Imbalance of Pulmonary Microvascular Endothelial Cell-Expression of Metalloproteinases and Their Endogenous Inhibitors Promotes Septic Barrier Dysfunction.

Sepsis is a life-threatening disease characterized by excessive inflammation leading to organ dysfunction. During sepsis, pulmonary microvascular endothelial cells (PMVEC) lose barrier function associated with inter-PMVEC junction disruption. Matrix metalloproteinases (MMP) and a disintegrin and metalloproteinases (ADAM), which are regulated by tissue inhibitors of metalloproteinases (TIMPs), can cleave cell-cell junctional proteins, suggesting a role in PMVEC barrier dysfunction. We hypothesize that septic PMVEC barrier dysfunction is due to a disruption in the balance between PMVEC-specific metalloproteinases and TIMPs leading to increased metalloproteinase activity. The effects of sepsis on TIMPs and metalloproteinases were assessed ex vivo in PMVEC from healthy (sham) and septic (cecal ligation and perforation) mice, as well as in vitro in isolated PMVEC stimulated with cytomix, lipopolysaccharide (LPS), and cytomix + LPS vs. PBS. PMVEC had high basal Timp expression and lower metalloproteinase expression, and septic stimulation shifted expression in favour of metalloproteinases. Septic stimulation increased MMP13 and ADAM17 activity associated with a loss of inter-PMVEC junctional proteins and barrier dysfunction, which was rescued by treatment with metalloproteinase inhibitors. Collectively, our studies support a role for metalloproteinase-TIMP imbalance in septic PMVEC barrier dysfunction, and suggest that inhibition of specific metalloproteinases may be a therapeutic avenue for septic patients.

Zinc binds non-cooperatively to human liver metallothionein 2a at physiological pH.

Maintenance of the homeostasis of zinc is very important in regulating bodily functions. There are over 300 Zn-dependent enzymes identified where Zn(II) plays a structural or catalytic role. However, an excess of Zn(II) in a cell is toxic and free Zn(II) is tightly controlled. Metallothioneins (MTs) are small cysteine rich proteins that can bind up to seven Zn(II) and act as a Zn(II) reservoir. The MT2a isoform is predominantly found in the liver. This study focused on designing an MT2a construct of recombinant human MT2a to determine the Zn(II) binding profile of MT2a in vitro. We analyzed the pH dependence of Zn-MT2a speciation from electrospray ionization mass spectral data. At physiological pH, Zn(II) is terminally bound to the cysteine thiols of MT2a, making bead-like structures (non-cooperative metal binding), while at low pH, Zn(II) formed Zn4S11-MT2a clusters involving bridged cysteinyl thiols to the Zn(II) (cooperative metal binding). The Zn(II) binding profile of MT2a was compared to Zn(II) binding profile of human kidney MT1a, which was reported in literature, and found that the Zn(II) binding profile of MT2a is similar to that of MT1a. The facility of forming bead-like structures at physiological pH for Zn5-MT2a means that Zn7-MT2a can donate up to two Zn(II) to Zn-dependent enzymes.