High Glucose Treatment Limits Drosha Protein Expression and Alters AngiomiR Maturation in Microvascular Primary Endothelial Cells via an Mdm2-dependent Mechanism.

Brian Lam,Emmanuel Nwadozi,Emilie Roudier,Olivier Birot,Tara L Haas

doi:10.3390/cells10040742

Abstract

Diabetes promotes an angiostatic phenotype in the microvascular endothelium of skeletal muscle and skin. Angiogenesis-related microRNAs (angiomiRs) regulate angiogenesis through the translational repression of pro- and anti-angiogenic genes. The maturation of micro-RNA (miRs), including angiomiRs, requires the action of DROSHA and DICER proteins. While hyperglycemia modifies the expression of angiomiRs, it is unknown whether high glucose conditions alter the maturation process of angiomiRs in dermal and skeletal muscle microvascular endothelial cells (MECs). Compared to 5 mM of glucose, high glucose condition (30 mM, 6–24 h) decreased DROSHA protein expression, without changing DROSHA mRNA, DICER mRNA, or DICER protein in primary dermal MECs. Despite DROSHA decreasing, high glucose enhanced the maturation and expression of one angiomiR, miR-15a, and downregulated an miR-15a target: Vascular Endothelial Growth Factor-A (VEGF-A). The high glucose condition increased Murine Double Minute-2 (MDM2) expression and MDM2-binding to DROSHA. Inhibition of MDM2 prevented the effects evoked by high glucose on DROSHA protein and miR-15a maturation in dermal MECs. In db/db mice, blood glucose was negatively correlated with the expression of skeletal muscle DROSHA protein, and high glucose decreased DROSHA protein in skeletal muscle MECs. Altogether, our results suggest that high glucose reduces DROSHA protein and enhances the maturation of the angiostatic miR-15a through a mechanism that requires MDM2 activity.

Highlights

Angiogenesis is a dynamic process through which endothelial cells proliferate, migrate and assemble to form new and mature capillaries
High glucose treatment significantly decreased DROSHA protein expression by 31% compared to normal glucose control conditions (5 mM, p = 0.0004, Figure 1A)
High glucose treat6 of 20 ment significantly decreased DROSHA protein expression by 31% compared to normal glucose control conditions (5 mM, p = 0.0004, Figure 1A)

Summary

Introduction

Angiogenesis is a dynamic process through which endothelial cells proliferate, migrate and assemble to form new and mature capillaries. MiR-126 can exert a pro-angiogenic effect by targeting the Sprouty-related protein SPRED-1, a negative regulator of VEGF-A signaling [10,12]. By downregulating SPRED-1 expression, miR-126 helps microvascular endothelial cells (MECs) sense VEGF-A signal and facilitates angiogenesis [12]. The polycistronic miR-17–92 cluster encodes for 7 mature miRs (miR17-3p, miR-17-5p, miR-18a, miR-19a, miR-20a, miR-19b and miR-92a-1) that regulate the expression of extracellular matrix proteins [13,14] In this cluster, miR-18a and miR-19a repress the expression of the angiostatic THBS-1 and enhance the angiogenic potential of microvascular endothelial cells [14,15]. MiR-15a can repress the expression of VEGF-A, its receptor VEGF-R2 and the endothelial-specific receptor tyrosine kinase Tie 2 [16,17], promoting angiostasis in MECs [18]

Methods

Results

Discussion

Conclusion