Vascular EC-SOD limits the accumulation, proinflammatory profibrotic reprogramming, and hyaluronan binding of  interstitial macrophages in hypoxia
Dysregulated redox signaling contributes to pulmonary hypertension (PH) and vascular-selective depletion of the redox enzyme EC-SOD (EC-SOD SMC KO) worsens chronic hypoxic PH. Given the important role of macrophages in vascular remodeling and PH, this study aimed to determine if interstitial macrophages (IMs) and their interactions with the extracellular matrix (ECM) component, hyaluronan, are modulated by vascular EC-SOD. Floxed wild-type (WT), EC-SOD SMC KO, and EC-SOD mimetic- or vehicle-treated mice were exposed to hypobaric hypoxia (~10% FiO2), for 4, 14, or 21 days. Using flow cytometry, we demonstrated that the transient increase in IMs at day 4 was exacerbated in EC-SOD SMC KO mice and prevented with EC-SOD mimetic (5 mg/kg, subcutaneous, day 1). Highlighting the importance of targeting vascular oxidative stress in the early response to hypoxia, pretreatment with this single dose of EC-SOD mimetic decreased right ventricular systolic pressure, right ventricular hypertrophy, and small vessel muscularization at day 21. To assess IM phenotypic reprogramming in this model, RNAseq was performed on flow-sorted IMs revealing baseline proinflammatory activation and enhanced activation of vascular and ECM remodeling pathways in response to hypoxia in EC-SOD SMC KO IMs compared to controls. To further investigate the ECM remodeling response, we quantified IMs expressing the hyaluronan receptor Lyve1, and IM-hyaluronan binding. Lyve1+IMs and Lyve1+HA+IMs were increased in response to hypoxia in EC-SOD SMC KO mice and accumulated in the perivascular space. In conclusion, vascular EC-SOD limits IM accumulation and proinflammatory profibrotic IM signaling, including perivascular accumulation of Lyve1+IMs and their binding to hyaluronan. Overall design: To investigate the effect of low vascular EC-SOD expression on pulmonary interstitial macrophage (IM) transcriptional responses to hypoxia, we exposed EC-SODloxp/loxp x Tgcre/SMMHC  and EC-SODloxp/loxp control mice to 4 days (peak inflammation) or 14 (resolution) days of hypobaric hypoxia. IMs were isolated from mouse lungs by FACS and RNAseq performed on each population. 4 male mice were pooled for each sample and n=3-4 samples analyzed per group.