The protein tyrosine phosphatase PTP-PEST mediates hypoxia-induced endothelial autophagy and angiogenesis via AMPK activation.

Global and endothelial loss of PTP-PEST (also known as PTPN12) is associated with impaired cardiovascular development and embryonic lethality. Although hypoxia is implicated in vascular remodelling and angiogenesis, its effect on PTP-PEST remains unexplored. Here we report that hypoxia (1% oxygen) increases protein levels and catalytic activity of PTP-PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry revealed that α subunits of AMPK (α1 and α2, encoded by PRKAA1 and PRKAA2, respectively) interact with PTP-PEST under normoxia but not in hypoxia. Co-immunoprecipitation experiments confirmed this observation and determined that AMPK α subunits interact with the catalytic domain of PTP-PEST. Knockdown of PTP-PEST abrogated hypoxia-mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). Absence of PTP-PEST also blocked hypoxia-induced autophagy (LC3 degradation and puncta formation), which was rescued by the AMPK activator metformin (500 µM). Because endothelial autophagy is a prerequisite for angiogenesis, knockdown of PTP-PEST also attenuated endothelial cell migration and capillary tube formation, with autophagy inducer rapamycin (200 nM) rescuing angiogenesis. In conclusion, this work identifies for the first time that PTP-PEST is a regulator of hypoxia-induced AMPK activation and endothelial autophagy to promote angiogenesis.

Hyperinsulinemia promotes endothelial inflammation via increased expression and release of Angiopoietin-2.

BACKGROUND AND AIMS: Angiopoietin-2 (ANG-2) mediates endothelial inflammation to initiate atherosclerosis and angiogenesis. Here we determined the serum levels of ANG-2 in hyperinsulinemic subjects and whether insulin increases its expression and release. METHODS: Healthy male subjects were recruited from the D-CLIP and CURES studies and, based on their fasting insulin levels, were classified as normoinsulinemic (n = 228) and hyperinsulinemic (n = 32). Serum proteins were estimated by ELISA. Endothelial inflammation was scored as the number of THP-1 monocytes adhered to HUVEC monolayer. Gene expression was determined with promoter reporter assays and semi-quantitative RT-PCR. Western blotting was used to assess changes in protein expression and activation. Immunofluorescence imaging and ChIP assay were used for nuclear localization and promoter binding studies, respectively. RESULTS: ANG-2 and sTIE2 levels were higher in hyperinsulinemic subjects. Hyperinsulinemic serum elicited endothelial inflammation, which was abrogated by an ANG-2 blocker antibody. Insulin (100 nM) increased mRNA and protein expression of ANG-2, and its release from HUVECs. It induced activation of p38 MAPK and an increase in protein levels and nuclear localization of cFOS. Binding of cFOS to the -640 to -494 promoter region mediated insulin dependent ANG-2 transcription. p38 MAPK inhibitor (25 µM) blocked insulin-induced nuclear localization of cFOS, expression of ANG-2 and ICAM-1, and release of ANG-2 into the culture medium. Spent medium collected from insulin treated cells enhanced endothelial inflammation, which was lost upon ANG-2 knockdown as well as upon p38 MAPK inhibition. CONCLUSIONS: ANG-2 levels are high in hyperinsulinemic subjects and insulin induces expression and release of ANG-2 from HUVECs through p38 MAPK-cFOS pathway to elicit endothelial inflammation.

Amarogentin, a secoiridoid glycoside, activates AMP- activated protein kinase (AMPK) to exert beneficial vasculo-metabolic effects.

INTRODUCTION: AMP-activated protein kinase (AMPK) is a drug target for treatment of metabolic and cardiovascular complications. Extracts of Gentianaceace plants exhibit anti-diabetic and anti-atherosclerotic effects, however, whether their phyto-constitutents activate AMPK remains to be determined. METHODS: Molecular docking of Gentiana lutea constituents was performed with crystal structure of human α2ß1γ1 trimeric AMPK (PDB ID: 4CFE). Binding of Amarogentin (AG) to α2 subunit was confirmed through isothermal titration calorimetry (ITC) and in vitro kinase assays were performed. L6 myotube, HUH7 and endothelial cell cultures were employed to validate in silico and in vitro observations. Lipid lowering and anti-atherosclerotic effects were confirmed in streptozotocin induced diabetic mice via biochemical measurements and through heamatoxylin and eosin, Masson's trichrome and Oil Red O staining. RESULTS: AG interacts with the α2 subunit of AMPK and activates the trimeric kinase with an EC50 value of 277 pM. In cell culture experiments, AG induced phosphorylation of AMPK as well as its downstream targets, acetyl-coA-carboxylase (ACC) and endothelial nitric oxide synthase (eNOS). Additionally, it enhanced glucose uptake in myotubes and blocked TNF-α induced endothelial inflammation. Oral supplementation of AG significantly attenuated diabetes-mediated neointimal thickening, and collagen and lipid deposition in the aorta. It also improved circulating levels of lipids and liver function in diabetic mice. CONCLUSION: In conclusion, AG exerts beneficial vasculo-metabolic effects by activating AMPK. GENERAL SIGNIFICANCE: Amarogentin, a naturally occurring secoiridoid glycoside, is a promising lead for design and synthesis of novel drugs for treatment and management of dyslipidemia and cardiovascular diseases.

Increased endothelial inflammation, sTie-2 and arginase activity in umbilical cords obtained from gestational diabetic mothers.

OBJECTIVE: The aim of this study was to determine subclinical inflammation in umbilical vein derived endothelial cells (HUVECs) obtained from Asian Indian subjects with gestational diabetes (GDM) and to determine levels of angiogenic factors and arginase activity in their cord blood. METHODS: This case-control study included 38 control and 30 GDM subjects. Subjects were confirmed as GDM based on 75g oral glucose tolerance test (OGTT) conducted in the second trimester of pregnancy. Angiogenic markers and arginase activity were measured in cord blood by ELISA and colorimetric methods respectively. Endothelial inflammation was assessed through adhesion of PKH26-labelled leukocytes onto HUVEC monolayer obtained from the study groups. Gene and surface expression of adhesion molecules were confirmed via reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry respectively. RESULTS: The study revealed increased adhesion of leukocytes to HUVECs isolated from GDM subjects compared to controls. HUVECs of babies born to GDM mothers had increased surface and mRNA expression of E-selectin. sTie2 levels were significantly higher in the cord blood for GDM subjects (3869 ± 370 ng/L) compared to controls (3045 ± 296 ng/L). Furthermore, arginase activity was higher in cord blood of GDM mothers as opposed to the control group (7.75 ± 2.4 µmoles of urea/ml/hour vs 2.88 ±0.49 µmoles of urea/ml/hour; p-value= 0.019). Spearman's correlation analysis revealed positive correlation of cord blood arginase activity with glucose intolerance (ρ=0.596, p=0.004) and post load glucose values (ρ=0.472, p=0.031) of mothers observed during the second trimester of pregnancy. CONCLUSIONS: HUVECs derived from Asian Indian GDM mothers, exhibit signs of sub-clinical endothelial inflammation along with increased levels of sTie2 and arginase activity in their cord blood serum.