Vascular Endothelial Cells and Innate Immunity

Abstract

In addition to the roles of endothelial cells (ECs) in physiological processes, ECs actively participate in both innate and adaptive immune responses. We previously reported that, in comparison to macrophages, a prototypic innate immune cell type, ECs have many innate immune functions that macrophages carry out, including cytokine secretion, phagocytic function, antigen presentation, pathogen associated molecular patterns (PAMPs)-, and danger associated molecular patterns (DAMPs)-sensing, pro-inflammatory, immune-enhancing, anti-inflammatory, immunosuppression, migration, heterogeneity, and plasticity. In this highlight, we introduce recent advances published in both ATVB and many other journals: 1) Several significant characters classify ECs as novel immune cells not only in infections and allograft transplantation but also in metabolic diseases; 2) Several new receptor systems including conditional DAMP receptors, non-pattern receptors, and homeostasis associated molecular patterns (HAMPs) receptors contribute to innate immune functions of ECs; 3) Immunometabolism and innate immune memory determine the innate immune functions of ECs; 4) a great induction of the immune checkpoint receptors (ICRs) in ECs during inflammations suggests the immune tolerogenic functions of ECs; and 5) association of immune checkpoint inhibitors with cardiovascular adverse events (CVAEs) and cardio-oncology indicates the potential contributions of ECs as innate immune cells.

1. Introduction

Under physiological conditions^{1, 2}, ECs are involved in the modulations of metabolic homeostasis (trophic functions), vascular hemodynamics (tonic functions)³, vascular permeability, coagulation, and cell extravasation (trafficking)². In a quiescent state, ECs balance the release of various vasodilating or vasoconstricting factors such as nitric oxide, prostacyclins, and endothelin to maintain vascular tone, blood pressure, and blood flow⁴. In addition, ECs secrete numerous cytokines and growth factors including interleukin-6 (IL-6)^5–7, thrombospondin, frizzled-related protein 3, insulin-like growth factor-1 (IGF-1), connective tissue growth factor (CTGF)⁸, bone morphogenetic protein (BMP)-9⁹, interleukin (IL)-1α^{10, 11}, IL-1β^{7, 12}, placental growth factor, leukemia inhibitory factor (LIF), Wnt family member 1 (WNT1)-inducible signaling pathway protein 1 (WISP-1), midkine, and adrenomedullin to facilitate cardiac performance and remodeling¹³. Furthermore, the endothelium is crucial in regulating coagulation, utilizing both anti-coagulation and pro-coagulation mechanisms^14–16. ECs have an essential role in modulating vascular permeability¹⁷. During states of acute and chronic inflammation¹⁸, hyperglycemia⁹, ECs display an excessive or prolonged increase in permeability, allowing for additional trafficking of immune cells and consequently deleterious effects resulting in tissue edema¹⁹. Of note, low dose mitochondrial reactive oxygen species (mtROS) generation, uncoupled from ATP production and promoted by proton leak^{20, 21}, drove upregulation of endothelial cell (EC) adhesion molecule, intercellular adhesion molecule-1 (ICAM-1)^20–23. This physiological ECs activation status may facilitate non-classical patrolling monocyte migration for immune-surveillance function in tissues²⁴. The inability of ECs to adequately carry out these functions, which is termed as endothelial dysfunction, causes an elevating risk of cardiovascular events^{11, 25–27}.

Under hypoxic conditions, thrombus-derived monocytes collected from patients with acute coronary artery disease could be transdifferentiated into ECs²⁸. ECs can also be transdifferentiated from fibroblasts via innate immune signaling of a glycolytic switch²⁹. In atherogenic processes, the endothelium is a source for plaque-associated mesenchymal cells through endothelial-to-mesenchymal transition (EndoMT)³⁰. A recent study also demonstrated the presence of EndoMT in human adipose tissue in obesity; and EndoMT reduced mitochondrial oxidative phosphorylation and glycolytic capacity of EC³¹. In addition, cardiovascular disorders, including atherosclerosis, are considered as premature aging³². The underlying mechanisms of a concept termed inflammaging³³ include genetic susceptibility, central obesity, increased gut permeability, changes to microbiota composition, cellular senescence, nucleotide-binding oligomerization domain-like (NOD)-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation, and oxidative stress. Chronic senescent cells lead to their deleterious effects through a secretory phenotype³⁴ known as the senescence-associated secretory phenotype (SASP)^{35, 36}. Proteomic analysis of endothelial particulate secretome represented by extracellular vesicles (EV) in the proinflammatory conditions exhibite the presence of pro-inflammatory and immune proteins involved in signal transduction, immune and inflammatory responses, and angiogenesis³¹.

ECs also have important immunological functions. The innate immune system³⁷ including ECs mediates non-specific immunity, which is immediate and antigen-independent. Innate immune interactions between the cardiovascular system and the immune system are a well-accepted mechanism underlying metabolic cardiovascular diseases, which has been emphasized by the success of CANTOS trial (Canakinumab Anti-Inflammatory Thrombosis Outcome Study), a therapeutic monoclonal antibody targeting IL-1β³⁸. Therefore, vascular ECs are innate immune cells¹ in many physiological and pathophysiological conditions, including infection, transplantation conditions^39–41 metabolic disorders such as hyperlipidemia^{42, 43}, hyperglycemia^{44, 45}, hyperhomocysteinemia^46–48, metabolic syndrome, obesity^{49, 50}, or hypertension, and cigarette smoke^{51, 52}. This review will highlight the recent publications to support that endothelial cells are multifunctional innate immune cells.

2. ECs are novel immune cells.

Historically, cardiovascular immunology has focused on the interactions between the cardiovascular and immune systems, which determine how immune cells promote^{53, 54} and suppress^55–58 cardiovascular diseases by modulating pathophysiological responses of cardiovascular cells. Additionally, immunological features of cardiovascular cells have been gradually recognized. Cell interactions have a few formats: i) resident cell-resident cell, ii) migrated cell-migrated cell, iii) migrated cell-resident cell, and iv) migrated cell-structural cell via direct and indirect (secretions and extracellular vesicles) manners⁵⁹. When investigators had to use microscopy to examine the morphology of migrated cell types in the inflammation sites and immunohistology, classical innate immune cells were identified including neutrophils, monocytes/macrophages, T cells, and mast cells⁶⁰, which emphasized the roles of cell migration in the cellular interactions during inflammation and immune processes. However, immune regulatory functions are not unique to migrated cells. The roles of structural cells and cardiovascular resident cells such as ECs in cellular interaction and immune regulation, when trans-endothelial migration of immune and inflammatory cells, have been under-appreciated for a long time. Besides the historical reasons, potential assumption that endothelial cells have no immune regulatory effects on migrated immune cells, inflammatory cells, vascular smooth muscle cells and other vascular cells may not be correct³. Now there are strong evidences⁶¹ that ECs and other structural cells such as lymphatic ECs^{62, 63}, epithelial cells^64–67, stromal cells^{66, 68–70}, Sca1⁺ progenitor cells⁷¹, vascular smooth muscle cells (VSMC)^72–78, Kupffer cells in the liver, adipocytes, and others⁷⁹ play significant roles in regulating innate and adaptive immune functions^{1, 40, 65, 80–82}. Of note, even adaptive immune cells such as CD8⁺ T cells⁸³, γδT cells⁸⁴, innate lymphoid cells⁸⁵, innate B cells⁸⁶, tissue-resident memory T cells⁸⁷, type 1 T helper cell (Th1)-like CD4⁺Foxp3⁺ regulatory T cells (Treg), Th2-like Treg, Th17-like Treg, and Tfh-like Treg⁸⁸, antigen-presenting cell (APC)-like Treg, have innate immune functions⁸⁹.

As we reviewed in 2013, eleven innate immune functions that macrophages carry out can also be performed by ECs, including cytokine secretion, phagocytic function, antigen presentation, PAMPs and DAMPs sensing, proinflammatory, immune-enhancing, anti-inflammatory, immunosuppression, migration, heterogeneity, and plasticity¹. A few principles in determining innate immune cell identity are summarized in Figure 1: First, the cells are capable of sensing the stimulations and danger signals by various PAMPs, DAMPs, proinflammatory and anti-inflammatory cytokines, growth factors, exosomes and extracellular vesicles⁹⁰; Second, in responding to stimuli, the cells are capable of secreting cytokines, chemokines, growth factors, other secretory proteins, microparticles⁹¹, exosomes⁹⁰, circular RNAs⁹², microRNAs^{49, 93–95}, and other noncoding RNAs^{49, 92, 96–98}, upregulating co-signaling receptors (co-stimulation and immune checkpoint receptors) and major histocompatibility complex II (MHC II) to directly or indirectly interact with adaptive immune cells^{81, 99}; Third, the cells are capable of presenting antigens via MHC II to CD4⁺ T helper cells^{80, 100, 101}; Fourth, the cells are capable of memorizing the challenges (trained immunity)^{102, 103} they encountered and enhancing response when encounter challenges again¹⁰⁴; and Fifth, the cells are capable of maintaining cellular homeostasis (trained immune tolerance)¹⁰⁵ from PAMPs, and DAMPs stimulations¹⁰⁶, which is similar to the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) immune function in bacteria and archaea from foreign DNA attacks¹⁰⁷. Of note, the class defining criteria of being able to present antigens via MHC II is not very restricted. It was recently reported using new single-cell RNA sequencing technique to separate antigenpresenting MHC II-high dendritic cell (DC) population from inflammatory function-high DC population¹⁰⁸, suggesting that not all the DC have high antigen presenting capacity. Other recent reports demonstrated that some professional innate immune cells including B cells, macrophages, natural killer cells (NK), monocytes, plasmacytoid dendritic cells (pDC), DC1 and DC2 have 1,300 innate immune gene expression differences, suggesting huge heterogeneities^{109, 110}. Therefore, it may not be optimal to use antigen-presenting capacity^{111, 112} as the essential criterion for judging the innate immune function of ECs.

Figure.1.

Schematic Model of Endothelial Cells Are Innate Immune Cells

ECs have classical DAMPs sensing systems. Traditional innate immune cells that patrol the blood, such as DCs, and Ly6C^low monocytes^{24, 113}, is equipped with a series of PAMPs receptors including Tolllike receptors (TLRs)¹¹⁴ and NOD-like receptors (NLRs)^{52, 115}. TLRs, NLRs, retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs), absent in melanoma 2 (AIM2)-like receptors (ALRs) and C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs). These receptors are part of the innate immune system and are known to be expressed on immune cells as well as non-immune cells¹¹⁶ including a few vascular cell types such as aortic ECs⁴³, angiogenic ECs⁴², Sca-1⁺ vascular progenitor cells⁷¹, and VSMCs^{72, 73}. PRRs can sense components of exogenous microbes as well as harmful endogenous components. These findings suggest a novel concept of conditional danger receptors that endogenous metabolites, when elevated to pathological concentrations, can trigger inflammation by binding to their intrinsic receptors rather than DAMPs/PAMPs such as TLRs or NLRs. This type of intrinsic receptors for elevated endogenous metabolites are conditional danger receptors since they carry out physiological signaling function when metabolites are in physiological concentrations^{117, 118}.

The cellular “receptors, trouble-detectors and metabolic sensors”⁷⁹, which can recognize the risk factors for atherogenesis¹¹⁹ such as hyperlipidemia¹²⁰ and hyperhomocysteinemia¹²¹, contribute significantly to the innate immune functions of ECs. The roles of PRRs have been characterized recently as bridging innate immune sensory systems for exogenous infectious agents and endogenous metabolic DAMPs to initiation of inflammation^{51, 122, 123}. In addition to TLRs and NLRs, four additional DAMP receptor categories have been characterized^{124, 125}: first, transmembrane C-type lectin receptors (dendritic cell natural killer lectin group receptor 1 (DNGR1, receptor for F-actin), macrophage-inducible C-type lectin (MINCLE, receptor for spliceosome-associated protein 130 (SAP130), β-glycosylceramide), Dectin-1 (receptor for N-glycans) and Dectin-2¹¹⁶; second, retinoid acid inducible gene I (RIG-I, melanoma differentiation-associated protein 5 (MDA5) and RIG-like receptor dsRNA helicase (LGP2)¹¹⁶), third, cytosolic DNA sensors such as AIM2 (absent in melanoma 2), cyclic GMP–AMP synthase (cGAS) and stimulator of interferon genes (STING)⁷⁶, and fourth, receptor for advanced glycation end products (RAGE, a receptor for AGEs, high mobility group box 1 (HMGB1)¹²⁶, S100s, β-amyloid (Aβ) and DNA)¹²⁷. Herein, we refer to the six categories above-mentioned as classical DAMP receptors. Early hyperlipidemia activates NLR/inflammasome-caspase-1 pathway in endothelial cells, which is responsible for increased atherogenesis⁴³, decreased angiogenesis⁴², and weakened progenitor cell vessel repair^{128, 129}. Oxidized LDL (oxLDL)-activated human aortic endothelial cells –(HAECs)- undergo EC inflammation and EC inflammatory cell death stage (pyroptosis) as judged by caspase-1 activation⁴³. In addition, ECs secrete the pro-inflammatory cytokine interleukin-8 (IL-8) in a NOD1-dependent response to microbial stimulation^{130, 131}, and NOD2-dependent response to bacterial peptidoglycan muramyl dipeptide^{132,89, 133}. IL-17 activates HAECs by inducing IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), chemokine (C-X-C) ligand 1 (CXCL1) and CXCL2¹³⁴, which may suggest an interaction mode between T cells and ECs. Besides, a very recent study revealed that in pulmonary vascular cells, EC-derived HMGB1 activate RAGE in ECs and SMCs via HIMF (Hypoxia-Induced Mitogenic Factor) to form a positive feedback loop, accelerating the secretion and release of more HMGB1 and fueling the vascular-immune milieu of PH development¹²⁶.

Inflamed endothelium and endothelium of atherosclerotic lesions have significant upregulation of TLR2 and TLR4 expression¹³⁵, and induce ECs responses such as the production of IL-1, IL-8, and monocyte chemotactic protein-1 (MCP-1) via TLR4^136–139. Similarly, LPS, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) can induce TLR2 expression¹⁴⁰. ECs also express CD14 which is also a known receptor for LPS¹⁴¹. TLR3, TLR7, and TLR8 are important in detecting viral RNA and activating innate immune responses against viruses. Human umbilical vein endothelial cells (HUVECs) do express TLR3. In fact, ECs also express IFN-α, which is an important cytokine in regulating innate immune responses against viruses and is shown to strongly induce TLR3 expression¹⁴². Moreover, ECs also express TLR9 which recognizes viral and bacterial DNA^{135, 143}. Aside from the expression of PRRs, ECs also express important downstream adaptor molecules for PRR signaling including myeloid differentiation-2 (MD2) and myeloid differentiation primary response protein 88 (MyD88)^{144, 145}. ECs express the lectin-like oxLDL receptor (LOX-1) at low levels. Strong evidence has suggested a pathological role of LOX-1 in atherosclerosis, a chronic autoimmune inflammatory disease in response to stimulation by oxLDL^{170, 171,146}, proinflammatory cytokines, and proatherogenic factors such as angiotensin II¹⁴⁷. LOX-1 is also important in endothelial-mediated vascular homeostasis and coagulation prevention under physiological conditions¹⁴⁸.

3. Receptors or signaling pathways contribute to innate immune functions of endothelial cells.

Following principles can be “extracted” underlying the current classification of DAMPs¹⁴⁹ and their receptors as: (1) endogenous metabolites released under the stresses could serve as the DAMPs; (2) DAMPs bind to specific receptors; (3) DAMPs receptor initiates proinflammatory transcription factor-mediated signaling¹⁵⁰; and (4) DAMPs receptor genes are evolutionally conserved from mice to humans¹⁵¹. However, the current classification and paradigm of DAMPs receptors have several problems. First, whether endogenous lysophospholipids (LPL) receptors that fit all the above-discussed principles can be classified as novel DAMPs receptors in initiating inflammation-modulating signaling; and second, the current DAMP receptor model emphasizes only the danger signals generated from endogenous metabolic processes but fails in recognizing the roles of potential endogenous metabolites in anti-inflammatory responses, inflammation resolution and maintenance of homeostasis. Therefore, LPLs can use their intrinsic receptors but not classical DAMP receptors to initiate innate immune signaling, which we termed conditional DAMP receptors^{118, 151}. Similarly, various types of non-PRR (non-pattern recognition) transmembrane proteins including TREMs (triggering receptors expressed on myeloid cells 1 (TREM1) and TREM2), G-proteincoupled receptors (N-formyl peptide receptor (FPR)1, FPR2, P2Y2 purinoceptor receptor (P2Y2R)⁵², P2Y6R, P2Y12R, calcium-sensing receptor (CaSR), G-protein-coupled receptor family C group 6 member A (GPRC6A)) and ion channels (transient receptor potential cation channel subfamily member 2 (TRPM2), other transient receptor potentials (TRPs), P2X7R) have been reported to sense DAMPs¹²⁵. Recently, significant progress has been reported in identifying additional membrane receptor systems in regulating EC activation in innate and adaptive immune responses as summarized in Table 1.

Table1.

Recent reports identified new evidence to support the model that endothelial cells have innate immune functions in metabolic diseases. (Part I)

Innate immune relevance	Associated metabolic situlations	Target and experimental model	Responses on ECs	Interaction between ECs and other cells	Reference
Complement system	Early ischemia Late ischemia	MBL (mannose-binding lectin)-deposition (MBL−/− mice)	Induces the binding of IL-1α with IL-1R1 Increases C3b and induces ICAM-1		10
	Atherogenesis	RGC-32 (response gene to complement 32) in resident in vascular cells, but not macrophage (RGC-32−/− mice)	induces ICAM-1, VCAM-1 through directly interacting with NF-KB	RGC-32 deficiency decreases TNF-α-induced monocyte-endothelial cell interaction	196
Reguatlion Of cytokines, chemokines and adipokines	Diabetic macular edema	BMP9/Alk1 (activin-like kinase receptor type I) (HUVECs and streptozotocin-induced mice)	Prevents VEGF-induced phosphorylation of VE-cadherin, induces occludin and strengthens vascular barrier functions		9
	Angiogenesis	BDNF (brain-derived neurotrophic factor) /NT-3 (neurotrophin-3) (ESCs, embryonic stem cells)		Promotes ESCs differentiation to ECs in a BDNF/NT3 receptors dependent way	197
	Abdominal aortic aneurysm (AAA)	FAM3D (Mouse Models of AAA, and FAM3D−/− mice)	FAM3D is upregulated in ECs by vascular pathogenic stimuli	Contributes to neutrophil recruitment via FPR-Gi Protein/β-Arrestin-Mac-1 Signaling	198
	Atherogenesis	IL-35 (HAECs and APOE−/− mice)	Inhibits mtROS-H3K14 acetylation-activator protein 1-mediated EC activation		199
	Vascualr inflammation	NOTCH1 signaling (HUVECs and RbpjiΔEC, NICD (Notch intracellular domain)iEC-OE mice)	Modulates the transcriptional response to inflammatory cytokines; Supports the expression of a subset of inflammatory genes at the enhancer level	Increases leukocyte recruitment to the inflamed lesion	200
	Endothelial regeneration	Cytokine-like protein dikkopf-3 (DKK3) (Human embryonic lung fibroblasts)		Drives human fibroblasts to differentiate to functional ECs via mesenchymal-to-epithelial transition and VEGF-microRNA-Stat3 pathways	201
	Atherosclerosis and arterial stiffness	SFRP5 (secreted frizzled-related protein 5) (HUVECs, HAECs and patients with type 2 diabetes)	Restors Wnt5 (wingless-type family member 5a)-reduced NO production via eNOS		202
Part II
Innate immune relevance	Associated metabolic situlations	Target and experimental model	Responses on ECs	Interaction between ECs and other cells	Reference
Receptor systems sensing (DMAPs)	Erosion-associated thrombosis	Neutrophil extracellular traps (HSVECs (human saphenous vein ECs) or HUVECs)	Augments ICAM-1, VCAM-1 and transcription factors through concerted action of IL-1α and cathepsin G, but not IL-1β		11
	Angiogenesis	Posttranslational proteolytic cleavage of VEGF receptors (HUVECs)	Upregulates neuropilin-1 (NRP1) species in an ADAM(a disintegrin and metalloproteinase)9/10-dependent manner resulting in inhibition of VEGF-induced EC motility and angiogenesis		203
	Angiogenesis	MicroRNA-199a-3p/5p (bovine aortic endothelial cells)	Redundantly decreases eNOS activity and induces its degradation, thereby supporting VEGF-induced endothelial tubulogenesis		97
	Atherogenesis	Dislipidemia and disturbed flow (HAECs and CD36−/− mice)	Increases oxLDL uptake and enhences endothelial stiffening via CD36		204
	Thrombophilia	Hypoxic trophoblasts derived HMGB1 (HUVECs and Pregnant mice)	Stimulates the generation and release of EC-oringin microparticles and enhances blood coagulation	Triggers neutrophil activation	205
	Thrombotic and inflammatory disorders	PolyP70 (Inorganic polyphosphate 70) (HUVECs)	Amplified HMGB1-mediated VWF release via binding to RAGE and P2Y1 receptors	Promotes VWF-platelet string formation on ECs	15
	Flow-dependent vascular remodeling	Lack of cystathionine γ-lyase (CSE−/− mice)	limits disturbed flow-induced ICAM-1, VCAM-1 through altering NO availability	Decreases monocyte infiltration	206
	Pulmonary Hypertension (PH)	HIMF Signaling (human pulmonary microvascular ECs and pulmonary arteries of patients with idiopathic PH and PH Models in mice and rats)	Triggers the HMGB1 pathway and RAGE	EC-derived HMGB1 induces an autophagic response, BMPR2 defects, and subsequent apoptosis-resistant proliferation in smooth muscle cells	126
	Pulmonary Hypertension	EC-specific caveolin-1 (Cav-1) depletion (EC-Cav1−/− mice)	exhibits a non-EC phenotype and contributes to vascular remodeling via TGF-β/pSmad2/3 signaling	With Increasing Cav-1+ extracellular vesicle shedding into the circulation and decreasing circulating monocytes	207
	Pulmonary Hypertension	Cigarette Smoke Exposure (PAECs and rats model)	Increases endothelial extracellular vesicles (eEV) generation and the spermine content in eEV	Triggers eEV migration into SMCs, and contributes to pulmonary artery smooth muscle constriction and proliferation via CaSR	208
	Vascular aging and acute myocardial infarction (AMI)	Switch in Lamb2 to Lamb1 (HUVECs and Mouse models of AMI)	Impaires the functional properties and phenotype of endothelial cell via integrin receptors		209
	ECs Dysfunction	Nine flavors added to tobacco products (HEACs)	Iimpairs eNOS agonist-stimulated NO production and triggers inflammation, even cell death, such as vanillin and eugenol.		5
	ECs Dysfunction	Exposure to fine particulate matter (PM2.5) (Endothelial progenitor cells, EPCs)		Impairs EPC abundance and function and prevents EPC-mediated vascular recovery after hindlimb ischemia via vascular VEGF resistance and a decrement in NO bioavailability	210
	Hypertension	GLP-1 (glucagon-like peptide-1) analogs (Global, EC-and myelomonocytic-specific Glp1r −/− mice)	Reduces blood pressure and protects endothelial function through endothelial but not myeloid cell GLP-1 receptor	Prevents Ly6G−Ly6C+ and Ly6G+ Ly6C+ cell infiltration to the vessel wall	211
Part III
Innate immune relevance	Associated metabolic situlations	Target and experimental model	Responses on ECs	Interaction between ECs and other cells	Reference
Receptor systems sensing (HAMPs)	Organ specificity	Cardiac ECs (transcriptome)	Highly expresses key regulators in fatty acid uptake, such as Meox2/Tcf15, Fabp4, and Cd36		212
	Pathological angiogenesis	EC-specific Atg5 deletion (HUVECs, HRMECs (human retinal microvascular ECs), MLECs (murine lung ECs) and Mice with EC-specific inactivation of Atg5)	Impairs mitochondrial function, diminishes production of mtROS, decreased oxidative inactivation of PTPs (phospho-tyrosine phosphatases)and hence, decreasing phosphorylation of the VEGFR2		167
	Atherogenesis	Atheroprotective pulsatile shear stress (HUVECs)	Activates ITPR3 transcription via KLF4-regulated H3K27ac (acetylation of histone 3 lysine 27) enrichment and chromatin accessibility, contributes to the Ca2+ - dependent eNOS activation and EC homeostasis.		168
	Atherogenesis and CAD (coronary artery disease)	JCAD, CAD-associated variants at 10p11.23, knockdown (HUVECs)	Decreases ICAM-1, VCAM-1 and Selectin E by negatively regulates YAP activity and Hippo signaling	Reduces monocyte adhesion	26
	Abdominal aortic aneurysm	Cilostazol, a selective inhibitor of phosphodiesterase III (PDEIII) (MAECs and APOE−/− mice)	Reduces MCP-1 and ICAM-1 via increasing intracellular cAMP	Reduces medial disruption and macrophage infiltration in angiotensin II-Induced AAA, but no effect on atherosclerosis	213
	Pulmonary hypertension	Hypoxia (HPMEC)	Induces SENP1 (sentrin-specific protease 1)and deprivates KLF15 by SUMOylation, lossing repression on arginase 2 promoter and impairing NO production		169
	Inflammation/Stress	A shift from AIP1A to AIP1B isoform (HUVECs)	Localizes to the mitochondria and augments TNFα-induced mtROS generation and EC activation		170
	Hypertension	Knockdown of SIRT3 (sirtuin 3) (EPCs)	Contributes to the decline in reendothelialization capacity	Results in mitochondrial oxidative damage, hyperacetylation of SOD2 (superoxide dismutase 2) in EPCs	171

The significance of solving these problems is that a new paradigm will encourage investigators¹⁵² to search for anti-inflammatory and homeostatic signals derived from endogenous metabolites. Recent progress in immunology has clearly demonstrated the well-published “two arms model.” This model states that there are several immunotolerance and anti-inflammatory mechanisms, including T cell coinhibition/immune checkpoint pathways¹⁵³, T cell anergy¹⁵⁴, Treg¹⁵⁵, and anti-inflammatory/immunosuppressive cytokines. Anti-inflammatory/immunosuppressive cytokines include transforming growth factor-β (TGF-β), IL-10, IL-35, and IL-37 as we and others reported^156–159, and specialized pro-resolving mediators (SPMs) such as lipoxins, E-series and D-series resolvins, protectins, and maresins¹⁶⁰, etc. Following the same logic of “two arms”, lysophosphatidylserine¹⁶¹, lysophosphatidylenthaolamine¹⁶² and IL-35¹⁶³ were identified as the signals that are generated from endogenous metabolic processes and have anti-inflammatory and homeostatic functions via pattern-dependent manners^{78, 164}. Therefore, HAMPs receptors are the receptors for binding to signals that are generated from endogenous metabolic processes and can initiate anti-inflammatory/homeostatic signaling and promote inflammation resolution^{151, 157, 165, 166}. Taking advantages of cell type-specific gene knockdown techniques and selective inhibitors, recent studies updated several molecules and receptors, as well as their mechanism in maintaining ECs homeostasis, such as Atg5 (autophagy protein 5)¹⁶⁷, ITPR3 (inositol 1,4,5-trisphosphate receptor 3)¹⁶⁸, GATA (GATA zinc finger transcription factor family)-6⁸, KLF (Kruppel-like factor)15¹⁶⁹, AIP1A (ASK1 [apoptosis signal-regulating kinase 1]-interacting protein-1 form A)¹⁷⁰ and sirtuin 3 (SIRT3)¹⁷¹. On the contrary, endothelial-specific deletion of the mineralocorticoid receptor protects against vascular inflammation in atherosclerosis in a sex-specific manner¹⁷². In addition, a novel model of endothelial dysfunction, that uses isogenic human induced pluripotent stem cell-derived cells harboring different alleles of the APOE gene and identifies ApoE4 expression by endothelial cells, results in cellular dysfunction. This new model exhibits a proinflammatory state and prothrombotic state, evidenced by enhanced secretion of Aβ (amyloid-β) 40 and 42, increased release of cytokines, and overexpression of the platelet-binding protein VWF (von Willebrand factor)¹⁷³.

4. Innate immune functions of endothelial cells are determined by immunometabolism and innate immune memory.

Highly ordered interactions between immune and metabolic responses are evolutionarily conserved and paramount for tissue and organismal health. Tissue immunometabolism emphasizes that tissue accessory cells such as immune cells, stromal cells and ECs communicate with their clients, tissue parenchymal cells, to optimize the metabolic process for environmental adaptation¹⁷⁴. Disruption of these interactions underlies the emergence of many pathologies, particularly chronic non-communicable diseases such as obesity and diabetes¹⁷⁵. The proinflammatory and anti-inflammatory functions of these immune cells are determined by the metabolic stage of the immune cells. The metabolic process of immune cells is called immunometabolism and its shift determined by inflammatory stimuli is called immunometabolic reprogramming¹⁷⁶. The recent report that 20 novel disease group-specific and 12 new shared macrophage pathways in eight groups of 34 diseases including 24 inflammatory organ diseases and 10 types of tumors¹⁷⁷, suggests that disease-related immune microenvironments shape immunometabolism and signaling pathways of immune cells.

In atherogenesis, cholesterol crystals and apolipoprotein B-peptides have been shown to activate macrophages and T helper cells, respectively¹⁷⁸. Meanwhile, lipoproteins are also important modulators of regulatory T cells that can hamper vascular inflammation^{178, 179}. As indicated in the comprehensive MetaCye Metabolic Pathway Dataset, which collected 2766 documented metabolic pathways, pro-inflammatory and anti-inflammatory pathways are highly specific and resulted from extensive metabolic remodeling and re-focusing¹⁸⁰. Indeed, two recent studies revealed unlike oxLDL/CD36 signaling in macrophages links dysregulated fatty acid metabolism to oxidative stress from the mitochondria, which triggers cell patrolling to drive chronic inflammation¹⁸¹, ECs present a CD36-independent regulation of a non-classical subset of monocytes, which function in an atheroprotective manner during early atherogenesis¹⁸². In diabetes, hyperglycemia and hyperlipidemia downregulate of TFEB (transcription factor EB) expression in aortic ECs, attenuating its anti-inflammatory effects via inhibiting IKK (inhibitor of nuclear factor kappa-B kinase) activity and increasing IκBα level to suppress NF-κB activity¹⁸³. Compared with the report numbers immunometabolism in macrophages (232 publications at PubMed), ECs immunometabolism is at the early stage (13 publications at PubMed). There is novel transcriptomic evidence that HAECs could be transdifferentiated into innate immune cells by exposing them to hyperlipidemia-up-regulated DAMP molecules, i.e. lysophospholipids. In RNA-seq analysis, lysophosphatidylcholine (LPC) up-regulated genes are involved in cholesterol biosynthesis, presumably through sterol regulatory element-binding protein 2 (SREBP2). Of note, SREBP2 activation mediates atheroprone flow-induced NLRP3 inflammasome function in ECs¹⁸⁴. By contrast, lysophosphatidylinositol (LPI) up-regulate gene transcripts critical for the metabolism of glucose, lipids, and amino acids. Of note, we reported that, in HAECs, LPC and LPI both induce adhesion molecules, cytokines, and chemokines, which are all classic markers of endothelial activation. Moreover, LPC and LPI share the ability to transdifferentiate HAECs into innate immune cells, including induction of potent DAMP receptors, such as CD36, T-cell costimulation-, coinhibition/immune checkpoint receptors, and MHC-II proteins. The induction of these innate-immunity signatures by lysophospholipids correlates with their ability to induce up-regulation of cytosolic calcium and mitochondrial ROS. Hence, lysophospholipids such as LPC and LPI induce innate immune cell transdifferentiation in HAECs, supporting a new concept that innate immune cells transdifferentiation of ECs confers a status of prolonged endothelial activation⁹⁹.

Trained immunity (also termed as innate immune memory) is an emerging concept describing a prolonged hyper-activation of the innate immune system after exposure to certain stimuli, leading to an augmented immune response to a secondary stimulus. Innate immune cells such as monocytes, macrophages, dendritic cells, and NK cells and some non-immune cells¹⁸⁵ have been shown to develop trained immunity by undergoing functional reprogramming when exposed to inflammatory stimuli, that elicit changed responses to subsequent inflammatory challenges. This long-term reprogramming depends on the rewiring of cell metabolism and epigenetic processes¹⁸⁶, and they stay at the basis of induction of both innate immune memory (also termed trained immunity) and innate immune tolerance¹⁸⁷. It has been identified that three metabolic pathways (trained immunity pathways, TIP) including glycolysis pathway, mevalonate pathway and acetyl coenzyme A (acetyl-CoA) generation are responsible for initiating innate immune memory formation. Inductions of trained immunity regulators are a new category of qualification markers for chronic disease risk factors and conditional DAMPs and potential mechanisms for acute inflammation transition to chronic ones. Increased acetylation of histone 3 lysine 14 (H3K14) in the genomic regions that encode TIP genes in comparison to that of endothelial activation genes such as ICAM-1¹⁰². Of note, among all the 26,625 compounds identified in the foods (http://foodb.ca/compounds) and environmental compounds (for example, more than 7,000 compounds in cigarette smoke (https://www.lung.org/stop-smoking/smoking-facts/whats-in-a-cigarette.html), therefore, only those compounds stimulations that induce trained immunity have potential to become cardiovascular disease (CVD) risk factors. This conceptual advance on identification of the key features of CVD risk factors will facilitate the future characterization of novel risk factors. Moreover, anti-inflammatory cytokines IL-10 and IL-35 inhibit endothelial activation gene expressions but spare the trained immunity enzyme gene expressions¹⁸⁸. The five new training programs have been reported including (i) β-glucan-induced, (ii) Bacillus Calmette-Guérin (BCG)-induced, (iii) oxLDL-induced, (iv) LPS-induced, and (v) aldosterone-induced¹⁰³. The future work will be needed to determine whether and how each of these training programs regulate innate immune functions of vascular cells in CVD¹⁰⁴.

5. Immune tolerogenic functions of ECs, immune checkpoint receptors (ICRs), and cardio-oncology.

Antigen-specific immunity requires regulated trafficking of T cells in and out of diverse tissues in order to orchestrate lymphocyte development, immune surveillance, responses, and memory. ECs serve as a unique barrier, as well as a sentinel, between the blood and the tissues, and as such, they play an essential locally tuned role in regulating T cell migration and information exchange. In addition to providing trafficking cues, intimate cell-cell interaction between lymphocytes and ECs provides instruction to T cells, which influences their activation and differentiation states¹⁸⁹. Aside from aiding T cells in playing a proinflammatory role in immune responses (also see the above-discussed sections on cytokines, chemokines, and secretory proteins), ECs can also have an immune tolerogenic function and induce suppressive immune function in T cells. Mouse ECs activated by IFN-γ and co-cultured with allogeneic CD4⁺ T cells are shown to induce the generation of immunosuppressive Treg¹⁹⁰. Furthermore, after contact with ECs, Treg upregulate the expression of ICR, programmed death-1 receptor (PD-1), and increase the production of anti-inflammatory cytokines IL-10 and TGF-β¹⁹¹.

Chronic kidney disease induces inflammatory CD40⁺ monocyte differentiation¹⁹², suggesting that reverse signaling via co-stimulation receptor CD40 promotes vascular inflammation. ECs and VSMCs upregulate 28 co-signaling receptors for T cell activation including 14 co-stimulation receptors (CSRs), 4 dual-function receptors and 10 co-inhibition receptors (CIRs) in pathologies^{81, 153}. ECs upregulate four CSRs such as inducible T cell costimulator ligand (B7-H2, CD275), CD40, Semaphorin 4A (SEMA4A) and CD112, and four CIRs including Galectin 9, TNF superfamily member 14 (HVEM, CD258), programmed cell death 1 ligand 2 (B7-DC, CD273), and programmed cell death 1 ligand 1 (B7-H1, PD-L1, CD274) after stimulation with TNF-α and IFN-γ¹⁹³. Forward and reverse signaling of three out of 18 CSRs, CD275, CD40 and SEMA4A (16.7%), play significant roles in vascular cells (including VSMCs) in response to proinflammatory cytokine TNF-α and IFN-γ stimulations. TNF-α and IFN-γ also upregulate five out of ten CIRs (50%) in ECs, suggesting that ECs play significant roles in immune tolerance, anti-inflammatory responses, and inflammation resolution⁸¹.

Recently, immune checkpoint inhibitors (ICIs) have been an important therapeutic advance in the field of cancer medicine, resulting in a significant improvement in survival of patients with advanced malignancies¹⁹⁴. Recent reports provided greater insights into the incidence of cardiovascular adverse events (CVAEs) with ICI use, which leads to the new development of cardio-oncology. Myocarditis is the most common CVAE associated with ICI. Pericardial diseases, Takotsubo syndrome, arrhythmias, and vasculitis constitute other significant adverse events (AEs)¹⁹⁵. ECs upregulation of multiple ICRs after the stimulation of proinflammatory cytokines TNF-α and IFN-γ make ECs as significant contributors to CVAEs associated with ICI therapies.

6. Summary

The current literature supports a conceptual innovation that ECs are innate immune cells not only in infections and allograft transplantation but also in metabolic cardiovascular diseases, other sterile inflammations and cancers. Continuous improvement of our understanding on innate immune features of vascular endothelial cells will lead to identification of novel targets for the development of the future therapeutics to treat cardiovascular diseases, cerebrovascular diseases, peripheral artery disease, metabolic diseases, inflammations, infections, neurodegenerative diseases and cancers.

Abbreviations

DAMPs

damage-associated molecular patterns

DC

dendritic cell

ECs

endothelial cells

HAECs

human aortic endothelial cells

HMGB1

high mobility group box 1

HUVECs

human umbilical vein endothelial cells

ICAM-1

intercellular adhesion molecule 1

PAMPs

pathogen associated molecular patterns

PRRs

pattern recognition receptors

SMCs

smooth muscle cells

VCAM-1

vascular cell adhesion molecule 1

VEGF

vascular endothelial growth factor