Modulation of the effect of vascular endothelial growth factor on endothelial cells by heparin: critical role of nitric oxide-mediated mechanisms.
BACKGROUND
In spite of intensive research, the actual role of heparin in endothelial cell (EC) biology remains incompletely understood. In particular, further insight is needed into the interaction of heparin with the potent heparin-binding angiogenic factor, vascular endothelial growth factor (VEGF). This study aimed to examine the effect of heparin on VEGF-mediated EC responses.


METHODS
Confluent bovine aorta EC were treated with high (HMWH) and low molecular weight heparin (LMWH). 3H-Thymidine (3H-Thy) uptake, flow cytometry, 51Cr-release, nitrites accumulation, and cytosolic free Ca2+ ([Ca2+]i), endothelial nitric oxide synthase (eNOS) mRNA expression and tissue factor (TF) concentration were measured.


RESULTS
HMWH and LMWH blocked VEGF proliferative actions and blunted VEGF-induced [Ca2+]i transients. However, the heparins did not block the VEGF protective effects on EC. These changes occurred in parallel with a potentiation of the VEGF-related NO production by both heparins. The Akt/PI3K inhibitor, LY 294002, blocked this potentiation, related to increased eNOS activity rather than eNOS expression. Connecting both effects, the NO antagonist, L-NAME, shifted the protective effects of VEGF to a cytotoxic mode.


CONCLUSIONS
HMWH and LMWH block the proliferative and [Ca2+]i-mobilizing effects of VEGF on EC, by a NO-dependent mechanism. On the contrary, VEGF-induced NO production is stimulated. The Akt/PI3K pathway at least in part mediates this effect. By changing the way the VEGF intracellular signaling is driven, heparin could act as a stabilizing factor for the endothelium, without stimulating vessel proliferation.
Yagüe
Susana
S
Medical Research Institute, Fundacion Jimenez Diaz, Universidad Autonoma, Instituto Reina Sofia de Investigacion Renal, Madrid, Spain.
Alvarez Arroyo
Victoria
V
Castilla
Angeles
A
González Pacheco
Francisco R
FR
Llamas
Pilar
P
Caramelo
Carlos
C
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D001011:Aorta / Q000166:cytology; D015152:Blotting, Northern; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006493:Heparin / Q000494:pharmacology*; D066298:In Vitro Techniques; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D012333:RNA, Messenger / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000187:drug effects / Q000378:metabolism*
D000925:Anticoagulants; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D006493:Heparin; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019331:NG-Nitroarginine Methyl Ester