An indirect co-culture study was performed over 14 days to test diffASC and mvEC behavior in the media in a more physiological co-culture and to elucidate a possible influence of the cell types on each other through soluble factors.
Dermal microvascular ECs (mvECs) were isolated from adult human skin (Klinik Charlottenhaus, Stuttgart) as described previously (Volz et al., 2017).
MvECs were seeded at 3.5 x [10.sup.3] cells/[cm.sup.2] in 24-well plates and expanded for three additional days in EGM-2mv.
In sepsis, multiple organ dysfunction, including ARDS, is presumed due to systemic inflammatory injury of the microvasculature, especially the MVEC [5-9].
Specifically, in ARDS, there are similar although more limited data to support pulmonary microvascular and MVEC injury and dysfunction.
This septic microvascular and MVEC dysfunction is especially characterized by impaired barrier function, with the septic hyperpermeability resulting in protein-rich tissue edema and PMN influx into organs.
To test this in MVEC, cells were treated with the caspase-8-specific inhibitor zIETD-fmk along with the PARP-1 inhibitor 3-ABA and exposed to TRAIL at pH 7.4 (Figures 3(a) and 3(c)) and pH 6.7 (Figures 3(b) and 3(c)).
To test the contribution of this executioner protein in TRAIL-induced necroptosis under acidic conditions, MLKL was silenced in MVEC using siRNA as confirmed by PCR and Western blot analyses (Figures 5(a) and 5(b)).
In contrast, our study shows that MVEC readily underwent classical necroptosis at pH 7.4 as well as "acidonecrosis" at pH 6-6.7 after TRAIL treatment (Figure 2).
EP-4 was almost undetectable in VSMC in culture in terms of mRNA (78-fold lower than in MVEC, not shown).
Our present results show that the expression levels of mPGES-1 correlate with the expression of vascular cell markers rather than with infiltrating macrophages markers suggesting that mPGES-1 is expressed in VSMC and MVEC as we reported before [17, 18, 26].
Taken together, our results support the concept that smoking substantially affects aortic vascular cells and suggest that MVEC are particularly affected.