Increased myocardial prevalence of C-reactive protein in human coronary heart disease: direct effects on microvessel density and endothelial cell survival

Abstract

Background: Elevated plasma C-reactive protein (CRP) is a biomarker of cardiovascular diseases (CVDs), but its potential roles as a participant of the disease process are not well defined. Although early endothelial cell injury and dysfunction are recognized events in CVD, the initiating events are not well established. Here we investigated the local myocardial CRP levels and cardiac microvessel densities in control and CVD tissue samples. Using in vitro methodologies, we investigated the direct effects of CRP on human endothelial cells.

Methods: Cardiac specimens were collected at autopsy within 4 h of death and were classified as normal controls or documented evidence of CVD. The regional prevalence of CRP and the cardiac microvessels (<40 μm) were investigated using immunohistochemistry. For in vitro experiments, human umbilical vein endothelial cells were incubated with CRP. Intracellular oxidant levels were assessed using 2',7'-dichlorofluorescein diacetate fluorescence microscopy, and cell survival was concurrently determined. Effects of chemical antioxidants on endothelial cell survival were also tested.

Results: Myocardial CRP levels were elevated in CVD specimens. This was associated with reduced cardiac microvessels, and this rarefaction was inversely correlated to adjacent myocardial CRP prevalence. CRP caused concentration-dependent increases in oxidant production and cell apoptosis.

Conclusions: These findings provide evidence supporting myocardial CRP as a locally produced inflammatory marker and as a potential participant in endothelial toxicity and microvascular rarefaction.

Figure 1

Increased prevalence of C-reactive protein levels in myocardium. Local myocyte production of CRP can be detected in human LV samples and myocardial CRP levels were significantly higher in the CVD samples (* - p<0.05).

Figure 2

Cardiac microvessel rarefaction in CVD. LV tissues were stained with CD-31, imaged at 400X magnification and microvessels were counted with automated macro. Significant reduction in LV tissue microvessel numbers was observed in the CVD samples as shown in the adjacent graph (* - p<0.05).

Figure 3

Increased myocardial CRP correlates with microvascular rarefaction. Statistically significant inverse correlation between relationship between local myocyte expression of CRP and prevalence of microvessels was observed in these human tissue samples.

Figure 4

CRP causes endothelial cell death at these clinically relevant concentrations. There was a concentration dependent increase in cell death (Panel A). Upon further investigations, the major mechanism of cell death was via increased apoptosis (Panel B). Data expressed as mean±SEM of 6-8 individual observations (* - p<0.05).

Figure 5

CRP treatment causes time dependent increase in cellular oxidants at clinically relevant concentrations. The oxidant levels are measured as DCF signal intensity (Y-axis) and time points are shown on X-axis. All data expressed as mean±SEM (* - p<0.05).

Figure 6

CRP cell toxicity can be prevented by chemical antioxidants. Cells were incubated with antioxidants in presence of CRP. Ascorbic acid and N-acetylcysteine demonstrated a cellular protective activity against CRP induced endothelial cell toxicity. Data expressed as mean±SEM of 8 individual observations (* - p<0.05).