S1P Signaling Research Articles

S1P1 deletion in oligodendroglial lineage cells: Effect on differentiation and myelination.

Sphingosine 1-phosphate (S1P) receptors are G protein-coupled receptors expressed by many cell types, including cells of oligodendrocyte (OLG) lineage. We had previously shown that targeted deletion of S1P1 in OLG lineage cells did not result in obvious clinical phenotype or altered number of OLGs at 3 months, but there were subtle abnormalities in myelin. In this study, we examined the role of S1P1 in developmental myelination and cell survival, focusing on age 3 weeks. We found that S1P1 deficiency led to delayed differentiation of OLG progenitors (OPCs) into OLGs that is independent of p38 phosphorylation. This was accompanied by decreased levels of myelin basic protein (MBP) but not of myelin-OLG glycoprotein (MOG), and slight decrease in myelin thickness in the corpus callosum of S1P1 conditional knockout (CKO) mice. S1P1 -deficient OLGs exhibited slower process extension, which was associated with attenuated phosphorylation of extracellular signal regulated kinases (ERKs) and p21-activated kinases (PAKs), and with upregulation of tropomodulin1. Basal levels of pAkt were not affected, though expectedly, no response to a selective S1P1 agonist SEW2871 was observed. S1P1 -deficient OLGs did not exhibit increased cell death in response to cuprizone, tumor necrosis factor-α, or deprivation of nutrients and growth factors. We conclude that S1P1 signaling regulates OLG development, morphological maturation and early myelination.

Curcumin Suppresses Phthalate-Induced Metastasis and the Proportion of Cancer Stem Cell (CSC)-like Cells via the Inhibition of AhR/ERK/SK1 Signaling in Hepatocellular Carcinoma.

Recent evidence indicating that phthalates promote cancer development, including cell proliferation, migration, and invasion, has raised public health concerns. Here, we show that bis(2-ethylhexyl) phthalate promotes the migration, invasion, and epithelial-mesenchymal transition of hepatocellular carcinoma cells. In addition, bis(2-ethylhexyl) phthalate increased the proportion of cancer stem cell (CSC)-like cells and stemness maintenance in vitro as well as tumor growth and metastasis in vivo. The various activities of curcumin, including anticancer, anti-inflammation, antioxidation, and immunomodulation, have been investigated extensively. Curcumin suppressed phthalate-induced cell migration, invasion, and epithelial-mesenchymal transition, decreased the proportion of CSC-like cells in hepatocellular carcinoma cell lines in vitro, and inhibited tumor growth and metastasis in vivo. We also reveal that curcumin suppressed phthalate-induced migration, invasion, and CSC-like cell maintenance through inhibition of the aryl hydrocarbon receptor/ERK/SK1/S1P3 signaling pathway. Our results suggest that curcumin may be a potential antidote for phthalate-induced cancer progression.

Abstract 067: Endothelial S1P/S1P1 Signaling is Critical Regulator of Blood Flow and Pressure

Background and objectives: Sphingosine-1-phosphate (S1P) is emerging as a pivotal signaling molecule within the vasculature. S1P has been known as a potent endothelial nitric oxide synthase activator through G-protein coupled receptor S1P1 and less abundant S1P3 on endothelial cells (EC). Although it has been recently shown that S1P/S1P1 signaling mediates EC alignment in response to flow, the role of S1P1 signaling in the pathogenesis of hypertension remains unknown. FTY720, an S1P1 functional antagonist, recently approved by the FDA to treat autoimmune conditions, induces a modest and transient decrease in heart rate in both animals and humans, suggesting that drugs targeting sphingolipid signaling affect cardiovascular functions in vivo. Thus, the aim of this study is to investigate whether S1P-S1P1 signaling pathway is crucial in blood flow and pressure regulation. Methods: The BP was evaluated in conscious mice with tail cuff system, whereas the vascular reactivity was examined with pressure myograph system in mesenteric arteries isolated from age matched WT and EC-S1P1-/- mice. Results. Here we report for the first time that S1P1-mediated signaling protects from hypertension. Administration of SEW 2871, an agonist of S1P1, to angiotensin-II-induced hypertensive mice lowered blood pressure to normotensive levels (110.7±3.1 vs. 160.7±2.4 mmHg, SEW 2871 vs. vehicle respectively). Pharmacological inhibition of S1P1 with W146 (100 nM and 1 μM) markedly reduced flow-induced vasodilation (Emax 25.2±3.8 and 2.5±2.3 vs. 48.6±4.9 μm, W146 100 nM and 1μM respectively vs. WT). These data were corroborated by using a genetic approach: mice EC-S1P1-/- were hypertensive (124.7±1.9 vs. 109±1.7 mmHg, EC-S1P1-/- vs. S1P1f/f mice) and showed a reduced response to flow-induced vasodilation (Emax 15.2±3.0 vs. 53.8±5.0 μm, EC-S1P1-/- vs. S1P1f/f mice mesenteric artery). Conclusion: Our study identifies S1P-S1P1 signaling as a new regulatory pathway of blood flow and pressure homeostasis, providing a novel therapeutic target for the treatment of hypertension.

Association of Sphingosine-1-phosphate (S1P)/S1P Receptor-1 Pathway with Cell Proliferation and Survival in Canine Hemangiosarcoma.

BackgroundSphingosine‐1‐phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs.Hypothesis/ObjectivesS1P/S1P1 signaling will contribute to the progression of hemangiosarcoma (HSA).AnimalsThirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 endothelial cell line derived from a dog with splenic hematoma were used.MethodsThis was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT‐PCR, immunohistochemistry, and immunoblotting were performed to examine S1P1 expression. S1P concentrations were measured by high‐performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca2+ mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining.ResultsCanine HSA cells expressed higher levels of S1P1 mRNA than nonmalignant endothelial cells. S1P1 protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P1, decreased S1P1 protein expression and induced apoptosis of HSA cells.Conclusions and clinical importanceS1P/S1P1 signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P1 or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA.

Corticotrophin-releasing factor participates in S1PR3-dependent cPLA2 expression and cell motility in vascular smooth muscle cells

This work is to investigate the role of CRF receptors in VSMC migration and the relevant mechanisms. We studied the role of CRF receptors in cell motility and found that S1P signaling pathway is involved in the regulation of cPLA2 induced by CRF. S1P is synthesized by Sphk1 and Sphk2 and binds to five GPCR designated S1P1-5. We observed that activation of CRFR1 resulted in increased cell migration, whereas activation of CRFR2 resulted in decreased cell migration. cPLA2 and iPLA2 were knocked down respectively to explore the corresponding effect on cell migration by means of shRNA interference. cPLA2 expression was increased by CRFR1 but decreased by CRFR2. On the contrary, iPLA2 expression was inhibited by CRFR1 but enhanced by CRFR2. The regulation of cPLA2 was in line with the regulation of Sphk1 and hence cell migration after the activation of CRFR1 or CRFR2. Consistently, S1P release was enhanced with CRFR1 activation. Both DMS (Sphk inhibitor) and CAY10444 (S1PR3 inhibitor) attenuated cPLA2 expression and thus decreased cell migration in response to CRF. In addition, CRF could not promote cell migration after S1PR3 silencing. Our results suggest the pro-migratory role of CRFR1–Sphk1–S1P–S1PR3–cPLA2 signaling pathway in VSMCs.

Terminology of CCN1-6 should not be applicable for their fragments and be limited to only full length CCN1-6

Terminology of CCN1-6 should not be applicable for their fragments and be limited to only full length CCN1-6

Exogenous S1P Exposure Potentiates Ischemic Stroke Damage That Is Reduced Possibly by Inhibiting S1P Receptor Signaling.

Initial and recurrent stroke produces central nervous system (CNS) damage, involving neuroinflammation. Receptor-mediated S1P signaling can influence neuroinflammation and has been implicated in cerebral ischemia through effects on the immune system. However, S1P-mediated events also occur within the brain itself where its roles during stroke have been less well studied. Here we investigated the involvement of S1P signaling in initial and recurrent stroke by using a transient middle cerebral artery occlusion/reperfusion (M/R) model combined with analyses of S1P signaling. Gene expression for S1P receptors and involved enzymes was altered during M/R, supporting changes in S1P signaling. Direct S1P microinjection into the normal CNS induced neuroglial activation, implicating S1P-initiated neuroinflammatory responses that resembled CNS changes seen during initial M/R challenge. Moreover, S1P microinjection combined with M/R potentiated brain damage, approximating a model for recurrent stroke dependent on S1P and suggesting that reduction in S1P signaling could ameliorate stroke damage. Delivery of FTY720 that removes S1P signaling with chronic exposure reduced damage in both initial and S1P-potentiated M/R-challenged brain, while reducing stroke markers like TNF-α. These results implicate direct S1P CNS signaling in the etiology of initial and recurrent stroke that can be therapeutically accessed by S1P modulators acting within the brain.

S1P signaling: new therapies and opportunities.

Development of sphingosine-1-phosphate receptor 1 (S1P1) modulators to dampen inflammation and its sequelae is becoming increasingly promising for treating medical conditions characterized by significant immunopathology. As shown by the non-selective S1P receptor modulator FTY720 (fingolimod [Gilenya®]) in the treatment of relapsing-remitting multiple sclerosis (MS), the ability to use S1P1 modulation to precisely block immune cell traffic—immunomodulation—while maintaining immunosurveillance, has opened therapeutic opportunities in various other immune-derived chronic pathologies, including inflammatory bowel disease (IBD), lupus, psoriasis, as well as, potentially, in early acute viral respiratory infection. Proof-of-concept studies across validated animal models with S1P receptor modulators highly selective for S1P1, such as BAF-312 (Siponimod), KRP-203, ONO-4641 (Ceralifimod), ponesimod and RPC-1063, and emerging clinical trials for safety and efficacy in humans, particularly in MS, ulcerative colitis (UC) and psoriasis, have set the stage for us to consider additional testing in various other autoimmune diseases.

CTGF/CCN2 exerts profibrotic action in myoblasts via the up-regulation of sphingosine kinase-1/S1P3 signaling axis: Implications in the action mechanism of TGFβ

The matricellular protein connective tissue growth factor (CTGF/CCN2) is recognized as key player in the onset of fibrosis in various tissues, including skeletal muscle. In many circumstances, CTGF has been shown to be induced by transforming growth factor beta (TGFβ) and accounting, at least in part, for its biological action. In this study it was verified that in cultured myoblasts CTGF/CCN2 causes their transdifferentiation into myofibroblasts by up-regulating the expression of fibrosis marker proteins α-smooth muscle actin and transgelin. Interestingly, it was also found that the profibrotic effect exerted by CTGF/CCN2 was mediated by the sphingosine kinase (SK)-1/S1P3 signaling axis specifically induced by the treatment with the profibrotic cue. Following CTGF/CCN2-induced up-regulation, S1P3 became the S1P receptor subtype expressed at the highest degree, at least at mRNA level, and was thus capable of readdressing the sphingosine 1-phosphate signaling towards fibrosis rather than myogenic differentiation. Another interesting finding is that CTGF/CCN2 silencing prevented the TGFβ-dependent up-regulation of SK1/S1P3 signaling axis and strongly reduced the profibrotic effect exerted by TGFβ, pointing at a crucial role of endogenous CTGF/CCN2 generated following TGFβ challenge in the transmission of at least part of its profibrotic effect. These results provide new insights into the molecular mechanism by which CTGF/CCN2 drives its biological action and strengthen the concept that SK1/S1P3 axis plays a critical role in the onset of fibrotic cell phenotype.

Fingolimod may support neuroprotection via blockade of astrocyte S1P and cytokine signaling cascades in Multiple Sclerosis

Fingolimod may support neuroprotection via blockade of astrocyte S1P and cytokine signaling cascades in Multiple Sclerosis

Correction: Memo Has a Novel Role in S1P Signaling and Crucial for Vascular Development

Correction: Memo Has a Novel Role in S1P Signaling and Crucial for Vascular Development

Sphingosine-1-phosphate (S1P) displays sustained S1P1 receptor agonism and signaling through S1P lyase-dependent receptor recycling

The sphingosine-1-phosphate (S1P) type 1 receptor (S1P1R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P1 receptor desensitization caused by synthetic S1P1 receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P1 receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P1 receptors. In recombinant S1P1 receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P1 receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P1 receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase – by facilitating S1P1 receptor recycling – is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates.

Targeting S1P1 Receptor Protects against Murine Immunological Hepatic Injury through Myeloid-Derived Suppressor Cells

Although FTY720 may alter migration and homing of lymphocytes via sphingosine-1-phosphate (S1P) receptors, our recent studies indicated that FTY720 directly controls the differentiation of Th1 cells to regulatory T cells (Tregs) by targeting S1P1. However, the pharmacological function of FTY720 in immunological hepatic injury remains unknown. In this study, the role and regulatory signaling pathway of S1P receptor were investigated using a pharmacological approach in immune-mediated hepatic injury (IMH). In the context of IMH, FTY720 significantly ameliorated mortality and hepatic pathology. In FTY720-treated mice, recruited CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs) mediate protection against IMH and are functional suppressive immune modulators that result in fewer IFN-γ-producing Th1 cells and more Foxp3(+) Tregs. In agreement, FTY720-treated MDSCs promote the reciprocal differentiation between Th1 cells and Tregs in vitro and in vivo. Mechanistically, FTY720 treatment induced inducible NO synthase expression and NO production in MDSCs. Pharmacologic inhibition of inducible NO synthase completely eliminates MDSC suppressive function and eradicates their inducible effects on T cell differentiation. Finally, the mTOR inhibitor, rapamycin, photocopies the effects of FTY720 on MDSCs, implicating mTOR as a downstream effector of S1P1 signaling. This study identifies MDSCs as an essential component that provides protection against IMH following FTY720 or rapamycin treatment, validating the S1P1-mTOR signaling axis as a potential therapeutic target in hepatic injury.

Treatment with the Immunomodulator FTY720 (Fingolimod) Significantly Reduces Renal Inflammation in Murine Unilateral Ureteral Obstruction

The S1P signaling pathway represents an important potential target for the modulation of tissue inflammation/injury. The immunomodulator FTY720, also known as fingolimod, is a potent agonist for multiple S1P receptors that was approved by the Food and Drug Administration to treat multiple sclerosis. We examined the therapeutic role of FTY720 for renal injury secondary to unilateral ureteral obstruction. CB57BL/6 mice underwent a sham procedure or unilateral ureteral obstruction and were treated with FTY720 by gavage for 1, 3 and 5 days. Control groups received vehicle. Ligated and unligated renal tissue was examined for histopathological changes, inflammatory and fibrotic markers, TGF-β1, α-SMA, and macrophage infiltration by Western blot and immunohistochemistry. Proinflammatory and profibrotic cytokines were profiled by quantitative reverse transcriptase-polymerase chain reaction. Pathological evaluation revealed that FTY720 treatment resulted in a significant reduction in inflammatory infiltration in obstructed kidneys compared to controls. Immunohistochemical and Western blot showed that TGF-β1 and α-SMA protein levels were similarly decreased, as was macrophage infiltration into the renal interstitial space, compared to untreated mice. In agreement with these observations quantitative reverse transcriptase-polymerase chain reaction revealed that inflammatory and fibrotic cytokines (MCP-1, IL-1β, CXCL1, TNF-α and TGF-β1) were also significantly decreased in the FTY720 group. This study suggests that in a murine ureteral obstruction model FTY720 significantly inhibited the production of inflammatory cytokines and factors regulating interstitial fibrosis and extracellular matrix accumulation. These findings were associated with decreased evidence of renal injury on pathological examination, suggesting that FTY720 or related compounds may be valuable modulators of obstruction induced renal injury.

MiRNA-95 Mediates Radioresistance in Tumors by Targeting the Sphingolipid Phosphatase SGPP1

Radiation resistance poses a major clinical challenge in cancer treatment, but little is known about how microRNA (miR) may regulate this phenomenon. In this study, we used next-generation sequencing to perform an unbiased comparison of miR expression in PC3 prostate cancer cells rendered resistant to fractionated radiation treatment. One miR candidate found to be upregulated by ionizing radiation was miR-95, the enforced expression of which promoted radiation resistance in a variety of cancer cells. miR-95 overexpression recapitulated an aggressive phenotype including increased cellular proliferation, deregulated G2-M checkpoint following ionizing radiation, and increased invasive potential. Using combined in silico prediction and microarray expression analyses, we identified and validated the sphingolipid phosphatase SGPP1, an antagonist of sphingosine-1-phosphate signaling, as a target of miR-95 that promotes radiation resistance. Consistent with this finding, cell treatment with FTY720, a clinically approved small molecule inhibitor of S1P signaling, sensitized miR-95 overexpressing cells to radiation treatment. In vivo assays extended the significance of these results, showing that miR-95 overexpression increased tumor growth and resistance to radiation treatment in tumor xenografts. Furthermore, reduced tumor necrosis and increased cellular proliferation were seen after radiation treatment of miR-95 overexpressing tumors compared with control tumors. Finally, miR-95 expression was increased in human prostate and breast cancer specimens compared with normal tissue. Together, our work reveals miR-95 expression as a critical determinant of radiation resistance in cancer cells.

Dynamic Cross Talk between S1P and CXCL12 Regulates Hematopoietic Stem Cells Migration, Development and Bone Remodeling

Hematopoietic stem cells (HSCs) are mostly retained in a quiescent non-motile mode in their bone marrow (BM) niches, shifting to a migratory cycling and differentiating state to replenish the blood with mature leukocytes on demand. The balance between the major chemo-attractants CXCL12, predominantly in the BM, and S1P, mainly in the blood, dynamically regulates HSC recruitment to the circulation versus their retention in the BM. During alarm situations, stress-signals induce a decrease in CXCL12 levels in the BM, while S1P levels are rapidly and transiently increased in the circulation, thus favoring mobilization of stem cells as part of host defense and repair mechanisms. Myeloid cytokines, including G-CSF, up-regulate S1P signaling in the BM via the PI3K pathway. Induced CXCL12 secretion from stromal cells via reactive oxygen species (ROS) generation and increased S1P1 expression and ROS signaling in HSCs, all facilitate mobilization. Bone turnover is also modulated by both CXCL12 and S1P, regulating the dynamic BM stromal microenvironment, osteoclasts and stem cell niches which all functionally express CXCL12 and S1P receptors. Overall, CXCL12 and S1P levels in the BM and circulation are synchronized to mutually control HSC motility, leukocyte production and osteoclast/osteoblast bone turnover during homeostasis and stress situations.

Identification of a novel cytokine amplification loop during influenza infection (P6142)

Abstract Pathogenic influenza viruses cause significant morbidity and mortality in humans. Influenza virus causes lung tissue damage by direct pulmonary epithelial injury and by its initiation of an aggressive innate inflammatory response. The sphingosine 1-phosphate (S1P) signaling system is well known for its role in modulation of immune cell trafficking. Using genetic and chemical tools to probe functions of the S1P1 receptor (S1P1-rec), we recently published that pulmonary endothelial cells (ECs) are central modulators of innate immune cell recruitment and cytokine responses early following influenza virus infection. Use of S1P1-rec agonist results in reduced influenza morbidity and mortality. While S1P1 receptor is found on ECs and lymphocytes within lung tissue, an S1P1-selective agonist suppressed cytokines and innate immune cell recruitment in wild-type and lymphocyte-deficient mice, clearly identifying pulmonary ECs as central regulators of cytokine storm. Now, we report a novel cytokine amplification loop requiring cellular cross-talk between pulmonary epithelial cells, ECs and plasmacytoid dendritic cells (pDCs). Cytokine amplification through this loop requires MyD88 and IRF7 signaling. Importantly, S1P1 signaling on ECs elicits a negative signal to pDCs leading to global suppression of cytokine amplification. These findings identify therapeutic control points to blunt pathology associated with influenza virus infection and further illuminate influenza pathogenesis.

Angiotensin II Drives the Production of Tumor-Promoting Macrophages

Macrophages frequently infiltrate tumors and can enhance cancer growth, yet the origins of the macrophage response are not well understood. Here we address molecular mechanisms of macrophage production in a conditional mouse model of lung adenocarcinoma. We report that overproduction of the peptide hormone Angiotensin II (AngII) in tumor-bearing mice amplifies self-renewing hematopoietic stem cells (HSCs) and macrophage progenitors. The process occurred in the spleen but not the bone marrow, and was independent of hemodynamic changes. The effects of AngII required direct hormone ligation on HSCs, depended on S1P(1) signaling, and allowed the extramedullary tissue tosupply new tumor-associated macrophages throughout cancer progression. Conversely, blocking AngII production prevented cancer-induced HSC and macrophage progenitor amplification and thus restrained the macrophage response at its source. These findings indicate that AngII acts upstream of a potent macrophage amplification program and that tumors can remotely exploit the hormone's pathway to stimulate cancer-promoting immunity.

Mrp1 is Essential for Sphingolipid Signaling to P-Glycoprotein in Mouse Blood–Brain and Blood–Spinal Cord Barriers

At the blood-brain and blood-spinal cord barriers, P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to central nervous system (CNS) pharmacotherapy. Recently, we showed that signaling through tumor necrosis factor-α (TNF-α), sphingolipids, and sphingosine-1-phosphate receptor 1 (S1PR1) rapidly and reversibly reduced basal P-glycoprotein transport activity in the rat blood-brain barrier. The present study extends those findings to the mouse blood-brain and blood-spinal cord barriers and, importantly, identifies multidrug resistance-associated protein 1 (Mrp1, Abcc1) as the transporter that mediates S1P efflux from brain and spinal cord endothelial cells. In brain and spinal cord capillaries isolated from wild-type mice, TNF-α, sphingosine, S1P, the S1PR agonist fingolimod (FTY720), and its active, phosphorylated metabolite, FTY720P, reduced P-glycoprotein transport activity; these effects were abolished by a specific S1PR1 antagonist. In brain and spinal cord capillaries isolated from Mrp1-null mice, neither TNF-α nor sphingosine nor FTY720 reduced P-glycoprotein transport activity. However, S1P and FTY720P had the same S1PR1-dependent effects on transport activity as in capillaries from wild-type mice. Thus, deletion of Mrp1 alone terminated endogenous signaling to S1PR1. These results identify Mrp1 as the transporter essential for S1P efflux from the endothelial cells and thus for inside-out S1P signaling to P-glycoprotein at the blood-brain and blood-spinal cord barriers.

Antagonism of the S1P Signaling Pathway Affects Placental and Fetal Development.

Antagonism of the S1P Signaling Pathway Affects Placental and Fetal Development.