Endothelial Secretome

Abstract

Endothelial cells produce huge proteomes from a relatively small total number of ECs. The ECs’ complex intercellular communication is possible through well-stored, classified, and compartmentalized secretory pathways, intermediated by the secretory vesicles and granules, with the purpose to maintain vascular homeostasis and integrity. Secreted proteins are involved in a myriad of cell communication processes. The local vascular microenvironment dynamically and constantly modifies the ECs’ secretome. We focus on the biological significance of secretome proteins in a healthy vascular microenvironment and under cardiovascular conditions. Vascular ECs crosstalk with other ECs, and other blood cells at a distance, with the circulating hematopoietic stem cells permitting adequate reactions to vascular injury, systemic or local inflammation, and viral or parasitic infections. Here, we overview current secretome biomarkers in vascular diseases, with a focus on their roles in diagnostic, prognostic, and therapeutics. Also, we highlighted some important pathological effects of exosome on cardiovascular disease. This chapter discusses current research directions characterizing vascular pathology conditioned secretomes, their regulation, and therapeutic pursuit. The overall aim of this chapter is to review current literature updates on endothelial secretome roles in endothelial homeostasis and in vascular disorders.

1. Introduction

Endothelium consists of approximately 10¹⁴ cells in all the vasculature [1]. Due to its versatile functions, the endothelium has been compared with a metabolic organ [1]. The ECs’ secretomes comprise all proteins secreted outside the cell, including enzymes, growth factors, and hormones. ECs have the most diverse regulatory roles, starting with a mechanical barrier, vascular tone, hemostasis, and thrombosis (including control of platelet response), inflammation, vascular permeability, and angiogenesis. The interactions of ECs with leukocytes are also mediated by constituents of the ECs’ secretome. The local microenvironment influences ECs’ secretome output. For instance, the presence or absence of certain constituents in the secretory vesicles or granules can be selectively changed by local inflammation or by shear stress [2]. Endothelial cells produce huge proteomes from a relatively small total number of ECs. This is possible by well-stored, classified, and compartmentalized polarized exocytosis [3]. Exchange information at a distance with other ECs, and with other blood cells, with the circulating hematopoietic stem cells. Vascular ECs crosstalk is intermediated by the secretory vesicles and granules with the purpose to maintain vascular homeostasis and integrity [4].

Despite the crucial role endothelial secretome is playing in EC function, we are only recently started to understand the molecular mechanisms governing the EC secretory function. In 2009, the first proteomic analysis on cultured HUVEC was conducted, and that study identified a number of 374 secreted proteins using nanoflow LC–MS/MS permitting the identification of angiogenic factors, extracellular matrix components, proteins involved in coagulation and inflammation, and in vascular tone, permeability and regeneration, and atherosclerosis and dissemination of metastasis [3]. More recently, 183 proteins were identified to be associated with the main secretory granules in quiescent HUVECS by proximity proteomics [5]. Meanwhile, a lot of progress was made in these regards, while some aspects are still under elucidation.

ECs intercellular crosstalk, which is mostly happening in the extracellular space, is highly controlled by ECs secretory pathways. It implies a donor (parent) cell which packs its contents into vesicles and a target (acceptor) cell that internalizes and uptake the vesicles. Endothelial cell secretory pathways occur via different size vesicles as follows: exocytosis of the principal secretory granules, Weibel–Palade bodies, and through smaller secretory granules, extracellular vesicles, and exosomes. Shedding of the ectodomain also provides many receptors and ligands for the target (recipient or acceptor) cell [6].

The main secretory storage granules of the ECs are Weibel–Palade bodies (WPBs), which represent the bulk source of stored, highly multimeric von Willebrand factor (vWF) [7]. The only other sources of vWF in the body are megakaryocytes and platelets α-granules, but they provide vWF in much smaller quantities only when stimulated [8]. WPBs are highly specialized organelles that ensure that ECs can promptly, and time-dependently respond to vascular injury or stress by enabling the controlled release of hemostasis and angiogenic factors, like vWF and not only to maintain vascular integrity. WPBs are large storage granules, their size ranges between 1 and 5 μm long and 100–300 nm wide, therefore, they are ideal for microscopy studies for secretion visualization from vascular endothelial cells.

Recent studies provide novel insights regarding endothelial secretome. Importantly, a recently published proximity proteomics study from Holthenrich et al. [5] provided interesting updates regarding WPB secretion regulation factors and the endothelial secretome.

EVs were first described in Peter Wolf as “platelet dust” and first characterized in 2011 by Gyorgy et al. [9] EVs are complex vesicular structures responsible for intercellular communication by transferring between cells: cytosolic proteins (e.g., enzymes and cytoskeletal proteins), lipids, mRNA, miRNA, and organelles from the parent cell.

EVs are released by virtually all cell types. EVs were identified in most body fluids and in the tissue matrix [10, 11]. Vesicles distinguish from one another based on size and density range and the mechanisms leading to their formation [12]. EVs originating from the cell membrane, by exposing to the exterior of the cytosolic side, by outward blebbing and budding, are named microvesicles, ectosomes, or microparticles [13, 14]. EVs that originate from the intracellular endocytic trafficking pathway is budding inward from the endosomal compartment, accumulate in multivesicular bodies (MVBs) in the form of many intraluminal vesicles [13] that upon fusion of the limiting membrane of MVBs with the plasma membrane, are released as exosomes [9, 14, 15]. EC-derived EVs biogenesis is lipid rafts-dependent and ADP-ribosylation factor 6 (ARF6)-dependent [16].

Several regulator molecules localized simultaneously on the EVs surface, and on the EC acceptor cells are known to be implicated in the delivery of cargo and uptake into the target cell, including integrins and integrin-associated proteins, tetraspanins, T-cell immunoglobulin, and mucin domain-containing protein-4 (TIM4), and lectins and heparan sulfate proteoglycans [4]. Tim4 is a receptor for TIM1 and phosphatidylserine on apoptotic cells. Without Tim4, macrophages cannot phagocytose apoptotic cells. These molecular pairs convey cargo delivery specifically to the vascular recipient cells, although it is not entirely clear how these processes occur [4].

EVs transfer their cargo from the parent cell to the target cell by: (1) docking to the target cell, (2) internalization of the EVs, and intracellular sorting through one of the endocytotic pathways; a pool of internalized EVs by the acceptor (target) cells are sent via endosomal escape, and (3) transfer of the EV content to the acceptor cell [4]. This way EVs influence the phenotypic traits of the recipient cell. Importantly, the released exosomes conserve many transmembrane proteins from the parent ECs.

ECs release into the extracellular space diverse types of EC-derived lipid membranal bilayer-enclosed structures in response to cellular activation or apoptosis, these microparticles have ambivalent functions (both favorable and detrimental) in vascular homeostasis.

In all, the aim of this chapter is to review current literature updates on endothelial secretome’s roles in endothelial homeostasis and in vascular disorders. We focus on the biological significance of secretome proteins in the vascular microenvironment in health and under different cardiovascular conditions. Secretome biomarkers in vascular diseases will be overviewed, with a focus on their roles in diagnostic, prognostic, and therapeutics. We highlight the important pathological effects of exosomes in cardiovascular disease. Most importantly, this chapter discusses vascular pathology conditioned secretomes, their regulation, and future therapeutic pursuit.

2. EC Secretome and exocytosis

2.1 Weibel: palade bodies (WPBs)–History, biogenesis, mechanisms, and pathogenesis

Discovery of WPBs - Edward Weibel and George Emil Palade were the first to describe WPBs in the early sixties by examining small arteries with a transmission electron microscope as “a hitherto unknown rod-shaped cytoplasmic component which consists of a bundle of fine tubules, enveloped by a tightly fitted membrane, was regularly found in endothelial cells of small arteries in various organs in rat and man.” [17] In TEM transversal section, WPBs are electrono-dense tubules packed in parallel bundles encapsulated in a lipid membrane. The tubules inside WPBs have a diameter of circa 12 nm and appear to be surrounded by a dense matrix, and sometimes they have a hinge at the end of the organelle. After WPBs discovery, it took approximately 20 years until the discovery of the main WPBs components. WPBs is the best example of a secretory organelle whose formation is dictated by vWF, its main storage constituent. vWF is secreted in the form of ultra-large multimers in response to the multitude of stimuli that activate ECs. vWF-deficient mice do not form EC WPBs [18]. Reversely, overexpression of vWF in other cell types leads to the formation of WPB-like granules with similar morphology: rod-shaped and striated granules [19, 20].

WPBs biogenesis - vWF is synthesized in the rough endoplasmic reticulum as a sequence of precursor conserved domains, as follows: D1-D2-D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK [21]. Consequently, furin, a pH-dependent enzyme, cleaves down the D1-D2 domain (approximately 100 kDa and ~ 750 aa) during the trans-Golgi network (TGN) processing step [21]. The sequence starting from the domain D′ and up to the cysteine knot (CK) domain represents the mature vWF [21, 22]. As vWF passes through the Golgi complex, it suffers more processing. At the level of the TGN, the presence of vWF leads to the biogenesis of WPBs [21]. In the TGN, vWF dimers go through the process of multimerization by bridging the D3 domains via interchain N-terminal disulfide bonds [22, 23]. In the TGN, WPBs go through a process of maturation and are directed, when needed, toward the basolateral surface in small vesicles [24] or stored in the highly multimerized form in WPBs [24] for later use via regulated secretion [21]. WPBs that protrude from the TGN have clathrin/AP1 coats on [25]. Overexpression of the dominant-negative construct AP180 and AP-1 inhibition with siRNA inhibits the formation of the clathrin coat. Therefore, it is considered that clathrin/AP1 are essential in the formation of vWF tubules and for their correct packaging [25]. Aftiphilin and ỿ-synergin are the downstream effectors of AP-1, which are recruited to the WPBs, as shown by their fluorescence microscopical partial colocalization with immature WPBs situated perinuclearly. Their depletion with siRNA does not modify the total number of WPBs or their intracellular distribution, but it increased the basal secretion of vWF and reduces the regulated vWF secretion [26]. vWF propeptide is necessary for WPBs biogenesis [20]. The vWF tubules are only forming at the level of TGN, not before, in the Golgi complex [25]. Immature (perinuclear) WPBs have different membrane protein sets than mature (cytoplasmic) WPBs. Upon maturation of WPBs, they lose their clathrin/AP-1 coat and its effectors aftiphilin and synergin [25, 26]. The tetraspanin CD63 is recruited to the mature WPBs with the help of the adaptor AP3, which targets proteins from endosome to the lysosome and related organelles. Lysosomal organelles, such as melanosomes and α-granules, have the characteristic to sort endosomal proteins into secretory granules after Golgi processing [27]. After the processing and maturation process, WPBs can be stored in the ECs for long times, for a few days [28].

WPBs exocytosis - vWF released from WPBs of vascular ECs has a crucial function in hemostasis and thrombosis. Under physiological conditions, WPBs have a low rate of basal secretion into the bloodstream that is thought to be required to maintain the blood level of VWF. The blood level of vWF is thought to be originating from the ECs exclusively, while the platelet α-granules and readily formed WPBs can supplement vWF released upon platelet activation. Under stimulation, the rate of WPBs can be rapidly increased by Ca²⁺- or by a cAMP-induced secretory mechanism. Upon activation of ECs, highly multimeric vWF is liberated from WPBs through the apical side of EC toward the bloodstream. Activated ECs lead to the regulated exocytosis of WPBs. As vWF is liberated from WPBs, it unfurls into long (1–5 μm) “strings” that platelets recognize via the A1 domain of vWF as “beads on the string” to initiate platelet adhesion and aggregation and the formation of a thrombus plug. Moreover, it vehiculated the idea that the contents of WPBs can be specifically altered, depending on where in the vasculature bed of which organs are ECs located. Ca²⁺ and cAMP-dependent controlled WPBs exocytosis regulate the local vascular environment to mediate the interconnected processes of hemostasis, vascular inflammation, and thrombosis. The molecular mechanisms controlling the trafficking of ECs secretory granules were only partially elucidated. We know that WPB priming and exocytosis are mediated by the N-ethylmaleimide factor, α-SNARE adaptor protein (α-SNAP), and SNARE receptors. WPB exocytosis depends on syntaxin 4, members of the SNARE complexes, synaptobrevin 3, synaptotagmin, and a sensor for Ca2 + −mediated exocytosis. In addition, nitric oxide (NO) was found to influence negatively NSF function by S-nitrosylation and inhibiting ECs exocytosis of vWF [29].

In addition, Weibel–Palade bodies contain P-selectin, other selectins, Rab27a, endothelin-1, endothelin-converting enzyme, angiopoietin, CD63, tissue-type plasminogen activator (tPA), interleukins (IL) IL-1, IL-8, CCL-2, eotaxin-3, osteoprotegerin, and calcitonin-gene related protein [30]. The question of cytokines incorporation into WPBs was recently revisited, Il1 and IL8 were found in WPBs but other cytokines are transported via other vesicles. These other components of WPBs like P-selectin, cytokines, and osteoprotegerin are incorporated during the processing phase in the TGN. P-selectin is a transmembranar protein, which is highly relevant for leukocyte rolling. Its big luminal domain is sufficient for the incorporation of P-selectin into the WPBs, even if truncated, probably because of its interaction with D′ and D3 domains of vWF [31, 32]. P-selectin is produced and stored in WPBs and the α-granules of platelets. P-selectin affects the formation of WPBs, andthe recruitment of leukocytes in vWF-deficient animals [33, 34].

The secretome of WPBs is characterized by dynamics and plasticity to meet the versatility of ECs pathophysiology [30]. Clotting factor VIII is stored in pulmonary ECs in association with vWF [35]. Osteoprotegerin is also stored in WPBs and its incorporation in WPBs is related to its interaction with vWF [7]. Osteoprotegerin (OPG) is one of the tumor necrosis factors (TNF) cytokine receptors [36]. Osteoprotegerin is involved in the inhibition of bone regeneration via OPG/the receptor activator of nuclear factor kappa-B (RANK)/receptor activator of nuclear factor kappa-B ligand (RANKL) axis, by decreasing the production of osteoclasts [36]. Another study focused on WPB resident proteins identified IGFBP7 by proteomic screening [7]. Angiopoietin-2 is part of a family of four growth factors whose activity is mediated via tyrosine kinase receptors (Tie) receptors 1 and 2. Angiopoietin-2 has different functions depending on the microenvironment conditions, an autocrine regulator, and an antagonist of Tie2. In activated ECs, vascular endothelial growth factor (VEGF) stimulation of angiopoietin-2 has proinflammatory effects. Angiopoietin-2 is incorporated into WPBs, at the level of TGN, if there is no P-selectin present because these two factors are mutually exclusive [37]. Angiopoietin-2 expression is induced by local inflammatory and prothrombotic factors, such as thrombin or under hypoxic conditions, and its release is highly regulated.

A recent study employed a new approach involving proximity proteomics to find proteins residing on the cytosolic face of the WPBs or in the vicinity of the organelle. In this case, biotinylating the target proteins in living cultured HUVECs followed by streptavidin pulldown and identification of the new proteins by tandem mass spectrometry [5]. The small GTPases Rab3b and Rab27 were previously identified in the proximity of WPBs on the cytoplasmic face of WPBs. In this study, the authors show that the two mentioned Rab small GTPases fused with peroxidase APEX2, and modified ascorbate peroxidase [5]. In this study, HUVECs were transfected with Rab3b tagged with FLAG-APEX2 construct. The resulted proteins were immunoblotted with anti-FLAG antibodies. Proteins associated with WPBs that were tagged, and upon adding biotin-phenol and peroxide of hydrogen for 1 minute there was peroxidase-catalyzed biotinylation of the protein in the vicinity of WPBs as shown by streptavidin pulldown from the pretreated HUVECs under resting conditions. The pulldowns were sent to tandem mass spectrometry.

This approach led to the identification of a total number of 183 proteins associated with WPBs and with one of the two Rab GTPases or with both constructs. Many of those were not identified in the vicinity or in association with WPB before. Importantly, these proteins were previously found to be related to membrane/protein transport or to organelle dynamics and plasticity. Vacuolar ATPase ATP6V, syntaxin binding protein 1, Rab 46, phospholipase D1, GBF1, and phosphatidylinositol 4-kinase were identified as WPBs constituents, some of them were previously known to be related to WPBs exocytosis [5]. Rab 7, one of the proteins identified by proximity proteomics was thought to regulate the transport from endosome to WPBs [5]. Some Golgin family members were also identified by the same approach. The secretory pathway Ca ²⁺ ATPase type 1 is another newly discovered constituent of WPBs, which is known to be involved in Ca²⁺ homeostasis [5]. The fact that well-known markers of WPBs, such as P-selectin, VAMP-3, CD63, MyRIP, and Slp4a, were recognized in this study verifies the specificity of their approach. A previous study showed that the interplay between Rab27A and its effectors Slp-4 and MyRIP controls histamine-induced vWF secretion [38]. CD63 is well known to be associated with WPBs.

Importantly, one new protein associated with WPBs by proximity ligation is the priming/tethering factor Munc 13-2, which is a positive regulator of histamine-induced WPB exocytosis of vWF [5]. Munc 13-2 was previously implicated in WPB exocytosis of angiopoietin-2 from the brain ECs [39]. After the initial proteomic screening and verification, Munc 13-2 localizes at WPB surface, and they also demonstrated that Munc 13-2 siRNA affects histamine-induced vWF secretion [5]. In addition, Munc 13-2 cooperates with Munc 13-4, which was found to be a plasma membrane priming and fusion factor for evoked WPBs exocytosis [5].

WPBs defects - Defects in the structure, packing, or sorting of vWF lead to von Willebrand disease (vWD), which has an incidence of approximately 1:1000 in general population and represents the most common inherited coagulopathy in humans. The inherited defects in vWF result in smaller and defective WPBs. Defects in the proteins that regulate WPB exocytosis also led to vWD. Reversely, excess of vWF released by activated ECs exposed to high shear stress or to vascular inflammation, or infection tilts the thrombotic propensity leading to disseminated intravascular coagulation and microvascular thrombosis.

2.2 Secretory granules, vesicles of 100–500 nm diameter which store cargo of smaller dimensions

The initial belief that cytokines reside exclusively in WPBs in vascular ECs from where they are released upon stimulation, that is, with histamine, was recently challenged [40]. The question of cytokines incorporation into WPBs was recently revisited, Il-1 and IL-8 were found in WPBs but other cytokines are transported via other vesicles. It was proven that cytokines originate and are also secreted from smaller vesicles by vascular ECs and that cytokines (monocyte-chemoattractant protein-1 [MCP-1], IL-6, and IL-8), EGFP, and tissue plasminogen (tPA) are much less efficiently stored in WPBs compared with vWF, but are present in “tPA and type 2 organelles.” [40] Chemokines are small cytokines that direct the movement of cells during embryogenesis, in order to maintain homeostasis or in pathological conditions. Their roles include cell proliferation, cell migration, cell differentiation, and implicit maintaining tissue and organs homeostasis by regulating the types and number of each cell produced. ECs express cytokines, such as interleukins (IL) IL-1, IL-5, IL-6, IL-8, IL-11, and IL-15, granulocyte/macrophage colony-stimulating factor (GM-CSF). Upon local or systemic vascular inflammation, secretion of a particular set of cytokines can attract specifically certain subtypes of leukocytes. ECs secrete chemokines, such as CCL2 (attracts monocytes), CCL5 (monocytes, eosinophils, and T cells), eotaxin-3/CCL26 (eosinophils), CXCL1 (to attract neutrophils), and CXCL10 (for T cells) [41, 42, 43]. Activated ECs secrete upon vascular injury by other coagulation agents, such as plasminogen activator inhibitor-1 (PAI-1) or other growth hormones like TGF-β [42].

Much of the intercellular communication performed by vascular ECs is done by means of soluble cytokines and chemokines. Upon endothelial cell activation during inflammation, cytokines are released from EC secretory vesicles, and there is vasodilation to lower the blood flow and recruitment of leukocytes at the site of infection or injury. The first step is the vasodilation of the blood vessel, which allows for better leukocytes interaction with the vascular ECs. Vasodilatation is partly a result of EC-induced mechanisms. The endothelium secretes increased the level of P- and E-selectins, intracellular adhesion molecules (ICAM), and integrins. 1) rolling adhesion: The initial low-affinity endothelial-leukocyte interactions involve an increased level of selectins. P-selectin is secreted from WPBs in minutes, in response to histamine released by mast cells or mediated by TNF-α or LPS. TNF-α is one type of molecule produced by macrophages in response to pathogen detection, causing endothelial activation. Histamine-mediated activated ECs rapidly externalize WPBs content, which includes preformed P-selectin. TNF-α or LPS induce the synthesis of E-selectin, which is exposed to the cell surface a few hours later. Selectins recognize Sialyl-LewisX on the rolling leukocytes across the endothelium, allowing them to adhere reversibly (tethering or capture) to the vascular wall. Without these initial interactions, the next steps of chemokine-dependent and chemokine-independent mechanisms leukocyte activation, and extravasation would not occur. These steps depend on the production and adequate release of P-selectins from WPB granules of vascular ECs. 2) Leukocyte-Endothelial tight binding relies on integrins (VLA-4, CD18, and CD11) on leukocytes and ICAMs on the surface of vascular ECs, such as TNF-α-induced endothelial ICAM-1, or ICAM-2, and VCAM-1 3, diapedesis is the step in which the leukocyte extravasate the endothelial wall. This step involves the interaction of integrins (CD11/CD18 and VLA-4) on leukocytes with ICAMs on ECs (i.e., ICAM-1 and VCAM-1), and with PECAM-1 (CD31).

2.3 Secretome trafficking via extracellular vesicles (EVs)

2.3.1 Secretome trafficking via endothelial microvesicles (MVs)

Endothelial MVs are plasma membrane-derived vesicles, they occur through blebbing and budding of the cell membrane starting intracellularly from the cytosol, budding toward the exterior of the cell [12]. Their size ranges from 100 to 2000 nm [15]. The majority of MVs come from platelets. Vesicles should be collected from plasma not from serum because activation of platelets leads to excessive release of platelets MVs, and contamination of the sample.

EVs reflect the status of the patent cell. EC-derived EVs can be released with the purpose to protect the endothelium from distress, therefore, they fulfill the function of gatekeepers, with cytoprotective and antiapoptotic effects.

One of the factors known to regulate the biogenesis of the microparticles is ARF6 [16].

EC-derived MVs carry markers/regulator proteins that are associated with a pathological state: vascular endothelial cadherin (VE-cadherin), endoglin (CD105), (c-Src kinase+, eNOS+ and caveolin1+, EPCR+). ECs-derived MVs from plasma of septic mice had increased levels of VE-cadherin+ and endoglin+ vesicles compared to sham control. EC-derived MVs applied in vitro on cultured ECs cause endothelial permeability dysfunctions by disturbing the adherens junctions and the cytoskeleton [44]. Platelet endothelial cell adhesion molecule-1 (CD31+ or PECAM-1), E-selectin, αv integrin (CD51), and intercellular cell adhesion molecule (ICAM)-1 (cytokine-stimulated HUVECs released increased levels of both factors; moreover, E-selectin-targeting to inflamed ECs help delivery of miRNAs from MVs, with anti-inflammatory effects [45]), or S-endo (CD146) [45], endothelial NO synthase, oxidated negatively charged phospholipids (infusion with man-made negatively charged phospholipid vesicles leads to severe thrombosis in primates and murine models [46]), and vascular endothelial growth factor receptor (VEGF-R2) [47]. High adhesion molecules level constitute a sign of endothelial dysfunction and decreased vessel elasticity [46].

Because the presence of EVs released by ECs in the circulation usually indicates a vascular or systemic disease, they can be used as markers of endothelial dysfunction. The endothelial origin of circulating MVs can be established by flow cytometry and other laboratory tests. One caveat is that, apart from E-selectin and VE-cadherin, these protein markers are not expressed exclusively by the vascular cells.

In the case of transfer of membrane-bound MVs by cocultures, recipient cells take some phenotypic characteristics of the MV-producing cells; sick or degenerative cell regain their normal phenotype, and a bidirectional membrane transfer is observed between cells. Circulating EVs can be distinguished by tissue source and disease state profiling. EVs are molecular heterogeneous and overlap a lot. There is also large heterogeneity in the mechanical properties of EVs that may dictate cellular behavior.

2.3.2 Secretome trafficking through exosomes

Exosomes are nanoscale vesicles with a diameter ranging between 30 and 150 nm. Exosomes can be released from any type of cell in the body, their release is higher from certain cell types. Exosomes originate from the intracellular endocytic trafficking pathway, during the endosome compartment maturation in MVBs; MVBs membrane fusion with the cell membrane allows the release of their intraluminal vesicle as exosomes, as shown by electron microscopic shots of exosomes that previously endocytosed colloidal gold [15].

Exosome biogenesis involves a two-step budding process: step 1) inward budding of the external plasma membrane through the endocytotic pathway to the endosomal compartment and intraluminal vesicles into MVBs and step 2) cytosolic MVBs secrete the exosome cargo. Importantly, the released exosomes conserve many transmembrane proteins from the parent ECs.

Silencing the endosomal sorting complex transport protein (ESCRT) members, ESCRT-0 and/or ESCRT-I, decreases exosome secretion [48], and modified the size of exosomes and their composition, and major histocompatibility complexes (MHC) levels, especially, impaired MHC II content, as shown by immunogold electron microscopy [48].

Several proteins are involved in exosome cargo sorting. Small GTPases Rab7a and Rab27b, found mainly on late endosomes, coordinate miRNA 143/miRNA150 export through nanovesicular trafficking, in response to the shear stress-inducible transcription factor Krüppel-like factor 2 (KLF2) overexpression, in cultured HUVECs, to levels that mimic shear stress levels [49]. The release of the exosome cargo goes through a Rab11- and Rab35-dependent regulatory pathway, which is involved in slow endocytic endosome recycling [49].

ShRNA knockdown of ESCRT-associated proteins, VTA, TSG101, VPS4, and ALG-2-interacting protein X increased exosome secretion, and increased MHC II proteic and mRNA content, as demonstrated on a 96-well plate screen of over 20 components of the ESCRT system and associated proteins [48].

Exosomes are characterized by: 1) expression of a set of integrins and tetraspanins (CD9, CD63, CD81, and CD82) for targeting and adhesion, 2) expression of proteins involved in membrane transport and fusion (annexins, Rab proteins, and flotillin), 3) expression of proteins associated with multivesicular body biogenesis (ALG-2-interacting protein X, TSG101, VPS4,a and VTA), 4) lipid-rafts (sphingolipids, sphingomyelinase, lipid ceramide, and cholesterol), 5) heat shock protein (HSP)-70 and − 90, as well as of 6) MHC I and II. 7) Another specific exosomal marker is a lysosomal-associated membrane protein-1 (Lamp1). 8) Importantly, EC-derived EVs contain miRNAs conveying immune responses.

Exosomes can be visualized on a NanoSight microscope. The uptake of endothelial exosomes, and the gain of function exosomes transfer can be measured.

Published data show that EC exosomes secreted in the circulation influence cellular behavior via paracrine signaling and can have huge biopotential: exosomes influence cell phenotypes, regulate protein synthesis, convey immune responses, stimulate angiogenesis, endothelial proliferation and migration, cell-free regeneration potential, and cardioprotective effects.

2.3.3 Caveolae

ECs also contain many caveolae, specialized endocytosis structures which are necessary for transcytosis of a variety of substances (i.e., albumin transport [5, 37] across the EC layer).

2.3.4 Tunneling nanotubules

Endothelial cells communicate with the help of tunneling nanotubes (TNT), which can be up to or more than 100 μm long and 50–200 nm in diameter. TNTs are composed of open-ended F-actin, nonadherent. TNTs form transiently for 30 minutes to 2 h and then retract and disappear.

2.4 Shedding of protein Ectodomains

Apart from the classical secretory pathways, about 2–4% of cell membrane proteins are released in circulation or into the extracellular space, in health or under pathological conditions, by ectodomain shedding, which is a proteolytic removal of a significant portion of the transmembrane protein ectodomain (the extracellular domain) [6]. Ectodomain shedding is relevant in growth factor signaling to cell adhesion, inflammation, cell survival, and cancer. The results of ectodomain shedding for ECs differ strongly depending on the type of shed transmembranar protein (i.e., adhesion molecules, growth factors, cytokines, and cell receptors). The shed enzymes are membrane-bound proteolytic enzymes that cleave, releasing the soluble ectodomains and leaving behind a protein fragment bound to the plasma membrane that can initiate signaling at this level or can be internalized in membrane-bound vesicles for unusual destination signaling in the nucleus to target gene activity or in the mitochondria.

ADAM17 and ADAM10 metalloproteases are the main sheddases expressed by ECs from the “a disintegrin and metalloprotease” (ADAM) family of sheddases. Upon inflammation, ADAM10 is responsible through a notch-dependent regulation for DII-1 and -4 expression and changes in Hes1 and Hey1 expression.

Among over 40 shedding substrates that ADAM10 has on resting and/or activated ECs, the most important ones include as follows: IL-6, Interleukin-6-receptor (IL-6R), IL-8 [50], CX3CL1, CD44, CXCL16, MCP-1, VEGFR2, sVCAM1 (on TNF-activated ECs) [51]. DLL4 and VE-cadherin (regulates endothelial permeability and transmigration [51, 52, 53, 54].

2.5 Organotypic EC secretome

Location dictates the function; it was demonstrated over the years that the characteristics of the secretome of different subtypes of ECs are dependent on their localization in the vasculature bed. Distinct subtypes of ECS secrete tissue-specific proteomes, which regulate specifically tissue homeostasis and regeneration and functional pathophysiology. There are key features specific to the secretome of the continuous, discontinuous, fenestrated, sinusoidal, Schlemm’s canal specialized ECs, and high endothelial venules. Brain, retina, and bones have organotypically differentiated ECs with specific morphological features that predict the functional particularities specific to the vessel bed and the tissue-specific EC secretome. Under physiological conditions, ECs have quiescent functions and phenotypic characteristics and they produce a different sets of vesicles and granules constituents upon activation. Quiescent ECs are not inactive though but under normal conditions they are inactive. They function as a gatekeeper in their microenvironment to control tissue function, homeostasis, and regeneration. As gatekeepers, ECs respond to different stimuli (inflammatory, infectious, metastasis, and high shear stress), they modify accordingly their phenotypes and functions to preserve vascular homeostasis. The molecular mechanisms controlling ECs vessel-bed specific differentiation and function are now emerging for protein preparation and secretion, and protein export into the extracellular microenvironment.

2.6 ECs apical and basolateral secretome

Endothelial cells have distinctive apical and basolateral secretomes. ECs polarize the secretion of small vesicles toward the apical side of ECs. For example, cytokines that are destined for blood circulation are secreted into the apical side of ECs. In contrast, the basolateral proteome is destined toward the components of the extracellular matrix, sharing their route with fibronectin and liprin-α1. The approach employed to dissect protein sorting in ECs to basolateral or apical compartments was to grow HUVECs on transwell inserts with separate collecting compartments for basolateral and for the apical secretory pathways.

2.7 miRNAs transfer functionally using EVs

A microRNA is RNA that binds with imperfect complementarity to its target mRNA, might be 6–7 nucleotides long sequences, with a lot of opportunities for binding at the end of the target mRNA, and many non-canonical mechanisms were described so far. The difference between siRNA (which was described first) and miRNA is the degree of complementarity with the target. miRNA bind to its target leads to shutting down of translation by several mechanisms: translational inhibition, deadenylation, and cleavage in certain situations. At the heart of the miRNA mechanism is arg0naute protein (Ago), which is part of the miRNA-mediated gene silencing complex (RISC). miRNA is loaded with the argonaute as it is made, mature acts as part of argonaute RISC complex.

Rab GTPases regulate membrane trafficking for EC-derived vesicular miRNA [49]. In cultured ECs, the miR-143 vesicular export occurs through a Rab7a/Rab27b-dependent mechanism, induced by overexpression of the transcription factor KLF2 at levels that mimic high shear stress [49].

3. Role of endothelial Secretome in endothelial dysfunction

Dynamic vasculature regulation correlates (updates) ECs secretome with ECs functional needs in health, and under inflammatory conditions, under high shear stress, or under abnormal angiogenic factors.

Upon injury, cells are recruited by exosome-mediated receptor-mediated interactions, variety of responses occur in the vasculature because of the diverse mechanisms of action. Internalization of EC exosomes by monocytes/macrophages can suppress systemic inflammation. In open wounds, exosome secretion of cytokine influences the cellular behavior of fibroblasts toward wound healing.

In pulmonary arterial hypertension (PAH), depletion of pulmonary caveolin-1 from the lungs is partially due to caveolin-1 positive extracellular vesicle (bigger than 100 nm) blebbing and shedding into the circulation [55]. Elevated levels of blood caveolin-1 + EVs correlated with TGF-β-induced microvascular remodeling and PAH [55]. In PAH, the vascular injury most probably induces EV release and caveolin-1 depletion from pulmonary ECs, while the “second hit” that promotes vascular remodeling might be chronic hypoxia [55]. In acute lung injury (ALI), EVs released by vascular ECs and epithelial cells in the lung has been shown to mediate cell-to-cell communication and transport bioactive molecules between cells. However, the role of bioactive proteins and lipid mediators carried by EVs in ALI pathophysiology is explored insufficiently. Mouse bronchoalveolar lavage fluid-derived EVs were found to contain high levels of cyclooxygenase, lipoxygenase, and cytochrome p450 metabolites, those levels increase during the acute inflammatory phase and decrease in the resolution phase of LPS-induced ALI.

Exosomes derived from apoptotic ECs have a particular secretome output, they contain self-made, self-tailored, non-coding RNAs and are highly immunogenic. Apoptotic ECs exosomes are carrying RNAs that are not usually found in healthy cells: viral-like endogenous retroelements (those are the most abundant), mitochondrial RNAs, U1 small nuclear RNA, and Y RNA that are involved in autoimmunity responses [56]. These nucleic acids are rich in U-bases and unstable folded, self-made RNAs of endothelial cells in apoptosis that can be recognized by toll-like receptors (TLR3, 7, and 8). They encompass a demonstrated role in murine models of inflammation and innate immune responses [56].

MiRNA-enriched EVs derived from monocytes can be transfer to quiescent, unstimulated ECs, which enhanced EC permeability and monocyte transmigration in a co-culture system condition.

In an in vitro study, Li et al. employed a co-culture system in which neutrophils extracted from healthy donor blood were co-cultured with brain microvascular ECs in presence of LPS. Exosomes purified from the LPS- exposed miR-122-5p rich neutrophils regulate oxidative stress, permeability, or apoptosis of capillary ECs in the brain [57].

miRNA-containing EVs originating from activated or apoptotic EC are able to communicate to their neighbors, are protecting the adjacent vascular ECs from apoptosis, and have potent anti-inflammatory effects [58]. Endothelial MVs transporting miR222 to the neighboring ECs are significantly reducing the ICAM-1 level in proximity cells, which have impaired monocyte adhesion assay [59]. In vitro, the pretreatment with EC-derived EVs (obtained from ECs stimulated with TNF-α) lead to reduced endothelial ICAM-1 expression at mRNA and protein levels, while did not influence VCAM1 [59]. In vivo, they were also able to demonstrate reduced ICAM-1 expression in APO E−/−mice, as shown by immunostaining assays of fragments of descending aorta from APO E−/−mice [59]. These protective effects of EC-derived EVs occurred by delivery of miRNA-222 to recipient endothelial cells [59]. The efficient delivery of miRNA-222 to the recipient cells was demonstrated by employing an assay developed from C. elegans, HCAEC were transfected with cel-miR-39 [59]. Next, they applied prediction methods questioning the target mRNA for miRNA222, and the most probable target was found by ICAM1 [59]. By using inhibitors of miRNA222 they were able to verify that functional miRNA222 targets ICAM1 and reduces its mRNA and protein expression in cultured cells [59, 60].

In an unstimulated, quiescent state, vascular ECs secrete extracellular vesicles containing anti-inflammatory microRNAs [57, 61]. They are transferring miRNA to monocytes and other vascular cells to prevent monocyte activation [62].

EC-derived EVs that can transfer MiR-10a to monocytic/macrophagic cells have anti-inflammatory effects. They inhibit the proinflammatory phenotype by inhibiting many proinflammatory genes by repressing the induction of the nuclear factor-kB (NF-kB) and IRF5 transcriptional pathways. MiR-10a negatively regulates effector proteins that destabilize I-kB. EC-derived EVs that can transfer MiR-10a to monocytic/macrophagic cells suppress a network of genes. One of these genes is interleukin-1-receptor-associated-kinase-4 (IRAK4) gene, which acts upstream of NF-kB signaling. MiR-10a suppressed β-TRC, and MAP3K7/TAK1 [62]. Loss of miR10 during atherogenesis has the opposite effect of activation of monocytes [63].

Increased/disturbed shear flow may lead ECs to deliver miRNAs miR126-3p, miR200a-3p to target cells like smooth muscle cells (SMC) by means independent of membrane-bound vesicles and argonaute-2-dependent, which is protecting miRNAs during delivery to the target cells [64] via VAMP-3/SNAP23-mediated pathway, leading to SMC hyperplasia.

Endothelial cell-specific MiR-126 decreased inflammatory-inducible expression of adhesion molecules in ECs, it decreased TNFα-induced VCAM-1 level in cultured primary human ECs, respectively, as shown by immunoblotting [65]. The presence of proinflammatory cytokines, such as TNF-a, induce the expression of VCAM-1 through the induction of NFkB and IRF1 pathway [65]. It has been suggested that miR126 is a target for VCAM-1 gene because of partial sequence matching in the 3’ UTR region position 619 to 625 within the human VCAM-1 transcript [65]. Transfection of HUVECs with antisense miR126 increased TNFα-induced VCAM-1 expression [65]. Overexpression of premiR-126, a precursor of miR126 increased endogenous miR126 and reduced VCAM1 expression [65]. Endothelial cell-specific MiR-126 negatively regulates leukocyte trafficking and adherence to TNF-α-activated HUVECs, through VCAM-1 expression inhibition [65]; the leukocyte rolling is VCAM-1 dependent, as shown by blocking VCAM-1 with an anti-VCAM-1 antibody [65, 66].

Primary rat hepatocyte-derived, CD81 and CD63 positive EVs were found to contain argininase-1, an enzyme that regulates the level of arginine, the substrate for eNOS nitric oxide synthetase [67, 68]. In vivo, these hepatic EVs obtained from the serum of rats under liver-damaging conditions with acetaminophen, or diclofenac treatment encapsulate more arginase-1 compared to untreated control [67], as shown by the untargeted blood metabolome approach [68]. Exposure of rat pulmonary artery ECs to hepatic EVs for 2 h provoked changes in ECs metabolome as compared to untreated control EVs, via an arginase-1-dependent mechanism [67]. Increased arginase activity led to nitric oxide defective synthesis and activity and ECs malfunction of pulmonary arteries in rats treated with liver-damaging drugs [67].

A screening (Taqman miRNA assay) for high levels of miRNAs in circulating vesicles collected from 180 patients with chronic coronary disease and circa 60 patients with the acute coronary syndrome, identified miRNA-92a-3p to be selectively increased in circulating vesicles isolated from plasma of patients with the coronary disease compared with control, as seen by RT-PCR [69], showing how atherosclerotic conditions selectively promote packaging of miRNAs, such as miRNA-92a-3p into circulating EVs [69]. The authors further explored the role of circulating vesicles carrying miRNAs on vascular ECs [69]. Functional miRNA-92a-3p was transferred from circulating vesicles into acceptor ECs [69]. MiR-92a-3p target is thrombospondin-1, which is increasing cell proliferation and migration, and inhibited angiogenesis and vessel-like networks [69].

Weilner et al. observed a higher rate of exosome secretion in senescent humans ECs compared with quiescent cells [70]. Circulating miR-31, encapsulated by senescent human EC-derived EVs, is upregulated in elderly donors and osteoporosis patients [70]. MiR-31-rich EV transfer in human mesenchymal stem cells inhibiting their differentiation toward osteogenesis, a switch from osteoblast genesis to osteoclast formation, which modifies the bone density [70]. Endothelial exosomes can transfer miR-503 to tumor cells, tumor cells can exert an antitumor effect via the transfer of miRNA from ECs, leading to decreased tumor growth and invasion [70].

4. Role of endothelial Secretome in vascular repair

EVs from ischemic tissues play a role in endothelial cell survival and in de novo, angiogenesis as shown in a murine model of ischemia after femoral artery ligation [47, 71]. Hussein et al. showed that injured ECs release caspase 3-rich EVs to protect the endothelial cells from complement-induced apoptosis in cultured HUVECs. Conversely, in the presence of inhibitors like staurosporine they showed that caspase 3 levels increase [71]. Thus, endothelial-derived MV contributes to the elimination of excess, stress levels of apoptotic agents, and to avoid apoptosis and cellular detachment [71].

Endothelial cells react to stressful conditions by releasing EVs, as a form of communicating the distress to the cells in proximity and to protect the endothelium [46]. Under lipid-induced oxidative stress conditions, endothelial cells release EVs containing endothelial NO synthase via AKT/eNOS -dependent signaling pathway, to protect the vessel from endothelial damage [46].

Secretory granules and vesicles can release mediators that are directly involved in the gatekeeper actions of EC by immediate, basal, or by evoked, rapid secretion according to the functional needs of the ECs. CD47 is an integrin-associated protein found often on EVs. CD47 role is to prevent EVs phagocytosis by macrophages, therefore, increasing EVs circulation time [15]. Moreover, activated protein-C (APC) interacts with endothelial protein-C receptor (EPCR) exposed both on the MVs surface and on ECs can cleave PAR-1 and trigger signaling leading to activation of S1P1 which via PI3 and AKT-dependent transactivation of KDR stimulate cell proliferation, and ultimately has an endothelial barrier and cytoprotective effects [72].

Endothelial-derived MV contributes to the sorting of several proapoptotic factors preventing cell detachment and apoptosis [71]. MVs carrying APC induced cytoprotective effects in a staurosporine-induced endothelial cell model of apoptosis assessed by APOPercentage assay and improved EC permeability percentage [72]. Activated protein-C (APC) binding to endothelial protein-C receptor (EPCR) exposed both on the MVs surface and on ECs can cleave PAR-1 and trigger signaling leading to activation of S1P1 which via PI3 and AKT-dependent transactivation of KDR, which, in turn, stimulate cell proliferation, with endothelial barrier protective and EC survival effects [72].

Endothelial exosomes are thought to be involved in angiogenesis. They incorporate and transfer delta-like-4 (Dll4), a notch ligand upregulated during angiogenesis, to neighboring endothelial cells via EVs, beyond cell–cell contact, conferring a tip cell phenotype to the detriment of stalk cells, resulting in a low level of notch signaling, loss of notch receptor and increased filopodia, branching formation that results in neovascularization [73].

Delivery of functional miRNAs by means of ECs-derived EVs to recipient ECs was also shown to help the process of vascular repair and angiogenesis [74]. In response to hypoxia, the expression of miRNAs, such as miR-210, is upregulated, leading to VEGF-induced chemotaxis to form capillary-like structures in cultured ECs. Upregulation of MiR-210 expression was also found in a surgical murine model of myocardial infarction [75].

5. Discussion and conclusions

By means of their vast secretome, ECs delivery platform sends messages at a distance to circulating blood cells, to other ECs, or to the normal or diseased cells of other organs. ECs complex intercellular communication is possible through the secretory pathways. ECs secretome output may vary, according to the specific vascular bed, whereas ECs phenotype is dictated by their function particularities. The secretome of WPBs is orchestrated by complex, dynamic secretory pathways to meet the versatility of ECs pathophysiology [30]. Novel protein members of the WPBs secretome were recently identified through a new approach of proximity proteomics, unveiling new facets of WPB exocytosis regulation.

EC-derived EVs are primary effectors in signaling pathways between vascular cells. EVs transfer their cargo from the parent cell to the target cell by: (1) docking to the target cell, and (2) internalization of EVs first, before releasing their content. A pool of internalized EVs by the acceptor (target) cells are sent through an endosomal escape system to unusual delivery destinations [13] and (3) release of EV cargo into the cytosol of the acceptor cell, followed by degradation or return to secretion circuit of the vesicles.

The EVs are secreted by virtually all kinds of eukaryotic cells and in all body tissues and fluids, including blood, saliva, urine, amniotic fluid, cerebrospinal fluid, and breast milk. EVs delivery to the target cells may occur using a cell-specific endocytotic mechanism, dependent on receptor-ligand interactions, via clathrin-dependent endocytosis, via clathrin-independent endocytosis lipid rafts-mediated or caveolae-mediated endocytosis, or by common targeting, occurring through pinocytosis, or phagocytosis. It can occur through membrane fusion or via intraluminal vesicles fusion with the endosomal limiting membrane. The sort of the uptake mechanism is given in part by the types of molecular regulators found both on EVs and the targeted plasma membrane of the acceptor cells, because this molecular pair influences the phenotypic traits and behavior of the recipient cells post EV content uptake. Upon shear stress, pH, pressure change, or shock, the ECs release a system of vesicles either into the extracellular space or they reach an additional target cell, which is transformed by this interaction, and that target cell takes on the characteristics of the shredded parent cell.

The ectodomain shedding by proteolytic cleavage of transmembranar protein exterior domain is the posttranslational modification that permits the appearance of new fragments, that function either as receptors or ligands, that control levels of signaling proteins in the recipient cells. This process is not an exemption for transmembranar protein, it rather occurs frequently and is a form of communication between cells. There are still many things unclear about the processes of ectodomain shedding, one question that remains to be elucidated is how the molecular pairing occurs between sheddases and substrates, timing, kinetics, and how sheddases alter the substrate’s function and we still must explore their potential as drug targets.

Further, we discussed some aspects related to the posttranscriptional-mediated miRNA regulation of gene expression programs of endothelial cells and their impact on vascular disease. Under disturbed shear flow, miRNAs may be delivered dependent on membrane-bound microparticles [66], or independent of membrane-bound microparticles, with the help of argonaute-2, which protects the miRNAs during delivery to the recipient cells [76].

EVs have beneficial effects, such as anti-inflammatory effects, inhibition of thrombus formation, or vascular repair and angiogenesis [77], but they might have detrimental effects leading to systemic inflammation, atherosclerosis, tilting vascular homeostasis, and thrombotic propensity. Therefore, they could serve as biomarkers of endothelial dysfunction.

EC-derived EVs are frequently found in patients with vascular conditions. EVs found in the plasma of these patients could be used as prognostic factors of vascular disease. Exosomes are still used only in investigational protocols. There are several clinical trials going on trying to prove applications in dermatology (burns) through regeneration and wound healing mediated by exosomes. Some tissular cells can be distinguished from the bloodstream circulating exosomes, but further investigations are needed to delineate the origin of the exosomes in circulation as well as different many facets involved in the creation of the exosomes, in the targeting, transport, and uptake mechanisms (ubiquitin and lipid-mediated) in physiological vs. specific cardiovascular disease condition.

The key advances of endothelial EVs and in particular exosomes for therapeutic purposes are: 1) exosomes can home, 2) can travel systemically without risk of clumping, 3) can travel via local or topical therapy, 4) exosomes cross the “blood–brain barrier,” 5) not perceived as foreign, and 6) they deliver miRNA and mRNA and signaling proteins to unite, to mobilize, to integrate, and cytoskeletal proteins to direct the focal adhesion, for matrix-directed acquisition, reduction of proliferation, and matrix production in the target tissue, and cell motion, cell-cycle, anti-apoptotic, and responses to oxidative stress are only a few of the things exosomes can do. 7) No first-pass lung effect, 8) easy to administer, store, and freeze, and 9) the dosage can be controlled.

Further investigations are required to subcategorize the exosomes depending on the cell type or lineage that they are secreted from, and their specific impact on the functions of the vasculature.

A dynamic endothelial cell secretome meets the vasculature bed functional needs through complex secretory pathways EC secretome’s constituents could be a readily accessible, rich source of non-toxic markers to monitor and properly assess the risk factors of vascular disease and prognosis. Most importantly, ECs secretome therapeutic potential is emerging for the treatment of various diseases and tissue injuries.