Extracellular vesicles (EVs) are lipid-bound vesicles released from cells under physiological and pathological conditions. Basing on biogenesis, dimension, content and route of secretion, they can be classified into exosomes, microvesicles (MVs) and apoptotic bodies. EVs have a key role as bioactive mediators in intercellular communication, but they are also involved in other physiological processes like immune response, blood coagulation, and tissue repair. The interest in studying EVs has increased over the years due to their involvement in several diseases, such as cardiovascular diseases (CVDs), and their potential role as biomarkers in diagnosis, therapy, and in drug delivery system development. Nowadays, the improvement of mass spectrometry (MS)-based techniques allows the characterization of the EV protein composition to deeply understand their role in several diseases. In this review, a critical overview is provided on the EV’s origin and physical properties, as well as their emerging functional role in both physiological and disease conditions, focusing attention on the role of exosomes in CVDs. The most important cardiac exosome proteomic studies will be discussed giving a qualitative and quantitative characterization of the exosomal proteins that could be used in future as new potential diagnostic markers or targets for specific therapies.

Human serum albumin (HSA) is associated with several physiological functions, such as maintaining oncotic pressure and microvascular integrity, among others. It also represents the major and predominant antioxidant in plasma due to the presence of the Cys34 sulfhydryl group. In this study, we assessed qualitative and quantitative changes in HSA in patients with heart failure (HF) and their relationship with the severity of the disease. We detected by means of mass spectrometry a global decrease of the HSA content in the plasma of HF patients in respect to control subjects, a significant increase of thio-HSA with a concomitant decrease in the reduced form of albumin. Cysteine and, at a lesser extent, homocysteine represent the most abundant thiol bound to HSA. A strong inverse correlation was also observed between cysteine-HSA and peak VO₂/kg, an index of oxygen consumption associated with HF severity. Moreover, in HL-1 cardiomyocytes incubated with H₂O₂, we showed a significant decrease of cell viability in cells treated with thio-HSA in respect to restored native-HSA. In conclusion, we found for the first time that S-thiolation of albumin is increased in the plasma of HF patients and induced changes in the structure and antioxidant function of HSA, likely contributing to HF progression.

Heart failure (HF) is a complex disease due to the intricate interplay of several mechanisms, which therefore implies the need for a multimarker strategy to better personalize the care of patients with HF. In this study, we developed a targeted mass spectrometry approach based on multiple reaction monitoring (MRM) to measure multiple circulating protein biomarkers, involved in cardiovascular disease, to address their relevance in the human HF, intending to assess the feasibility of the workflow in the disease monitoring and risk stratification. In this study, we analyzed a total of 60 plasma proteins in 30 plasma samples from eight control subjects and 22 age- and gender- matched HF patients. We identified a panel of four plasma proteins, namely Neuropilin-2, Beta 2 microglobulin, alpha-1-antichymotrypsin, and complement component C9, that were more abundant in HF patients in relation to disease severity and pulmonary dysfunction. Moreover, we showed the ability of the combination of these candidate proteins to discriminate, with sufficient accuracy, HF patients from healthy subjects. In conclusion, we demonstrated the feasibility and potential of a proteomic workflow based on MRM mass spectrometry for the evaluation of multiple proteins in human plasma and the identification of a panel of biomarkers of HF severity.

Cigarette smoking is a major independent risk factor for cardiovascular diseases (CVD). The underlying mechanisms, however, are not clearly understood. Lungs are the primary route of exposure to smoke, with pulmonary cells and surfactant being the first structures directly exposed, resulting in the leakage of the immature proteoform of surfactant protein B (proSP-B). Herein, we evaluated whether proSP-B joined the cargo of high-density lipoprotein (HDL) proteins in healthy young subjects (n = 106) without any CVD risk factor other than smoking, and if HDL-associated proSP-B (HDL-SPB) correlated with pulmonary function parameters, systemic inflammation, and oxidative stress. At univariable analysis, HDL-SPB resulted significantly higher in smokers (2.2-fold, p < 0.001) than in non-smokers. No significant differences have been detected between smokers and non-smokers for inflammation, oxidation variables, and alveolar-capillary diffusion markers. In a multivariable model, HDL-SPB was independently associated with smoking. In conclusion, HDL-SPB is not only a precocious and sensitive index of the acute effects of smoke, but it might be also a potential causal factor in the onset of the vascular damage induced by modified HDL. These findings contribute to the emerging concept that the quality of the HDL proteome, rather than the quantity of particles, plays a central role in CVD risk protection.

Advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs), particularly carboxymethyl-lysine (CML), have been largely proposed as factors involved in the establishment and progression of heart failure (HF). Despite this evidence, the current literature lacks the comprehensive identification and characterization of the plasma AGEs/ALEs involved in HF (untargeted approach). This work provides the first ex vivo high-resolution mass spectrometry (HR-MS) profiling of AGEs/ALEs occurring in human serum albumin (HSA), the most abundant protein in plasma, characterized by several nucleophilic sites and thus representing the main protein substrate for AGE/ALE formation. A set of AGE/ALE adducts in pooled HF-HSA samples was defined, and a semi-quantitative analysis was carried out in order to finally select those presenting in increased amounts in the HF samples with respect to the control condition. These adducts were statistically confirmed by monitoring their content in individual HF samples by applying a targeted approach. Selected AGEs/ALEs proved to be mostly CML derivatives on Lys residues (i.e., CML-Lys12, CML-Lys378, CML-Lys402), and one deoxy-fructosyl derivative on the Lys 389 (DFK-Lys 389). The nature of CML adducts was finally confirmed using immunological methods and in vitro production of such adducts further confirmed by mass spectrometry.