Effect of inorganic polyphosphate on microvascular endothelial permeability and its mechanism: focus on AMP kinase signaling pathway

Background and Aim : The present study integrates systems and molecular biology approaches to investigate the regulatory role of AMPK activator, metformin, on polyP-induced hyper-permeability in different organs in three different models including local, systemic short- and systemic long-term approaches in mice. Methods : PolyP-induced hyper-permeability was performed according to three different models including local (50-200 µg polyP/Mouse) administered intra-dermally, systemic short-term (4-400 µg polyP/Mouse injected in the intraperitoneal cavity, and systemic long-term (4 and 40 µg polyP/Mouse/day ip) approaches in mice. The protocol used for each group were evaluate vascular permeability in different groups, the Evans Blue (EB) dye (20mg/ml) was directly injected into the tail vein of anesthetized mice intravenously (iv) and tissues were isolated and maintained in formamide solution. PolyP-triggered leakage of organs’ vessels was measured based on flux of EB dye-bound albumin and monitored by spectrophotometer at 620 nm. The harvested tissues were fixed and stained with haematoxylin and eosin or Trichrome. The oxidative stress was evaluated via measuring the level of MDA, total thiol, catalase and superoxide dismutase (SOD) activities. Evaluation of the enzymatic activity of matrix metalloproteinases (MMP-2 and -9) was performed via gelatin zymography. Protein levels of IFN-γ and TNFα were quantified using a commercially available ELISA kit.Results : Our results showed that polyP disrupts endothelial barrier integrity in skin, liver, kidney, brain, heart, and lung in all three study models which are all abrogated in the presence of metformin. We also showed that activation of AMPK signaling pathway, regulation of oxidant/anti-oxidant balance, suppression of NF-κB signaling, down regulation of tissue necrosis factor alpha (TNF-α) as well as decrease in inflammatory cell infiltration are molecular mechanisms by which metformin elicits it’s protective responses against polyP-induced hyper-permeability.Conclusion : These results support the clinical value of AMPK activators including a FDA-approved drug metformin, which is widely utilized for type 2 diabetes in attenuating vascular damage in polyP-associated inflammatory diseases.

Keywords:

polyP-induced hyper-permeability , AMPK , polyP , metformin