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Hyperosmolarity caused by high levels of glucose and mannitol induces angiogenesis by activating the Tonicity-responsive cis-acting elements, aquaporin 1- and Na+/H+ exchanger 1 and COX-2 and MMPs in human endothelial cells

Cardiovascular Pharmacology and Pharmacotherapy


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Madonna
  Presenter
Presenter | see Discussant report Play presentation webcast
Madonna, Rosalinda
(U.S.A.)
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List of Authors: 

R. Madonna, E. Montebello, Yong-Jian Geng and R. De Caterina

Abstract:


Background and Objective:In overt diabetes, persistent hyperglycemia increases the plasma osmolarity, leading to adaptive cellular responses, such as expression of heat-shock proteins and proinflammatory factors, including cycloxygenase-2 (COX-2), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). As a key enzyme in prostaglandin metabolism and angiogenesis, COX-2 plays a role in regulation of plaque stability, and often it co-localizes with matrix metalloproteinases (MMP)-2 and -9.
This study aimed to test the hypothesis that hyperosmotic stress promotes angiogenesis through activation of COX-2 and MMP, which orchestrates endothelial sprouting and migration.

Methods and Results:Human aortic endothelial cells (HAEC) and dermal microvascular endothelial cells (HMVEC) were incubated with 5.5 mmol/L glucose (normoglycemia, basal), high glucose (HG) at 12.5, 25 and 45 mmol/L, or with the hyperosmolar controls mannitol (HM) at 12.5, 25 and 45 mmol/L, or equimolar concentrations of sodium chloride. Both HG and HM increased expression of COX-2 (HG 2.5±0.03 fold; HM 1.8±0.05 fold, p<0.01) and MMP proteins (HG 1.8±0.01 fold; HM 1.6±0.03; n=5, P<0.01) and also induced MMP gelatinolytic activities as shown by zymography. At electrophoretic mobility shift assay, treatment with HG and HM (or sodium chloride) for 1 hour, but not equimolar concentrations of saccharose or tumor necrosis factor (TNF)-α (10 ng/mL), increased the binding of Tonicity enhancer binding protein (TonEBP) to the putative Tonicity enhancer element located in the 5’-flanking region of the COX-2 and MMP genes. This was associated with a time-dependent increase in NFkB DNA-binding activity in cells treated with HG (1 and 3 hours) and TNF-α (up to 24 h). At any time points mannitol, saccharose and sodium chloride did not activate NFκB. HG and HM also induced endothelial migration determined by fluorimetric assay (HG 1.3±0.02 fold; HM 1.1±0.01 fold; n=3, P<0.05) and tubulization in Matrigel (HG 3.4±0.2 fold; HM 4±0.5 fold; n=3, p<0.01) (Figure 1). Upstream and downstream targeting of the osmosignaling pathway by gene silencing with small interfering RNA to osmosensing water channels aquaporin-1 (AQP1) and sodium/hydrogen exchanger-1 (NHE-1), and to the osmoinducible transcription factor TonEBP reverted the inducing effects of HG- and HM on expression of COX-2 and MMPs and angiogenic activities

Abstract Madonna - Fig 1

Upstream and downstream targeting of the osmosignaling pathway by gene silencing with small interfering RNA to osmosensing water channels aquaporin-1 (AQP1) and sodium/hydrogen exchanger-1 (NHE-1), and to the osmoinducible transcription factor TonEBP reverted the inducing effects of HG- and HM on expression of COX-2 and MMPs and angiogenic activities.

Conclusion: By activating the tonicity-responsive cis-acting elements binding to TonEBP and the water channels AQP1 and NHE-1, the hyperosmolarity caused by HG or HM induced COX-2 expression, MMP zymoactivity and angiogenesis in human endothelial cells. Targeting osmosignaling pathways may represent a novel strategy to reduce inflammatory angiogenesis in diabetes and related vascular complications, such as plaque rupture and micro-vascular dysfunction.

Abstract Madonna Fig2


 

References


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The content of this article reflects the personal opinion of the author/s and is not necessarily the official position of the European Society of Cardiology.