In recent decades, it has been suggested that exosomes may contribute to the onset and progression of cardiovascular diseases7. Plasma exosomes participate in the inflammatory immune response in acute myocardial infarction20. Acute myocardial infarction patients' protein expression in the blood is distinct from that of patients with SAP21. Exosomes of MSCs isolated from human umbilical cord blood have cardioprotective effects in animal models of AMI and cardiomyocyte hypoxia injury22, as reported by Marta Zarà et al22. This study used plasma profiling proteomic technique and found that in AMI and SAP, SERPIND1, MASP1, FCN2, and AMBP were upregulated whereas HLA-C was found to be downregulated. Compared to SAP, SERPIND1, MASP1, FCN2, and AMBP were upregulated, and HLA-C was downregulated as well. These proteins may contribute to the progression of coronary atherosclerosis.
Serine protease inhibitors (serpins) are the largest superfamily of protease inhibitors in the body. They are distributed throughout the whole body. Thus, they can play different biological functions due to their different distribution positions23. Serpind1 is an important member of the serpin superfamily24 and is also known as heparin cofactor II (HCII). Studies have shown that this gene is related to the coagulation system, vascular injury, inflammatory response, and tumors25, 26. Heparin cofactor II (HCII) and antithrombin III (ATIII) are two circulating serpins that are crucial for preventing thrombosis by inhibiting thrombin in a glycosaminoglycan-dependent way. Improved plasma HCII activity independently lowers the prevalence of angiographic restenosis, as revealed by Takamori et al27. Huang PH et al. showed that individuals with coronary heart disease or acute coronary syndrome have elevated plasma HCII activity, which correlated significantly with endothelial vasodilation and hence can predict future cardiovascular events28. After hospitalization, plasma HCII activity was evaluated in the blood of 110 consecutive AMI patients examined by Shao-Sung Huang et al29. Each patient was assigned to one of three categories based on their HCII levels: high, normal, or low. The incidence of major adverse cardiovascular events (MACE) was reduced in subjects without diabetes who had higher plasma HCII activity. Plasma HCII activity was an independent predictor of future MACE in a Cox multiple regression evaluations involving all patients. These findings suggested that plasma HCII activity may predict subsequent cardiovascular events following AMI. More importantly, HCII activity may considerably contribute to atherothrombosis29. Biglycan, through the heparin cofactor II pathway, was shown to cause severe atherosclerosis in mice30. After injuring the femoral artery with a cuff or a wire, HCII (+/-) mice showed rapid intimal hyperplasia and frequent thrombosis, as described by Ken Ichi Aihara31. Recent research has linked serpind1 (HCII) to an increased risk of cardiovascular problems such as heart attacks and strokes. Based on our findings, serpind1 expression in plasma exosomes is 1.09 times higher in patients with MI compared to those with SAP and 1.12 times higher than those without coronary heart disease. These results implicated that serpind1 may considerably contribute to myocardial infarction development.
Mannose-binding lectin 2 (MBL2) is one of the glycoproteins of the complement system. It is the most important first-line anti-infective immune molecule in host nonspecific immunity. It can recognize and bind to a variety of pathogens and play an anti-infective role by regulating cell phagocytosis and activating the complement system. MBL2 needs to bind to serine proteases related to mannan-binding lectins (masp-1 and masp-2) to activate the lectin cascade, start the complement system and resist whole-body infection32. Mannan binding lectin linked serine protease (MSAP) complements the lectin cascade of MSAP − 1 and MSAP − 2 and contributes to thrombosis33. V Frauenknecht33 found that the level of MASP-1 was the highest in patients with subacute myocardial infarction and the lowest in patients with acute stroke, suggesting that the level of MASP-1 may change in vascular diseases. The next year, Charlotte B. Holt showed that although MASP-1 levels were higher in MI patients, they were unrelated to their prognosis. According to these data, plasma levels of MASP-1 are much greater in MI patients than in healthy control individuals, although they are not related to immediate outcomes as determined by the salvage index and final infarct34. Rui-Xia Song35 found that plasma MASP-1 levels were high in children with Kawasaki disease who were recovering from coronary artery lesions. The above studies support that MASP-1 is linked with coronary artery disease and MI. However, there has been no study on the expression of MASP-1 in plasma exosomes after myocardial infarction. This study found that the plasma level of MASP-1 in patients with MI and SAP was elevated than that in the control group (ratios of 1.54 and 1.41, respectively), which provides a new basis for MASP-1 as a biomarker of myocardial infarction.
Ficolin-2 (FCN-2) is one of the few molecules that may stimulate the lectin complement cascade. It can form a complex with lectin-related serine proteases (MASPs)-1, -2, and − 3, resulting in various biological effects36. Ficolin-2 can bind calreticulin in phagocytes through its collagen-like domain to regulate bacterial phagocytosis and the apoptosis of cells36. Combined with the role of MASP-1 in myocardial infarction, we speculate that FCN may be related to arteriosclerosis and myocardial infarction. FCN was found to be substantially expressed in intracranial blood during human ischemic stroke, according to Benton Maglinger's research37, suggesting that FCN2 may play a role in atherosclerotic variations. According to research by Jaydeep A. Badarukhiya38, FCN2 variants and haplotypes are linked to rheumatic heart disease. Proteomics was applied by Yilong Pan39, who found new plasma biomarkers for STEMI and NSTEMI patients. High FCN2 levels were confirmed by ELISA during the course of AMI. This was the first study to specifically address that patients with AMI have elevated levels of FCN2 expression in their peripheral blood. We observed that patients with MI had elevated levels of FCN2 expression in their plasma exosomes than patients with SAP or the control group (AMI/SAP ratio = 1.33, AMI/CON ratio = 1.69, SAP/CON ratio = 1.29). Our findings validate Yilong Pan's findings at the exosome level.
α1-Microglobulin/bikunin precursor (AMBP) is a glycoprotein synthesized and secreted by hepatocytes that is related to tissue injury, the regulation of immunity, and defense against cell oxidation40. Reported studies have revealed that the expression of AMBP is increased in elderly patients with cardiovascular diseases41. Salaheldin Ahmed42 discovered that the expression of AMBP in heart failure with maintained ejection fraction and pulmonary hypertension was greater than in pulmonary arterial hypertension. The amount of AMBP was elevated in the coronary artery section with significant atherosclerotic stenosis, as determined by RT-qPCR43. Proteinuria is a major cardiovascular disease risk factor in diabetic people. AMBP is favorably connected with proteinuria in diabetic individuals, however, no correlation has been identified between AMBP and coronary calcification44. Joo Pedro Ferreira45 observed in the EXAMINE study that troponin and AMBP added predictive value for MI (cardiovascular death, heart failure, all-cause death, and combinations of these outcomes). The above studies are the latest literature in recent years that suggest that AMBP is related to heart disease and myocardial infarction. Our research revealed that there was a considerable increase in the expression of AMBP in the blood exosomes of myocardial infarction patients, which presents a more solid theoretical foundation for the investigation of AMBP's role in myocardial infarction.
HLA-C is a locus of human leukocyte antigen (HLA) located on chromosome 6. Mature HLA-C protein will interact with β2 microglobulin to form a heterodimer and is anchored to the cell surface as a receptor. There are different conclusions regarding the role of HLA-C in coronary artery disease. After investigating the link between the HLA-C locus and restenosis during coronary balloon angioplasty, Y Watanabe46 found that CW1 was inversely connected with restenosis at the HLA-C locus. Therefore, HLA-C may be a helpful diagnostic for predicting restenosis following percutaneous transluminal coronary angioplasty (PTCA). By examining methylation patterns in coronary artery disease, Shyamashree Banerjee found that HLA-C was stimulating the inflammatory cascades47. Recent genome-wide association studies (GWAS) have shown new data regarding the molecular cascades that are connected to atherosclerosis and its progression38. CAD risk is linked to loci relevant to inflammation38 which include CXCL2, which encodes atherosclerotic protective chemokines, and the region near HLA-C in the main histocompatibility site on chromosome 6p2148. A new CAD locus located on chromosome 6p21.3 was identified in the major histocompatibility complex (MHC) between hcg27 and HLA-C and achieved genome-wide significance in joint analysis49. Our results agree with these studies. We found that in plasma and plasma exosomes, HLA-C was decreased by more than 50% in acute myocardial infarction patients (AMI/CON ratio = 0.48) and SAP patients (SAP/CON ratio = 0.53).
In conclusion, The expression of four plasma exosomes biomarkers in AMI patients and stable angina pectoris (SAP) was higher than that in noncoronary heart disease (NCHD) patients, and HLA-C was found to be downregulated not only in exosomes, but also in plasma. The obtained results serve as new candidate targets for the detection and therapy of AMI.
Limitations
Our research is a preliminary exploration and has not been applied in a clinic, which needs large-scale multicentric studies for safeguarding and validation research. Therefore, the specific mechanism and its relationship with prognosis need to be further explored.