It is now well recognized that CRP is a major risk factor for a wide variety of cardiac and vascular disorders, including cardiomyopathies, myocarditis, and vasculopathies. CRP is now shown to play a major role in many cardiovascular events and has complex pathobiology. CRP is known to colocalize with complement in the atherosclerotic plaque due to its local production. CRP also contributes to perpetuation and amplification of inflammatory responses (17
). Given the importance of CRP as a critical participant in CVD, the mechanistic understanding of its vascular endothelial toxicity is critical. Recent studies have suggested that CRP has specific vascular effects that include apparent impairment in vascular endothelial function. These vascular changes may be explained by either direct toxicity to the endothelium in vivo or indirect effects related pro inflammatory pathways associated or other cytokines. There is some controversy in the literature regarding the direct toxic effects of CRP. It has been thought that CRP induced augmentation of endothelial cell activation requires CD-14 as a cofactor(18
). Conversely, Devraj et. al. have demonstrated that native CRP alone is capable of inducing HAEC activation (19
). Molins et. al. demonstrated that monomeric CRP has more potent prothrombotic effects as compared to native CRP (20
). Given these complexities in vivo
, we tested the hypothesis that CRP has direct actions on vascular endothelial cells. Direct toxicity of CRP on endothelial cells has been previously investigated in regards to cellular phenotypes and nitric oxide synthase regulation. However, there have been few studies investigating the direct mechanistic aspects of CRP-induced vascular endothelial cell toxicity. One limitation of many in-vitro studies is the concentrations of CRP used which range from 5mg/L to 100mg/L. In a clinical setting 3-5mg/L CRP level is considered to be moderate to high risk. Low-grade chronic elevation in CRP levels may be more significant for direct effects of CRP as compared to acute increases. We have therefore used clinically relevant concentrations of CRP to investigate the direct effects on endothelial cell survival.
We demonstrated for the first time that there is a measurable level of CRP in cardiac tissue from human LV samples with evidence of CVD. In a parallel set of experiments, the presence of CRP in the cardiac tissues was confirmed by western blotting (data not shown here). This observation suggests that CRP might be produced locally in cardiomyocytes. Indeed many recent studies have demonstrated that CRP is produced in multiple sites including kidney and neuronal cells. These increased levels of CRP were directly correlated to reduction in local microvessel density. In-vitro investigations with recombinant human CRP caused endothelial cell death and significantly increased intracellular oxidant production. By using general antioxidants, we showed that it prevented CRP induced cell death, indicating the critical role of ROS in CRP induced EC death. Thus, we have demonstrated, that CRP, at concentrations known to predict adverse vascular outcomes in vivo, has direct toxic effects on endothelial cells, and that the toxicity is mediated via reactive oxidant species. These adverse effects of CRP on endothelial cell survival may play a critical role in coronary microvessel rarefaction observed in patients with CVD. It is important to note that many confounding factors may also contribute to the coronary microvascular changes observed in this patient population. Myocardial hypertrophy, chronic ischemia and atherosclerosis have all been individually associated with reduction in coronary reserve and may cause rarefaction (21
). However, our study establishes a direct association between the increased myocardial CRP levels and decreased microvessel numbers. In this setting, CRP may act as a direct toxicant to the coronary microvascular endothelium and thus act as the major contributor to accelerate the microvascular changes caused by CVD in these patients.
Using a general live/dead assay for adherent endothelial cells, we detected significant loss of cell viability at clinically relevant concentrations of CRP (5 and 10μg/ml) after 48h of incubation. These studies were carried out in low-serum conditions in order to limit the cell growth during incubation period. Control cells in these studies were incubated in low serum in absence of CRP. These concentrations of CRP are within the range of ‘moderate to high risk’ plasma concentrations in patients with proven CVD disease risk. We then evaluated potential mechanisms of cell death (e.g., early apoptosis vs. necrosis), and found that after CRP exposure, significant increases in apoptotic cell death was detected in a concentration dependent manner. In contrast, the incidence of necrosis was relatively low (<5%) and not related to CRP concentration. Under the same experimental conditions and identical concentrations, heat denatured CRP did not show any significant effect on cell survival. There have been several reports in the literature suggesting that the cell death observed with CRP treatment is solely due to the azide content in the commercially available protein (22
). Our data with the heat-denatured protein suggests that potential contamination in commercial CRP is not a possible mechanism of our observation. Our findings of CRP induced cellular apoptosis are consistent with those of Zhang et al (25
) although they have utilized higher CRP exposure levels (up to 25μg/ml) as compared to our investigations. We have utilized lower CRP concentrations in our studies because it has been previously demonstrated that even small changes in CRP levels can significantly increase disease risk (26
Increased reactive oxygen species are often seen in various settings of endothelial dysfunction, and in previous investigations we have demonstrated that this cell type is an avid producer of reactive oxygen and nitrogen species when stressed (28
). Therefore, we next evaluated the effects CRP with respect to intracellular oxidant production by using live cell imaging. We observed significant increases in reactive species (as measured by the nonspecific detector of reactive nitrogen and oxygen species, DCF) at 48 h post CRP incubation at 5 and 10 μg/ml. It is important to note that in these experiments the incubation medium containing drug is removed at the time of oxidant detection (e.g., cells are washed and loaded with dye); thus, direct chemical oxidant production from CRP is not a possible mechanism of our observations.
Although at this time the source of intracellular oxidants is unclear, potential sites may include disruption of the mitochondrial electron transport chain or activation of various oxidase enzymes. In previous studies we have demonstrated that antioxidants such as NAC and ascorbic acid can significantly reduce drug induced oxidant production (29
). Given these previous observations we evaluated the effects of this combined treatment (CRP plus ascorbic acid and CRP plus NAC) with respect to cell survival. Our aim was to distinguish between two possibilities: first, increased production of cellular ROS and cell death are two separate events induced by CRP, or second, that ROS production is a major cause of CRP induced cell death. We found that the addition of antioxidants significantly attenuated the CRP-induced endothelial cell death at 48 h. These observations are consistent with recent clinical reports suggesting that vitamin C supplementation yielded a 24.0% reduction in plasma CRP levels of healthy individuals exposed to active or passive smoking (30
). Moreover these data are consistent with a mechanism of CRP-induced oxidant production, leading to initiation of apoptotic death sequences. Further characterization of the pathways involved in this process and targets involved in initiation are clearly warranted.
Fujii et. al. (31
) have recently demonstrated that CRP, at concentrations known to predict cardiovascular events, may serve to impair endothelial progenitor cell (EPC) antioxidant defenses, and promote EPC sensitivity toward oxidant-mediated apoptosis and telomerase inactivation. Thus it would be of clinical relevance to further investigate the possible therapeutic value of the use of antioxidants supplementation in patients with elevated plasma CRP levels.