Endothelial Repair and Endothelial Cell-Derived Secretome

Growing evidence supports the hypothesis that endothelial cell-derived microparticles (MPs) might contribute to the pathogenesis of cardiovascular (CV) disease. Endothelial cell-derived MPs play a pivotal role in the regulation of the endogenous repair system, thrombosis, coagulation, infl ammation, immunity and metabolic memory phenomenon. There is evidence that the MPs are secreted actively accompanied to other regulatory molecules. All these actively synthetizing and secreting factors include proteins, adhesion and intercellular signal molecules, peptides, lipids, free DNAs, microRNAs, and even microparticles (MPs) are defi ned as cellular secretome. The proteomic profi le of secretome is under tightly control of genetic and epigenetic mechanisms, which may altered a secretion of the proteins involved into MPs’ organization. Finally, this may contribute the modifi cation of MP’s after their secretion and throughout transfer to the target cells. As a result, communicative ability of endothelial cell-derived MPs may suffi ciently worse. Subsequently, cross talk between some components of secretome might modulate delivering cargos of MPs and their regenerative and proliferative capabilities via intercellular signaling networks. The aim of the review is to discuss the effect of various components of secretome on MP-dependent effects on endothelium. Short Review


INTRODUCTION
For last decade, elevated\circulating level of microparticles (MPs) produced by various types of blood cells have been de ined in the patients with established cardiovascular (CV) disease, as well as in individuals at higher risk of CV events and CV diseases [1][2][3][4]. The endothelial damage occurring in CV disease is considered as an initial step to CV complications, as well as early stage of asymptomatic target organs damage irrespectively to the CV mortality and morbidity. The ultimate goal is to identify useful diagnostic and predictive biomarkers and/or target tools in the vulnerable patient population.
The current challenges in CV medicine have been addressing to improve of actual screening methods and predictive patients' platform. There is suggestive evidence that a number of circulating endothelial cell-derived MPs (EMPs) might be a clinically useful biomarker, which may pretty accurate predict CV complications in general populations and clinical outcomes patients with known CV disease [5][6][7].
Although an origin of endothelial cell-derived MPs from activated or apoptotic cells is crucial for realizing tissue repair, degenerative processes modulation, immune mediation, and directly/indirectly vascular injury [8], there are several controversies regarding an involvement of MPs in pathogenesis of CV disease [9]. The irst controversy affects the pathophysiological properties of MPs. Indeed, the MPs secreted by activated endothelial cells may contribute to tissue reparation, restore endothelial function, mediate progenitor cell mobbing and differentiation, whereas apoptotic MPs are able directly injury endothelial cells and via a transfer of several proteins, active molecules, chromatin compounds including microRNAs and DNAs, regulate in lammation, coagulation, and immune response [10]. The next controversy relates a different presentation of endothelial cell-derived MPs in plasma of healthy individuals and changing of their numbers in various CV diseases and CV risks. Interestingly, circulating number of MPs originated from apoptotic endothelial cells increases in patients with CV risk factors, after newly CV events and in individuals with established CV disease. However, the ability of activated endothelial cells to active secret MPs progressively decreases depending on CV risk presentation, i.e. diabetes mellitus, abdominal obesity, insulin resistance, renal disease, and is due co-existing endothelial disintegrity [11][12][13]. Unfortunately, although there is strong association between circulating number of activated endothelial cell-derived MPs and CV risk, elevated level of apoptotic endothelial cell-derived MPs appears to be much more accurate predictive biomarker relating to CV death and CV diseases progression [14]. Another controversy is that the endothelial cell-derived MPs are constitutive biomarker of endothelial dysfunction playing a pivotal role in in lammation, vascular injury, angiogenesis, and thrombosis, but their predictive value on CV manifestation and progression is beyond severity of endothelial dysfunction. In fact, the imbalance between number of circulating endothelial cell-derived MPs distinguished their origin (activated or apoptotic endothelial cells) can be applied as more promising routine tests to improve CV risk prediction [15,16]. Whether "impaired phenotype" of endothelial cell-derived MPs as a causality factor contributed the vascular "competence" in CV disease is a predominantly pre-existing phenomenon associated with genetic/ epigenetic performances or is resulting in various metabolic and age-dependent factors is not clear. There is a large body of evidence regarding that the biological abilities of EPCs are likely under posttranslational control of their secretome and that restore endothelial integrity after vascular injury by EPCs might relate to components of secretome, especially signature of MPs. Probably, variable effect of endothelial cellderived MPs might relate to secretome particularities and the triggers, which induced cell mechanisms of synthesis and secretion of secretome. The main mechanisms involve secretome in the regulation of EPC' recruitment, migration, differentiation, and release of pro-angiogenic factors are not fully investigated and require more explanations.
The aim of the review is to discuss the effect of various components of secretome on endothelium through MP-dependent mechanisms.

Defi nition of Secretome
The variety spectrum of paracrine factors secreted by cells is due speci ic and non-speci ic triggers and exerted biological effects on target cells are determined secretome. By now, the secretome is considered a collection of factors consisting of transmembrane proteins and other components secreted by cells into the extracellular space. All these actively synthetizing and secreting factors include proteins, adhesion and intercellular signal molecules, peptides, lipids, free DNAs, microRNAs, and extracellular vesicles (i.e. exosomes and MPs). A signi icant portion (roughly 20%) of the human secretome consists of secretory proteins incorporated into MPs. Interestingly, the proteomic pro ile of secretome is under tightly control of genetic and epigenetic mechanisms, which may altered a secretion of the proteins involved into MPs' organization. Nevertheless, the secretome of endothelial cells and endothelial progenitors could be altered by ischemia, hypoxia, in lammation, endotoxemia, none-speci ic cell activation, and coagulation [17]. Finally, all this may contribute the modi ication of MP's after their secretion and throughout transfer to the target cells [18]. Although the innate mechanisms of alterations in the proteome of MPs with different origins are completely clear, the cellular secretome with preferential up-regulation and down-regulation mechanisms of broad spectrum of proteins (i.e. matricellular proteins, cell-cell adhesion molecules, pro-coagulants, growth factors) incorporated into MPs is considered a powerful trigger of tissue response to injury [17][18][19]. Indeed, the cell-to-MPs' cooperation depends on presentation of the surface of the vesicles and target cells appropriate recognizing antigens, which interact with secreted proteins and consequently regulate in different manner the tissue response via switching speci ic transmembrane signal systems to nuclear transcriptional factors (i.e., NK-kB) [20]. Recently it has been clear shown that the components of the secretome might be attributes of overactive immune system during antigen stimulation and probably act to counterbalance the release of many in lammatory and damageable substances and associates with vascular injury and tissue damages [21]. Overall, the role of MPs as component of cell secretome with post-transcriptionally modi ied regulatory activity is under wide discussion.
Thus, mature endothelial cells and endothelial progenitor cells may communicate with each other by means of exchange of MPs independently of direct cell-cell contacts with secreted proteins or diffusible soluble stimulatory factors secreted by other cells, i.e. activated mature endothelial cells, progenitor cells, and circulating antigen presenting cells, immune cells [22].

Defi nition of microparticles
MPs are large and very variable on their shapes and dimensions (predominantly 100-1000 nm) population of microvesicles, which are shedding from plasma membranes of parent cells in response to cell activation, injury, and/or apoptosis [23,24]. Under normal physiological condition a phospholipid bilayer of plasma membrane of cells represented phosphatidylserine and phosphatidy lethanoalamine in inner lea lets, whereas phosphatidylcholine and sphingomyelin represent in the external lea lets. The asymmetrical distribution of phospholipids in the plasma membrane is supported by activity of three major intracellular ATP-dependent enzyme systems, i.e. lippase, loppase, and scramblase. Because aminophospholipids are negatively charged, but phospholipids exhibit neutral charge, the main role of intracellular enzyme systems is supporting electrochemical gradient. Both lippase and loppase belong to family of ATP-dependent phospholipid translocases. The lippase translocates phosphatidylserine and phosphatidylethanoalamine from the external lea lets to the inner one. The loppase transports phospholipids in the opposite direction. Finally, scramblase being to Ca 2+ -dependent enzyme system exhibits unspeci ically ability of moving of phospholipids between both lea lets of plasma membrane [25].
Importantly, disappearing of the asymmetrical phospholipid distribution in the bilayer of the cell membrane is considered a clue for vesiculation and forming of MPs. Indeed, both processes of apoptosis or cell activation are required asymmetry in phospholipid distribution that leads to cytoskeleton modi ications, membrane budding and MPs release. The mechanism of vesiculation closely affects genome and may mediate by some triggers including in lammation, while in some cases there is a spontaneous release of MPs from stable cells or due to injury from necrotic cells or from mechanically damaged cells [18].
Recently MPs are considered a cargo for various molecules. Indeed, MPs carry proteins, RNA, micro-RNA, and DNA fragments from their cells of origin to other parts of the body via blood and other body luids. Within last decade it has become to know that MPs would act as information transfer for target cells [17]. However, the difference between innate mechanisms affected the release of MPs from stable cells, activated cells or apoptotic cells is yet not fully investigated and requires more studies [26].

Endothelial cell-derived MPs
Endothelial cells-derived MPs are released by inducer like angiotensin II, lipopolysaccharide, and hydrogen peroxide leading to the worsening of endothelial integrity, endothelial dysfunction, development and progression of microvascular in lammation. All these processes relate to atherosclerosis, thrombosis, heart failure and lead to major CV events. However, there are multiple physiological pathways for endothelial cells-derived MPs generation like NADPH oxidase derived endothelial ROS formation, Rho kinase pathway, and mitogen-activated protein kinases. Figure 1 is reported the role of secretome in the regulation of MP's function.
Endothelial cells release phenotypically and quantitatively distinct MPs in activation and apoptosis. As a result, MPs are suf iciently distinguished each other on their ability to contain some antigen presentation [26] and inner components, i.e. matrix metalloproteinases (MMP)-2, MMP-9, MT1-MMP, chromatin, active molecules (heat shock proteins), some hormones (angiotensin II), growth factors (transforming factor-beta) [26][27][28][29]. It is suggested that the epigenetic modi ication of the parent cells might directly impact on functionality of secreted MPs and their ability to in luence various biological effects [30]. Indeed, the endothelial cell-derived MPs isolated from the serum of patients with diabetes mellitus, chronic kidney disease, heart failure and atherosclerosis are defective in induction of vascular relaxation, maturation of progenitor cells and endothelium repair [31,32]. As factors contributing in the response of the target cells after MP stimulation they could be pointed in lammatory cytokines (tumor necrosis factor-alpha, interleukin-[IL] 4, IL-17), glucose, advanced glycation end-products, uremic toxins, free DNA, products of lipid peroxidation [33]. Nonetheless, hypoxia-modi ied endothelial cell-derived MPs are able to carry reactive oxygen species and thereby may impair target cells by promoting apoptosis and oxidative stress [34]. They cannot be excluded the role of metabolomics-regulated microenvironments of target cells as a causative factor modifying the response after MPs' cooperation [35,36]. It has been postulated that activation of p53 subunit, Akt/ GSK-3beta and JAK2/STAT3 signaling pathways are involved in the regulation of MPs' synthesis and that these molecular targets are under close control of various metabolites and intermediates, as well as epigenetics' mechanisms [37]. Thus, secretome of endothelial cells included metabolites, proteins, intermediates, DNAs, reactive oxygen radicals, active molecules, may probably modify and even alter a communicative ability of MPs secreted by endothelial cells [38].

Relation between secretome and endothelial cell functionality
Endothelial cell-derived MPs are not only delivery of intra-vesicular cargo and information, but they may directly modulate vascular function autocrine and paracrine effects on target cells through surface interaction, and cellular fusion. Subsequently, they have in vitro investigated that the MPs and other fractions of secretome might On the one hand, the pro-in lammatory secretome from endothelial cells is reported to have deleterious effects in vasculature in animal models [39,40]. It is known that the broad spectrum of pro-in lammatory cytokines predominantly IL-6 and tumor necrosis factor-alpha via NF-kB activation might be stimulated and sustained secretion of modi ied MPs mediating mobbing and differentiation of endothelial progenitor cells, as well as exaggerated extravascular collagen deposition, leading to adverse vascular remodeling.
On the other hand, it has been suggested that enhancing of the target cell mobility and differentiation through MP production could be worse, inverted or even suf iciently abandoned [41]. Indeed, secretome of apoptotic peripheral blood cells may induce cytoprotection effect instead expected worsening of tissue remodeling in animal model of acute myocardial infarction [42,43]. Additionally, this effect is probably due to the activation of pro-survival signaling cascades in the cardiomyocytes and the increase of homing of regenerative cells through stimulation of metabolite modi ied MPs [44]. Therefore, in clinical settings angiogenic early outgrowth endothelial progenitor cells have been reported to contribute to endothelial regeneration, CV risk and to limit neointima formation after vascular injury through cooperation with metabolically modi ied MPs [45].
Thus, there is a large body of evidence regarding being of modifying effect of secretome components on MPs' ability for tissue regeneration or injury. Moreover, regenerative potency of apoptotic cell secretome was even higher than those in activated cells. However, new phenomenon opens serious perspective to clinical implementation of MPs as not just diagnostic tool with predictive possibilities, but as transfer system with therapeutic potentials [46,47].
Whether endothelial cell-derived MPs are capable to induce variable effects on target cells depending on proteomic of MPs or functional in nature of secretome is not fully understood [48]. In fact, cross talk between some components of secretome including MPs might modulate delivering cargos of MPs through involving the intercellular signaling networks and thereby modify their regenerative and proliferative capabilities [49][50][51].
Overall, the hypothesis regarding that the developing functional EPC incompetence could impede vascular repair via worsening secretome of them through maladaptive transfer of MPs appears to be promising for explanation of some phenomenons, i.e. metabolic memory in diabetes, adipocytokines' regulation in obese and heart failure, insulin resistance in metabolic syndrome, developing of endothelial dysfunction at early stage of CV diseases, vascular aging.

CONCLUSION
The endothelial cell secretome has most commonly been investigated in preclinical settings as a source of regulating factors that in luence target cells and emerges as a crucial player of EMP function. However, the interaction of different components of secretome may modify the MPs' structure and functionality. It has been hypothesized that endothelial regeneration is under tight control of autocrine and paracrine mechanisms affecting parent endothelial cells through modi ication of their secretome. Although there is the commonly used clinically validated biomarkers to identify endothelial injury and dysfunction, lack of endothelial reparation biomarkers, which are assessable in routine clinical practice, is crucial for an assay of probability to respond of the therapy of CV diseases. Through the pooling of data, discovery of the novel biomarkers of endothelial reparation could be improve our scoring of the patients requiring personalized CV care. The matter of metabolic modi ication of one is uncertain and requires more investigations in future. Greater understanding of the underlying mechanisms of impaired endothelial repair system could be a network of creating a novel platform for the evaluation of the novel treatment strategies of CV diseases.