driven by tissue factor.

The procoagulant protein tissue factor (F3) is a powerful growth promoter in many tumors, but its mechanism of action is not well understood. More generally, it is unknown whether hemostatic factors expressed on tumor cells influence tissue factor-mediated effects on cancer progression. In this study, we investigated the influence of tissue factor, endothelial cell protein C receptor (EPCR, PROCR), and protease activated receptor-1 (PAR1, F2R) on the growth of malignant pleural mesothelioma (MPM), using human MPM cells that lack or express tissue factor, EPCR or PAR1, and an orthotopic nude mouse model of MPM. Intrapleural administration of MPM cells expressing tissue factor and PAR1 but lacking EPCR and PAR2 (F2RL1) generated large tumors in the pleural cavity. Suppression of tissue factor or PAR1 expression in these cells markedly reduced tumor growth. In contrast, tissue factor overexpression in nonaggressive MPM cells that expressed EPCR and PAR1 with minimal levels of tissue factor did not increase their limited tumorigenicity. More importantly, ectopic expression of EPCR in aggressive MPM cells attenuated their growth potential, whereas EPCR silencing in nonaggressive MPM cells engineered to overexpress tissue factor increased their tumorigenicity. Immunohistochemical analyses revealed that EPCR expression in tumor cells reduced tumor cell proliferation and enhanced apoptosis. Overall, our results enlighten the mechanism by which tissue factor promotes tumor growth through PAR1, and they show how EPCR can attenuate the growth of tissue factor-expressing tumor cells.

Phylogeography of mitochondrial DNA and Y-chromosome haplogroups reveal asymmetric gene flow in populations of Eastern India.

Polymorphisms in mitochondrial (mt) DNA and Y-chromosomes of seven socially and linguistically diverse castes and tribes of Eastern India were examined to determine their genetic relationships, their origin, and the influence of demographic factors on population structure. Samples from the Orissa Brahmin, Karan, Khandayat, Gope, Juang, Saora, and Paroja were analyzed for mtDNA hypervariable sequence (HVS) I and II, eight Y-chromosome short tandem repeats (Y-STRs), and lineage-defining mutations diagnostic for Indian- and Eurasian-specific haplogroups. Our results reveal that haplotype diversity and mean pairwise differences (MPD) was higher in caste groups of the region (>0.998, for both systems) compared to tribes (0.917-0.996 for Y-STRs, and 0.958-0.988 for mtDNA haplotypes). The majority of paternal lineages belong to the R1a1, O2a, and H haplogroups (62.7%), while 73.2% of maternal lineages comprise the Indian-specific M*, M5, M30, and R* mtDNA haplogroups, with a sporadic occurrence of West Eurasian lineages. Our study reveals that Orissa Brahmins (a higher caste population) have a genetic affinity with Indo-European speakers of Eastern Europe, although the Y-chromosome data show that the genetic distances of populations are not correlated to their position in the caste hierarchy. The high frequency of the O2a haplogroup and absence of East Asian-specific mtDNA lineages in the Juang and Saora suggest that a migration of Austro-Asiatic tribes to mainland India was exclusively male-mediated which occurred during the demographic expansion of Neolithic farmers in southern China. The phylogeographic analysis of mtDNA and Y-chromosomes revealed varied ancestral sources for the diverse genetic components of the populations of Eastern India.