Sathish, Venkatachalem

Neurotrophins (NTs), which play an integral role in directing proper neuronal development and function, have been found in non‐neuronal tissue (including lung), but their role is still under investigation. We previously found NTs such as brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT3) and their receptors to be expressed in human airway smooth muscle (ASM). In this study, we hypothesized that NTs contribute to regulation of ASM function via regulation of mechanisms that control intracellular Ca 2+ ([Ca 2+ ] i ). Human ASM cells isolated from lung samples incidental to patient surgery were incubated for 24 or 48 h in medium (control), 10 nM BDNF or NT3 in the presence or absence of inhibitors of signaling pathways such as MAP kinases (PD98059), PI3/Akt (Wortmannin) and NFκB (SN50). In fluo‐4 loaded cells, [Ca 2+ ] i responses to ACh and histamine were significantly higher following BDNF and NT3 exposure: effects that were blunted by SN50 and PD98059, but not Wortmannin. Western analysis of cell lysates showed higher expressions of Ca 2+ regulatory proteins such as Orai1, TRPC3, and SERCA with 1 or 10nM BDNF exposure, but decreased expression by NT3. These data show that NTs activate signaling pathways in human ASM that lead to enhanced [Ca 2+ ] i responses, and could thus contribute to airway hyperresponsiveness. Supported by NIH grants HL088029, HL090595 and FAMRI.

Smoking contributes to or exacerbates diseases such as asthma and COPD, where airway hyperreactivity is a key feature. Enhanced Ca 2+ in airway smooth muscle contributes to airway hyperreactivity. We previously determined that the cytokine TNFα upregulates expression of Ca 2+ regulatory molecules such as membrane bound bifunctional enzyme CD38 and Ca 2+ influx regulatory TRPC3 in human airway smooth muscle cells (HASM). We hypothesized that cigarette smoke (CS) is an inflammatory stimulus that upregulates Ca 2+ regulatory mechanisms in HASM. Western analysis showed increased expression of CD38, TRPC3 and TRPC6 in lungs from mice following 4 week in vivo CS exposure. These proteins were also upregulated in HASM cells incubated in 1 or 2% CS extract (CSE), as well as in HASM samples from patients with COPD (compared to controls). In fura‐2 loaded HASM cells, exposure to CSE enhanced histamine‐mediated Ca 2+ mobilization as well as store‐operated calcium entry: effects substantially blunted by TRPC3 siRNAs. Finally, in living lung slices of mice exposed for 4 weeks to CS, ACh‐induced airway contractility was substantially greater, compared to controls. These results indicate that CS upregulates expression and function of Ca 2+ regulatory proteins, thus contributing to airway hyperreactivity in smoking related diseases. Supported by the Annenberg Foundation (MEW), HL090595 (CMP), FAMRI (RV, YSP).

Enhanced airway contractility by inflammatory cytokines involves increased intracellular Ca 2+ levels in airway smooth muscle (ASM). In this study, we examined the role of the bidirectional plasma membrane Na + /Ca 2+ exchanger (NCX) in human ASM Ca 2+ regulation following exposure to the pro‐inflammatory cytokines tumor necrosis factor alpha (TNFα) or interleukin‐13 (IL‐13). Both cytokines significantly increased NCX1 expression, with these effects inhibited by dexamethasone (NFκB inhibitor) or PD98059 (MAPK inhibitor). In human ASM cells loaded with the Ca 2+ indicator fura‐2, or the intracellular Na + indicator SBFI, NCX‐mediated Ca 2+ influx (with simultaneous Na + efflux) was enhanced by both cytokines. NCX‐mediated Ca 2+ efflux (with simultaneous Na + influx) was also enhanced, but not to the same extent. The NCX inhibitor KBR7943 as well as NCX1 siRNA inhibited cytokine enhancement of both modes NCX and of Ca 2+ responses to bronchoconstrictor agonists. These data indicate that a) NCX normally operates in human ASM; b) cytokines increase NCX expression, leading to greater NCX‐mediated Ca 2+ influx, thus contributing to enhanced Ca 2+ in inflamed ASM. Supported by NIH grants HL74309 (GCS), HL090595‐S2 (VS) and HL090595 (CMP).

The incidence, severity, and mortality of ongoing coronavirus infectious disease 19 (COVID-19) is greater in men compared with women, but the underlying factors contributing to this sex difference are still being explored. In the current study, using primary isolated human airway smooth muscle (ASM) cells from normal males versus females as a model, we explored the effect of estrogen versus testosterone in modulating the expression of angiotensin converting enzyme 2 (ACE2), a cell entry point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using confocal imaging, we found that ACE2 is expressed in human ASM. Furthermore, Western analysis of ASM cell lysates showed significantly lower ACE2 expression in females compared with males at baseline. In addition, ASM cells exposed to estrogen and testosterone for 24 h showed that testosterone significantly upregulates ACE2 expression in both males and females, whereas estrogen downregulates ACE2, albeit not significant compared with vehicle. These intrinsic and sex steroids induced differences may help explain sex differences in COVID-19.