Endothelial autophagy deficiency induces IL6 - dependent endothelial mesenchymal transition and organ fibrosis.

Macroautophagy/autophagy plays a vital role in the homeostasis of diverse cell types. Vascular endothelial cells contribute to vascular health and play a unique role in vascular biology. Here, we demonstrated that autophagy defects in endothelial cells induced IL6 (interleukin 6)-dependent endothelial-to-mesenchymal transition (EndMT) and organ fibrosis with metabolic defects in mice. Inhibition of autophagy, either by a specific inhibitor or small interfering RNA (siRNA) for ATG5 (autophagy related 5), in human microvascular endothelial cells (HMVECs) induced EndMT. The IL6 level was significantly higher in ATG5 siRNA-transfected HMVECs culture medium compared with the control HMVECs culture medium, and neutralization of IL6 by a specific antibody completely inhibited EndMT in ATG5 siRNA-transfected HMVECs. Similar to the in vitro data, endothelial-specific atg5 knockout mice (Atg5 Endo; Cdh5-Cre Atg5 flox/flox mice) displayed both EndMT-associated kidney and heart fibrosis when compared to littermate controls. The plasma level of IL6 was higher in Atg5 Endo compared to that of control mice, and fibrosis was accelerated in Atg5 Endo treated with a HFD; neutralization of IL6 by a specific antibody inhibited EndMT and fibrosis in HFD-fed Atg5 Endo associated with the amelioration of metabolic defects. These results revealed the essential role of autophagy in endothelial cell integrity and revealed that the disruption of endothelial autophagy could lead to significant pathological IL6-dependent EndMT and organ fibrosis. Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; EndMT: endothelial-to-mesenchymal transition; HES: hematoxylin and eosin stain; HFD: high-fat diet; HMVECs: human microvascular endothelial cells; IFNG: interferon gamma; IL6: interleukin 6; MTS: Masson's trichrome staining; NFD: normal-fat diet; siRNA: small interfering RNA; SMAD3: SMAD family member 3; TGFB: transforming growth factor ß; TNF: tumor necrosis factor; VEGFA: vascular endothelial growth factor A.

TM5441, a plasminogen activator inhibitor-1 inhibitor, protects against high fat diet-induced non-alcoholic fatty liver disease.

Recent evidences showed that elevation of plasminogen activator inhibitor 1 (PAI-1) was responsible in mediating obesity-induced non-alcoholic fatty liver disease (NAFLD) and metabolic disorders. Here, we investigated the effect of TM5441, an oral PAI-1 inhibitor that lacks of bleeding risk, on high-fat diet (HFD)-induced NAFLD. HFD-fed C57BL/6J mice was daily treated with 20 mg/kg TM5441. To examine the preventive effect, 10-week-treatment was started along with initiation of HFD; alternatively, 4-week-treatment was started in mice with glucose intolerance in the interventional strategy. In vivo study showed that early and delayed treatment decreased hepatic steatosis. Particularly, early treatment prevented the progression of hepatic inflammation and fibrosis in HFD mice. Interestingly, both strategies abrogated hepatic insulin resistance and mitochondrial dysfunction, presented by enhanced p-Akt and p-GSK3ß, reduced p-JNK signaling, along with p-AMPK and PGC-1α activation. Consistently, TM5441 treatment in the presence of either PAI-1 exposure or TNF-α stimulated-PAI-1 activity showed a restoration of mitochondrial biogenesis related genes expression on HepG2 cells. Thus, improvement of insulin sensitivity and mitochondrial function was imperative to partially explain the therapeutic effects of TM5441, a novel agent targeting HFD-induced NAFLD.

A very-low-protein diet ameliorates advanced diabetic nephropathy through autophagy induction by suppression of the mTORC1 pathway in Wistar fatty rats, an animal model of type 2 diabetes and obesity.

AIMS/HYPOTHESIS: The efficacy of a low-protein diet (LPD) on diabetic nephropathy is controversial. We aimed to investigate the renoprotective effects of an LPD and the underlying molecular mechanism in a rat model of type 2 diabetes and obesity. METHODS: Diabetic male Wistar fatty (fa/fa) rats (WFRs) were treated with a standard diet (23.84% protein) or an LPD (5.77% protein) for 20 weeks from 24 weeks of age. We investigated the effect of the LPD on renal function, fibrosis, tubular cell damage, inflammation, mitochondrial morphology of proximal tubular cells (PTCs), apoptosis, autophagy and activation of mammalian target of rapamycin complex 1 (mTORC1). RESULTS: Kidney weight, albuminuria, excretion of urinary liver-type fatty acid binding protein, levels of plasma cystatin C and changes in renal histology, including fibrosis, tubular cell damage and inflammation, were aggravated in WFRs compared with non-diabetic Wistar lean rats (WLRs). Fragmented and swelling mitochondria accumulated in PTCs and apoptosis were enhanced in the kidney of WFRs. Immunohistochemical staining of p62 and p-S6 ribosomal protein (p-S6RP) in the tubular lesions of WFRs was increased compared with WLRs. The LPD intervention clearly ameliorated damage as shown by the assessment of renal function and histology, particularly tubulointerstitial damage in diabetic kidneys. Additionally, the 5.77% LPD, but not the 11.46% LPD, significantly suppressed p-S6RP levels and increased microtubule-associated protein light chain 3-II levels in the renal cortex. The LPD intervention partially decreased HbA1c levels in WFRs, and no differences in mean BP were observed among any of the groups. CONCLUSIONS/INTERPRETATION: A very-low-protein diet improved advanced diabetic renal injuries, including tubulointerstitial damage, by restoring autophagy through the suppression of the mTORC1 pathway.