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After an pathogen invades a host, it undergoes a series of phases that eventually lead to multiplication of the pathogen.
- Outline the stages of disease: incubation, prodromal, acute and convalescence periods
- The first phase is characterized by complete lack or very few symptoms.
- As the pathogen starts to reproduce actively, the symptoms intensify. Bacterial and viral infections can both cause the same kinds of symptoms but there are some differences too.
- The last phases are characterized by decline in symptoms severity until their disappearance. However, even if the patients recover and return to normal, they may continue to be a source of infection.
- subclinical: Of a disease or injury, without signs and symptoms that are detectable by physical examination or laboratory test; not clinically manifest.
- clinical latency: The period for which an infection is subclinical.
- viral latency: A form of viral dormancy in which the virus does not replicate at all.
Stages of Disease
After an infectious agent invades a host (patient), it undergoes a series of phases (stages) that will eventually lead to its multiplication and release from the host.
STAGE 1: INCUBATION PERIOD
This refers to the time elapsed between exposure to a pathogenic organism, and from when symptoms and signs are first apparent. It may be as short as minutes to as long as thirty years in the case of variant Creutzfeldt–Jakob disease. While the term latency period is used as synonymous, a distinction is sometimes made between incubation period, the period between infection and clinical onset of the disease, and latent period, the time from infection to infectiousness. Whichever is shorter depends on the disease.
A person may be a carrier of a disease, such as Streptococcus in the throat, without exhibiting any symptoms. Depending on the disease, the person may or may not be contagious during the incubation period. During clinical latency, an infection is subclinical. With respect to viral infections, in clinical latency the virus is actively replicating. This is in contrast to viral latency, a form of dormancy in which the virus does not replicate.
STAGE 2: PRODROMAL PERIOD
In this phase, the numbers of the infectious agents start increasing and the immune system starts reacting to them. It is characterized by early symptoms that might indicate the start of a disease before specific symptoms occur. Prodromes may be non-specific symptoms or, in a few instances, may clearly indicate a particular disease. For example fever, malaise, headache and lack of appetite frequently occur in the prodrome of many infective disorders. It also refers to the initial in vivo round of viral replication.
STAGE 3: ACUTE PERIOD
This stage is characterized by active replication or multiplication of the pathogen and its numbers peak exponentially, quite often in a very short period of time. Symptoms are very pronounced, both specific to the organ affected as well as in general due to the strong reaction of the immune system.
Viral infections present with systemic symptoms. This means they involve many different parts of the body or more than one body system at the same time; i.e. a runny nose, sinus congestion, cough, body aches, etc. They can be local at times as in viral conjunctivitis or “pink eye” and herpes. Only a few viral infections are painful, like herpes. The pain of viral infections is often described as itchy or burning.
The classic symptoms of a bacterial infection are localized redness, heat, swelling and pain. One of the hallmarks of a bacterial infection is local pain, pain that is in a specific part of the body. For example, if a cut occurs and is infected with bacteria, pain occurs at the site of the infection. Bacterial throat pain is often characterized by more pain on one side of the throat. An ear infection is more likely to be diagnosed as bacterial if the pain occurs in only one ear.
After the pathogen reaches its peak in newly-produced cells or particles (for viruses), the numbers begin to fall sharply. Symptoms are still present but they are not as strong as in the acute illness phase.
STAGE 4: CONVALESCENCE PERIOD
The patient recovers gradually and returns to normal, but may continue to be a source of infection even if feeling better. In this sense, “recovery” can be considered a synonymous term.
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- Structural and organizational aspects of bone and cartilage including molecular and cellular biology, biochemistry, physiology and biomineralization with respect to osteoblasts, osteoclasts, chondrocytes, connective tissue cells and other cells in the marrow environment in both normal and pathological conditions.
- Basic and clinical studies of calciotropic hormones and paracrine factors involved in bone and cartilage biology in humans and animal models, metabolic bone diseases [e.g., osteoporosis, osteopenia, osteogenesis imperfecta, Paget’s disease of bone, and chronic kidney disease (CKD) induced metabolic bone diseases (CKD-MBD)], and diseases of mineral ion homeostasis associated with abnormalities of parathyroid hormone, Vitamin D, calcitonin and other hormonal and paracrine factors.
- Immunological aspects of bone diseases.
- In vitro and animal models of molecular pathogenesis and biology of osteosarcoma and chondrosarcoma, assessing functional effects of primary tumors and metastasis to bone.
- Regulation of biomineralization of the extracellular matrix of skeletal and connective tissues structure and organization of matrix components cell matrix interaction and signaling in normal and pathological conditions.
- Tooth and enamel development .
- Mechanisms of craniofacial and skeletal patterning, lip and palate development, including gene discovery, gene expression and genetic linkage studies in humans and animal models cellular proliferation, lineage commitment and differentiation chondrodysplasias, osteodysplasias and cellular aspects of aging of the skeleton using in vitro and in vivo models.
10.3B: Disease Development - Biology
After decades of playing second fiddle, RNA has now acquired its coveted role in all aspects of biological research.
Transcriptome profiling has identified key features unique to the retina, including non-coding RNAs that are likely linked to both development and disease pathology.
Alternative splicing is a hallmark of transcriptomes of developing mammalian retina, and especially of photoreceptors. At least 50% of the retina-expressed genes exhibit an altered structure compared to the reference.
Diverse and functionally characterized neurons in the retina provide a unique model to dissect the function of distinct RNA molecules in biological processes and evaluate novel therapeutic paradigms.
For decades, RNA has served in a supporting role between the genetic carrier (DNA) and the functional molecules (proteins). It is finally time for RNA to take center stage in all aspects of biology. The retina provides a unique opportunity to dissect the molecular underpinnings of neuronal diversity and disease. Transcriptome profiles of the retina and its resident cell types have unraveled unique features of the RNA landscape. The discovery of distinct RNA molecules and the recognition that RNA processing is a major cause of retinal neurodegeneration have prompted the design of biomarkers and novel therapeutic paradigms. We review here RNA biology as it pertains to the retina, emphasizing new avenues for investigations in development and disease.
S1P (sphingosine 1-phosphate) and lysophosphatidic acid are secreted lipid mediators produced by metabolism of membrane phospholipids.
S1P and lysophosphatidic acid interact with specific G-protein–coupled receptors to regulate vascular development, physiology, and cardiovascular diseases.
S1P/lysophosphatidic acid signaling axis intersects with fundamental developmental systems such as Wnt (int/wingless family)/β-catenin.
Metabolic and signaling genes in the S1P/lysophosphatidic acid system show cardiovascular disease-specific heterogeneity.
The endothelium is a dynamic and heterogeneous organ system that responds and adapts to various stimuli during embryonic development and postnatal homeostasis. During early development of the vascular system, endothelial cells (EC) undergo vascular network formation in response to hypoxic stimuli and cooperate with other developmental events in organogenesis. Arteries, capillaries, and veins, each with their repertoire of constituent cell types, undergo vessel-specific EC differentiation. Moreover, the vasculature of each organ system exhibits heterogeneity in structure and function, a phenomenon referred to as organotypic EC specialization. Even within the same vessel segment or vascular bed, there is local heterogeneity in EC structure, gene expression, and function. This allows for plasticity, adaptability, and resilience of the vascular system to the changing environments that the organism encounters, thus providing optimal responses for vascular barrier function, tone (contraction/relaxation), inflammation and resolution, thrombus formation, directional blood flow, and the transport of molecules and cells between blood and tissues. 1,2
The diversity of endothelial subtypes is in part determined by a multitude of cell surface receptors that respond to local or systemic factors. Well-characterized EC receptors include receptor tyrosine kinases, such as the Ang (angiopoietin) receptor TIE2, VEGF (vascular endothelial growth factor) receptors VEGFR (VEGF receptor)1–3, as well as a variety of GPCRs (G-protein–coupled receptors). GPCR expression in individual ECs varies depending on the vascular bed, vessel type, flow, and developmental context. 3–6 Such GPCRs respond to a variety of ligands including small peptides (endothelins, bradykinin, neuropeptides, apelin), morphogenetic factors (Wnt [int/wingless family] proteins), proteases (thrombin, trypsin), ECM (extracellular matrix), chemokines, 17β-estradiol, mechanical forces, metabolites, protons (pH), and bioactive lipids (lysolipids, eicosanoids, etc). In this review, we focus on lysolipid GPCRs, in particular those that respond to metabolites of the membrane phospholipids—S1P (sphingosine 1-phosphate) and lysophosphatidic acid (LPA)—as central orchestrators of vascular development and tractable therapeutic targets in cardiovascular, autoimmune, and pathogen-induced diseases.
Lysolipids in the Vascular System
S1P and LPA Metabolism
S1P and LPA are the 2 well-characterized bioactive lysolipid species. These molecules have hydrophobic backbones and polar phosphate head groups, which makes them impermeant to cellular membranes. S1P is less water-soluble than LPA, thus necessitating carrier proteins (termed chaperones) in extracellular compartments. 7,8 S1P circulates at concentrations of ≈0.7 to 1 µmol/L in the chaperone-associated form. Circulating LPA concentrations are much lower (estimated to be in the ten to hundreds of nM reviewed in Yung et al 7 and Yanagida and Hla 8 ). The majority of circulating S1P (≈65%) is bound to the ApoM on HDL (high-density lipoprotein) particles, whereas the remainder is associated with albumin. 9,10 In contrast, ≈65% of LPA is associated with albumin while the remainder is predominantly HDL-bound. 11,12 Thus, HDL and albumin are the primary chaperones for both S1P and LPA. Notably, murine development and survival proceeds in the absence of both ApoM and albumin, whereas in such situations S1P associates with other macromolecules in vivo, such as ApoA4. 13
Albumin-bound S1P and LPA are short-lived in circulation (t1/2 [half-life] <20 minutes), 11,14,15 and their concentrations in blood are determined by substrate availability and the activities of metabolic enzymes. 8 Two sphingosine kinase enzymes, SPHK1 (sphingosine kinase) and SPHK2, phosphorylate intracellular sphingosine to generate S1P, 16 although a fraction of secreted SPHK1 is present in plasma. 17 Erythrocyte-mediated S1P export to blood by the MFSD2B (major facilitator family transporter 2b) accounts for the majority of circulatory chaperone-bound S1P. 18 Meanwhile, S1P synthesized in blood and lymphatic vessel EC is transported to blood or lymph by another transporter—SPNS2 (spinster 2). 19–22 Endothelial SPNS2-mediated S1P transport accounts for ≈10% of plasma S1P 23 and at least 80% of lymph S1P. 21,24,25 Thus, S1P in lymph is primarily generated in lymphatic endothelium, whereas S1P in blood is primarily derived from red blood cells and, to a lesser extent, EC. Restricted expression of Spns2, Sphk1, and Mfsd2b in endothelial or erythroid cell types has been corroborated by single-cell RNA-sequencing from mouse embryos (Figure 1).
Figure 1. Expression of lysolipid metabolic and signaling genes during embryogenesis and in postnatal endothelium.A and B, Expression of genes encoding lysolipid metabolic enzymes or transporters (Sphk1 [sphingosine kinase], Sphk2, Lpp3, Mfsd2b, Spns2 A) or receptors (S1pr1–5, Lpar1–6 B) in selected cell types and embryonic structures. Single-cell RNA-sequencing (RNA-seq) data are from a publicly available database (https://oncoscape.v3.sttrcancer.org/atlas.gs.washington.edu.mouse.rna/landing) provided by the authors. 26 C, Expression of S1P (sphingosine 1-phosphate) and lysophosphatidic acid receptors from RNA-seq of freshly isolated endothelial cells (EC) from postnatal day 7 mouse brain, liver, lung, or kidney. 6 TPM indicates transcripts per million.
LPA synthesis is regulated by ATX (autotaxin), a secreted phospholipase D that removes the choline moiety from lysophosphatidylcholine to generate LPA. 27 Though lysophosphatidic acids are derived from phospholipids of variable chain lengths, the most abundant circulating species in mammals is 18:1 oleoyl-LPA. 27 A significant portion of circulating LPA is synthesized in blood upon ATX-catalyzed hydrolysis of circulating lysophospholipids, which are present in lipoproteins (LDL [low-density lipoprotein] and VLDL [very low-density lipoprotein]) and other carriers. 27 During embryonic development, ATX is widely expressed and exhibits particularly high expression in osteoblasts, chondrocyte progenitors, endothelium, early mesenchyme, and megakaryocytes (Figure 1).
The influence of diet and microbiome composition on sphingolipid levels and liver health is an active area of research. Dairy products and eggs are enriched with S1P precursors such as sphingomyelin. 28 However, hydrolytic enzymes in the small intestine and colon, including sphingomyelinase, ceramidase, and glucoceramidase catabolize most ingested sphingolipids to free fatty acids and sphingosine. Nonetheless, sphingolipid-enriched diets have been shown to improve liver health, reduce lipid accumulation in tissue as well as circulating cholesterol in rodent models, 29–31 as well as inhibit atherosclerosis. 32 Bacteroides, the major genus in the human gut after weaning, 33 expresses serine palmitoyltransferase enzymes that participate in de novo sphingolipid synthesis. 34 Notably, bacterial-derived sphingolipids transfer to host gut epithelium and hepatic portal vein tissue. 34 Links between gut microbiota, sphingolipids, dyslipidemia, and cardiovascular health warrant further investigation.
Vascular Lysolipid Receptors
S1P and LPA are high-affinity ligands that bind their respective receptors with apparent nanomolar dissociation constants. 35–37 There are 6 known LPAR1–6 (LPA receptors 1–6) 38 and 5 known S1PR1–5 (S1P receptors 1–5). 39 Single-cell RNA-sequencing of mice at embryonic day (E) 9.5 to E13.5 corroborated previous studies 3,40–42 demonstrating that S1pr1 is abundantly expressed in ECs (Figure 1). S1pr2–5 are expressed at relatively low levels in ECs both during embryogenesis and postnatally (Figure 1). Among the S1PR-deficient mice that have been generated, only S1pr1-knockout animals die embryonically 42 (Table).
Table. Vascular Phenotypes Observed in Mice With Altered Lysolipid Signaling
Atx indicates autotaxin CAG, chicken alpha actin Cdh5, cadherin 5 DKO, double knockout E, embryonic day EC, endothelial cells GPCR, G-protein–coupled receptors KO, knockout LPA, lysophosphatidic acid Lpar, LPA receptors LPP3, lipid phosphate phosphatase 3 Rac, small GTPase Rac RBC, red blood cell RhoA, small GTPase Rho A S1P, sphingosine 1-phosphate S1pr, S1P receptors Sphk, sphingosine kinase and Tg, transgenic.
Lpar1, Lpar4, and Lpar6 are expressed in embryonic cardiovascular cells including endothelial, smooth muscle, and cardiomyocyte lineages (Figure 1). Among these cell types, Lpar2 and Lpar5 are not significantly expressed, and Lpar3 expression is limited to cardiac muscle lineages. Although mice lacking any single LPAR can survive to term, only Lpar4 −/− mice exhibit partially penetrant embryonic lethality as a result of defective vasculature. 7,43 This phenotype is fully penetrant and more severe in Lpar4 −/− Lpar6 −/− double-knockout mice, 44 suggesting that these receptors have redundant functions during embryogenesis.
S1P and LPA Regulate Vascular Development
Discrete functions of S1P and LPA receptors are attributable in part to their unique distribution, ligand availability, heterotrimeric G-protein coupling, and activation of Rho family small GTPases (reviewed in Yung et al 7 and Taha et al 37 ). Endothelial S1PR1 expression and signaling through Gi/Rac (small GTPase Rac) is restricted to perfused, S1P-containing vessels and is essential for vascular stabilization and inhibition of VEGF-induced hypersprouting 23,40,41,45,46 (Figure 2). In the absence of endothelial S1P signaling, most, if not all, developing vascular networks exhibit excessive sprouts, branches, and hemorrhagic areas that are incompatible with life after E14.5. Developing capillaries and veins in S1PR-deficient retinas fail to express specialized components of neurovasculature and instead upregulate migration- and tip cell-associated genes, such as Esm1, Angpt2, and Apln. 46 These findings suggest that S1PR signaling is needed for vascular stability, patterning, and organotypic specialization during organ development through pathways conserved from tight coevolution which integrate signaling fitness, mechanical and metabolic inputs. 47
Figure 2. Schema of lysophosphatidic acid (LPA) and S1P (sphingosine 1-phosphate) signaling during retinal sprouting angiogenesis. The angiogenic front of a developing vascular plexus, composed of ≈6–10 rows of endothelial cells (EC), is surrounded by high levels of VEGF (vascular endothelial growth factor). 1 Here, LPA activates EC LPAR6 (LPA receptor 6 and possibly LPAR4), promoting proliferation and migration downstream of Gα12/13 and Rho family GTPases in cooperation with VEGF receptor signaling. S1PR1 (S1P receptor 1) expression is relatively low in these EC. Cell-cell contacts are stronger in more mature vascular regions and are speculated to be enriched with active LPP3 (lipid phosphate phosphatase 3), which degrades LPA and limits EC LPAR signaling. Meanwhile, as these maturing vascular regions acquire S1P from flowing blood (black arrows), EC express high levels of S1PR1. S1P-S1PR1 signaling contributes to vascular stability (eg, stabilization of adherens junctions) and permits organotypic vascular specialization in the retina. These S1PR1-mediated events are likely downstream of RAC1 activation. Growing vascular networks undergo remodeling to optimize tissue perfusion (black arrows). We speculate that poorly perfused EC are selected for pruning in part because they become deficient in S1P-S1PR1 signaling. Resultant vascular instability decreases cell-cell adhesions and reduces LPP3 activity, thereby creating microdomains of LPA that activate LPAR6 on remodeling EC to promote incorporation into well-perfused capillaries. Rac indicates small GTPase RAC1.
Unlike S1P/S1PR1 signaling, which is generally perfusion-dependent for receptor expression and ligand availability, LPA appears to be more constitutively available and particularly important for LPAR signaling in ECs of nonperfused vascular sprouts 44,48–50 (Figure 2). LPAR4 and LPAR6-mediated activation of Gα12/13/Rho GTPase is required for endothelial proliferation, vascular branching, and network expansion. 44,48,51 These findings suggest that S1P signaling is engaged in ECs undergoing integration within the endothelium, maturation, and stabilization, whereas LPA signaling occurs in ECs undergoing migration or proliferation. In addition, LPA signaling appears to be involved in vessels undergoing regression. 49
Accumulating evidence demonstrates that the integral membrane protein LPP3 (lipid phosphate phosphatase 3) inhibits endothelial LPAR signaling specifically at cell membrane regions that participate in cell-cell contact, 44,48,51–54 thereby restricting LPAR signaling to noncontact sites. 51 During vascular development, ECs deficient in cell-cell contacts are found primarily in blind-ended sprouts 55 and remodeling vessels 56 (Figure 2). LPP3, a lipid phosphate phosphatase with an extracellular mode of action, is likely an essential cell-autonomous regulator of EC LPAR signaling because EC-specific LPP3-knockout embryos die by E11.5 with severe vascular defects in extraembryonic vasculature and in the embryo proper. 52,57 Some of these defects may be a consequence of LPP3 loss of function destabilizing β-catenin which has been demonstrated to vary with cell density. 58 Because β-catenin is a key intracellular regulator of adherens junctions and Wnt signaling pathways, this nexus may be the point at which lysolipid and Wnt signaling pathways intersect.
S1P Signaling in EC
In human umbilical vein EC (HUVECs), S1P binding to S1PR1 induces Ca 2+ mobilization, activation of the GTPase Rac, actin polymerization of the cortical cytoskeleton, and adherens junction assembly. 59,60 Concomitantly, S1PR1 inhibits adenylyl cyclase activity by coupling to Gαi, 59 thus reducing intracellular cAMP levels. Other downstream targets of S1PR1 activation include PI3K (phosphoinositide 3-kinase)/Akt activation, PLC (phospholipase C) activation, and increased p-Src (phosphorylated Src). 37,61
Vascular S1P signaling results in pleiotropic yet protective EC changes. S1PR1-mediated actin rearrangement and adherens junction assembly increases endothelial barrier function in vitro and is likely a key mechanism by which S1PR1 enhances vascular integrity and perfusion in lung, trachea, and retina tissues. 40,46,62–66 In the postnatal brain, endothelial S1PR1 limits leakage of small (<3 kDa) molecules 67 and also functions in neural progenitor and glial cells to inhibit postnatal hemorrhage in germinal matrix. 68 In large arteries, the S1PR1/Gαi/Akt axis regulates blood pressure and vascular tone via activation of endothelial NO synthase, which produces NO to induce vasorelaxation. 66,69–71 During embryonic development, the S1PR1/Rac signaling axis is essential as either S1P deficiency, EC-specific S1PR1 deficiency, or EC-specific Rac1 deficiency results in severe vascular defects and embryonic lethality. 16,40,41,45,46,72,73
S1P-deficient embryos lacking sphingosine kinases (Sphk1 −/− Sphk2 −/− ) exhibit severe hemorrhage, dilated blood vessels, and do not survive after E13.5. 16 Similarly, global or EC-specific S1pr1 deletion results in severe hemorrhage, disruption of adherens junctions, and hyperbranching of distal vascular beds (eg, brain and retinal vessels) and major proximal arteries (eg, dorsal aorta), which is not compatible with life after E14.5. 40,41,45,46 These animals also exhibit widespread vascular hypersprouting, which is characterized by increased tip cell frequency and filopodia density in developing vasculature consistent with a failure to eliminate excess cells. 40,41,46 Deletion of Sphk1 in the erythroid lineage of Sphk2 −/− mice revealed that red blood cells generate ≈95% of the S1P content in embryonic tissue, which is required for survival after E13.5. 23 In addition to severe hemorrhaging and vascular malformations in the head and aorta, yolk sacs of these S1P-deficient embryos exhibited disorganized, hyperbranching capillary networks. 23 Maternal administration of the S1PR1 agonist SEW2871 rescued lethality in embryos lacking red blood cell–derived S1P, further demonstrating the essential role of S1PR1 in developmental S1P signaling. 23 However, these embryos lack Sphk2 in platelets, and more recent analysis suggested that red blood cells and platelets have redundant functions as suppliers of embryonic S1P needed for proper vascular development. 74
Detailed insights into EC S1PR signaling have been obtained using the retina as a model of vascular network formation and maturation. Over the first 9 days of postnatal murine life, vessels grow radially and form a network of arteries, capillaries, and veins extending to the retina periphery. In perfused vessels of the nascent vascular network, S1P/S1PR1 signaling promotes endothelial maturation and adherens junction assembly. 40,41,46 ECs at the angiogenic front, including tip cells of blind-ended sprouts, are very different from those in the nascent network as they are poorly perfused and lack S1PR1 expression. 40,41,46 Angiogenic front ECs engage in VEGFR signaling which drives expression of JunB, c-Jun, and other tip cell genes that contribute to EC proliferation, migration, full integration into the endothelial monolayer, and proper patterning. 6,46,75–77 Evidence suggests a mechanism by which S1PR-dependent VE-cadherin (vascular endothelial cadherin) assembly promotes endothelial maturation in the nascent vascular network through adherens junction assembly as well as suppression of AP-1 46 and FOXO1 78 transcription factors.
In addition, EC S1PR signaling supports vascular maturation through positive regulation of proteins critical for neurovascular specialization (discussed below). 6,46,79–81 Concomitantly, EC S1PR signaling suppresses expression of migration-promoting genes often observed in tip cells, including Esm1, Igfbp3, and Angpt2. 6,75–77,82 Ectopic expression of tip cell genes in the nascent network goes hand-in-hand with hypersprouting. 40,41,46,76 Inversely, inducible S1PR1 over-expression suppressed vascular sprouting and tip cell frequency, resulting in hypovascularization. 40 Genetic mosaic studies showed that S1PR1-expressing ECs tend to incorporate into the mature regions of the vascular network rather than adopt a tip cell position, 41 further supporting the notion that S1PR1 facilitates vascular maturation in a context-dependent cell-autonomous manner.
Expression of Notch pathway components and target genes, which also inhibit hypersprouting, 76 was unaffected in S1PR-knockout retinal ECs. 40,41,46 In addition, aortic hyperbranching was observed in S1pr1 −/− and EC-specific S1PR1-knockout embryos but not in Dll4 +/- or EC-specific Rbpj-knockout embryos. These distinct outcomes downstream of S1P or Notch inhibition suggest that these ligands inhibit hypersprouting through discrete mechanisms. 40,41,46 However, S1PR and Notch signaling pathways may intersect or cross-regulate each other in additional biological contexts, such as biomechanical signaling. 83,84 This framework may also be understood in the context of interactions between different cell competition pathways, but these hypotheses are only now beginning to be tested. 47,85
LPA Signaling in Vascular Development
Endothelial responses to LPA and S1P are very different, if not in direct opposition. In HUVECs, LPA signals through LPAR6 and rapidly induces actin stress fibers that decrease cell-cell adhesion and cause intercellular gaps. 51,86 After binding LPA, LPAR6 couples with Gα12/13 and activates the GTPase RhoA (small GTPase Rho A) and its target ROCKI/II (Rho-kinase I/II). 51,86 This is the mechanism by which LPA reduces endothelial barrier integrity and promotes vascular leak. 51,57,86 Inhibition of any individual component in LPA signaling, including LPAR6, Gα13, RhoA, or ROCK abrogates LPA induction of stress fibers. 51,86 Like LPAR6, LPAR4 activates the Gα12/13/Rho/ROCK pathway 36 but can also couple with Gq/11, Gi/o, and Gs. 36 Overlapping signaling components downstream of LPAR4 and LPAR6 in embryonic endothelium may contribute to their functional redundancy during embryogenesis. 44 Indeed, all Lpar6 −/− mice and most Lpar4 −/− mice survive to term, but all Lpar4 −/− Lpar6 −/− double-knockout mice die by E10.5 due to vascular defects. 43,44 LPA-deficient Atx −/− mice, EC-specific Gα13-knockout, and Rock1 −/− Rock2 −/− mice each die embryonically with impaired vascular development, 87–91 suggesting that these genes encode essential components for EC LPAR signaling. Endothelial RhoA, however, is dispensable as EC-specific RhoA-knockout animals develop normally, 92 suggesting that EC LPARs signal through one or more of the ≈18 Rho GTPases expressed in endothelium. 93
Phenotypes associated with Lpar4 −/− embryos include pericardial effusion, severe edema, general fragility, subcutaneous hemorrhage, and lethality (≈35% penetrant). 43 Lpar4 −/− Lpar6 −/− phenotypes include pericardial effusion, severe developmental delay, poor vascular network formation in the head and intersomitic regions, absence of blood vessels in the yolk sac, and death by E10.5 (100% penetrant). 44 Both global and EC-specific Gα13-knockout mice die between E9.5 and E11.5 and fail to form normal yolk sac vasculature. 87,88 This phenotype was also seen in Rock1 −/− Rock2 −/− embryos, which die between E8.5 and E9.5. 89 Thus, the LPAR/Gα13/ROCK signaling axis is essential for early vascular development.
In contrast to the hypersprouting phenotypes in S1PR1-deficient retinas, EC-specific LPAR4/LPAR6 double-knockout (Lpar4:Lpar6 iΔEC ) mice exhibit hyposprouting with reduced vascular density and branching. 44 Tip cells in Lpar4:Lpar6 iΔEC retinas were few in number and exhibited defects including reduced filopodia length and frequency. 44 This hypovascular phenotype was reproduced in Lpar6 −/− retinas, suggesting that LPAR4 and LPAR6 are not entirely redundant during sprouting angiogenesis and that LPAR6 may be the primary mediator of LPA-induced EC proliferation during retinal development. 48
ATX is widely expressed during embryogenesis and is essential for embryonic development. 90,94 Atx −/− mice are LPA-deficient and have vascular defects in the embryo proper, lack yolk sac vasculature, and die from circulatory failure by E10.5. 90,91 These phenotypes were reproduced in embryos harboring a biallelic single amino acid substitution rendering ATX catalytically inactive, suggesting that enzymatic formation of LPA (or a related molecule) is the primary defect. 95 This is in stark contrast to LPP3, for which there is substantial in vitro and in vivo evidence for an EC-autonomous LPA/LPP3/LPAR axis that controls the subcellular location of LPAR signaling.
Encoded by the gene Ppap2b, LPP3 is a glycoprotein with a channel-like structure composed of 6 putative transmembrane domains. 96 LPP3 is best known for catalyzing dephosphorylation of phosphatidic acid, C1P (ceramide 1-phosphate), S1P, and LPA to generate diacylglycerol, ceramide, sphingosine, and monoacylglycerol, respectively. 96 Biochemical analysis showed that human LPP3 has highest affinity (1/Km) and catalytic efficiency (Vmax/Km) for LPA and PA, whereas these values were 3× to 4× lower for S1P and C1P. 97 Reduction of LPP3 activity can result in local or systemic accumulation of LPA, phosphatidic acid, C1P, and S1P. 98 For example, cardiac-specific LPP3-knockout mice harbor ≈3-fold higher (LPA) in circulation relative to wild-type counterparts. 99
Lpp3 is expressed in many cell types and structures during embryogenesis including, but not limited to, endothelial, cardiac, vascular, nervous, and mesenchymal tissues 100 (Figure 1). Both global and EC-specific LPP3-knockout mice die by E10.5 and exhibit hemorrhage of the embryo proper, defective yolk sac vasculature, and failure to form a chorioallantoic placenta. 52,100
In HUVECs, LPP3 appears to partition LPAR signaling between regions of strong and weak cell-cell contact. 44,48,51–54 LPP3 knockdown enhanced sensitivity to LPA-induced stress fibers. 51 This LPA response was more robust in subconfluent HUVECs, indicating that cell-cell contacts are involved in inhibition of LPAR signaling. After treatment with forskolin to enhance cell-cell adhesion, LPP3 localized to sites of cell-cell contact and the LPA-induced stress fiber response was abolished, suggesting cross-talk with cAMP-regulated signaling pathways. 51 However, forskolin-treated HUVECs deficient in LPP3 were sensitive to LPA, suggesting that LPP3 inhibits LPAR signaling in ECs with strong cell-cell adhesion. 51 In scratch assays, LPP3 localized to sites of cell-cell contact but was absent from noncontact sites in leader cells at the monolayer edge. 51 The leading edges (noncontact sites) of these cells showed robust stress fiber responses after LPA treatment, 51 suggesting that LPAR signaling is permitted in membrane compartments that lack cell-cell contact and, therefore, also lack LPP3 activity. Studies using LPP3-knockout mouse embryonic ECs 52 or LPP3-knockout human aortic ECs 53 reported that LPP3 promotes adherens junction assembly 52,53 and inhibits intercellular gap formation. 53 LPP3-deficient human aortic ECs showed reduced barrier function, 54 suggesting that LPP3 promotes endothelial barrier function, perhaps by attenuating LPA-dependent Rho GTPase activation.
These results demonstrate that LPP3 can localize to sites of EC-cell contact and inhibit LPA/LPAR signaling, restricting high levels of LPAR signaling to non–cell-cell contact sites. Noncontact sites of EC membranes have important migration-associated functions in tip cells and remodeling capillaries undergoing regression, 56 a process promoted by LPA/LPAR signaling 51 (Figure 2).
EC-specific LPP3-knockout embryos die at or before E10.5 with hemorrhagic areas in the embryo proper, abnormalities of the aortic sac, outflow tract, irregular intersomitic vasculature, defective yolk sac vasculature, failure to form a chorioallantoic placenta, as well as decreased cardiac trabeculation and growth of the compact myocardial wall. 52,57,100 These findings suggest that LPP3 function leads to spatial restriction of lysolipid receptor signaling to regulate vascular development (Figure 2).
In summary, mechanistic studies that reveal the in vitro differences between S1P and LPA signaling explain, at least in part, the in vivo functions of these lysolipids. S1P signaling-deficient mice exhibit vascular hypersprouting, which is characterized by hyperbranching, disorganized vascular networks that are poorly perfused and lack barrier integrity. In contrast, LPA signaling-deficient embryos exhibit vascular hyposprouting with notable absence or apparent lack of blood vessels in some tissues, particularly in the yolk sac. Conversely, S1P1/Gi/Rac signaling promotes cell-cell contact, adherens junction assembly, and vascular stability (Figure 2).
Whether endothelial LPARs exhibit spatial expression gradients during vascular development remains to be determined. Our understanding of in vivo LPAR and S1PR distributions, including subcellular localization, has been limited by the lack of widely available high-quality antibodies and the technical challenges associated with precise measurement of lysophospholipid gradients in complex tissues. 101
Developmental Studies Suggest Lysolipid and Wnt Connections
As outlined above, many of the core processes influenced by lysolipid signaling have also been observed to be regulated, to some extent, by the central pathways of early development: Notch, BMP, and in particular Wnt signaling. This is interesting given the dependence of Wnt signals on lipids and lipoproteins, 102 and the intricate relationships between Wnt signals, cell polarity, intercellular junctions, cytoskeletal structure, and metabolism. 85,103,104 Wnt signaling’s role in central decisions is illustrated by the diverse effects of mutations in Wnt pathway genes during development and disease. 105 Importantly, there are often parallels between the effects of Wnt gain of function and loss of function which suggest a tuning of a wide range of signaling pathways, although in many instances, this may simply reflect our limited understanding of the relevant biology. 103,106 There are several key downstream effectors of Wnt including the β-catenin destruction complex, the planar cell polarity pathway, and a complex noncanonical pathway that appears to modulate the multifaceted effects of calcium signals throughout the cell. 103,105
Specific lipids and lipoproteins are required for the transmission of Wnt signals between cells and inability to package Wnt ligands in appropriate lipoprotein essentially abolishes the downstream effects in receiving cells. 102 Classical Wnt effects are restricted to only a few cell diameters in range, but recent evidence of signaling roles for circulating lipoproteins in higher vertebrates raises the possibility that Wnt signals may also operate at a distance. 107,108 The lipid requirements for Wnt signal transduction are poorly understood, but specific lipids or lipoproteins may be necessary for endocytosis in the receiving cell of the ligand-receptor/coreceptor complexes or subsequent gating of the signals by intraorganelle pH or other factors. 104,109–111 The parallels between lysolipid pathways and Wnt in development warrant further studies to elucidate underlying mechanisms. Some recently described mechanisms are discussed in detail below.
S1P and Wnt Signaling in Blood-Retina-Barrier Development
Like lysolipid signaling, Wnt signaling is transduced by a family of receptors with 7 transmembrane domains, some of which have been shown to couple to heterotrimeric G proteins. 112 The protein Norrin (Ndp), a TGF (transforming growth factor)-β family member produced by glia, is a high-affinity Wnt-like ligand for its EC receptor FZD4 (Frizzled4). 113 Norrin/FZD4 signaling increases the activity of β-catenin and TCF/LEF TFs (transcription factors), 114 which leads to induction of proteins that promote a functional blood-retina-barrier (BRB) and suppression of proteins that cause vascular leakage or fenestration. 114,115 The developing BRB becomes dysfunctional upon loss of Norrin, or EC-specific deletion of Fzd4, Cttnb1, and S1pr1. 40,41,46,114,115 There are shared albeit distinct outcomes of endothelial S1P/S1PR and Norrin/FZD4/β-catenin signaling in BRB development, which suggests both convergent and divergent signaling, possibly as a function of underlying metabolic states, microenvironmental, or humoral factors. 104
Wnt-deficient retinas are hypovascular, 114–117 whereas S1PR1 signaling-deficient retinas are hypervascular. Thus, Wnt signaling promotes EC proliferation and vascular branching 117 while S1P signaling stabilizes blood vessels. 40,41,46 An in vivo reporter of canonical Wnt signaling was active throughout the developing retinal vascular ECs, 81 suggesting that β-catenin and TCF/LEF factors are active in blind-ended vascular sprouts as well as in ECs undergoing maturation. Whether Wnt signaling is involved in expression of tip cell genes at the vascular front is not known. However, there is evidence suggesting that both Wnt and S1P signaling suppress a tip cell gene expression program in the nascent vascular network.
Deficiency of either Wnt or S1P signaling results in vascular leakage and hemorrhage, 40,46,114 which can increase the concentration of VEGF and activation of VEGFRs. In fact, EC VEGFR signaling is required for expression of the prototypical tip cell genes Esm1, Apln, and Igfbp3, 75 which are induced in retinal ECs that lack Wnt or S1P signaling (Figure 3). Although tissue hypoxia and high VEGF might contribute to ectopic expression of tip cell genes in Ndp-knockout and S1pr-knockout retinal ECs, it is also possible that VEGFR-independent mechanisms contribute to S1P- and Wnt signaling-mediated suppression of these genes in the nascent vascular network. Indeed, tissue hypoxia might be anticipated to interrupt Wnt signaling, possibly explaining some of the selective phenomena observed. 104,110
Figure 3. Gene expression in Norrin-deficient and S1PR (sphingosine 1-phosphate receptor)-deficient retinal endothelial cells (EC).A–E, Expression of selected genes in Norrin knockout (Ndp-KO) and wild-type (WT) retinal EC were acquired from https://jacobheng.shinyapps.io/cnshypoxia/. 118 For control and S1PR-deficient retinal EC, data were acquired from GEO accession GSE141440. 46 A and B, Expression of neurovascular-enriched transcripts that encode tight junction components (A), transporters or transcription factors (B). C, Expression of genes that are enriched in tip cells. D, Expression of Plvap. E, S1pr1 or Fzd4 expression in Ndp-KO or S1PR-deficient retinal EC, respectively. FACS indicates fluorescence-activated cell sorting and scRNA-seq, single-cell RNA-sequencing.
Yanagida et al 46 reported that 97 neurovasculature-enriched transcripts were down-regulated in S1PR-deficient retinal ECs. Ndp-knockout retinal ECs also down-regulated many of these transcripts (Figure 3A and 3B). 118 For example, both Wnt and S1P signaling are required for normal expression of tight junction components (Lsr, Ocln), transporters (Mfsd2a, Tfrc), and transcription factors (Lef1, Tcf7, Zic3) that are enriched in the vasculature of the central nervous system 46 (Figure 3). Expression of Cldn5 and Plvap, well-characterized targets of Wnt signaling in the BRB, were not significantly affected in S1PR signaling-deficient retinal ECs (Figure 3A and 3D). 46 Therefore, S1P and Wnt signaling regulate common and distinct gene sets that determine neurovascular structure and function. For example, suppression of Plvap (fenestrae) and induction of Cldn5 (tight junctions) are likely 2 mechanisms by which Wnt signaling, but not S1P signaling, promote BRB integrity (Yanagida et al 46 and Figure 3). However, S1PR1 signaling is upstream of CLDN5 expression in lymphatic ECs during developmental lymphangiogenesis. 119 Thus, S1PR1 and other EC surface receptors do not exhibit a one-size-fits-all model of signaling and transcriptional outputs but rather have unique functions according to developmental stage, vascular bed, microenvironmental context, and EC subtype.
Any epistatic relationship between endothelial Wnt and S1P signaling is anticipated to be multifaceted and insights will likely require analysis of novel mouse strains, 103 such as Ctnnb1 flex3/+ S1p1 f/f Cdh5-Cre ERT2 , which would address whether increasing β-catenin activity is sufficient to rescue maturation defects in S1PR1-knockout retinal ECs. The Ctnnb1 flex3 allele encodes a β-catenin protein that resists degradation and is sufficient for induction of a BRB-like endothelial phenotype (MFSD2A+, LEF1+, CLDN5+, PLVAP−) in the vasculature of circumventricular (eg, neuroendocrine) organs. 81 Additionally, R26-8xTCF/LEF-LSL-H2B-GFPS1p1 f/f Cdh5-Cre ERT2 mice would likely address the effect of EC-specific S1pr1-knockout on β-catenin and TCF/LEF transcriptional activity in retinal ECs. 81
Mosaic deletion of Fzd4 in BRB and BBB vasculature showed that induction of CLDN5 and suppression of PLVAP occurs in an FZD4-dependent, cell-autonomous fashion and is unlikely secondary to hypoxia or high VEGF levels. 81 Similar mosaic experiments in S1PR signaling-deficient mice might reveal cell-autonomous S1PR versus hypoxia-dependent effects on expression of BRB-enriched genes, such as Mfsd2a, Tfrc, and Lef1. Alternatively, sFlt1 (fms-like tyrosine kinase-1) that blocks VEGF signaling may be a useful reagent to investigate the role of this pathway in Wnt- and S1P signaling reporter mice. Because VEGF itself can induce vascular leak, 120 adherens junctions (VE-cadherin) should also be examined to determine the role of aberrant VEGF signaling in junctional breakdown observed in S1PR-knockout retinal vasculature. 40,41,46 These experiments may provide novel mechanistic insights into S1PR- versus VEGFR-mediated gene expression and vascular function during BRB development in the context of Wnt signaling.
LPP3, Lysolipids, and Wnt Signaling in Gastrulation and Axial Patterning
Some Lpp3 −/− embryos exhibited defective gastrulation with axis duplication at E7.5 (30% penetrance), a phenotype reminiscent of ectopic Wnt signaling. 100 Over-expression of Xwnt3a or Xwnt8 induces axis duplication in Xenopus embryos, 100 as does over-expression of Cwnt8C 121 or ablation of the Wnt signaling inhibitor Axin 122 in mouse embryos. At E7.0, expression of brachyury, a WNT3 target gene, is restricted to the primitive streak 100 however, Lpp3 −/− embryos with axis duplication have 2 brachyury-expressing primitive streak structures. 100 In addition, expression of the Wnt signaling antagonist Dkk1 is markedly reduced in Lpp3 −/− embryos. 100 Importantly, axis duplication induced by injection of Xwnt3a or Xwnt8 mRNA was inhibited by coinjection with mouse Lpp3 mRNA, demonstrating that LPP3 can inhibit signaling by these Wnt ligands. 100
There are several open questions related to LPP3-mediated axial patterning, including whether specific lysolipid receptors are involved. To date, axis duplication has not been reported in S1PR- or LPAR-deficient mice, suggesting that LPP3-mediated axial patterning does not require signaling by individual lysolipid receptors. This notion is supported by insights from Drosophila, which lack orthologs of vertebrate G-protein–coupled lysolipid receptors but express 2 LPP3 homologs, Wun and Wun2 (wunen and wunen-2) that are essential for gastrulation. 96,100 In somatic cells, wun and wun2 produce phospholipid metabolites that serve as guidance cues by repelling germ cells (GCs), a process that is required for bilateral sorting of GCs away from the ventral midline. 96,123 Wun-deficient embryos have scattered GCs and high frequency of GC death. 124 Interestingly, a Drosophila GPCR called Tre1 is required for GC migration towards high concentrations of Wun-generated phospholipids in a Gαo-dependent manner. 125 In support of the notion that Tre1 binds Wun metabolites, the human Tre1 homolog, GPR84, binds medium-chain fatty acids. 126 Thus, Tre1 might be the primary receptor for Wun-generated phospholipids, though this would require confirmation by receptor signaling assays.
Tre1, in addition to guiding GC migration, is required for Rho-mediated protein polarization in GCs. 125 Drosophila Tre1 may have conserved functions that are split among multiple proteins in vertebrates. 125 The presence of compensatory mechanisms in vertebrates may explain why defective gastrulation is ≈30% penetrant in Lpp3 −/− mice but 100% penetrant in Wun-null Drosophila embryos. In addition, the phosphatase activity of wun2 is essential for GC survival. 127 Human LPP3 can rescue GC death in wun2-deficient embryos, suggesting an evolutionarily conserved phosphatase function for LPP3 in gastrulation. 127
Evidence to date strongly suggests that the phospholipids metabolized by LPP3 must be spatially compartmentalized to ensure normal axial patterning. Given that lipoprotein-derived precursors feed into lysolipid metabolic pathways, LDL, VLDL, and other lipoproteins may be involved in spatial control of lysolipid signaling. However, identification of the specific lipids, the means of compartmentalization, and clarification of downstream signaling mechanisms all remain a major challenge. Isolation of these molecules will provide mechanistic insight into Wun and LPP3-mediated axial patterning, cell survival and may also reveal how phospholipids regulate Wnt signaling in vertebrates or invertebrates. 128 A detailed mechanistic understanding of embryonic Wun and LPP3 metabolites and cognate receptors may inform exploration of LPP3-mediated endothelial functions during development, postnatal homeostasis, and disease.
Lysolipids in Cardiovascular Disease
Insights From Human Studies
In the 1970s, landmark studies reported negative correlations between coronary artery disease (CAD) severity and circulating HDL levels. 129–131 Subsequent biochemical analyses of HDL particles have uncovered significant heterogeneity. 132 For example, on a stoichiometric basis, 1 in 10 HDL particles contains S1P. 133 Although only a minority of studies in the HDL field have focused on lysolipids, recent work has linked HDL-S1P to CAD. For example, HDL-S1P (1) correlates inversely with the severity of coronary atherosclerosis, 134 (2) is an independent predictor of coronary in-stent restenosis, 135 (3) is lower in patients with stable CAD than in healthy individuals, 136,137 and (4) correlates negatively with the occurrence of CAD independently of HDL-cholesterol 137 (reviewed in Levkau 138 ). In HUVECs, HDL isolated from CAD patients was ineffective at stimulating S1PR1-dependent vasoprotective signaling events, including vasodilation, which was rescued by providing exogenous S1P. 139
Lipoprotein(a), which is highly predictive of cardiovascular diseases (CVD) in humans, was shown to supply autotaxin and therefore involved in local signaling of LPA via its receptors. In calcific aortic valve disease, lipoprotein(a)-derived autotaxin directly induces valve fibrosis and calcification, presumably via a Rho GTPase signaling pathway. 140,141 Inhibitors of this signaling axis may be useful in the medical management of aortic valve diseases.
S1PR1 genomic heterogeneity may contribute to CVD risk. For example, single-nucleotide polymorphisms (SNPs) in the N-terminal cap region of S1PR1 were associated with multivessel CVD in a patient cohort, suggesting a potential regulatory function of this receptor. 142 Asthma is characterized by a chronic inflammatory process with increased vascular permeability. Several SNPs (rs2038366, rs3753194, rs59317557) in the putative enhancer and promoter regions of S1pr1 associate with increased risk of asthma development. 143 The SNP rs2038366 was notable for conferring S1pr1 downregulation by luciferase assay in human pulmonary artery ECs. 143
S1PR1: Pharmacological considerations
The Food and Drug Administration–approved S1PR functional antagonist FTY720 (fingolimod) is prescribed as an immunomodulatory agent to treat relapse-remitting multiple sclerosis. 144 This drug’s mechanism of action is downregulation of lymphocyte S1PR1 in lymphoid tissue, which sequesters lymphocytes by preventing chemotaxis towards high (S1P) in circulation. 145 Although this lymphocyte-targeted drug has clear immunologic benefits, endothelial S1PR1 is also being explored as a therapeutic target in autoimmune and fibrotic conditions. Studies using experimental disease models (discussed below) suggest that endothelial S1PR1 agonism mitigates inflammation through at least 2 mechanisms: (1) enhancing the vascular barrier and (2) attenuating endothelial inflammatory responses, each limiting leukocyte recruitment to tissue parenchyma. 146
The notion that pharmacological or endogenous agents elicit varying degrees of biased engagement of S1PR1 with either Gi/Rac or β-arrestin pathways has both clarified and added complexity to our understanding of S1P signaling. 39,107,147 For example, arterial ECs of the thoracic aorta express S1PR1 mRNA 148 and protein 107 in a homogenous manner and are exposed to circulatory S1P. However, S1PR1/β-arrestin coupling is heterogeneous in aortic arterial endothelium of adult mice, 107,148 and the frequency of S1PR1/β-arrestin coupling increases with temporal transition from early postnatal to young adult, which coincides with upregulation of thrombospondin-1. 107,148 Despite high expression and the functional importance of S1PR1 in early postnatal mouse retinal ECs, we observed relatively low levels of S1PR1/β-arrestin coupling in these cells (unpublished observation). In contrast, developing embryonic lymphatic vessels show high levels of S1PR1/β-arrestin coupling. 119 Although these data suggest a spectrum of S1PR1/β-arrestin signaling among ECs, we lack in vivo evidence for such a spectrum of S1PR1/Gi signaling. We can hypothesize that cells highly engaged in β-arrestin signaling are relatively low in S1PR1/Gi activity. Endogenous mechanisms that skew S1PR1 towards β-arrestin versus Gi are unclear but may involve concomitant signaling pathways, such as LPARs, 149 VEGFRs, junctional signals, shear force responses, or S1PR1 association with presently unknown cofactors. A prototypical example of cofactor-dependent signaling in vascular endothelium occurs in the central nervous system when ECs respond to WNT7 ligands with the multi-protein complex of FZD4/GPR124/RECK/LRP6. 150–153
Patients taking FTY720 risk complications from lymphopenia, which is a direct result of β-arrestin–mediated S1PR1 internalization in lymphocytes. 145,154 A similar mechanism in ocular endothelium may underpin macular edema that occurs in a small subset (0.8%–1.5%) of patients. 155 Vascular development is apparently unaffected in mice expressing 2 mutant S1PR1 alleles (S1pr1 S5A/S5A ) that encode a β-arrestin coupling- and internalization-defective receptor. 154 Furthermore, S1pr1 S5A/S5A mice are more resistant to FTY720-induced lung vascular leakage and S1PR1 degradation. 62 Therefore, endothelium-targeted S1PR1 agonists would likely provide maximal therapeutic benefit if biased towards activation of Gαi/Rac to limit β-arrestin recruitment (ie, mimic S1pr1 S5A/S5A ) and avoid receptor degradation and lymphopenia. A compound matching these criteria was recently described and showed therapeutic efficacy in preclinical models of coronary endothelial damage and renal ischemia/reperfusion injury. 147 In a recent phase 1 clinical study of diabetics, this compound, SAR247799 stimulated myocardial perfusion without inducing lymphopenia in diabetics. 156
Taken together, these studies of S1PR1 signaling highlight an important consideration for drug development and remind us that S1P measurement in patient fluids or tissue does not inform on S1PR1 expression or the extent of β-arrestin versus Gi/Rac signaling, which may have more functional significance than S1P levels alone.
S1P in Experimental Disease Models
Rheumatoid arthritis and systemic lupus erythematosus, although etiologically complex, share the pathophysiologic mechanisms of neutrophil activation and immune complex deposition in tissues with resultant end-organ damage. S1PR1 agonism limits vascular barrier leakage associated with immune complex deposition. 65 Inversely, genetic inactivation of EC S1PR1 or pharmacological S1PR1 antagonism resulted in more vascular leak and pulmonary neutrophil accumulation relative to control animals. 65
After organ damage, endothelial S1PR1 promotes recovery, regeneration, and limits fibrosis. In a hydrochloric acid-induced model of lung injury, EC S1PR1 protected against vascular leak and limited fibrosis. 157 Following partial hepatectomy, EC S1PR1 protected against fibrosis and improved liver vascular function, perfusibility, tissue regeneration, and animal survival. 158 Perhaps it is not surprising that S1PR1 is important for tissue regeneration as the vasculature is a critical component of most major organ systems and S1PR1 is a central regulator of vascular network formation, but other roles for lysolipid signals in endothelial or epithelial biology may be involved.
In the murine Apoe −/− high-fat diet model, EC-specific S1PR1 deficiency exacerbated disease severity and macrophage infiltration into atherosclerotic plaques. 107 Although S1P signaling in ECs seems protective in the context of atherosclerosis, S1P regulation of macrophage phenotypes is more complex. S1PR2 159 or S1PR3 160 deficiency attenuated foam cell accumulation into lesions—an effect that is myeloid cell-intrinsic, as evidenced by bone marrow chimera experiments. In contrast, S1PR1-specific agonists confer an anti-inflammatory macrophage phenotype in vitro. 161 Thus, we propose a model describing pro- and anti-inflammatory effects of S1P as first compartmentalized among cell types (vascular versus myeloid), 162 secondarily compartmentalized between different S1P receptors (S1PR1 versus S1PR2/3), and at a tertiary level when considering S1PR1 association with Gi/Rac versus β-arrestin pathways.
S1PR1 signaling is engaged in arterial ECs of the aorta intima as well as in adventitial lymphatic endothelium. 148 In homeostasis, S1PR1 attenuates expression of proinflammatory transcripts in arterial (eg, Cx3cl1/fractalkine, Vcam1, Ptgs2) and lymphatic ECs (Ccl21, Irf8, Il7). Furthermore, S1PR1 is a critical regulator of developmental lymphangiogenesis. 119 Therefore, future studies might parse out blood vascular versus lymphatic S1PR1 signaling in mitigation of inflammation.
Consistent with an anti-inflammatory S1PR1 function, Teijaro et al 163 demonstrated that the S1PR1 agonist CYM-5442 mitigates influenza virus–induced pulmonary cytokine storm and leukocyte infiltration. These effects of CYM-5442 were observed in Rag2 −/− mice, which lack mature B and T cells, suggesting a minor role for lymphocyte S1PR1. 163 Lung ECs from influenza virus–infected mice showed reduced CCL2 and CXCL10 expression in response to CYM-5442, 163 suggesting that EC S1PR1 attenuates cytokine amplification during influenza virus infection. The expression of cytokines has been linked to Wnt-Ca 2+ signaling in ECs, and there is initial evidence of bidirectional cross-talk between TLR2/4, inflammasome activation, and canonical Wnt signals, 164–166 although the detailed molecular mechanisms have not been studied.
The primary Mendelian forms of atherosclerosis result from mutations in a small number of genes which cause familial dyslipidemias and premature vasculopathy. These genes (Ldlr, ApoB, Lrp6, Ldlrap, Pcsk9) not only all share the defining vascular phenotypes but also participate in different aspects of Wnt signaling, implying some commonality to the underlying mechanisms of atherosclerosis, but to date defining any shared mechanism has proven elusive. 109,167,168 Activation of canonical Wnt signaling has been observed in the endothelium of murine models before the emergence of focal atherosclerotic lesions and has been attributed to flow effects. 169 Disruption of physiological endothelial-smooth muscle interactions with proliferation of subjacent smooth muscle is associated with local canonical Wnt activation in reporter mice. 170 In later stages of the atherosclerotic process, several aspects of Wnt signaling have been directly implicated in vascular calcification, including evidence that LRP6 mitigates calcification in Ldlr −/− diabetic mice. 171 Ongoing work exploring the role of Wnt and lysolipids in the pathophysiology of atherosclerosis spans the full repertoire of Wnt signaling, but unifying generalizable insights have yet to emerge. 172
LPA and Vascular Disease
In patients with acute coronary syndromes, culprit coronary arteries showed elevated LPA levels relative to the peripheral circulation. 173 LPA accumulates in the lipid core region of human and mouse atherosclerotic lesions, 174–176 and unstable plaques show high frequency of ATX immunostaining in the necrotic core. 177
In addition to disruption of endothelial junctions (discussed above), LPA also induces NFκB signaling and expression of downstream proinflammatory molecules in ECs. 178 Furthermore, LPA has been shown to positively regulate monocyte/macrophage uptake of oxidized-LDL, 179,180 expression of the proinflammatory molecule IL (interleukin)-1β, 180 and inhibit apoptosis, 181 which may inhibit macrophage clearance from subendothelial space. These effects are likely mediated by LPAR1 or LPAR2. 182,183 Importantly, the LPA/LPAR5 signaling axis induces platelet activation, which may contribute to atherothrombosis. 184,185 Finally, a pharmacological inhibitor of LPAR1/3 reduced plaque burden and myeloid infiltrate 186,187 in 2 different models of murine atherosclerosis, whereas injection with LPA20:4 increased plaque burden and myeloid infiltrate. 187 Collectively, these data imply that LPA enhances atherothrombosis and subendothelial foam cell accumulation, although careful studies of signaling events intrinsic to specific cell types are lacking in this field.
Consistent with human genetic studies suggesting a protective role for LPP3 in endothelium, 54,188–190 EC LPP3 expression protects against lung vascular leakage in homeostatic and endotoxemic conditions in mice. 57 Global reduction of LPP3 in the postnatal period accelerates atherosclerosis development in a mouse model, which is largely attributable to the role of LPP3 in smooth muscle cells but unlikely related to LPP3 function in myeloid cells. 191 Inhibition of LPA signaling by an ATX inhibitor or pan-LPAR inhibitor rescued the exacerbated lung vascular leakage in EC-specific LPP3-knockout animals. Conversely, mice with low levels of circulating ATX (and likely reduced LPA content) were resistant to LPS-induced lung vascular leakage. 57 Intradermal LPA injection–induced permeability of skin vasculature in a dose-dependent fashion, 57 consistent with HUVEC responses to LPA. 51,86 In endotoxemia models, EC-specific LPP3-knockout mice showed ≈3-fold increases in plasma (IL-6) and peritoneal leukocyte recruitment after thioglycolate injection, suggesting that EC LPP3 attenuates inflammatory responses. 57 Collectively, these data suggest that endothelial LPP3 attenuates LPA/LPAR-mediated vascular permeability and inflammation.
Several multiethnic genome-wide association studies found evidence of a strong association between CAD and an SNP (rs17114036) in intron 5 of Ppap2b (which encodes the LPP3 protein). 188–190 In human aortic ECs, rs17114036 is within a ≈1.2 kb peak of high chromatin accessibility and histone modification (H3K27ac, H3K4me2 [dimethyl modified histone H3 at lysine 4]) associated with active enhancers. 54 Other cell types (K562, GM12878, NHEK [normal human epidermal keratinocytes]) lacked indications of active chromatin, suggesting a unique role for this ciselement in ECs. 54 Luciferase assays demonstrated significant enhancer activity for the ≈1.2 kb region at rs17114036. 54 CRISPR-Cas9–mediated deletion of a ≈66 bp region enclosing rs17114036 attenuated LPP3 expression. 54 Consistently, mutagenesis of the risk allele (T/T) to the protective genotype (T/C) resulted in a ≈6-fold increase in enhancer activity, establishing a causal relationship between the T/T variant and attenuated LPP3 expression. 54 Taken together with information from genome-wide association studies, these experiments suggest that strategies that promote endothelial LPP3 activity or expression may have therapeutic benefit in CVD.
Outstanding Questions and Perspectives for Future Research
S1P and LPA receptors, and molecules that regulate lysolipid bioavailability, are emerging as tractable targets for a range of pathologies that stem from autoimmune diseases, tissue injury, and pathogen infection. Animal models have yielded insights regarding downstream outcomes of single and collective receptor signaling, namely, S1PR1 as the primary endothelial S1PR promoting stabilization and maturation, whereas LPAR4 and LPAR6 promote EC proliferation and vascular front expansion. Further studies of subcellular location-specific LPAR activation, such as LPP3-regulated restriction of signaling to regions devoid of cell-cell contacts, warrant further study. Similarly, we lack mechanisms to describe S1PR1 biased signaling towards Gi/Rac or β-arrestin pathways. Understanding of these pathways may facilitate rational drug design as well as development of assays that, in addition to lipid measurements, will more precisely inform on the lysolipid signaling status of patients.
We lack a mechanism to explain why the rs17114036 SNP, which appears to regulate EC LPP3 expression, is strongly correlated with CVD risk. Is the mechanism as straightforward as aberrant LPAR signaling in coronary arteries downstream of increased local LPA levels? or do individuals with this genetic variant have developmental defects, perhaps involving Wnt signaling, that compound with other factors and manifest as CVD? As we learn more about the basic science of lysolipid signaling using biochemical, cell culture, and animal models, we will be better prepared for rational design of therapeutic agents and evaluation of their efficacy.
Post-transcriptional regulation of lysolipid receptors in cell type-specific contexts is an active area of investigation. Several studies have identified microRNAs that directly or indirectly downregulate S1pr1 expression in human cancer cell lines, including miR (micro RNA)-148a in ovarian cancer, 192 miR-149 in hepatocellular carcinoma, 193 and miR-133b in nasopharyngeal carcinoma. 194 At least 2 studies have shown that miR-155 targets S1pr1 in lymphocytes. 195,196 miR-24 downregulates S1pr1 expression in HUVECs and human kidney epithelial cells, 197 miR-24 antagonism improved survival and tissue vascularization in a model of renal ischemia/reperfusion injury. 197 Similarly, the miR-17–92 cluster negatively regulates normal and ischemia-responsive arteriogenesis around mouse limbs, likely via inhibition of Wnt signaling components including FZD4 and LRP6. 198 As compared to vascular S1P receptors, less is known about microRNA regulation of Lpar4 and Lpar6, though one study showed miR-139-5p targets Lpar4 in human umbilical cord mesenchymal stem cells. 199 MicroRNA signaling in vascular biology has been reviewed elsewhere, 200–202 and additional insights are likely to arise from unbiased profiling in a human 3-dimensional–culture angiogenesis model. 203 Currently, data indicate that antagonism of endothelial microRNAs that downregulate S1pr1 may improve vascular function after tissue injury.
The enzyme SPL (S1P lyase) irreversibly catabolizes S1P to phosphoethanolamine and hexadecenal. 204 Along with LPP3 205 and SPNS2, 20 SPL is critical for T-cell egress from lymphoid organs to circulation. 206 Mechanistically, SPL expressed by thymic dendritic cells causes parenchymal S1P sinks that promote T-cell chemotaxis towards relatively high (S1P) in the bloodstream. 207 Ongoing research aims to determine whether SPL-targeted therapies may provide immunomodulatory benefits similar to those of FTY720 (fingolimod) without inhibiting vascular-protective S1P signaling. 207–210
Finally, understanding the complex and often contradictory effects of Wnt and lysolipid signaling in vascular biology is likely to require a much deeper exploration of the developmental effects of these convergent pathways on endothelial biology. Accumulating evidence suggests that the potential to connect mechanotransduction, metabolism, cell polarity, and intercellular communication or competition with the fundamental processes of aging remains high, but mechanistic molecular models will require unraveling many of the most complex temporal and regional signaling hierarchies in development. 85,103,105