Treatment with captopril abrogates the altered expression of alpha1 macroglobulin and alpha1 antiproteinase in sera of spontaneously hypertensive rats

Proteome Science Treatment with captopril abrogates the altered expression of alpha1 macroglobulin and alpha1 antiproteinase in sera of spontaneously hypertensive rats Norhaniza Aminudin 0 Nur-Atiqah H Abdullah 0 Hasni Misbah Saiful A Karsani 0 Ruby Husain See Z Hoe Onn H Hashim 0 Institute of Biological Sciences, Faculty of Science, University of Malaya , 50603 Kuala Lumpur , Malaysia Background: Proteins that are associated with hypertension may be identified by comparing the 2-dimensional gel electrophoresis (2-DE) profiles of the sera of spontaneously hypertensive rats (SHR) with those generated from normotensive Spraque-Dawley rats (SDR). Results: Five proteins of high abundance were found to be significantly altered when the 2-DE serum profiles of the SHR were compared to those that were similarly generated from the SDR. Analysis by mass spectrometry and database search identified the proteins as retinol binding protein 4, complement C3, albumin (19.9 kDa fragment), alpha1 macroglobulin and alpha1 antiproteinase, which are all known to be associated with hypertension. The altered expression of the two latter proteins was found to be abrogated when similar analysis was performed on sera of the SHR that were treated with captopril. Conclusion: Our data suggests that serum alpha1 macroglobulin and alpha1 antiproteinase are potentially useful complementary biomolecular indicators for monitoring of hypertension. hypertension; biomarker; spontaneously hypertensive rats; serum proteins; proteomics - Background Spontaneously hypertensive rats (SHR) have been widely used as an animal model to investigate primary hypertension and its relationship to cardiovascular diseases. The SHR strain was generated in the 1960s by Okamoto et al. by selective breeding of the Wistar-Kyoto rats with high blood pressure [1]. The blood pressure of SHR usually rises at around 5-6 weeks of age and the systolic pressure of an adult SHR may reach a value of between 180 and 200 mmHg. The SHR usually develops characteristics of cardiovascular diseases like hypertrophy of the heart and blood vessels, which start at around 40 weeks of age [2]. Hypertension has been known to cause the altered levels of serum or plasma proteins. A considerable number of proteins have been previously reported to be altered in levels in the sera of both animals and human subjects [3-6]. While some of the proteins were thought to be involved as anti-inflammatory and protective response [4,7], others were related to endothelial vascular repair [5], arterial smooth muscle cell growth [6] and some may instead be the contributing factors of hypertension. In the present study, we have investigated the simultaneous expression of the high abundant serum proteins in the normotensive Spraque-Dawley rats (SDR) and compared it with those expressed in the sera of SHR as well as the SHR that were treated with captopril. Table 1 Mean systolic blood pressure of control and captopril-treated rats Systolic blood pressure (mmHg) SDR - Sprague Dawley rats, SHR - spontaneously hypertensive rats, C-SHR captopriltreated SHR. Data are expressed as mean S.E.M. Values of p < 0.05 were considered significant. Results Monitoring of rat blood pressure Table 1 demonstrates the blood pressure of control rats and those treated with 60 mg/kg body weight/day of captopril. The blood pressure of the control SDR rat group showed stable normal systolic pressure. The hypertensive group of rats (SHR) also showed an unchanged high level of systolic blood pressure. However, a significant reduction of the blood pressure was observed in the captopril-treated SHR. Profiling of rat serum samples Figure 1 demonstrates a typical representative 2-DE protein profile that was generated from a serum sample obtained from control non-treated SHR. Serum proteins were separated on the basis of their different pIs and molecular weights when subjected to 2-DE. Hundreds of highly-resolved protein spots were detected when the 2DE gels were silver-stained. Due to the broad dynamic range of proteins that are present in the serum, only the highly abundant proteins were detected by silver staining. Similar profiles were generated when serum samples of the SDR and captopril-treated SHR were subjected to 2-DE. Image analysis of rat serum proteins When image analysis was performed on the silverstained 2-DE gels generated using serum samples obtained from the SDR and compared to similar profiles generated from the SHR, the expression of 13 protein spots was initially found to be significantly altered (p < 0.05). However, only five of the protein spots, i.e. 1, 50, 85, 117, and 211, were truly altered when the p-values Figure 1 Image of a representative silver-stained 2-DE gel showing the high abundant proteins detected in an SHR serum. Spots 1, 50, 85, 117 and 211 were significantly altered in levels when image analysis comparing the expression of 2-DE protein spots of SDR serum samples with those of the SHR. Acid side of the gel is to the left and relative molecular mass declines from the top. were corrected for false significant results using the method of Benjamini and Hochberg [8] (Figure 2). Using the serum proteins expressed in the sera of normotensive SDR as a standard reference, the levels of two proteins were found to be significantly lowered (spots 1 and 117) and three proteins (spots 50, 85 and 211) were apparently enhanced in the sera of the SHR. However, only spots 1, 50 and 85 maintained to be significantly altered in levels when the serum 2-DE profiles of SHR that were treated with captopril were compared those of Figure 2 Relative expression of protein spots of interest in sera of SDR, SHR and captopril-treated SHR. The percentage of volume contribution was determined using the Image Master 2D Platinum Software 7.0. Relative volumes of spots 1, 50, 85, 117 and 211 were significantly different when comparison was made between SHR and SDR. However, only spots 1, 50 and 85 were significantly different when the 2-DE images of captopriltreated SHR were compared to those obtained from SDR. Y axis of histogram indicates percentage of volume contribution. Asterisks denote significantly different values relative to the SDR (p< 0.00233). the normotensive SDR (Figure 2). The expression of spots 117 and 211 was apparently no longer significantly different and appeared to be normalised. Cropped images of the five serum protein spots in 2-DE gels of control and experimental rats are shown in Figure 3. Identification of differentially-expressed rat serum proteins The five differentially expressed protein spots were confidently identified by mass spectrometry using the 4800 Plus MALDI TOF/TOF analyzer and MASCOT database search (Table 2). They were retinol binding protein 4 (RBP4; spot 1), complement C3 (C3; spot 85), albumin (ALB; spot 50), alpha1 macroglobulin (A1MG; spot 117) and alpha 1 antiproteinase (A1AP; spot 211). The albumin spot was apparently resolved in th (...truncated)