Continuously-infused human C-reactive protein is neither proatherosclerotic nor proinflammatory in apolipoprotein E-deficient mice.

Studies of human native C-reactive protein (nCRP) in mice have shown effects ranging from proatherogenic, to antiatherogenic, to no effect. It is likely that these disparities are related to (a) the use, in some studies, of contaminated nCRP, or to (b) variation in CRP levels associated with either its episodic administration or the use of CRP-transgenic mice. In our study, 12-week-old male apolipoprotein E-deficient (apoE (-/-)) mice, maintained on a Western diet, received azide- and endotoxin-free nCRP (n = 23) or placebo (n = 23) continuously via osmotic pumps (20.4 microg/day) for 4 weeks. CRP-treated and control mice developed similar atherosclerotic lesions in whole aortas (nCRP: 10.4 +/- 4.7% vs. controls: 11.7 +/- 4.4%, P = 0.76) and aortic roots (nCRP: 65.0 +/- 7.8% vs. controls: 64.7 +/- 9.7%, P = 0.94). No differences were observed in macrophage or T-lymphocyte infiltrates and there was no meaningful change in VCAM-1 or IL-6 expression, in the levels of soluble VCAM-1, or in circulating proinflammatory (IL-1 beta, IL-6, IL-12p40, IL-12p70, TNF-alpha, and INF-gamma), or anti-inflammatory (IL-4 and IL-10) cytokines. We conclude that continuous infusion of uncontaminated nCRP in apoE (-/-) mice is not associated with increased atherosclerosis, does not alter systemic or local inflammation, and does not affect endothelial activation. These observations suggest that alternative approaches to study CRP (perhaps using different pentraxins in the mouse model or using a rabbit model instead of a mouse model) are needed to evaluate the effects of pentraxins on atherosclerosis.

Conduct unbecoming: C-reactive protein interactions with a broad range of protein molecules.

BACKGROUND: C-reactive protein (CRP), a pentamer composed of five identical 23-kd subunits, is a member of a highly conserved family of proteins known as pentraxins. CRP has been recognized as a risk factor for the development of both the native and transplant-associated forms of atherosclerosis. Understanding the biology of CRP may be relevant to understanding atherosclerosis development and progression. METHODS: Using Western-blotting techniques, we examined the interactions between native, monomeric and mutationally and chemically modified CRP and a variety of antibodies, monoclonal and polyclonal. RESULTS: CRP in its denatured monomeric form, but not in its native pentameric conformation, associates promiscuously with IgG molecules, including normal human IgG, as well as with a number of other proteins. This behavior is intrinsic to CRP and is not noted with other pentraxins such as serum amyloid P component or the long pentraxin, PTX3. Monomeric CRP co-localizes with vitronectin in human heart tissue sections. CONCLUSIONS: We present these findings as cautionary advice, to indicate that characterization of monomeric CRP can be complicated by the propensity of the molecule to interact with a variety of immunoglobulins and other proteins. We also suggest that it is possible that such interactions could serve to eliminate excess of monomeric CRP and/or to scavenge altered, damaged and denatured proteins. These reactivities may be part of a regulatory mechanism to limit inflammation in the arterial wall.

SU1498, an inhibitor of vascular endothelial growth factor receptor 2, causes accumulation of phosphorylated ERK kinases and inhibits their activity in vivo and in vitro.

SU1498, an inhibitor of vascular endothelial growth factor receptor 2, has been used successfully to study the physiological manifestations of receptor functions. Here we report that in addition to its anti-receptor activity, SU1498 stimulates accumulation of phosphorylated ERKs in human umbilical vein endothelial cells and in human aortic endothelial cells in a manner that is dependent on the functioning of the upstream components of the MAPK pathway, B-Raf, and MEK kinases. The enhanced accumulation of phospho-ERKs is observed only in cells that have been stimulated with sphingosine 1-phosphate or protein growth factors; SU1498 by itself is ineffective. We show that the inhibitor acts by blocking the kinase activity of phospho-ERK both in a direct assay and in immunoprecipitates from cells treated with the compound. The data reveal a novel and unique way in which MAPK signaling pathway may be blocked in human endothelial cells.

Migration of vascular smooth muscle cells induced by sphingosine 1-phosphate and related lipids: potential role in the angiogenic response.

The bioactive lipids sphingosine 1-phosphate (SPP), sphingosylphosphorylcholine, and lysophosphatidic acid play an important role in angiogenesis as a result of their effects on both the migration of endothelial cells (ECs) and the integrity of EC monolayers. Here we show that extremely low concentrations of serum and nanomolar concentrations of these biologically active lipids stimulate migration of human aortic smooth muscle cells (SMCs). However, at dosages most effective in promoting EC migration and in enhancing EC monolayer integrity, serum and SPP potently inhibited SMC migration; SPP also blocked the migration induced by protein growth factors. Treatment of SMCs with SPP induced transient phosphorylation of a 175- to 185-kDa protein corresponding to the PDGF receptor, indicating transactivation of this receptor. SPP and related lipids may play a key role in angiogenesis by coordinating the migration of both endothelial cells and vascular smooth muscle cells in response to the changing gradients of these bioactive lipid messengers.

Safety Evaluation of Glucose Isomerase Derived from Flavobacterium arborescens and Used in Production of High Fructose Corn Syrup.

Flavobacterium arborescens is a common rod-shaped, gram-negative bacterium which, when cultivated in a nutrient medium, is an efficient source of glucose isomerase (GI). GI is then used in the production of high fructose corn syrup. Studies were conducted to assure product safety and establish GRAS status for GI derived from F. arborescens . A viable cell suspension of F. arborescens and the cell-free medium in which the organism was cultured were administered i.v. to rats and rabbits. For feeding studies, the cells were immobilized using polycationic polymers and a crosslinking agent (i.e., chitosan, polyethylenimine and glutaraldehyde). GI, in the whole cell immobilized form, was offered at concentrations of 0, 1.5, 3.0 or 5.0% (wt/wt) of the diet to dogs for a minimum of 90 consecutive days and to rats over three generations. Animals were observed daily for signs of toxicosis; body weight and food consumption were monitored; biochemical tests, hematologic determinations, and urinalyses were done on blood and urine samples; and thorough gross and microscopic tissue examinations were performed at terminations. There were no signs of infection or toxicosis following i.v. administration of F. arborescens or the cell-free supernatant fluid. This, and the lack of toxicity in dogs and rats which received daily dietary concentrations of GI many times above the projected highest possible human exposure level, suggest that there should be virtually no risk of toxicity associated with the consumption of food and beverages containing high fructose syrup produced by GI derived from F. arborescens .