ET 1 is a high-yield vasoactive substance that is primarily synthesized by the EC of the blood and lymphatic trees [28]. However, ET 1 effects are not limited to the vascular tree 2 and have a high affinity to ET_A, ET_B, and ET_C receptors, >60% of endothelin heart receptors are ET_A and vice versa in the lung, liver, and kidney, predominant ET_B [29]. The presence of various isotypes of endothelin receptors makes ET 1 a universal active substance, ET 1 receptors are present in the cardiovascular system (endothelium, VSMCs, cardiomyocytes), urinary system (renal medulla), nervous system (neurons), immune system and skin (macrophages, leukocytes, mast cells, Kupffer cells), respiratory system (tracheal epithelium, airway epithelial cells), other tissues and cells (fibroblasts, hepatic sinusoids) [30, 31]. Physiologically, four types of endothelin receptors are present in the organism (ET_A, ET_B1, ET_B2, ET_C); however, only two of them have been well studied. In fact, endothelial-endothelin production is enhanced by the activation of activator protein-1, GATA-2, smad, hypoxia-inducible factor-1, and NF-κB transcription factors [30, 32]. Normally, each organ has a higher concentration of specific isotype of the endothelin receptor and accordingly the endothelial of these organs produce the more required endothelin subtype. For instance, the kidney EC, mostly synthesize ET₁ and express ET_B receptors, to scavenge ET-1 from the plasma and reduce salt and water reabsorption in renal tubules [29]. Endothelin contributes to the extracellular matrix production, and proliferation and migration on vascular EC, vascular smooth muscle cells, fibroblasts, and macrophages/immune cells [33]. Activation of endothelin receptor-B (ET-B) on the EC leads to vasodilation by releasing specific vasodilator mediators and oppositely on the vascular smooth muscle cells where ET-B receptor activation results in vasoconstriction. The ET-A receptor is specific for vascular smooth muscle cells [34]. The biosynthesis of endothelin 1 by the EC is promoted by hormones (epinephrine, cortisol, angiotensin II, vasopressin, insulin), xenobiotics (A23187, cyclosporine), peptides (cytokines; IL-1, TGF-β, endotoxin, ET 1), psychochemical stimuli (hypoxia, shear stress; low), blood components (thrombin, glucose, oxidised LDL) as well as by inhibition of prostacyclin, NO, ANP, BNP, CNP, heparin, shear stress; high [30, 35]. The ET 1 degraded by lysosomal cathepsin A and its inactivation disorders lead to elevation of arterial blood pressure. In the kidney, ET1 induces sodium excretion (diuresis), exerts its effect through ET-B receptors on the epithelial cells of the collecting duct [29]. Several clinical data showed a relationship between ET-1 and the pathogenesis of pulmonary arterial hypertension, vasculopathy, and fibrosis of the skin, lungs, and other organs in systemic sclerosis as well as the potential therapeutic role for endothelin antagonists in these conditions in systemic sclerosis.

Auxiliary vasoconstrictor substances are thromboxane A2, superoxide, their release is usually inflammation-dependent. Both exert their effect in an auto-paracrine manner on their specific EC receptors. However, their effects and receptors are not limited to the ECs, but they can interact directly with VSMCs and induce extra actions (Table 2).

Normally, in the state of inflammation or high blood pressure, the EC shrink, while VSMCs relax to increase interendothelial space and vascular diameter, which is accompanied by hyperpermeability and lower blood pressure. Prostacyclin I2, NO, urotensin, CNP, adrenomedullin, adenosine, purines, reactive oxygen species, and H2S all are vasodilator molecules. NO is a free nitrogen radical released as a byproduct of several cellular activities, including the electron transport chain and under hypoxia and anoxia. Several cells release NO under unfavorable conditions including EC. NO is the most active vasodilator substance and controls the synthesis and activity of vasodilator molecules in the EC. Suggesting, a balance is kept between the endogenous NO production and ET-1/ETA activity to maintain the systematic vascular tone. Endothelial-derived NO release can inhibit the effect of adrenergic innervation on vascular smooth muscle cells [36]. Therefore, the sympathetic tone is predominant in the case of endothelial dysfunction. Currently, it is widely accepted that all adrenergic receptors (β1, 2, 3) contribute to the regulation of vascular tone. However, adrenergic receptors exert their effect on the vessels through the regulation of NO synthase activity, suggesting a NO-dependent vasomotor regulation. Furthermore, the β2 receptor is the predominant vasodilator in large vessels, α2 vasoconstrictor in the peripheral vessels, while β3 was found to enhance eNOS activity in the cardiac chambers and coronary microcirculation, indicating a vasoprotective role [37]. β2 expression is required to sustain a sufficient NO plasma level which is required for maintaining vascular homeostasis, whereas β2 gene silencing in vitro induced oxidative stress and impaired endothelial NO production, confirmed by antioxidant administration superoxide dismutase, reversed the vasoconstriction [37]. The stimulation of the β3 receptor induces angiogenesis. Accumulating evidence, hydrogen peroxide (H₂O₂) is directly involved in vasorelaxation, whereas, endothelium-dependent relaxation (EDR) involves both pertussis toxin-sensitive G_i include; responses to α2-adrenergic agonists, serotonin, and thrombin, and pertussis toxin-insensitive G_q include; adenosine diphosphate and bradykinin coupling proteins. EDR is promoted by newly discovered stimulators such as insulin, adiponectin [38].

Auxiliary, EC respond to high shear stress by releasing peptide hormone called adrenomedullin, due to activation of the endothelial mechanosensitive cation channel PIEZO1, which induces endothelial NO synthesis by binding of adrenomedullin to its G_s-coupled receptor protein in EC [39]. PIEZO1 activation induces G_q/G11-coupled purinergic P2Y2 receptor activation via an ATP-dependent manner. This required to phosphorylate eNOS at serine 1177/633 which is involved PECAM-1, VE-cadherin, and vascular endothelial growth factor receptor-2 (VEGFR-2)/VEGFR-3 mechanosensitive complex activation [39]. Therefore, adrenomedullin is a marker of hypertension and a guide for the treatment of sepsis and acute heart failure since it shows the severity of endothelial dysfunction including endothelial barrier function disturbance. Adrenomedullin is degraded by neprilysin which is blocked by valsartan, after 22 minutes of lifelong, adrenomedullin is degraded or cleared through binding to its cell surface receptor and internalization then degradation [40]. Finally, NO itself, under certain conditions such as hypoxia, can induce biased activation of soluble guanylyl cyclase promoting the synthesis of cyclic inosine monophosphate instead of cGMP and enhancing contraction rather than relaxation of the beneath VSMCs.

Angiogenesis is a complex process that requires the cooperation of various cell types through signaling exchange between these cell types, aimed at forming new blood vessels from old ones, vascular stem cells, or EC. Angiogenesis or lymphangiogenesis are critical in the progression of many diseases including tumor metastasis and ischemic heart disease [41]. Indeed, EC proliferation-induced-angiogenesis is triggered by several growth factors that are released by the underlying matrix including; FGF-2, TGF-β, TNF-α, IGF-1, and VEGF. However, VEGFR-2 activation, separately able to induce EC proliferation as well as migratory and sprouting activity and to help promote EC to form tubule-like structures. The VEGF combination with chemokines such as angiopoietin-1 and integrins can determine the neovessels’ destination by ephrins migration regulation. The VEGF is required for the initiation of angiogenesis and the formation of immature vessels, while Ang1 and ephrinB2 are responsible for the maturation of initial immature blood vessels. Furthermore, Ang1 expression probably is required for the maintenance of vasculature cells’ identity. On the curbing of EC proliferation is mediated by thrombospondin-1, canstatin, arrestin, tumstatin, angiostatin, and endostatin which are also released by the beneath matrix [6]. Also, the receptor for the vascular endothelial protein tyrosine phosphatase is allocated on the endothelial cell, involved in angiogenesis regulation and membrane fluidity, as well as homeostasis maintaining of the inflamed area through the regulation of the tyrosine kinase receptor Tie-2 and VE-cadherin substrates regulation [42]. Undoubtedly, EC are also involved in vascularization regulation of vascularization.

The endothelial released molecules are also involved in the regulation of angiogenesis such as activated protein c (APC) and vWF [43]. More recently, novel investigations have shown that suppression of vWF expression increases endothelial proliferation, migration, and angiogenesis through binding to integrin αvβ3, and components of Weibel-Palade bodies, such as angiopoietin-2 and galectin-3, whose storage is regulated by vWF, whereas endothelial protein C receptor probably suggests hematopoietic stem cells dormancy and multilineage reconstitution potential [44, 45]. Angiopoietin Ang2 (ANGPT2) binds to tyrosine kinase Tie2 and destabilize blood vessels and synergize with VEGF to promote angiogenesis [46]. Additionally, Gal-3, via the interaction with VEGFR-2 and integrin αvβ3 endothelial cell vWF domains induces angiogenesis, whereas, Gal-3 inhibition, reduces proangiogenesis activity [47]. The Gal-3 regulates cell-cell adhesion which is probably an important step for proangiogenesis [48]. Recent clinical data suggest that an imbalance between Ang1 and Ang2 activity probably underlying the development of the vascular lesions, and maintaining a balance between these growth factors is required for healthy vasculature [49].

Protein c and s are primary anticoagulant peptides synthesized by the liver and platelet as well as EC. Protein c activation requires thrombin-thrombomodulin complex formation on the EC surface, involves proteolysis at Arg169 in EPCR-bound protein c EPCR [50, 51]. Once protein c has been activated, protein s synergistically with calcium and phospholipids cleavage specific arginine polypeptide of proaccelerin and activated antihemophilic factors to inactivate coagulation and promote fibrinolysis by inhibition of tenase and prothrombinase complex [50, 52]. The APC activities are enhanced by protein S, high-density lipoprotein, and glucosylceramide [53]. Furthermore, in animal models, APC induces ERK1/2 (extracellular signal-regulated kinase) during EC proliferation and angiogenesis to promote early growth response factor-1 gene expression through PAR1/S1P1-dependent but EPCR-independent mechanisms solely inhibit TNFα-induced apoptosis in EC [51]. Moreover, in rheumatoid synovial fibroblasts, APC decreases the TNFα-induced phosphorylation of p38 MAPK and JNK [54]. Deficiency of protein s and or c results in DIC syndrome, while activated PC exerts a neuroprotective and neurogenesis effect through G-protein–coupled receptor, protease-activated receptor 1 (PAR1), PAR3, and EPCR that produce signaling at Arg46 site (biased signaling) [53]. The APC binds to EPCR and can cleave PAR-1 and PAR-3, which induce β-arrestin-2–biased PAR1 signaling that results in protection of the vascular endothelium from apoptosis while promoting barrier function. APC reduces the release of neutrophil extracellular trap release [55].

TM is a receptor on the surface receptor of blood and lymphatic vessels thrombin EC, but not only in EC, consisting of 5 domains, each of which has a role in norm and pathology [56]. Thrombomuldin regulates coagulation activity and the release of inflammatory molecules, as well as EC migration via fibronection and ezrin. Besides, its cytoprotective effects interfere with the nuclear translocation of NF-κB and activation of AP-1 [51, 56, 57]. In murine aortic EC, the interaction of the EGF5 domain of TM with G-protein coupled receptor 15 promotes angiogenesis and cell survival through activation of ERK and BCL-2 pathway, suggesting a cytoprotective and angiogenic role of TM on EC [58]. Moreover, endothelial CD39/NTPDase-1 poses an anticoagulant effect by degrading the platelet agonist ADP. TM modulates the duration of pERK nuclear retention and cell proliferation in response to inflammatory stimuli. TM enhances smooth muscle proliferation by activating the ERK pathway through the EGFR axis that negatively affects the growth arrest-specific gene 6, whereas, TM knockdown causes smooth muscle calcification in animal models [59]. Low TM expression by the EC is probably correlated with atherosclerosis development, via the activation of p38 and JNK pathways, where the oxidized low-density lipoprotein reduces the levels of nuclear transcription factors RARβ, RXRα, Sp1, and Sp3 and their binding to TM promoter [51].

When the continuity of endothelium is disrupted or the EC are glycosylated, the coagulation cascade is initiated. EC play a significantly crucial role in maintaining an average level of coagulability, not hyper or hypo clotting. This feature is achieved through the balance between the endothelial released hemostatic factors and the expression of pro and anticoagulant cell surface receptors. In favor of clot formation, EC induce the expression of procoagulant and suppress the expression of anticoagulant factors. Where TNF reduces the formation of TM, endothelial anticoagulant cofactor, and induces procoagulant mechanisms. EC release tissue-type plasminogen activator, plasminogen activator inhibitor type I, platelet activating factor, and vWF into plasma. Exposure to phosphatidylserine induces the release and binding of vWF to collagen fibers and subsequent platelet adhesion to vWF through GP Ib. oxidative stress improves the adhesiveness of vWF, auto association of vWF is regulated primarily by ADAMTS13 protease and is influenced by the level of LDL and HDL, as well as thrombospondin-1 [60, 61]. The arrest by the EC was firstly described in 1882 by Giulio Bizzozero, but later on, appeared that leukocytes roll on the endothelial surface as a secondary phenomenon for coagulation.

The maintenance of transport between the bloodstream and tissues is carried out by EC. Usually, EC perform transcellular (intracellular) or paracellular (intercellular) transport for the fluids, molecules, and leukocytes, paracellular is the dominant form. Intracellular transport requires the formation of caveolae and, through cellular endocytosis, the transported material is internalised, then the intracellular cytoskeleton leads it from the luminal to the abluminal side and in the opposite direction. However, intercellular transport is primarily through specific intercellular tight junctions (e.g., occludins associated with ZO-1, ZO-2, and cingulin proteins) or gap junctions, VE-cadherins (intracellular cytoplasmic proteins and catenins connected to the actin-based microfilament system) [19]. The rearrangement of cadherins and occludins is required to regulate permeability. To note, the VE-cadherins functionality is regulated by small molecules of GTPases that belong to the Rho family, protein phosphorylation, and through interaction with other transmembrane proteins. Several molecules that affect the transport intensity of intercellular transport include; physical forces (e.g., fluid shear stress) and biological mediators (VEGF, FGF, Angiopoietin-1, histamine, sphingosine-1-phosphate) as well as the balance of adhesive forces between EC and between cells and the extracellular matrix and contractile forces generated by the actomyosin system. The loss of barrier function usually arises after disturbance in the regulation mechanisms of the trans or intercellular transport. For instance, under the state of inflammation, the high level of inflammatory mediators in particular histamines induces the intercellular transport of macro and microparticles from the bloodstream to the plasma that results in edema [19].

Endoglin is a TGF-β receptor that positively regulates ALK-1 signaling to Smad1, 5, and 8 but negatively regulates ALK-5-induced Smad2 and Smad3 pathways in EC. ALK-1-mediated activation of Smad1, 5, and 8 EC require the phosphorylation of endoglin in Ser646 and Ser649 by TβRI. The (L)-endoglin comprises a long cytoplasmic tail of 47 amino acid residues, while (S)-endoglin with a short cytoplasmic tail of only 14 amino acid residues. Enhancement of signaling through ALK-1 requires L-endoglin to activate Id1 expression [62]. Whereas, signaling via ALK-5 requires S-endoglin to induce expression of the plasminogen activator inhibitor-1 (PAI-1). The PDZ-binding motif of the cytoplasmic tail of L-endoglin interacts with GAIP interacting protein, carboxyl terminus (GIPC), a scaffolding protein that controls the cell-surface receptor expression and trafficking [62]. When TGF-β-independent interacts with endoglin, GIPC induces phosphorylation of Smad1, 5, and 8 as well as promotes cell-surface retention of endoglin. Furthermore, endoglin binds bone morphogenetic proteins and enhances their signaling via Smad1, 5, and 8. Pathophysiologically, endoglin gene results in deleterious defects in the cardiovascular system, whereas homozygous endoglin gene silencing results in hereditary hemorrhagic telangiectasia type I [62]. Elevation of endoglin is found in preeclampsia [56].

Thromboxane is well known as a vasoconstrictor, through the regulation of endothelial NO synthetase activity. TPr antagonist leads to decrease phosphorylation of eNOS at Ser1177 and Akt at Ser473. Whereas, TPr activation enhances reduction of the Rho-associated kinase- phosphatase and tension homolog deleted on chromosome 10-Akt-eNOS axis, suggesting TPr role in endothelial dysfunction [63]. TPr is involved in the impairment of endothelial function via reduction of NO synthesis and decreased intercellular (EC) gap junction, in turn, this contributes to the development of atherosclerosis and diabetes mellitus as well as hypertension [64].

Not surprising that the EC poses receptors for the angiotensin II, exerts its effect through the G_q receptor signaling cascade, believed that angiotensin regulates the release of vasodilator substances from the EC. Furthermore, angiotensin reduces EC growth and regulates fibrinolysis through the AT2 receptor. Recent findings concluded a direct relationship between angiotensin II receptor type 1 and EC dysfunction through reduction of the endothelial NO synthase phosphorylation via AT 1 R Nox/ROS/PP2A axis, confirmed by AT1 receptor antagonists administration that normalized the NO level via blocking phosphorylation of PP2Ac Tyr307 as well as proved the role of AT-II by knockdown down the p22phox gene, also decreases the phosphorylation of PP2Ac Tyr307 [65].

The EC do pose a cell surface receptor for the serotonin; 1A, 1B, 3A, and 6. A recent investigation suggested an inhibitory role for these receptors, where 1A, 1B, receptors agonist reduced transendothelial cell migration, particularly, allergy-inducing leukocytes [66]. However, EC modify the serotonin-vascular tone activity via regulating the content of serotonin in the vascular bed. Monoamine oxidase type 1 is present in the EC that are directly related to the regulation of the vascular smooth muscle cells.

Recent data were showing that each organ’s EC has its isotype of the muscarinic (M) receptor, however, the M5 receptor approximately is expressed in all EC. The activation of the M3 receptor in healthy endothelium is responsible for vasodilation and oppositely; vasoconstriction in dysfunctional EC [67]. M1 receptors that have been found in cerebral microcirculatory endothelial cells (CMEC) have a relatively low functionality, whereas, highly expressed M3 and M4 receptors [68]. However, the study concluded that all M subtype receptors are expressed by CMECs, particularly in brain vessels and especially M3>M2>M4>M1>M5. This adds more complexity to the surface-expressed receptors of the EC in various organs, probably the same receptors present in the whole vascular tree or organ-specific!

More recently, new data have explained the role of acetylcholine released by the endothelial cell (ACh) in the flow-mediated vasorelaxation, suggesting a stimulatory effect for ACh in calcium channels to release internally stored calcium and induce NO release that leads to vasorelaxation through the previously cAMP pathway [69]. However, ACh production by the EC requires transferring of coenzyme, acetyl‐CoA to choline by a polarized mitochondrial membrane electron transporting complexes, besides, the external supply of choline precursors such as pyruvate was sufficient to induce flow‐evoked ACh production [69].

More recently, new investigations have addressed, scavenger receptor class B type I is an extremely important endothelial receptor, involved in the regulation of HDL transport in the liver, and its dysregulation is believed to be responsible for dyslipidemia and atherosclerosis development [70]. Furthermore, recent investigations were showing that the vitamin D endothelial receptor is directly involved in vasoactivity regulation, particularly in cerebral vessels, where the knockdown of the endothelial vitamin D receptor gene was accompanied by severe vasospasm [71].

Additionally, a small, highly conserved, intracellular calcium-binding protein termed CaM present in the EC. CaM regulates intracellular calcium influx, involved in several physiopathological processes including inflammatory and EC migration inhibition. CaM is required to activate transient receptor potential 6 (TRPC6) channels that later activate the TRPC5 channels to enhance intracellular calcium influx [72]. Also, CaM is involved in the leukocyte paraendothelial migration (TEM) via activating the IQ‐domain GTPase‐activating protein 1 (IQGAP1) and particularly CaM‐binding isoleucine‐glutamine (IQ), inhibition of CaM, theoretically can be used as an anti-inflammatory approach (Table 2) [73].

A bunch of fundamental biological distinctions is present between the lymphatic and blood EC despite the topographical similarities, more than 400 differences in the expressed genes, returned to the difference in the functions of each (Table 3) [78]. The lymphatic EC perform a semi-professional antigen-presenting to the lymphatic circulatory T cytotoxic CD8 cells through invadosome-like protrusion (MHC-I) and express specific surface antigens and receptors such as podoplanin, VEGFR-3, or lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) [79, 80].

Remarkable evidence concluded that sigma receptor-1 of the lymphatic EC is responsible for the regulation of the tone and barrier function as well as the permeability of the lymphatic tree through regulation of nitroxide biosynthesis signaling pathway [20].