Remote ischemic conditioning-induced hyperacute and acute responses of plasma proteome in healthy young male adults: a quantitative proteomic analysis

Abstract

Background: 

Long-term remote ischemic conditioning (RIC) has been proven to be beneficial in multiple diseases, such as cerebral and cardiovascular diseases. However, the hyperacute and acute effects of a single RIC stimulus are still not clear. Quantitative proteomic analyses of plasma proteins following RIC application have been conducted in preclinical and clinical studies but exhibit high heterogeneity in results due to wide variations in experimental setups and sampling procedures. Hence, this study aimed to explore the immediate effects of RIC on plasma proteome in healthy young adults to exclude confounding factors of disease entity, such as medications and gender.

Methods: 

Young healthy male participants were enrolled after a systematic physical examination and 6-month lifestyle observation. Individual RIC sessions included five cycles of alternative ischemia and reperfusion, each lasting for 5 min in bilateral forearms. Blood samples were collected at baseline, 5 min after RIC, and 2 h after RIC, and then samples were processed for proteomic analysis using liquid chromatography-tandem mass spectrometry method.

Results: 

Proteins related to lipid metabolism (e.g., Apolipoprotein F), coagulation factors (hepatocyte growth factor activator preproprotein), members of complement cascades (mannan-binding lectin serine protease 1 isoform 2 precursor), and inflammatory responses (carboxypeptidase N catalytic chain precursor) were differentially altered at their serum levels following the RIC intervention. The most enriched pathways were protein glycosylation and complement/coagulation cascades.

Conclusions: 

One-time RIC stimulus may induce instant cellular responses like anti-inflammation, coagulation, and fibrinolysis balancing, and lipid metabolism regulation which are protective in different perspectives. Protective effects of single RIC in hyperacute and acute phases may be exploited in clinical emergency settings due to apparently beneficial alterations in plasma proteome profile. Furthermore, the beneficial effects of long-term (repeated) RIC interventions in preventing chronic cardiovascular diseases among general populations can also be expected based on our study findings.

Registration: 

ClinicalTrials.gov, NCT03105141.

Introduction

Remote ischemic conditioning (RIC) is the intriguing phenomenon whereby brief, reversible episodes of ischemia and reperfusion applied in one vascular bed, tissue, or organ confers global protection, rendering remote tissues and organs resistant to ischemia/reperfusion injury. Upper limb RIC has been widely applied for post-stroke rehabilitation in clinical settings.^[1-4] RIC has shown promising therapeutic benefits in preventing severe damages by ischemia and reperfusion injury in patients with cerebrovascular diseases in clinical trials.^[5] Moreover, the user-friendly design (easy to carry on, non-invasive, and cost-effective) of the RIC system makes its application straightforward in most cases.^[6-8] Mechanistically, RIC-mediated neuroprotective events involve the activation of anti-oxidant, neuronal, and immune response-related signaling pathways.^[9-11]

Our team demonstrated that repeated sessions of bilateral arm RIC might have long-lasting clinical advantages in acute ischemic stroke (AIS) patients undergoing thrombectomy,^[1] intracranial atherosclerotic stenosis (ICAS),^[2,3] and cerebral small vessel disease (CSVD).^[4] Zhao et al^[1] enrolled patients with AIS who were suspected of having an emergent large-vessel occlusion in the anterior circulation and who were scheduled for endovascular treatment within 6 h of ictus. Once daily RIC was performed before recanalization, immediately following recanalization, and once daily for the subsequent seven days. No serious RIC-related adverse event was found. Meng et al^[2,3] demonstrated that long-term RIC (300 days) could improve cerebral perfusion and reduce recurrent strokes in ICAS patients. Wang et al^[4] found that patients with CSVD reduced white matter hyperintensities and improved cognitive function after twice daily RIC for one year.

More interestingly, the clinical outcomes of single versus multiple sessions of RIC in preclinical and clinical studies have been comprehensively reviewed by Landman et al^[5]. In animal brains, a single session of RIC can activate time-dependent protective mechanisms, namely short-lasting (2 h) protection immediately after RIC induction,^[12] and delayed protective phase, starting at 12–24 h post-RIC and lasting till 48–72 h.^[13] In human brains, Hougaard et al^[14] conducted a tissue survival analysis in AIS patients who received a single RIC session during transportation to the hospital, and subsequently underwent thrombolysis within 4.5 h, suggesting the immediate neuroprotective effect of prehospital RIC intervention. England et al^[15] have reported that single RIC treatment within 24 h post-AIS can improve the prognosis reflecting a lower 90-day score on the National Institutes of Health Stroke Scale (NIHSS). Guided by these facts, we sought to further investigate the protective mechanisms activated immediately after RIC, which could be critical for emergency and prehospital patients.

In recent years, proteomic techniques have been used to better understand changes in the plasma protein expression profiles in response to RIC stimulation [Table 1].^[16-21] However, there are high levels of heterogeneity among study findings due to wide variations in study model design, selection of disease type, sampling time, and RIC frequency and effect duration [Table 1]. Long-term intervention (5 weeks, twice a week) of RIC in the experimental AIS rhesus model has shown activation of complement, endovascular homeostasis, anti-coagulation mechanism, and regulation of lipid metabolism (anti-atherogenesis).^[18] Only one study focused on the post-RIC effect in healthy male adults, with the exclusion of probable proteomic changes under a certain diseased condition.^[19] Hepponstall et al^[19] have demonstrated that a single RIC could upregulate fibrinogen beta chain (FGB), fibrinogen gamma (FGG), and apolipoprotein A (ApoA)-mediating signaling as early as 15 min after RIC, and sustain up to 24 h. However, the inflation/deflation pressures of RIC, were not clearly mentioned in the study.

Table 1 - A systematic review of plasma proteomic study after RIC stimulus based on mass spectrometry sampling.

							RIC intervention method
Study	Country	Study type	Population status	Patients/animal model number	Age∗	Gender ratio (female/male)	Frequency	Duration (bout × amount)	Cuff pressure	Location	Sampling time
Lang et al (2005)	Germany	Experimental	Wild-type Wistar rats with sustained myocardial ischemia†	Sham (n = 6) Coronary preconditioned (n = 6) Renal preconditioned (n = 6)	8–10 weeks	0/18	Once	Coronary preconditioned group: 10 min (1 bout for 5 min coronary occlusion followed by 5 min of reperfusion repeated for three times) Renal preconditioned group: 30 min (1 bout for 10 min renal artery occlusion followed by 20 min of reperfusion)	Not mentioned	Coronary occlusion or renal artery occlusion	Baseline Immediately after preconditioning
Hepponstall et al [19]	Australia	Clinical	Healthy male adults	5	36.2 ± 6.3 years	0/5	Once	40 min (4 bouts‡ × 10 min)	Not mentioned	Bilateral upper limbs	Baseline, 15 min post-RIC, 2 h post-RIC
Hibert et al [20]	France	Experimental	Wild-type Wistar rats	RIC 5 min (n = 10) RIC 10 min (n = 10) Control (n = 10)	8–10 weeks	0/30	Once	10 min (1 bout for 10 min of ischemia alternating with 5 min of reperfusion) 20 min (1 bout for 10 min of ischemia alternating with 10 min of reperfusion)	Determined by a change in skin color and a decrease in subcutaneous limb temperature§	Upper right femoral artery	Baseline, 5 min post-RIC, 10 min post-RIC
Nikkola et al [16]	United States	Clinical	aSAH	13	51.2 ± 13.6 years	8/5	Four RIC sessions over 2–12 days	40 min (4 bouts‡ × 10 min)	20 mmHg over the patient's baseline systolic blood pressure||	Unilateral lower limb	Baseline, post-four RIC session
Chao de la Barca et al (2016)	France	Experimental	Wild-type Wistar rats	MI (n = 8) RIC before MI (n = 8)	8–10 weeks	0/16	Once	40 min (4 bouts‡ × 10 min)	Not mentioned	Upper right femoral artery	Immediately after preconditioning
Saber et al [17]	United States	Experimental	Male C57BL/6 mice with diffuse TBI	Sham (n = 8) TBI (n = 10) Sham RIC (n = 8) TBI RIC (n = 13)	8–10 weeks	0/39	Once (1 h after TBI)	40 min (4 bouts‡ × 10 min)	Pressure was applied by twisting the wire tie approximately four times clockwise	Left hind limb	Baseline
Thorne et al [21]	United Kingdom	Clinical	Human kidney transplant recipients	Control (n = 6) RIC (n = 6)	Control: 48.0 (30.1–70.3) years RIC: 52.2 (43.6–60.5) years	Control: 1/5 RIC: 1/5	Once	40 min (4 bouts‡ × 10 min)	Not mentioned	At the thigh on the opposite side used for transplantation	Baseline, 30 min post-RIC, 90 min post-RIC, Day 1 post-RIC, Day 5 post-RIC
Song et al [18]	China	Experimental	Rhesus monkey with ischemic stroke	2	8–10 years	0/2	Two RIC sessions every week for 5 weeks	50 min (5 bouts‡ × 10 min)	200 mmHg	Bilateral upper limbs	Baseline, Week 1 post-RIC, Week 2 post-RIC, Week 3 post-RIC, Week 4 post-RIC, Week 5 post-RIC

^∗Age was reported as either mean ± standard deviation or median (range), if not otherwise specified.

^†All groups were subjected to a long period of sustained myocardial ischemia (45 min), followed by 2 h reperfusion.

^‡Each bout for 5 min of ischemia alternating with 5 min of reperfusion.

^§No specific cuff pressure to achieve ischemia was provided. Limb ischemia was confirmed by a change in skin color and a decrease in subcutaneous limb temperature. Following limb reperfusion, the skin color turned back to pink, and the under-skin temperature reached the baseline temperature.

^||Cuff pressure was originally inflated at 20 mmHg over the patient's baseline systolic blood pressure, then it was increased until the dorsalis pedis pulse was abolished, as confirmed by a Doppler ultrasonography. This pressure was maintained for 5 min throughout the duration of the inflation cycle. aSAH: Aneurysm subarachnoid hemorrhage; MI: Myocardial ischemia; RIC: Remote ischemic conditioning; TBI: Traumatic brain injury.

Therefore, we included only healthy young male adults to eliminate confounding factors contributing to different protein expressions, such as older age, gender, and comorbidities. We aimed to investigate immediate changes in the proteomic profile after single RIC stimulation in healthy young male adults, which was in consistence with our previous RIC intervention parameters.^[1-4]

Methods

Subject selection

The Xuanwu Hospital Ethics Committee (No.: Clinical research 2015-006) approved the study protocol, and participants provided written informed consent.

Only young male participants were included in this study to avoid the influence of sex hormones and aging on the hemostasis/inflammation system. Meanwhile, we did not include elder people due to the high incidence of other morbidities (e.g., diabetes, hypertension, obesity, cardiovascular disease, lung disease, or chronic kidney disease). A healthy lifestyle was closely evaluated for consecutive six months. We defined a healthy lifestyle based on five factors: never smoking, body mass index (18.5–24.9 kg/m²), moderate to vigorous physical activity (>30 min/day), moderate alcohol intake (male 5–30 g/day), and diet quality score measured by Alternate Healthy Eating Index (AHEI; an AHEI score in the top 40% of the initial enrolled participants).^[22,23] After a six-month observation of a healthy lifestyle and without a history of any chronic disease, medication, and/or substance abuse, the selected participants were then fasted overnight and received single RIC stimulation [Figure 1A]. The experimental design of sampling after overnight fasting is to exclude the possible influence of food on regulating the expression of proteins related to lipid metabolism.

Figure 1: Participants’ inclusion process and sample collection scheme. I: Ischemia; R: Reperfusion; RIC: Remote ischemic conditioning.

Limb RIC intervention protocol

All participants underwent only one cycle of RIC using an automatic RIC device (patent number ZL200820123637.X, Beijing Renqiao Cardiovascular and Cerebrovascular Disease Prevention Research Jiangsu Co., Ltd., China) which executed programmed blood pressure cut-off and on cycles. The device could simultaneously block the venous and arterial blood flows in bilateral upper limbs with 200 mmHg cuff pressure for 5 min and then deflate for 5 min for reperfusion. Each cycle consisted of five bouts, lasting for a total of 50 min. This RIC intervention protocol (five times of limb ischemia/reperfusion) was based on our previous findings of the beneficial effects of this protocol in the Chinese population with cerebrovascular diseases.^[1-4]

Blood sampling

Blood was drawn from the same arm median cubital vein from all participants at three time points, including baseline (pre-RIC), 5 min after RIC, and 2 h after RIC, respectively. Figure 1A presents the whole experimental scheme. The time selection of samples was based on our previous study.^[24]

Safety measures

Vital signs, including respiration, blood pressure, and heart rate, were closely monitored to ascertain procedural safety during RIC intervention. Local skin integrity was evaluated by examining the indications of erythema, swelling, and ecchymosis.

Sample analysis

We analyzed a total of 12 plasma samples in this study (four participants and three time-points). Plasma samples were first pre-treated using Pierce^™ Top2 protein depletion kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), then 100 μL of cleared plasma sample of each subject were mixed with polyethylene glycol (PEG) 6000 to a final concentration of 12% for the mass spectrometric analysis [Figure 1B].

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Overnight on-column trypsinization at 37°C was performed by adding trypsin at a ratio of 1:30 (enzyme:protein) to denature plasma samples (95°C). Columns were centrifuged and washed, and digested protein fragments were eluted at pH 10 with gradually increasing percentage of acetonitrile (ACN) (6%, 9%, 12%, 15%, 18%, 21%, 25%, 30%, and 35%) through an in-house C18 conversion cartridge (Beijing Genomics Institute, China). Finally, samples were pulled together by each time-point and equally distributed (500 ng each) into three parts for vacuum dry (Speedvac, Eppendorf). Then, dried samples were mixed with 5 μL of trifluoroacetic acid (TFA, 0.1%) and a saturated solution of α-cyano-4-hydroxy-trans-cinnamic acid in 50% ACN at a 1:1 ratio to ensure resuspension. Then, 1 μL of the mixture was spotted onto a stainless-steel plate.

We used the nLC-Easy1000-Orbitrap Fusion Tribid MS system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) to analyze peptide mass. Buffer A was composed of 0.1% formic acid (FA), and buffer B was prepared by mixing buffer A with ACN. Samples were separated through a gradient of 5–31% ACN in 0.1% FA (buffer B) on the ReproSil-Pur Basic C18 column (120 mm × 150 μm, 1.9 μm particle size; Dr. Maisch HPLC GmbH, Ammerbuch, Germany) at 250 nL/min flow rate. The MS parameter setup was performed as described in our previous studies.^[18]

Proteomics data analysis

The MaxQuant software (v1.3.1; https://www.maxquant.org) was used for raw data review and analysis, as described elsewhere.^[16] MaxQuant and MaxLFQ algorithms were used to extract label-free quantitation (LFQ) values following log₂ transformation.

Parametric one-way analysis of variance (ANOVA) was applied to compare the variations in protein expressions (considering the normal physiological distribution of quantitative MS data) at different time points (log₂ of the difference in total intensities/target protein), which are presented as volcano plots.

Three databases were used for functional annotation and classification of unique sequences assembled, such as (1) Gene Ontology (GO) database to identify the biological functions of target proteins by dividing the functions into three categories, namely biological process (BP), molecular function (MF), and cellular component (CC); (2) Eukaryotic Orthologous Group (KOG) classification to evaluate the effectiveness of the annotation process;^[25] (3) Kyoto Encyclopedia of Genes and Genomes (KEGG) database to predict pathway involvement with respect to a systematic, network-based description of the molecular interactions between genes.^[26] A detailed illustration of the difference between GO, KOG, and KEGG analysis was presented in Supplementary Table 1 (https://links.lww.com/CM9/B416).

Differences were considered significant at a two-sided P value < 0.05. Analyses were performed using R software (version 3.6.2 [2019-12-12]).

Results

A total of 200 male participants were first subjected to a thorough physical examination, including that of neurological, cardiovascular, respiratory, and gastrointestinal systems. After a six-month observation of a healthy lifestyle, four adult male volunteers with age of 24.5 ± 1.3 years and normal blood routine test results were finally enrolled [Supplementary Table 2, https://links.lww.com/CM9/B416].

Differential plasma protein expression profiling between baseline and post-RIC groups

We observed significant correlations between abundant plasma proteins in four participants at designated time points, indicating downstream differential profiling was reliable [Supplementary Figures 1A–1C, https://links.lww.com/CM9/B416].

We noticed significant differential upregulation of five proteins and downregulation of two proteins between the baseline and post-RIC groups [Figure 2 and Supplementary Tables 3–5, https://links.lww.com/CM9/B416]. Differential expression profiles were visualized in a volcano plot [Figure 3]. Mannosyl-oligosaccharide 1,2 alpha-mannosidase IA (MAN1A1) was upregulated at 5 min and 2 h post-RIC samples. Precursor proteins were likely to be regulated after single RIC stimulation. The levels of carboxypeptidase N catalytic chain precursor (CPN precursor) and mannan-binding lectin serine protease 1 isoform 2 precursor (MASP1 isoform 2 precursor) were immediately increased at 5 min after RIC. While hepatocyte growth factor activator preproprotein (HGF activator preproprotein) and apolipoprotein F precursor (ApoF precursor) proteins were upregulated at 2 h after RIC. However, compared with the proteomic changes at 5 min after RIC, differentially downregulated proteins, including complement C1q subcomponent subunit C precursor (C1qc precursor) and fibronectin isoform 5 preproprotein (FN preproprotein) were found to show decreased levels at 2 h after RIC.

Figure 2: Profiling of the differential plasma protein expression between baseline and the post-RIC groups. The three groups of blood samples referred to samples taken from the same arm at baseline (i.e., no intervention except blood sampling), and at 5 min and 2 h after the completion of a single time RIC stimulus, demonstrating an immediate and early global proteomic response to the RIC stimulus. The expression level of the differential plasma protein was visually categorized on a scale based on chromatic aberration changes (from red to blue), with the color of red indicating the Log₂FC value of 2 and color of blue indicating the Log₂FC value of -2. Every color block represents the value interval of 0.5. ApoF precursor: Apolipoprotein F precursor; C1qc precursor: Complement C1q subcomponent subunit C precursor; CPN precursor: Carboxypeptidase N catalytic chain precursor; FC: Fold change; FN preproprotein: Fibronectin isoform 5 preproprotein; HGF activator preproprotein: Hepatocyte growth factor activator preproprotein; MAN1A1: Mannosyl-oligosaccharide 1,2-alpha-mannosidase IA; MASP1 isoform 2 precursor: Mannan-binding lectin serine protease 1 isoform 2 precursor; RIC: Remote ischemic conditioning.

Figure 3: Volcano plot demonstrating the differential plasma protein expression between the baseline and post-RIC groups. Volcano plot showing plasma protein level changes between: baseline and 5 min after RIC (A), baseline and 2 h after RIC (B), 5 min and 2 h after RIC (C). X-axis: protein level difference indicated by log₂ fold change; Y-axis: statistical significance indicated by −log10 (P value). In total, three, three, and two proteins had fold changes >2 (at a P value < 0.05) for (A), (B), and (C), respectively. Upregulated proteins are highlighted in red and downregulated proteins are green. ApoF precursor: Apolipoprotein F precursor; C1qc precursor: Complement C1q subcomponent subunit C precursor; CPN precursor: Carboxypeptidase N catalytic chain precursor; FN preproprotein: Fibronectin isoform 5 preproprotein; HGF activator preproprotein: Hepatocyte growth factor activator preproprotein; MAN1A1: Mannosyl-oligosaccharide 1,2-alpha-mannosidase IA; MASP1 isoform 2 precursor: Mannan-binding lectin serine protease 1 isoform 2 precursor; RIC: Remote ischemic conditioning.

Functional annotation and classification of the assembled unique sequences

GO analysis

GO database analysis identifies specific functions of proteins based on their standardized vocabulary. GO classifications were categorized as MF, BPs, and CCs. Table 2 and Supplementary Tables 6–8 (https://links.lww.com/CM9/B416) show the comparison of differentially expressed proteins between baseline and post-RIC time-points. The immediate upregulated proteins, compared with the baseline, were mainly enriched in samples of 5 min and 2 h post-RIC groups. However, immediate downregulated proteins were observed in significantly higher proportions at 2 h post-RIC, compared with 5 min post-RIC time-point.

Table 2 - Major upregulated/downregulated proteins of four enrolled healthy young male adults after RIC based on GO analysis.

Protein	Modulation	Main function	Major changing time
Mannosyl-oligosaccharide 1,2-alpha-mannosidase IA	Upregulated	Post-translational protein modification, mannosidase activity	5 min, 2 h
Carboxypeptidase N catalytic chain precursor	Upregulated	Bradykinin catabolic process, metallocarboxypeptidase activity	5 min
Mannan-binding lectin serine protease 1 isoform 2 precursor	Upregulated	Regulation of complement activation, lectin pathway	5 min
Hepatocyte growth factor activator preproprotein	Upregulated	Serine-type endopeptidase activity, blood coagulation	2 h
Apolipoprotein F precursor	Upregulated	Cholesterol metabolic process	2 h
Complement C1q subcomponent subunit C precursor	Downregulated	Complement activation, classical pathway	5 min vs. 2 h
Fibronectin isoform 5 preproprotein	Downregulated	Cell adhesion, cell motility	5 min vs. 2 h

GO: Gene ontology; RIC: Remote ischemic conditioning.

At 5 min after RIC, three upregulated proteins (namely MAN1A1, CPN precursor, and MASP1 isoform 2 precursor) were identified in the following dominant sub-categories: “biological regulation” “cellular process” and “immune system process” in BPs; CCs included “cellular anatomical entity” and “intracellular components”; and MF sub-category indicated “binding” and “molecular function regulator”. Hence, the biological regulation, intracellular metabolic mechanism, and immunological process regulation were major components in the GO classification of these annotated sequences.

At 2 h after RIC, increased levels of MAN1A1, ApoF precursor, and HGF activator preproprotein were observed when compared with baseline proteomic level. They were significantly involved in the BPs (“metabolic process”), CCs (“cellular anatomical entity” and “intracellular components”), as well as in “binding” and “catalytic activity” sub-categories among MF.

The only two downregulated proteins (C1qc precursor and FN preproprotein) were found at 2 h after RIC, compared with 5 min post-RIC group. “Cellular process,” “binding,” and “molecular function regulator” were mainly involved.

KOG analysis

To further evaluate the biological functions of these differentially regulated proteins, we performed KOG-based functional classification [Supplementary Tables 9 and 10, https://links.lww.com/CM9/B416]. We detected three proteins that were differentially regulated following RIC stimulation, including MAN1A1 and related glycosyl hydrolases, trypsin, and fibrillins and related Ca²⁺-binding EGF-like domain-containing proteins which were reportedly involved in carbohydrate transport, amino acid transport, and signal transduction mechanisms, respectively [Table 3].

Table 3 - Main functions of enriched proteins of four enrolled healthy young male adults after RIC based on KOG analysis.

Protein	KOG ID	Functional categories	Definition	Major changing time
MAN1A1 and related glycosyl hydrolases	KOG2204	Metabolism	Carbohydrate transport and metabolism	5 min, 2 h
Trypsin	KOG3627	Metabolism	Amino acid transport and metabolism	5 min
Fibrillins and related proteins containing Ca2+-binding EGF-like domains	KOG1217	Cellular process and signaling	Signal transduction mechanisms	2 h

ID: Identification; KOG: Eukaryotic Orthologous Group; RIC: Remote ischemic conditioning; MAN1A1: Mannosyl-oligosaccharide alpha-1,2-mannosidase.

KEGG analysis

This pathway analysis facilitates the identification of the interaction network of the target protein to dissect the biological functions of the respective protein entity. Supplementary Tables 11–13, https://links.lww.com/CM9/B416, show four major differentially enriched pathways, including N-glycan biosynthesis, protein processing in endoplasmic reticulum, and complement and coagulation cascades, involving altered proteins between the baseline and post-RIC groups [Table 4].

Table 4 - Major enriched pathways of four enrolled healthy young male adults after RIC based on KEGG analysis.

Pathway	Pathway ID	Major changing time
N-glycan biosynthesis	hsa00510	5 min, 2 h
Complement and coagulation cascades	hsa04610	5 min, 5 min vs. 2 h
Protein processing in endoplasmic reticulum	hsa04141	2 h

ID: Identification; KEGG: Kyoto Encyclopedia of Genes and Genomes; RIC: Remote ischemic conditioning.

Discussion

Our study revealed the hyperacute (5 min) and acute (2 h) proteomic changes following single RIC stimulation in healthy young male adults. A strict sample selection process was implemented, including systematic physical examinations and 6-month observation of a healthy lifestyle. Moreover, we excluded confounding factors of age, gender, comorbidities, and different RIC intervention parameters, which may provide more reliable results in the effects of RIC. The blood samples were collected at three time points: baseline (pre-RIC), 5 min after RIC, and 2 h after RIC. GO, and KOG analyses and KEGG network identification were then systematically performed, which yielded seven highly altered protein factors and at least four major differentially enriched pathways.

Complement cascades regulation

The complement cascade was ranked one of the major enriched pathways. More specifically, two differentially regulated proteins were found to be related to this pathway. MASP1 isoform 2 precursor protein, a member of the lectin pathway, was upregulated at 5 min after RIC. However, this factor has an inhibitory effect on the activation of the lectin pathway in the complement cascades. C1qc precursor protein level was decreased at 2 h after RIC, compared with that at 5 min after RIC. C1qc precursor is an essential part of C1q which combines with C1r and C1s proenzymes to form the C1 complex, the first component of the serum complement system. Taken together, the immediate effect of one-time RIC might be involved in the inhibition of complement cascades in either lectin (fast-regulated) or classical pathway (slow-regulated). These findings were consistent with that of previous reports. Hepponstall et al^[19] have reported an increase in C3 level at 15 min after RIC and a decrease of C1r, C4B, and C8 levels at 24 h after RIC. However, the RIC intervention protocol was different from ours, such that four cycles of 5 min ischemia alternating with 5 min reperfusion, no cuff pressure value provided.^[19] The RIC stimulus in our study was more intense and longer. This might explain how a one-time application of RIC in our study could induce faster downregulation of complement cascades.

Moreover, although we did not observe any differentially expressed proteins or enriched pathways in the acquired immune system after a single RIC intervention, the consensus in immunology is not changed that activation of the innate immune system can induce enhanced responsiveness to subsequent triggers in the acquired immune system.^[27,28] Therefore, the effects of RIC in the acquired immune system should be further explored in the future.

Coagulation/fibrinolytic system regulation

The hemostasis pathway was also found to be significantly enriched in our experimental analysis. HGF activator preproprotein was successfully identified to be upregulated at 2 h after RIC. HGF acts as the stimulator of mitogenesis, matrix invasion, and cell motility, playing a central role in tumorigenesis, angiogenesis, and tissue regeneration.^[29] Surprisingly, HGF is also a structural homolog of plasminogen, a key enzyme in the fibrinolytic system. The activity of HGF is mainly regulated by the serum-derived protease, HGF activator preprotein, which could efficiently transform HGF to its active form. Besides, the HGF activator preprotein itself could activate the downstream signaling in the blood coagulation cascade, suggesting a crosstalk between tissue damage and HGF activity.^[29] However, Hepponstall et al^[19] have found that levels of FGB and FGG were upregulated as early as 15 min after RIC and sustained up to 24 h. Collectively, a single RIC intervention could induce both stimulatory effects on coagulation and fibrinolytic processes to achieve the hemostatic balance.

Lipid metabolism regulation

ApoF, a structurally unrelated factor to classical apolipoproteins, was upregulated at 2 h after RIC. Since it can inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins, it could serve as the lipid transfer inhibiting factor (LTIF). ApoF is believed to be a high-density lipoprotein (HDL)-associated protein. Like ApoE, ApoF's plasma concentration is about 80 μg/mL, of which 60 μg/mL is associated with HDL. However, ApoF depletion could not alter either plasma lipid concentrations or HDL size.^[30]

The structure and composition of HDL were underestimated before. Recent proteomic analyses revealed more than 50 unique bona fide protein components in HDL.^[31-33] Moreover, many of these protein components have reportedly no known role in inter- or intracellular lipid mobilization, rather involved in diverse cellular processes like innate immunity, protease inhibition, platelet function, complement activation, and inflammation.^[33] It raised the question of whether ApoF could modulate HDL functionality to regulate lipid metabolism.

Previous proteomic studies also indicated the modulation of apolipoprotein expressions after RIC. Hepponstall et al^[19] have demonstrated that ApoA-I, A-II, and D levels are upregulated during the early response (15 min) and late response (24 h) phases after one-time RIC stimulation in healthy young adults. Previously, we showed that long-term use of RIC might elevate the ApoA-II level in the primate ischemic stroke model.^[18] Thus, we speculated that the induced elevation of multiple apolipoproteins post-RIC might be consistent with the effects of lipid metabolism regulation by repeated RIC stimulations.

Others

Although CPN precursor and MAN1A1 were found significantly upregulated, their related pathways (inflammation and protein glycosylation, respectively) were not enriched in KEGG analysis. This might be due to insufficient elicitation of these cellular pathways by single RICs.

CPN precursor was found to increase at 5 min after RIC. Previous studies have demonstrated that CPN could protect the body from potent inflammatory and vasoactive peptide proteins containing C-terminal Arginine or Lysine (such as kinins or anaphylatoxins) released into the circulation.^[34-36] The increased level of CPN precursor might indicate an increase in the matured CPN level, which could induce an anti-inflammatory process. This is consistent with our previous proteomic analyses in the primate stroke model. It has been shown that anti-inflammatory pathways are enriched after long-term (5-week) RIC intervention.^[18] Thus, we may speculate that healthy adults subjected to long-term RIC may benefit from induced anti-inflammatory responses and prevention from various vascular diseases, such as coronary artery disease and cerebral stenosis.

Our study, however, had several limitations. First, more blood sampling time-points would be ideal for comparing the related proteomes in post-RIC intervention in healthy young male adults. For instance, adding the proteomic analyses at 15 min and 24 h after RIC could improved the reliability of the data. Second, the inclusion of different RIC parameters (e.g., cuff pressure [220 mmHg vs. 200 mmHg vs. 180 mmHg], inflation/deflation cycle time [5 min vs. 10 min], and bouts frequency [5 times vs. 4 times per week]) might induce more diverse and intense effects on plasma proteome. Future studies are needed to explore more on these questions. Additionally, we included only young male adults in this study to exclude other confounding factors, such as the estrogen/progesterone effect on the inflammatory process in women and the comorbidity of chronic diseases or vascular risk factors in elderly people. The findings should be interpreted with caution due to the limited sample size and population. In the future, we could further evaluate the effect of RIC in plasma proteome in a population with certain disease entities and gender variation. Clinical studies with larger sample sizes are needed to confirm the results obtained in this study. Moreover, epigenetic and metabolic reprogramming events should also be investigated.

In conclusion, after a single RIC stimulation in healthy young male adults, several differentially modulated proteins and their respective enriched pathways were observed in quantitative proteomic analysis. Anti-inflammation (inhibitory effect on complement cascades in terms of both classical and lectin pathways and upregulated levels of CPN precursor), balanced hemostasis (stimulatory effect on coagulation and fibrinolysis), and lipid metabolism regulation (upregulation of ApoF) were the major effects induced by one-time application of RIC. Thus, a single RIC intervention may exert protective effects in the initial phase, which facilitates our understanding of the application of RIC in the emergency or prehospital settings. Moreover, we suggest the beneficial effect of repeated RIC interventions in preventing vascular diseases among general populations.

Funding

This work was supported by the National Key R&D Program of China (2017YFC1308400), the National Natural Science Foundation (81371289), and the Beijing Natural Science Foundation (7212047), and Capital Medical Development Scientific Research Fund (2022-2-2015).

Conflicts of interest

None.