Levels associate with severely decreased eNOS activity, resulting in vascular dysfunction; especially that these mice also demonstrate vascular inflammation [42,43,44]. In an in vitro model of IH in HMVEC eNOS mRNA levels were decreased, suggesting that even short-term exposure to IH causes changes similar to those described earlier in OSA patients and OSA in vitro model [6,45,46]. However, eNOS mRNA levels did not Title Loaded From File change in HCAEC following exposure to IH, indicating that EC from distinct vascular beds respond differently to the same hypoxic insult. In addition to its impact on eNOS, IH, a critical component of OSA, promotes oxidative stress within the vasculature, causing vascular and systemic inflammation that culminates in vascular remodeling and atherosclerosis. Several 24195657 studies reported increased levels of proinflammatory molecules in OSA patients [7,47,48,49,50,51]. We confirmed that the systemic inflammatory response associated with OSA was also Title Loaded From File observed in severely hypoxemic patients’ skin biopsies, as evaluated by increased mRNA levels of VCAM-1. Similarly, we noted some increase in VCAM-1 mRNA levels in aortas of mice exposed to IH compared to mice placed under IA, and in HCAEC exposed to IH compared to a normoxic control. Beyond supporting existing data [52,53], these results validate our mouse and cell culture models of OSA, as they demonstrate the expected inflammatory response to hypoxic insult. Moreover, we analyzed expression levels of the NF-kBdependent and NF-kB inhibitory protein A20 [25,33,54,55]. We have previously shown that A20 exerts protective, anti-inflammatory and anti-apoptotic functions in EC [33,55,56]. Our data show that A20 mRNA was significantly increased in skin biopsies of severely hypoxemic compared to mildly hypoxemic OSA patients, which indicates that the inflammatory insult associated with mild hypoxemia is not sufficient to upregulate A20 transcription. A20 mRNA levels were also increased in our in vitro models of OSA. Elevation of A20 in response to IH reveals the presence of an inflammatory milieu associated with chronic OSA, and is in agreement with observed upregulation of other NF-kB-dependent genes, such as VCAM-1. Alternatively, upregulation of A20 could result from hypoxia-induced increase in A20 transcription throughactivation of a hypoxia-response element (A/(G)CGTG) recently identified in the A20 promoter in glioblastoma cell-lines [57]. We also analyzed the expression of HIF-1a, a transcriptional regulator of oxygen homeostasis, and its downstream target VEGF [58,59,60], in skin biopsies of OSA patients, and in mouse aortas and EC cultures exposed to IH. Both HIF-1a and VEGF mRNA levels were higher in skin of severely hypoxemic OSA patients compared to mildly hypoxemic group. VEGF expression was also upregulated in aortas of mice exposed to IH compared to their respective controls. These findings indicate that in those tissues only a significant hypoxic insult exerts a response to hypoxia. The different effects of IH on HIF-1a mRNA levels in HMVEC and HCAEC further highlight heterogeneity among EC originating from different vascular beds, mainly in terms of their susceptibility to IH. While many studies support the hypothesis that IH upregulates HIF-1a, some reports show no impact of OSA on HIF-1a expression [61]. Although we have not confirmed that HIF-1a mRNA levels translate into protein, we have indirect evidence that HIF-1a protein levels likely parallel mRNA levels [62]. VEGF prom.Levels associate with severely decreased eNOS activity, resulting in vascular dysfunction; especially that these mice also demonstrate vascular inflammation [42,43,44]. In an in vitro model of IH in HMVEC eNOS mRNA levels were decreased, suggesting that even short-term exposure to IH causes changes similar to those described earlier in OSA patients and OSA in vitro model [6,45,46]. However, eNOS mRNA levels did not change in HCAEC following exposure to IH, indicating that EC from distinct vascular beds respond differently to the same hypoxic insult. In addition to its impact on eNOS, IH, a critical component of OSA, promotes oxidative stress within the vasculature, causing vascular and systemic inflammation that culminates in vascular remodeling and atherosclerosis. Several 24195657 studies reported increased levels of proinflammatory molecules in OSA patients [7,47,48,49,50,51]. We confirmed that the systemic inflammatory response associated with OSA was also observed in severely hypoxemic patients’ skin biopsies, as evaluated by increased mRNA levels of VCAM-1. Similarly, we noted some increase in VCAM-1 mRNA levels in aortas of mice exposed to IH compared to mice placed under IA, and in HCAEC exposed to IH compared to a normoxic control. Beyond supporting existing data [52,53], these results validate our mouse and cell culture models of OSA, as they demonstrate the expected inflammatory response to hypoxic insult. Moreover, we analyzed expression levels of the NF-kBdependent and NF-kB inhibitory protein A20 [25,33,54,55]. We have previously shown that A20 exerts protective, anti-inflammatory and anti-apoptotic functions in EC [33,55,56]. Our data show that A20 mRNA was significantly increased in skin biopsies of severely hypoxemic compared to mildly hypoxemic OSA patients, which indicates that the inflammatory insult associated with mild hypoxemia is not sufficient to upregulate A20 transcription. A20 mRNA levels were also increased in our in vitro models of OSA. Elevation of A20 in response to IH reveals the presence of an inflammatory milieu associated with chronic OSA, and is in agreement with observed upregulation of other NF-kB-dependent genes, such as VCAM-1. Alternatively, upregulation of A20 could result from hypoxia-induced increase in A20 transcription throughactivation of a hypoxia-response element (A/(G)CGTG) recently identified in the A20 promoter in glioblastoma cell-lines [57]. We also analyzed the expression of HIF-1a, a transcriptional regulator of oxygen homeostasis, and its downstream target VEGF [58,59,60], in skin biopsies of OSA patients, and in mouse aortas and EC cultures exposed to IH. Both HIF-1a and VEGF mRNA levels were higher in skin of severely hypoxemic OSA patients compared to mildly hypoxemic group. VEGF expression was also upregulated in aortas of mice exposed to IH compared to their respective controls. These findings indicate that in those tissues only a significant hypoxic insult exerts a response to hypoxia. The different effects of IH on HIF-1a mRNA levels in HMVEC and HCAEC further highlight heterogeneity among EC originating from different vascular beds, mainly in terms of their susceptibility to IH. While many studies support the hypothesis that IH upregulates HIF-1a, some reports show no impact of OSA on HIF-1a expression [61]. Although we have not confirmed that HIF-1a mRNA levels translate into protein, we have indirect evidence that HIF-1a protein levels likely parallel mRNA levels [62]. VEGF prom.
