Carbon monoxide and nitric oxide mediate cytoskeletal reorganization in microvascular cells via vasodilator-stimulated phosphoprotein phosphorylation: evidence for blunted responsiveness in diabetes.
OBJECTIVE
We examined the effect of the vasoactive agents carbon monoxide (CO) and nitric oxide (NO) : n the phosphorylation and intracellular redistribution of vasodilator-stimulated phosphoprotein (VASP), a critical actin motor protein required for cell migration that also controls vasodilation and platelet aggregation.


METHODS
We examined the effect of donor-released CO and NO in endothelial progenitor cells (EPCs) and platelets from nondiabetic and diabetic subjects and in human microvascular endothelial cells (HMECs) cultured under low (5.5 mmol/l) or high (25 mmol/l) glucose conditions. VASP phosphorylation was evaluated using phosphorylation site-specific antibodies.


RESULTS
In control platelets, CO selectively promotes phosphorylation at VASP Ser-157, whereas NO promotes phosphorylation primarily at Ser-157 and also at Ser-239, with maximal responses at 1 min with both agents on Ser-157 and at 15 min on Ser-239 with NO treatment. In diabetic platelets, neither agent resulted in VASP phosphorylation. In nondiabetic EPCs, NO and CO increased phosphorylation at Ser-239 and Ser-157, respectively, but this response was markedly reduced in diabetic EPCs. In endothelial cells cultured under low glucose conditions, both CO and NO induced phosphorylation at Ser-157 and Ser-239; however, this response was completely lost when cells were cultured under high glucose conditions. In control EPCs and in HMECs exposed to low glucose, VASP was redistributed to filopodia-like structures following CO or NO exposure; however, redistribution was dramatically attenuated under high glucose conditions.


CONCLUSIONS
Vasoactive gases CO and NO promote cytoskeletal changes through site- and cell type-specific VASP phosphorylation, and in diabetes, blunted responses to these agents may lead to reduced vascular repair and tissue perfusion.
Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida, USA.
Sergio
S
Li Calzi
Daniel L
DL
Purich
Kyung Hee
KH
Chang
Aqeela
A
Afzal
Takahiko
T
Nakagawa
Julia V
JV
Busik
Anupam
A
Agarwal
Mark S
MS
Segal
Maria B
MB
Grant
D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism* / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D003925:Diabetic Angiopathies / Q000378:metabolism* / Q000473:pathology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D008833:Microcirculation / Q000166:cytology / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D009569:Nitric Oxide; D002248:Carbon Monoxide