Ascorbate protects against vascular leakage in cecal ligation and puncture-induced septic peritonitis

Abstract

Vascular leakage in multiple organs is a characteristic pathological change in sepsis. Our recent study revealed that ascorbate protects endothelial barrier function in microvascular endothelial cell monolayers through inhibiting serine/threonine protein phosphatase 2A (PP2A) activation (Han M, Pendem S, Teh SL, Sukumaran DK, Wu F, Wilson JX. Free Radic Biol Med 48: 128-135, 2010). The present study addressed the mechanism of protection by ascorbate against vascular leakage in cecal ligation and puncture (CLP)-induced septic peritonitis in mice. CLP caused NADPH oxidase activation and endothelial nitric oxide synthase (eNOS) uncoupling to produce superoxide, increased NO production by inducible NOS (iNOS) and neuronal NOS (nNOS) activity, and elevated 3-nitrotyrosine (a product of peroxynitrite) formation and PP2A activity in the hindlimb skeletal muscles at 12 h after CLP. The increase in PP2A activity was associated with decreased levels of phosphorylated serine and threonine in occludin, which was immunoprecipitated from freshly harvested endothelial cells of the septic skeletal muscles. Moreover, CLP increased the vascular permeability to fluorescent dextran and Evans blue dye in skeletal muscles. An intravenous bolus injection of ascorbate (200 mg/kg body wt), given 30 min prior to CLP, prevented eNOS uncoupling, attenuated the increases in iNOS and nNOS activity, decreased 3-nitrotyrosine formation and PP2A activity, preserved the phosphorylation state of occludin, and completely inhibited the vascular leakage of dextran and Evans blue. A delayed ascorbate injection, given 3 h after CLP, also prevented the vascular permeability increase. We conclude that ascorbate injection protects against vascular leakage in sepsis by sequentially inhibiting excessive production of NO and superoxide, formation of peroxynitrite, PP2A activation, and occludin dephosphorylation. Our study provides a scientific basis for injection of ascorbate as an adjunct treatment for vascular leakage in sepsis.

Evaluation of Vitamin C for Adjuvant Sepsis Therapy

“…The increased production of peroxynitrite may also result from increased superoxide synthesis by uncoupled iNOS. Taken together, these findings support using cultures of microvascular endothelial cells to study in vitro the mechanisms underlying septic microvascular dysfunction, because CLP similarly raises Nox1, iNOS, peroxynitrite, protein nitration, and plasma protein extravasation levels in vivo (40,64,68).…”

“…Further, ascorbate injection blocks the increase by CLP of iNOS mRNA expression in microvascular endothelial cells at 3 h after CLP, apparently due to inhibition by ascorbate of NADPH oxidase activity (63,65). Parenteral ascorbate also blocks the increases in total NOS, iNOS, and neuronal NOS (nNOS) activities in skeletal muscle of CLP mice (68) (Fig. 3).…”

“…of ascorbate (200 mg/kg), given 30 min prior to CLP, prevents eNOS uncoupling, iNOS and nNOS activation, peroxynitrite formation, PP2A activation, occludin dephosphorylation, and dextran (70 kDa) and plasma protein extravasation at 12 h post-CLP. A delayed ascorbate injection, given 3 h after CLP, also attenuates the endothelial barrier dysfunction (68).…”

“…CLP causes NADPH oxidase activation and endothelial NOS (eNOS) uncoupling that produce superoxide, and CLP also induces activations of iNOS and nNOS that generate NO, in skeletal muscles (68). The superoxide and NO combine to form peroxynitrite, which then nitrates multiple proteins (Fig.…”

“…Immunoblot analysis of freshly harvested endothelial cells of the septic skeletal muscles reveals dephosphorylation of serine and threonine in occludin (68), similar to the observation made in microvascular endothelial cell cultures exposed to LPS + IFNc (24). Moreover, the systemic inflammatory response of mice to CLP is associated with a very reproducible extravasation of fluorescent dextran (average molecular mass 70 kDa) in cremaster muscle and Evans blue-conjugated albumin in hindlimb skeletal muscles within 12 h, at which time survival is 100% (64,68) (Fig. 6).…”