Fig 1: Nicotine vapor inhalation increases ACE2 mRNA and protein expression in male, but not female, lung. (a-b) After five consecutive days of vapor exposure, lung tissue was analyzed for the expression of ACE2 mRNA. (a) Increased ACE2 mRNA expression was found in lung tissue of male mice exposed to nicotine vapor, as compared to control (no vapor exposure) and vehicle vapor exposure. (b) In females, no significant differences were found in ACE2 mRNA expression among exposure conditions. (c-d) Lung tissue was also examined for ACE2 protein expression. (c) Quantification of the number of ACE2-positive cells in lung tissue revealed that nicotine vapor significantly increased ACE2 protein expression in male mice, as compared to the control and vehicle vapor groups. (d) The number of ACE2-positive cells did not differ among groups in female lung tissue. Individual data points are represented on each graph, and graph bars express as the mean ± SEM. ##p < 0.01 vs vehicle vapor, ###p < 0.001 vs vehicle, **p < 0.01 vs control, ***p < 0.001 vs control. (e-j) Representative images of ACE2 immunoreactivity (red) in lung tissue following exposure. In males, high levels of ACE2 immunoreactivity can be evidenced in lungs exposed to nicotine vapor (i), as compared to control (e) and vehicle vapor (g). For females, representative images demonstrate similar levels of lung ACE2 protein expression for the control (f), vehicle vapor (h) and nicotine vapor (j) groups. DAPI: Blue. Scale bar =50 µm.
Fig 2: Acute and chronic administration of DIZE prevents the onset of cognitive impairment in young pre-symptomatic Tg2576 mice. a Associative recognition memory was assessed in younger WT (n = 20) and Tg2576 mice (9–12 months of age) that received either acute (10 days; n = 17) or chronic (10 weeks n = 11) DIZE administration. Tg2576 vehicle mice (n = 16) showed an age-dependent impairment in OiP performance (^^^p < 0.001), which was not observed in either the acute or chronic DIZE-administered Tg2576 mice (ps > 0.05). Tg2576 vehicle mice were also impaired compared to WT vehicle (***p < 0.001), Tg2576 acute (•••p < 0.001) and Tg2576 chronic DIZE administered mice (+++p < 0.001). b–d Aß levels were quantified by ELISA. Soluble Aß40 level was reduced in both chronic (*p < 0.05) and acute DIZE-administered Tg2576 mice (**p < 0.01) compared to Tg2576 vehicle mice. Soluble Aß43 was reduced in Tg2576 mice that received acute DIZE administration (*p < 0.05) compared to Tg2576 vehicle mice. e–g ACE2 and ACE1 activities were determined by enzyme activity assays. ACE2 activity was significantly increased in the hippocampus of Tg2576 mice administered DIZE either acutely (**p < 0.01) or chronically (p < 0.05) compared to WT vehicle mice. Despite a numerical reduction in ACE1 activity in DIZE administered mice, no significant changes were reported, p > 0.05; however, the ratio of ACE2:ACE1 was significantly increased in Tg-chronic mice compared to WT vehicle (*p < 0.05). h IL-1ß level was increased in Tg2576 vehicle mice compared to WT vehicle controls (***p < 0.001) but was significantly reduced after acute (*p < 0.05) and chronic (**p < 0.01) DIZE administration in Tg2576 mice compared to Tg vehicle mice. Behavioural data were analysed using mixed measures ANOVA. Significant interactions were further analysed by tests for simple main effects with Bonferroni corrections for multiple comparisons. ACE2 and IL-1ß analyses were performed using one-way ANOVA with post hoc Tukey analysis. Amyloid analysis was performed using non-parametric Kruskal–Wallis one-way ANOVA with multiple comparisons. Error bars represent the SEM
Fig 3: DIZE-mediated restoration of the recognition associative memory deficit in Tg2576 mice is mediated by enhanced ACE2 and is associated with reduced soluble Aß42. a Associative recognition memory was impaired in Tg vehicle mice (n = 12) compared to WT vehicle mice (n = 21) (***p < 0.001) and Tg2576 DIZE-administered mice (n = 13) (^^^p < 0.001). DIZE improved the cognitive performance of Tg2576 mice compared to pre-DIZE administration performance (•••p < 0.001). Co-administration of DIZE + C16 in Tg2576 mice (n = 11) prevented the improvement compared to pre-administration performance (p > 0.05). DIZE + C16 mice were also impaired compared to WT vehicle (***p < 0.001) and Tg2576 DIZE mice (+++p < 0.001). b Hippocampal ACE2 activity was increased in Tg2576 DIZE-administered mice (n = 22) compared to WT vehicle (n = 33) (**p < 0.01) and Tg2576 vehicle mice (n = 23) (###p < 0.001; n = 23). Although numerically reduced, no difference was reported in ACE2 activity in Tg2576 mice co-administered DIZE + C16 (p > 0.05; n = 11). There was no significant difference in ACE1 activity across groups but the ratio of ACE2:ACE1 was significantly elevated in Tg2576 DIZE mice compared to Tg2576 vehicle mice (p < 0.01). These changes were not apparent in Tg2576 mice co-administered DIZE and C16, p > 0.05. c Soluble and insoluble levels of Aß40, Aß42 and Aß43, were measured by ELISA. Soluble Aß42 was significantly lower in Tg2576 DIZE mice compared to mice co-administered DIZE + C16 (*p < 0.05). Insoluble Aß42 and 43 was significantly reduced in both Tg2576 DIZE mice and Tg2576 mice co-administered DIZE + C16. Insoluble Aß40 was unchanged between Tg-DIZE and Tg-DIZE + C16 mice. Behavioural data were analysed using mixed measures ANOVA. Significant interactions were further analysed by tests for simple main effects with Bonferroni corrections for multiple comparisons. ACE2 and Aß analysis was performed using one-way ANOVA with post hoc Tukey analysis. Error bars represent the SEM
Fig 4: Proposed mechanisms for increased risk for severe COVID-19 cardiorenal outcomes in normal women and women with PCOS. Top panels: Women with PCOS have increased body weight, fat mass, and cardiac and renal hypertrophy. Lower panels: Androgens differentially regulate the cardiac and renal expression of SARS-CoV-2 viral entry proteins including the SARS-CoV-2 receptor ACE2 and several cellular proteases that promote viral entry and propagation. HFD exacerbates androgens-mediated obesity and cardiorenal hypertrophic response and modulates androgens’ effect on the expression of the different cellular proteases. The red stars indicates the parameters that are exacerbated with HFD.
Fig 5: Correlations between renal androgen receptor mRNA expression and SARS-CoV-2 viral entry proteins. Animals were treated with dihydrotestosterone (DHT) or vehicle (Veh) and maintained in low (LFD) or high (HFD) fat diet for 90 days. Androgen receptor and SARS-CoV-2 viral entry proteins renal mRNA expression was quantified by RT-qPCR. Pearson’s correlation coefficients (r) were calculated between androgen receptor and Ace2 (A), Tmprss2 (B), Tmprss4 (C), furin (D), cathepsin L (E), and ADAM17 (F) mRNA expression.
Supplier Page from Abcam for Mouse ACE2 ELISA Kit