P588Diabetes alters bone marrow vascular niche by Akt impairment and increased vascular permeability throughout Src activation

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Abstract

Background

Systemic microvascular complications are frequent and life threatening in patients with diabetes. We demonstrated tha tmicroangiopathy endangers stem cells (SCs) of the bone marrow (BM) endosteal niche, by altering the perfusion gradient across the marrow and determining profound environmental changes that disturb haematopoiesis. We also described distinct alterations in BM endothelial cells (ECs) in vivo and ex vivo, which might lead to altered SC trafficking. New evidence indicates that BMECs supportSC homeostasis by angiocrine mechanisms involving the Akt signaling pathway, but whether this signaling is altered by diabetes remains unknown.

Objective

To investigate new molecular mechanisms implicatedin BM endothelial dysfunction in diabetes.

Methods and Results

Using whole-genome Illumina gene arrays, we found that primary BMECs from Type-1 diabetic (T1D) mice differ from non-diabetic BMECs with respect to gene clusters that control cell cycle, migration, adhesion, oxidative stress, and glucose metabolism. In particular, among the Akt regulated genes, FGF2, Jag1, Jag2, and Dll4 were downregulated, whereas Ang2 was upregulated. Moreover, among migration/adhesion related genes, SDF-1, adherens and tight junctions components were downregulated, whereas ICAM-1, VCAM-1, E and P selectins were upregulated. Using an Akt activity assay, we showed that diabetes leads to Akt inactivation in T1D BMECs (P < 0.01 vs.controls). To investigate the molecular and functional mechanisms of vascular permeability, we studied immortalized human BMECs (hBMECs) in a trans-well insert permeability assay. We found that moderate increases in glucose are sufficient to induce endothelial permeability, which was inhibited by the ROS scavenger N-acetyl-cysteine and the Src inhibitor SU6656. By Western Blot, we confirmed that high glucose triggers the phosphorylation of Pyk2, a Src kinase co-actor, and VE-cadherin (Y658 andY731 sites), implicated in disassembly of adherens junctions. An in vivo permeability assay was performed on tibiae and femura by systemically injected fluorescent-labelled dextrans. T1D mice (14 weeks) showed an increase in BM vascular permeability (1.42 fold in femura and 1.31 fold in tibiae, both P < 0.01). This effect was preserved by continuous insulin supplementation (P < 0.01).

Conclusion

Here, we newly demonstrate that two important mechanisms involved in endothelial-dependent maintenance of SC homeostasis (Akt) and trafficking (Src/Pyk2/Ve-cadherin) are altered in diabetes. We also show that metabolic control is crucial to preserve BMvascular barrier function in diabetes.

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