Patients with Coronary Artery Disease Have Lower Levels of Antibody to Heat-Stressed Fibroblast Derived Proteins, versus Normal Subjects

Abstract

Cellular stress response plays an important role in the pathophysiology of coronary artery disease (CAD). Inhibition of cellular stress may provide a novel clinical approach regarding the diagnosis and treatment of CAD. Fibroblasts constitute 60-70% of cardiac cells and have a crucial role in cardiovascular function. Hence, the aim of this study was to show a potential therapeutic application of proteins derived from heat-stressed fibroblast in CAD patients. Fibroblasts were isolated from the foreskin and cultured under heat stress conditions. Surprisingly, 1.06% of the cells exhibited a necrotic death pattern. Furthermore, heat-stressed fibroblasts produced higher level of total proteins than control cells. In SDS-PAGE analysis, a 70 kDa protein band was observed in stressed cell culture supernatants which appeared as two acidic spots with close pI in the two-dimensional electrophoresis. To evaluate the immunogenic properties of fibroblast-derived heat shock proteins (HSPs), the serum immunoglobulin-G (IgG) was measured by ELISA in 50 CAD patients and 50 normal subjects who had been diagnosed through angiography. Interestingly, the level of anti-HSP antibody was significantly higher in non-CAD individuals in comparison with the patient's group (p < 0.05). The odds ratio for CAD was 5.06 (95%CI = 2.15-11.91) in cut-off value of 30 AU/mL of anti-HSP antibody. Moreover, ROC analysis showed that anti-HSP antibodies had a specificity of 74% and a sensitivity of 64%, which is almost equal to 66% sensitivity of exercise stress test (EST) as a CAD diagnostic method. These data revealed that fibroblast-derived HSPs are suitable for the diagnosis and management of CAD through antibody production.

Figure 1

Overall study design flowchart. Among 134 potentially patients who referred for angiography, fifty patients with coronary atherosclerosis (stenosis ≥ 50%) and fifty healthy individuals (no plaque) were selected via the gold standard method. Serum was separated, and anti-HSP IgG was measured by ELISA assay. ^∗NEG, POS, and con stand for negative, positive, and concentration, respectively.

Figure 2

SDS-PAGE and 2-DE pattern of heat-stressed fibroblasts derived proteins. (a) The single protein band with a relative molecular weight of 70 kDa in silver-stained 12% SDS-PAGE which was carried out with two different samples (s1 and s2) prepared from heat-stressed fibroblast supernatants (lanes 2 and 4). This band was not detectable in the culture supernatant of nonstressed cells (lane 3, negative control). Lane 1 is the molecular weight size marker. (b) 2-DE gel of heat-stressed fibroblasts derived protein with Coomassie Blue staining. The arrow shows two protein spots with a relative molecular weight of 70 kDa in the acidic region of the gel.

Figure 3

Morphology and cell death pattern of heat-stressed fibroblasts. There is no significant morphological difference between fibroblasts before and after heat shock. (a) Fibroblasts before and (b) after heat shock stress. (c) Forward- and side-scatter plot (left plot) and dot plot graph (quadrant curve) of heat-stressed fibroblasts (right plot). Apoptosis and necrosis cell death pattern were 19% and 1%, respectively (right plot).

Figure 4

The serum levels of anti-HSP antibody in CAD patients and normal control which shows a diagnostic value of this type of antibody. (a) The anti-HSP IgG was measured by ELISA in serum of CAD patients (n = 50) as well as in normal control (n = 50). Results are expressed in arbitrary units per milliliter (AU/mL), and the horizontal lines indicated the median. p value was equal to 0.0006 when two groups compared with the Mann–Whitney U test. (b) The receiver operating characteristic (ROC) curve analysis showed 64% sensitivity and 74% specificity for a cut-off value of 30.5 (AU/mL). The area under the curve (AUC) was 0.74 (p < 0.05).