Lymphatic and Blood Endothelial Extracellular Vesicles: A Story Yet to Be Written

Extracellular vesicles (EVs), such as exosomes, microvesicles, and apoptotic bodies, are cell-derived, lipid bilayer-enclosed particles mediating intercellular communication and are therefore vital for transmitting a plethora of biological signals. The vascular endothelium substantially contributes to the circulating particulate secretome, targeting important signaling pathways that affect blood cells and regulate adaptation and plasticity of endothelial cells in a paracrine manner. Different molecular signatures and functional properties of endothelial cells reflect their heterogeneity among different vascular beds and drive current research to understand varying physiological and pathological effects of blood and lymphatic endothelial EVs. Endothelial EVs have been linked to the development and progression of various vascular diseases, thus having the potential to serve as biomarkers and clinical treatment targets. This review aims to provide a brief overview of the human vasculature, the biology of extracellular vesicles, and the current knowledge of endothelium-derived EVs, including their potential role as biomarkers in disease development.


The Human Vasculature and Endothelial Cell Heterogeneity
The human vascular system can be broadly separated into the blood and the lymphatic system. The blood vasculature is responsible for the active supply and distribution of blood and its components, whereas the lymphatic system removes accumulating interstitial fluid to ensure tissue homeostasis. Thus, the formation, maintenance, and remodeling of a functioning vascular network is essential for oxygen and nutrient supply as well as lymphatic fluid drainage in the healthy human body [1]. Vasculogenesis describes the formation of new vessels from endothelial progenitor cells [2]. In contrast, angiogenesis refers to the formation of new vessels splitting and sprouting from preexisting ones [3]. Endothelial cells (ECs) arise from the mesodermal stem cell lineage. Mesodermal progenitor cells develop to angioblasts under the influence of a group of transcription factors including E26 transformation-specific (ETS) [4]. Specification into endothelial and hematopoietic lineage is primarily driven through the expression of transcription factors Etv2 and Npas4l [5]. During this process, activation of distinct signaling networks leads to further differentiation into arterial, venous, and lymphatic subtypes [1]. The specification into arterial or venous fate is further driven by regulators such as SoxF transcription factors, Notch receptor proteins, bone morphogenic proteins (BMPs), and transforming growth factor-beta (TGF-ß) [5]. The upregulation of Notch has been shown to lead simultaneously to the expression of arterial markers and the downregulation of venous ones [6]. By suppressing of Notch signaling, the nuclear receptor Coup-TFII acts as a key regulator of venous

Biology of Extracellular Vesicles
Extracellular vesicles (EVs) have been found to influence a variety of pathological and physiological processes, such as inflammation, coagulation, or atherosclerosis, specifically in the context of vascular biology. However, their small subfractions (<200 nm) especially, together with possible differences between different vascular origins, remain poorly understood to this day. In general, EVs are defined as lipid membrane-enclosed vesicles with cellular origin that transport bioactive cargo including lipids, proteins, and nucleic acids. Not considering their cellular origin or cargo, they are commonly differentiated based on their size and release pathway into exosomes (30-150 nm), microvesicles (100 nm-1 µm), and apoptotic bodies (50 nm-5 µm) as the three main subcategories [10]. The small subset of EVs, most often termed exosomes, are vesicles that are actively secreted via an endosomal pathway. After the inward budding of the plasma membrane during endocytosis, membrane-bound proteins as well as extracellular components are internalized [11]. Subsequently, after scission from the membrane, the internally formed vesicles, now termed early endosomes, experience further regulated inward budding and cargo sorting during the maturation to late endosomes. The regulated inward budding and transport of cytoplasmic cargo leads to the formation of intraluminal vesicles (ILVs), which hallmarks the transition of late endosomes to multivesicular bodies (MVBs) [12].
These sorting and vesicle-formation processes are a topic of ongoing research that has revealed the high complexity and involvement of a variety of different proteins such as the endosomal sorting complex required for transport (ESCRT) or RAS-related protein RAB31. Mature MVBs are either fused with lysosomes for content degradation by hydrolytic enzymes through a ubiquitin-and clathrin-dependent manner or are trafficked to the cell membrane, where the subsequent fusion causes the release of the ILVs into the extracellular space in which they are then termed exosomes [12][13][14][15][16][17][18]. The commonly larger species of EVs, termed microvesicles (MV), originate, in contrast to exosomes, from the budding of the plasma membrane into the extracellular space and subsequent dissociation from the membrane. Specific adaptation of physical properties by changes of lipid and protein components thereby allows the remodeling of the cellular membrane, leading to separation and release of the MVs. Various molecules such as the ARF6 GTPase or vesicle-associated membrane protein 3 (VAMP3) have been shown to facilitate the active transport of cargo, such as proteins, enzymes, as well as nucleic acids, to the membrane prior to the budding process [19][20][21]. Although similar in size, apoptotic bodies show distinct differences to their exosomal and microvesicular counterparts. Apoptotic bodies are generally released through internal changes of the cytoskeleton and increasing hydrostatic pressure during cell death. These vesicles mainly function as a means of disposal of the dying cell, resulting in a highly similar proteomic profile to the cell lysate itself. The release of apoptotic bodies has been shown to influence various biological processes, such as the activation and modulation of the immune system by transferring antigens, increasing cell proliferation in stem cells as well as tumor niche formation [10,[22][23][24][25]
EndoEVs as liquid biopsy-derived biomarkers are increasingly coming into focus as their abundance and cargo provide valuable information on EC response to certain stimuli and can be pathognomonic for EC damage. EndoEVs can be characterized by markers inherent to their biogenesis or parental cells or those accumulating under certain conditions ( Figure 1). Contrary to common assumptions, phosphatidylserine (PS) is not only found in the outer membrane leaflet of MVs but also exosomes [42]. PS on endoEVs facilitates tethering to distant ECs and conveys pro-coagulant effects, as was shown by tissue factor (CD142)-bearing BEC-EVs [43,44]. Recent evidence suggests an apoptosisrelated subtype of exosomes (apoExos) that in BECs are shed in a caspase 3-dependent manner [45]. ApoExos, alongside exosome-specific tetraspanin CD63, express the lysosomal marker LAMP1 (CD107a), heat-shock protein 70 (HSP70), and sphingosine 1-phosphate receptors 1 and 3 (S1PR1, S1PR3) [46,47]. BEC apoExos also enrich pro-inflammatory non-ribosomal non-coding viral-like RNAs [45].

The Role of EndoEVs in Pathology
The secretion of EVs represents one form of cell-cell communication and is thus an important component in physiological and pathological processes [5,83]. Although little is known about the exact identity of endoEVs, recent studies suggest a high degree of heterogeneity and plasticity influencing cells, tissues, and organs both locally and systemically [5,84]. EndoEVs exhibit molecular patterns, which suggests that they contribute to the maintenance of tissue homeostasis, including cell survival and protection, angiogenesis, and an anti-inflammatory state. Moreover, through the production of nitric oxide (NO) ECs are able to control vasodilation, vasoconstriction, and thrombogenesis. In this context, it has been shown that the endothelial NO release triggered by high shear stress can impair the release of endoEVs [83]. A recent study investigated the effect of impaired blood flow on endothelial MV release in healthy subjects using an occlusion cuff model. In comparison to the control arm, significantly higher levels of CD62E+ and CD31+/CD42b-endoMVs were observed [85]. Accordingly, the levels of circulating endoEVs in the physiological state are considered to be rather low [83,86]. Nonetheless, EndoEVs exhibit molecular patterns that suggest that they contribute to the maintenance of tissue homeostasis, including cell survival and protection, angiogenesis, and an anti-inflammatory state. Other studies provide evidence that exosomes derived from ECs exposed to hypoxia and inflammatory cues contain proteins and mRNA that indicate the state of the cell of origin [87]. In addition, ECs have been shown to actively protect themselves from apoptosis and cellular stress by releasing endoMVs. This is achieved by the encapsulation of intercellular caspase-3 into endoMVs and their subsequent release from ECs, which results in a reduction of respective molecule levels within the cell [88]. Moreover, by presenting the endothelial protein C receptor, endoMVs can accelerate the activation of protein C, which may have anti-inflammatory and anti-apoptotic effects and thereby promote cell survival [89]. By transferring miRNA-rich microvesicles, protein and mRNA expression in recipient cells has been shown to be influenced by endoEVs [90]. For example, the horizontal transfer of miR-126 can inhibit the expression of SPRED-1 (an intracellular inhibitor of angiogenic signaling), leading to the promotion of angiogenesis [91]. Additionally, endoMVs were shown to express the urokinase-type plasminogen activator (uPA) and its respective receptor (uPAR). Therefore, endoMVs may contribute to the generation of plasmin, which in turn can have beneficial effects on tissue remodeling and in vitro tube formation [78]. Matrix metalloproteinase-2 and -9 in secreted endothelial vesicles can be associated with degradation of the surrounding extracellular matrix, release of growth factors, and thereby promotion of angiogenesis [67]. Some studies indicate that these effects are dose-dependent, with low (physiological) concentrations of endoMVs appearing to favor the formation of capillary-like structures and high concentrations inhibiting tube formation [78]. EndoEV-dependent transfer of functional miRNA-222 into ECs can decrease the expression of ICAM-1, which appears to have anti-inflammatory effects [92]. Moreover, endoEVs containing anti-inflammatory miRNAs are able to limit monocyte activation [93]. In contrast to the observation that rather low levels of endoEVs can be detected in physiological conditions, elevated levels of endoEVs may be indicative of diseases associated with endothelial dysfunctions [83]. Increased amounts of endoEVs are produced and released upon activation or apoptosis of endothelial cells. These EVs may play a role in the onset and progression of various vascular diseases [83]. Activating stimuli for ECs include proinflammatory cytokines such as TNF-a, bacterial lipopolysaccharide, reactive oxygen species (ROS), plasminogen activator inhibitor, thrombin, C-reactive protein, low shear stress [94], hypoxia, cell injury, and senescence [95]. EndoEVs released in response to proinflammatory signals may further drive inflammation through paracrine signal transduction, thereby promoting endothelial dysfunction [5]. These findings were further demonstrated by showing that endoMVs were able to activate human pulmonary microvascular endothelial cells and to induce the production of proinflammatory cytokines [96]. EndoEVs released upon TNF-α stimulation showed a significant change in the amount of proinflammatory mRNA, such as IL-8, MCP-1, IL-32, and VCAM-1, the latter being involved in the mobilization of leukocytes, which in turn also favors a proinflammatory state [83,97]. Endothelial dysfunction is a critical feature of type 2 diabetes and is considered a major cause of diabetic cardiovascular complications [98]. Koga et al. observed that significantly elevated levels of CD144 + endoMVs were found in patients suffering from diabetes mellitus compared to nondiabetic controls. In addition, they described that the CD144 + endoMVs levels in diabetes mellitus patients with coronary artery disease (CAD) were significantly higher than in diabetic patients without CAD [98].
In addition, endothelial dysfunction can be associated with the development of atherosclerosis [90]. EndoEVs carrying miR-155 were able to enhance the activation of monocytes and shift the balance from an anti-inflammatory to a pro-inflammatory phenotype, causing these EVs to contribute to atherosclerosis [99]. Partially, endoEVs released by inflammatory stimuli can be characterized by co-expression of tissue factor (TF) and phosphatidylserine (PS) on their outer surface, which facilitates the binding of EVs and provides them with procoagulant properties. Thus, the release of endoEVs seems to be associated with the activation of coagulation cascades and the formation of thrombi, suggesting that they might be also be linked to the development of stroke [86,90]. Simak et al. showed higher PS + endoMVs levels in patients with acute ischemic stroke compared to controls. A correlation between endo-EV level and lesion volume and clinical outcome was observed [100]. Additionally, it was found that elevated plasma levels of endoMVs can be linked to acute coronary syndrome, which includes myocardial infarction, angina pectoris, and myocardial ischemia. [55].

Conclusions
Taken together, increasing evidence supports the potential roles of EVs derived from blood and lymphatic endothelial cells in order to maintain physiological homeostasis as well as their roles in pathological settings. Further studies specifically addressing the vascular bed-specific molecular cargos in endoEVs will be needed to clarify their various roles and functions throughout biological processes. To achieve this goal, special attention must be given to the isolation and characterization of EVs, which demands general standards in EV research. As importantly corroborated by the International Society of Extracellular Vesicles (ISEV), publishing guidelines to help researchers in this fast-growing field studies on crucial issues, such as the comparability of EV, are of importance beyond any specificity of EVs in the vascular system [101].