Gideon M. Hirschfield

C-reactive protein: a critical update

In the mid 1990s, immunoassays for C-reactive protein (CRP), with greater sensitivity than those previously in routine use, revealed that increased CRP values, even within the range previously considered normal, strongly predict future coronary events. These findings triggered widespread interest, especially, remarkably, in the US, where the clinical use of CRP measurement had been largely ignored for about 30 years. CRP production is part of the nonspecific acute-phase response to most forms of inflammation, infection, and tissue damage and was therefore considered not to provide clinically useful information. Indeed, CRP values can never be diagnostic on their own and can only be interpreted at the bedside, in full knowledge of all other clinical and pathological results. However, they can then contribute powerfully to management, just as universal recording of the patient’s temperature, an equally nonspecific parameter, is of great clinical utility. The present torrent of studies of CRP in cardiovascular disease and associated conditions is facilitated by the ready commercial availability of automated CRP assays and of CRP itself as a research reagent. However, unlike the earlier rejection in the US of CRP as an empirical test because of its perceived lack of specificity, the current enthusiasm over CRP in cardiovascular disease is widely characterized by failure to recognize appropriately the nonspecific nature of the acute-phase response, and by lack of critical biological judgment. Quality control of the source, purity, and structural and functional integrity of the CRP, and the relevance of experimental design before ascribing pathophysiological functions, are also often ignored. This article provides information about CRP as a protein and an acute-phase reactant, and a knowledge-based framework for interpretation and analysis of clinical observations of CRP in relation to cardiovascular and other diseases. We also review the properties of CRP, its possible role in pathogenesis of disease, and our own observations that identify it as a possible therapeutic target.

Targeting C-reactive protein for the treatment of cardiovascular disease

Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement, increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively. Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement. Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.

Inflammation and Endothelial Function Direct Vascular Effects of Human C-Reactive Protein on Nitric Oxide Bioavailability

Background—Circulating concentrations of the sensitive inflammatory marker C-reactive protein (CRP) predict future cardiovascular events, and CRP is elevated during sepsis and inflammation, when vascular reactivity may be modulated. We therefore investigated the direct effect of CRP on vascular reactivity. Methods and Results—The effects of isolated, pure human CRP on vasoreactivity and protein expression were studied in vascular rings and cells in vitro, and effects on blood pressure were studied in rats in vivo. The temporal relationship between changes in CRP concentration and brachial flow-mediated dilation was also studied in humans after vaccination with Salmonella typhi capsular polysaccharide, a model of inflammatory endothelial dysfunction. In contrast to some previous reports, highly purified and well-characterized human CRP specifically induced hyporeactivity to phenylephrine in rings of human internal mammary artery and rat aorta that was mediated through physiological antagonism by nitric oxide (NO). CRP did not alter endothelial NO synthase protein expression but increased protein expression of GTP cyclohydrolase-1, the rate-limiting enzyme in the synthesis of tetrahydrobiopterin, the NO synthase cofactor. In the vaccine model of inflammatory endothelial dysfunction in humans, increased CRP concentration coincided with the resolution rather than the development of endothelial dysfunction, consistent with the vitro findings; however, administration of human CRP to rats had no effect on blood pressure. Conclusions—Pure human CRP has specific, direct effects on vascular function in vitro via increased NO production; however, further clarification of the effect, if any, of CRP on vascular reactivity in humans in vivo will require clinical studies using specific inhibitors of CRP.

Human C-Reactive Protein Does Not Protect against Acute Lipopolysaccharide Challenge in Mice

The physiological and pathophysiological functions of C-reactive protein (CRP), the classical acute-phase protein, are not well established, despite many reports of biological effects of CRP in vitro and in model systems in vivo. Limited, small scale experiments have suggested that rabbit and human CRP may both protect mice against lethal toxicity of Gram-negative bacterial LPS. However, in substantial well-controlled studies in C57BL/6 mice challenged with Escherichia coli O111:B4 LPS, we show in this work that significant protection against lethality was conferred neither by an autologous acute-phase response to sterile inflammatory stimuli given to wild-type mice 24 h before LPS challenge, nor by human CRP, whether passively administered or expressed transgenically. Male mice transgenic for human CRP, which mount a major acute-phase response of human CRP after LPS injection, were also not protected against the lethality of LPS from either E. coli O55:B5 or Salmonella typhimurium. Even when the acute-phase human CRP response was actively stimulated in transgenic mice before LPS challenge, no protection against LPS toxicity was observed. Indeed, male mice transgenic for human CRP that were pretreated with casein to stimulate an acute-phase response 24 h before LPS challenge suffered significantly greater mortality than unstimulated human CRP transgenic controls. Rather than being protective in this situation, human CRP may thus have pathogenic proinflammatory effects in vivo.

C-Reactive Protein Alters Vascular Reactivity by Increasing Nitric Oxide Production

ELAM (nglml) investigated the role of NO in the development of tolerance to prolonged administration of alpha-2 adrenergic agonists. Method: Tolerance was assessed by the attenuation of the inhibitory effect of dexmedetomidine (DEX), a highly specific alpha-2 adrenergic agonist, on prostaglandin E-1 (PGE-I) stimulated adenylyl cyclase (AC) in the mouse neuroblastoma X rat glioma hybrid cell (NG108-15) line (neuronal cells which express the alpha2 8 adrenergic receptor). Cells were chronically exposed to 0.1 pM DEX for 2, 4, 8, 14 hours and DEX + 100 pM N-nitro-L-arginine, a NO synthase (NOS) inhibitor, for 2 hours. The cells were also exposed to L-arginine (a NO donor) for 20 minutes. The cells were then washed three times and acutely exposed to varying doses of DEX (IpM to I pM) and the AC activity measured. Results: Cells exposed to DEX for 2 hours or longer were desensitized as evidenced by a 10 fold shift to the right of the DEX dose response curve. The cells exposed to DEX and N-nitro-Larginine for 2 hours showed no such desensitization. Cells exposed to L-arginine (without DEX) demonstrated a 6 fold shift to the right in the DEX dose response curve. Summary: Tolerance to DEX can be induced in an in-vitro model. This is demonstrated by the right shift in the dose response curve, after 2 hours exposure to DEX. This effect can be reproduced by NO exposure after only 20 minutes, and prevented when NOS is inhibited during prolonged DEX exposure.