The protective mechanism underlying remote ischemic preconditioning (RIPC) is unclear. This study aims to verify whether the protein expression profile in the serum could be altered by RIPC and to detect potential protein mediators. Transient limb ischemia consisting of three cycles of 5-min ischemia followed by 5-min reperfusion was performed on sixty healthy volunteers. Serum samples were collected at 30 min before transient limb ischemia and at 1 hour (h), 3 h, 8 h, 24 h, and 48 h after completion of three cycles. Changes in the serum protein profile were analyzed by two-dimensional gel electrophoresis and proteins were identified by MALDI-TOF/TOF mass spectrometry. Fourteen differentially expressed proteins were identified and, respectively, involved in immune system, lipid binding and metabolism, apoptosis, and blood coagulation. Complement C3, vitronectin, and apolipoprotein A-I were further confirmed by western blotting, and the results showed that their contents decreased significantly after transient limb ischemia. It is concluded that transient limb ischemia alters the serum protein expression profile in human being, and that reduction of serum contents of complement C3 and vitronectin may represent an important part of the mechanism whereby RIPC confers its protection.
Ischemic preconditioning (IPC), induced by exposing tissues to transient nonfetal ischemia prior to a prolonged ischemic insult [
However, the mechanism through which the protective signal is conveyed from the preconditioned limb to the remote organs is unclear, although the neural pathway [
We designed and conducted this study to investigate whether serum proteins could be altered by transient limb ischemia in human beings and to explore whether there existed any potential protein mediators in the serum that facilitate the protection induced by RIPC. In this study, transient limb ischemia was conducted in healthy volunteers and the approach of comparative proteomics was applied to identify serum proteins before and after transient limb ischemia. Proteins whose expressions were altered after transient limb ischemia were further studied and some of these proteins were validated by western blotting.
We recruited sixty healthy volunteers and obtained written informed consent from them. Volunteers’ characteristics were shown in Table
Healthy volunteers’ characteristics*.
Age (years) | 22 (1.8) |
Male | 30 (50%) |
Mean arterial pressure (mmHg) | 85 (9) |
Pulse (bmp†) | 75 (12) |
Pulse oxygen saturation (%) | 99 (1) |
Body mass index (Kg/m2) | 20.49 (2.30) |
†bmp: beats per minute.
Transient limb ischemia was achieved by three cycles of ischemia and reperfusion, and each cycle consisted of 5-min ischemia followed by 5-min reperfusion of the nondominant arm. Ischemia and reperfusion were induced by a 12 cm-wide blood pressure cuff placed on the nondominant upper arm inflated to 200 mmHg for 5 min and then deflated for 5 min.
At 30 min before transient limb ischemia and at 1 h, 3 h, 8 h, 24 h, and 48 h, respectively, after the the completion of three cycles of transient limb ischemia, blood (10 mL) was collected from the contralateral arm into tubes and was processed according to a standard operating procedure. The tubes were labeled and transported to the laboratory on ice within 15 min. The blood was centrifuged at 2500 rpm at 4°C for 10 min. Serum samples of each volunteer at all the time points were then collected, aliquoted, and stored at −80°C. Each serum sample underwent only two freeze/thaw cycles during all the following experimental protocols.
Samples of 3 volunteers at all the time points were randomly selected from the samples of 60 volunteers to undergo the 2D-gel study. Each serum sample (1 mL) was processed by using reagents provided by the commercial ProteoMiner Protein Enrichment Kits (Bio-Rad) to decrease high-abundance proteins and to enrich low-abundance proteins. After enrichment, serum was purified by ReadyPrep 2D Cleanup Kit (Bio-Rad). Protein concentration was determined by the Bradford assay with the BSA standard (Bio-Rad).
A purified serum sample containing 150
Stained gels were scanned by densitometric scanning (Typhoon-9200, Amersham Company, Sweden) and the scanned images were exported into Image Master 2D Elite 5.0 software (Amersham Biosciences, Buckinghamshire, UK) for analysis. Differentially expressed protein spots in the gels, which were the spots expressed more than 1.5-fold differences in expression level as compared with the samples before transient limb ischemia at any time point after transient limb ischemia, were excised. The excised spots were immediately washed in redistilled water and then washed in 50% (v/v) acetonitrile in 100 mmol/L amine carbonate and then digested with 20
Based on their functional relevance and potential significance with regard to IR injury and organ protection, three proteins of complement C3, vitronectin, and apolipoprotein A-I (apoA-I) were selected to undergo western blotting. Original serum samples from all sixty volunteers were analyzed by western blotting to confirm expressions of the three proteins. Protein concentration was determined by the Bradford assay with the BSA standard (Bio-Rad), and then equal amounts of total protein (25
Quantitative data were analyzed with statistical package SPSS 16.0 (Chicago, IL). Data were presented as mean ± standard deviation of the mean. Repeated-measures analysis of variance (RMANOVA) was used for serial measurements. Statistical significances were evaluated at a two-tailed significance level of 0.05. This trial was registered with ClinicalTrials.gov, no.
Differentially expressed protein spots in the gels were identified as fourteen different proteins by MALDI-TOF/TOF mass spectrometry and database search (Figure
Differentially expressed proteins in sera after RIPC compared with that in sera before RIPC.
Spota | Protein name | Accession numberb | Protein MW (Da) | Protein PIc | Sequence coveraged (%) | Fold changee | Protein scoref |
---|---|---|---|---|---|---|---|
(i) |
|
||||||
1 | Complement component 4B | IPI00887154 | 192627.5 | 6.89 | 2 |
|
158 |
2 | Complement C1q subcomponent subunit B | IPI00477992 | 26704.5 | 8.83 | 22 |
|
84 |
3 | Complement C3 | IPI00783987 | 187029.9 | 6.02 | 15 |
|
121 |
4 | C4b-binding protein alpha chain | IPI00021727 | 66989.4 | 7.15 | 9 |
|
71 |
5 | Ficolin-3 | IPI00419744 | 31657.4 | 6.36 | 18 | ↑1.521 h | 107 |
6 | Interalpha-trypsin inhibitor heavy chain H4 | IPI00896419 | 103293 | 6.51 | 18 | ↑2.511 h | 97 |
7 | Vitronectin | IPI00298971 | 54271.2 | 5.55 | 4 |
|
70 |
| |||||||
(ii) |
|
||||||
8 | Apolipoprotein A-I | IPI00021841 | 30758.9 | 5.56 | 25 |
|
79 |
9 | Apolipoprotein L1 | IPI00514475 | 43946.9 | 5.6 | 28 | ↑1.681 h | 88 |
10 | Apolipoprotein J | IPI00291262 | 52461 | 5.89 | 20 |
|
219 |
| |||||||
(iii) |
|
||||||
11 | Desmoplakin | IPI00013933 | 331568.7 | 6.44 | 8 | ↑1.6248 h | 67 |
12 | Gelsolin | IPI00026314 | 85644.2 | 5.9 | 16 | ↑2.211 h | 131 |
| |||||||
(iv) |
|
||||||
13 | Antithrombin-III | IPI00032179 | 52657.8 | 6.11 | 23 |
|
180 |
14 | Heparin cofactor 2 | IPI00879573 | 57034.2 | 6.41 | 17 | ↑2.321 h | 64 |
bAccession number from IPI (International Protein Index) database of matched proteins.
cPI refers to isoelectric point.
dPercent of number of observed amino acids in sequence length (%).
e“
fCombined scores of all observed mass spectra matched to amino acid sequences used for protein identification.
Representative images of SYPRO-Ruby-stained 2-DE gels. Representative images of SYPRO-Ruby-stained 2-DE gels at various time points ((a) before transient limb ischemia, (b) 1 h after transient limb ischemia, (c) 3 h after transient limb ischemia, (d) 8 h after transient limb ischemia, (e) 24 h after transient limb ischemia, and (f) 48 h after transient limb ischemia). The high-abundant proteins such as albumin and immunoglobulins were depleted from serum using the multiple-affinity column, as described in Section
Peptide mass fingerprinting spectrum and a typical MS/MS map of complement C3. (a) Peptide mass fingerprinting spectrum of complement C3. The arrow indicates the peptide detected at
Among these fourteen proteins, seven proteins were related to immune response processes, including complement component 4B, complement C1q subcomponent subunit B, complement C3, C4b-binding protein alpha chain and ficolin-3 in complement pathway, vitronectin in immune response, and interalpha-trypsin inhibitor heavy chain H4 in acute phase response. Among the altered proteins, ficolin-3 and interalpha-trypsin inhibitor heavy chain H4 were upregulated, while the other proteins were downregulated. Besides, three proteins involved in lipid metabolic process were affected, namely, apoA-I and apolipoprotein J downregulated and apolipoprotein L1 upregulated. These proteins were also related to immune response. Then, two proteins involved in cell apoptosis were all upregulated, including desmoplakin and gelsolin. Finally, two proteins related to blood coagulation were also affected, antithrombin-III downregulated, and heparin cofactor 2 upregulated.
The contents of the three proteins (complement C3, vitronectin, and apoA-I) in the volunteers’ sera (
Western blotting data of complement C3, vitronectin, and apoA-I. The results were presented as mean ± SD. Error bars were SD. Repeated-measures analysis of variances was performed to evaluate statistical significance.
Baseline relative density of complement C3 was defined as 1. The reduction of complement C3 was significant at 1 h (
Baseline relative density of vitronectin (75 KDa) was defined as 1. The reduction of vitronectin (75 KDa) was significant at 8 h (
Baseline relative density of vitronectin (65 KDa) was defined as 1. The reduction of vitronectin (65 KDa) was not significant at 1 h (
Baseline relative density of apoA-I was defined as 1. The reduction of apoA-I was significant at 3 h (
There were hardly any studies conducted amongst healthy volunteers in order to detect clinically relevant protein mediators that facilitate the protective effects of RIPC. Isolated buffer-perfused animal hearts in vitro and animal models in vivo have been extensively used for investigating the protective effect and mechanism of RIPC. Although these models have been shown to be reproducible and efficient, isolated hearts could not reflect the effects of nervous and circulatory system in the whole body, and animal disease models cannot represent the complex human clinical setting very well. Therefore, conducting a controlled human study is a crucial translational step from bench to bedside. A recent human study by Hepponstall et al. found that the RIPC stimulus modified the plasma protein content in blood, but this study enrolled only five healthy volunteers [
In this study, we compared serum proteins profiles after transient limb ischemia with that before transient limb ischemia in healthy volunteers by a proteomic approach and showed that there existed changes of serum proteins induced by transient limb ischemia. The changed proteins were mainly involved in the inflammatory system and also involved in the lipid metabolic system, cell apoptosis, and coagulation system.
In the 2-DE results of our study, we observed that complement component 4B, complement C1q subcomponent subunit B, C4b-binding protein alpha chain, and complement C3 were all decreased after transient limb ischemia.
Furthermore, we confirmed the changes of three proteins of complement C3, vitronectin (75 KDa), and apoA-I after transient limb ischemia by western blotting. The downregulation of complement C3 could persist for one day or perhaps even longer, the content of vitronectin (75 KDa) decreased significantly at 8 h, 24 h, and 48 h after transient limb ischemia, and the content of apoA-I decreased significantly at 3 h and 8 h after transient limb ischemia.
The pathway through which RIPC protects organs is unclear, but three possible mechanisms have been suggested [
The complement cascade has been shown to be a key mediator of IR injury [
Complement C3 is the central molecule of the complement system, at which the classical, lectin, and alternative pathways converge. Complement C3 is also associated with myocardial infarction, and it is more significant than any other traditional risk factors [
Vitronectin is a multifunctional glycoprotein present in plasma, extracellular matrix, and blood platelets. It is found in two molecular forms in human blood, which are a single chain (75 kDa) and a clipped form of two chains (65 and 10 kDa) [
ApoA-I is the major protein of high-density lipoprotein. Besides its role in cholesterol metabolism, it possesses antiatherosclerotic, antioxidant, anti-inflammatory, and antithrombotic activities [
This study was designed as a self-control study to confirm whether the composition of serum proteins was changed after transient limb ischemia in human beings. Time points for blood sample collection were selected in consideration of early and late phase of protection induced by RIPC. The early phase lasts for up to 3 h after ischemic preconditioning, whereas the late phase starts at 12–24 h after ischemic preconditioning [
It should be noted that it is very complicated to elucidate the mechanism of RIPC. Nevertheless, this study just finds some changes of serum proteins at limited time points which should serve to facilitate future extensive work aimed to elucidate the RIPC protective mechanism.
All authors declared no conflicts of interests.
Ting Pang, Yang Zhao, and Nan-Rong Zhang contributed equally to this study. San-Qing Jin and San-Qiang Pan designed the experiment, and San-Qing Jin supervised the whole process of the study and revised the paper draft. San-Qiang Pan helped analyzing the data. Ting Pang and Yang Zhao did the experiment (including sera collection, 2-DE, and western blotting), analyzed the data, and wrote the paper draft. Nan-Rong Zhang recruited volunteers and performed transient limb ischemia on volunteers. All authors have seen and approved the final version of this paper.
The authors thank the Natural Science Foundation from Department of Science and Technology of Guangdong province for its support (no. 07001664). The authors thank Hong-Bin Feng, Yan Zhou, and Dan Hu at Department of Anesthesiology in the Sixth Affiliated Hospital of Sun Yat-Sen University for recruiting volunteers and collecting blood and Su-Mei Li and Hong-Hai Yuan at Department of Anatomy in Medical College of Jinan University for their assistance in western blotting and protein identification.