RNA profiling of laser microdissected human trophoblast subtypes at mid-gestation reveals a role for cannabinoid signaling in invasion.

Human placental architecture is complex. Its surface epithelium, specialized for transport, forms by fusion of cytotrophoblast progenitors into multinucleated syncytiotrophoblasts. Near the uterine surface, these progenitors assume a different fate, becoming cancer-like cells that invade its lining and blood vessels. The latter process physically connects the placenta to the mother and shunts uterine blood to the syncytiotrophoblasts. Isolation of trophoblast subtypes is technically challenging. Upon removal, syncytiotrophoblasts disintegrate and invasive cytotrophoblasts are admixed with uterine cells. We used laser capture to circumvent these obstacles. This enabled isolation of syncytiotrophoblasts and two subpopulations of invasive cytotrophoblasts from cell columns and the endovascular compartment of spiral arteries. Transcriptional profiling revealed numerous genes, the placental or trophoblast expression of which was not known, including neurotensin and C4ORF36. Using mass spectrometry, discovery of differentially expressed mRNAs was extended to the protein level. We also found that invasive cytotrophoblasts expressed cannabinoid receptor 1. Unexpectedly, screening agonists and antagonists showed that signals from this receptor promote invasion. Together, these results revealed previously unseen gene expression patterns that translate to the protein level. Our data also suggested that endogenous and exogenous cannabinoids can affect human placental development.

Gravidity-dependent associations between interferon response and birth weight in placental malaria.

BACKGROUND: Maternal malarial infection leads to poor perinatal outcomes, including low birth weight from preterm delivery and/or fetal growth restriction, particularly in primigravidas. In placental malaria, Plasmodium falciparum-infected red blood cells cause an inflammatory response that can interfere with maternal-fetal exchange, leading to poor growth. The type I interferon (IFN-I) pathway plays an immunomodulatory role in viral and bacterial infections, usually by suppressing inflammatory responses. However, its role in placental malaria is unknown. This study examines the cytokine responses in placental tissue from subsets of malaria-infected and uninfected women, and attempts to correlate them with particular birth outcomes. METHODS: 40 whole placental biopsy samples were obtained from pregnant women at least 16 years of age recruited to a larger prospective chemoprevention trial against malaria. These were patients at Tororo District Hospital in Uganda, an area of high malaria endemicity where approximately 40% of women have evidence of malaria infection at delivery. They were regularly followed at a local clinic and monitored for fever, with blood smears performed then and at time of delivery to diagnose malaria infection. Placenta biopsies were taken for histological diagnosis of placental malaria, as well as quantitative PCR analysis of genes in the IFN-I pathway (IFN-ß, IL-10 and MX-1). Parameters such as infant birth weight and gestational age were also recorded. RESULTS: Histological analysis revealed placental malaria in 18 samples, while 22 were found to be uninfected. RT-PCR analysis showed a four-fold increase in IFN-ß and IL-10 expression in multigravidas with placental malaria when compared to gravidity-matched, uninfected controls. This effect was not observed in primigravidas. Interestingly, linear regression analysis showed a positive association between IFN-ß levels and higher birth weights (ß = 101.2 g per log2-fold increase in IFN-ß expression, p = 0.042). This association was strongest in primigravidas with placental malaria (ß = 339.0, p = 0.006). CONCLUSIONS: These results demonstrate differential regulation of the IFN-I pathway in placental malaria according to gravidity, with the greatest anti-inflammatory response seen in multigravidas. The association between IFN-ß levels and higher birth weight also suggests a protective role for IFN-I against fetal growth restriction in placental malaria.

Cytotrophoblast extracellular vesicles enhance decidual cell secretion of immune modulators via TNFα.

The placenta releases large quantities of extracellular vesicles (EVs) that likely facilitate communication between the embryo/fetus and the mother. We isolated EVs from second trimester human cytotrophoblasts (CTBs) by differential ultracentrifugation and characterized them using transmission electron microscopy, immunoblotting and mass spectrometry. The 100,000  g pellet was enriched for vesicles with a cup-like morphology typical of exosomes. They expressed markers specific to this vesicle type, CD9 and HRS, and the trophoblast proteins placental alkaline phosphatase and HLA-G. Global profiling by mass spectrometry showed that placental EVs were enriched for proteins that function in transport and viral processes. A cytokine array revealed that the CTB 100,000  g pellet contained a significant amount of tumor necrosis factor α (TNFα). CTB EVs increased decidual stromal cell (dESF) transcription and secretion of NF-κB targets, including IL8, as measured by qRT-PCR and cytokine array. A soluble form of the TNFα receptor inhibited the ability of CTB 100,000  g EVs to increase dESF secretion of IL8. Overall, the data suggest that CTB EVs enhance decidual cell release of inflammatory cytokines, which we theorize is an important component of successful pregnancy.

Racial/ethnic and geographic differences in polybrominated diphenyl ether (PBDE) levels across maternal, placental, and fetal tissues during mid-gestation.

Prenatal polybrominated diphenyl ether (PBDE) exposures are a public health concern due to their persistence and potential for reproductive and developmental harm. However, we have little information about the extent of fetal exposures during critical developmental periods and the variation in exposures for groups that may be more highly exposed, such as communities of color and lower socioeconomic status (SES). To characterize maternal-fetal PBDE exposures among potentially vulnerable groups, PBDE levels were examined in the largest sample of matched maternal serum, placenta, and fetal liver tissues during mid-gestation among a geographically, racially/ethnically, and socially diverse population of pregnant women from Northern California and the Central Valley (n = 180; 2014-16). Maternal-fetal PBDE levels were compared to population characteristics using censored Kendall's tau correlation and linear regression. PBDEs were commonly detected in all biomatrices. Before lipid adjustment, wet-weight levels of all four PBDE congeners were highest in the fetal liver (p < 0.001), whereas median PBDE levels were significantly higher in maternal serum than in the fetal liver or placenta after lipid-adjustment (p < 0.001). We also found evidence of racial/ethnic disparities in PBDE exposures (Non-Hispanic Black > Latina/Hispanic > Non-Hispanic White > Asian/Pacific Islander/Other; p < 0.01), with higher levels of BDE-100 and BDE-153 among non-Hispanic Black women compared to the referent group (Latina/Hispanic women). In addition, participants living in Fresno/South Central Valley had 34% (95% CI: - 2.4 to 84%, p = 0.07) higher wet-weight levels of BDE-47 than residents living in the San Francisco Bay Area. PBDEs are widely detected and differentially distributed in maternal-fetal compartments. Non-Hispanic Black pregnant women and women from Southern Central Valley geographical populations may be more highly exposed to PBDEs. Further research is needed to identify sources that may be contributing to differential exposures and associated health risks among these vulnerable populations.

Association of polybrominated diphenyl ether (PBDE) levels with biomarkers of placental development and disease during mid-gestation.

BACKGROUND: Polybrominated diphenyl ether (PBDE) exposures have been associated with adverse pregnancy outcomes. A hypothesized mechanism is via alterations in placental development and function. However, we lack biomarkers that can be used as early indicators of maternal/fetal response to PBDE exposures and/or perturbations in placental development or function. METHODS: To evaluate the relationship between PBDE levels and placental biomarkers during mid-gestation of human pregnancy (n = 62), we immunolocalized three molecules that play key roles in cytotrophoblast (CTB) differentiation and interstitial/endovascular uterine invasion-integrin alpha-1 (ITGA1), vascular endothelial-cadherin (CDH5), and metalloproteinase-1 (MMP1)-and assessed three morphological parameters as potential indicators of pathological alterations using H&E-stained tissues-leukocyte infiltration, fibrinoid deposition, and CTB endovascular invasion. We evaluated associations between placental PBDE levels and of biomarkers of placental development and disease using censored Kendall's tau correlation and linear regression methods. RESULTS: PBDEs were detected in all placental samples. We observed substantial variation in antigen expression and morphological endpoints across placental regions. We observed an association between PBDE concentrations and immunoreactivity of endovascular CTB staining with anti-ITGA1 (inverse) or interstitial CTBs staining with anti-CDH5 (positive). CONCLUSIONS: We found several molecular markers that may be sensitive placental indicators of PBDE exposure. Further, this indicates that placental biomarkers of development and disease could be useful barometers of exposure to PBDEs, a paradigm that could be extended to other environmental chemicals and placental stage-specific antigens.

Up-regulated cytotrophoblast DOCK4 contributes to over-invasion in placenta accreta spectrum.

In humans, a subset of placental cytotrophoblasts (CTBs) invades the uterus and its vasculature, anchoring the pregnancy and ensuring adequate blood flow to the fetus. Appropriate depth is critical. Shallow invasion increases the risk of pregnancy complications, e.g., severe preeclampsia. Overly deep invasion, the hallmark of placenta accreta spectrum (PAS), increases the risk of preterm delivery, hemorrhage, and death. Previously a rare condition, the incidence of PAS has increased to 1:731 pregnancies, likely due to the rise in uterine surgeries (e.g., Cesarean sections). CTBs track along scars deep into the myometrium and beyond. Here we compared the global gene expression patterns of CTBs from PAS cases to gestational age-matched control cells that invaded to the normal depth from preterm birth (PTB) deliveries. The messenger RNA (mRNA) encoding the guanine nucleotide exchange factor, DOCK4, mutations of which promote cancer cell invasion and angiogenesis, was the most highly up-regulated molecule in PAS samples. Overexpression of DOCK4 increased CTB invasiveness, consistent with the PAS phenotype. Also, this analysis identified other genes with significantly altered expression in this disorder, potential biomarkers. These data suggest that CTBs from PAS cases up-regulate a cancer-like proinvasion mechanism, suggesting molecular as well as phenotypic similarities in the two pathologies.

Trisomy 21 is Associated with Caspase-2 Upregulation in Cytotrophoblasts at the Maternal-Fetal Interface.

Impaired placentation is implicated in poor perinatal outcomes associated with Trisomy 21. Earlier studies revealed abnormal cytotrophoblast differentiation along the invasive pathway as a contributing mechanism. To further elucidate the causes, we evaluated Caspase-2 expression at the protein level (immunolocalization and immunoblot) in samples from Trisomy 21 (n = 9) and euploid (n = 4) age-matched placentas. Apoptosis was investigated via the TUNEL assay. An immunolocalization approach was used to characterize Caspase-3, Fas (CD95), and Fas ligand in the same samples. Caspase-2 was significantly overexpressed in Trisomy 21 placentas, with the highest expression in villous cores and invasive cytotrophoblasts. Immunolocalization showed that Caspase-3 had a similar expression pattern as Caspase-2. Using the TUNEL approach, we observed high variability in the number of apoptotic cells in biopsies from different regions of the same placenta and among different placentas. However, Trisomy 21 placentas had more apoptotic cells, specifically in cell columns and basal plates. Furthermore, Caspase-2 co-immunolocalized with Fas (CD95) and FasL in TUNEL-positive extravillous cytotrophoblasts, but not in villous cores. These results help explain the higher levels of apoptosis among placental cells of Trisomy 21 pregnancies in molecular terms. Specifically, the co-expression of Caspase-2 and Caspase-3 with other regulators of the apoptotic process in TUNEL-positive cells suggests these molecules may cooperate in launching the observed apoptosis. Among trophoblasts, only the invasive subpopulation showed this pattern, which could help explain the higher rates of adverse outcomes in these pregnancies. In future experiments, this relationship will be further examined at a functional level in cultured human trophoblasts.

Defective decidualization during and after severe preeclampsia reveals a possible maternal contribution to the etiology.

In preeclampsia (PE), cytotrophoblast (CTB) invasion of the uterus and spiral arteries is often shallow. Thus, the placenta's role has been a focus. In this study, we tested the hypothesis that decidual defects are an important determinant of the placental phenotype. We isolated human endometrial stromal cells from nonpregnant donors with a previous pregnancy that was complicated by severe PE (sPE). Compared with control cells, they failed to decidualize in vitro as demonstrated by morphological criteria and the analysis of stage-specific antigens (i.e., IGFBP1, PRL). These results were bolstered by global transcriptional profiling data that showed they were transcriptionally inert. Additionally, we used laser microdissection to isolate the decidua from tissue sections of the maternal-fetal interface in sPE. Global transcriptional profiling revealed defects in gene expression. Also, decidual cells from patients with sPE, which dedifferentiated in vitro, failed to redecidualize in culture. Conditioned medium from these cells failed to support CTB invasion. To mimic aspects of the uterine environment in normal pregnancy, we added PRL and IGFBP1, which enhanced invasion. These data suggested that failed decidualization is an important contributor to down-regulated CTB invasion in sPE. Future studies will be aimed at determining whether this discovery has translational potential with regard to assessing a woman's risk of developing this pregnancy complication.

The human chorion contains definitive hematopoietic stem cells from the fifteenth week of gestation.

We examined the contribution of the fetal membranes, amnion and chorion, to human embryonic and fetal hematopoiesis. A population of cells displaying a hematopoietic progenitor phenotype (CD34++ CD45low) of fetal origin was present in the chorion at all gestational ages, associated with stromal cells or near blood vessels, but was absent in the amnion. Prior to 15 weeks of gestation, these cells lacked hematopoietic in vivo engraftment potential. Differences in the chemokine receptor and ß1 integrin expression profiles of progenitors between the first and second trimesters suggest that these cells had gestationally regulated responses to homing signals and/or adhesion mechanisms that influenced their ability to colonize the stem cell niche. Definitive hematopoietic stem cells, capable of multilineage and long-term reconstitution when transplanted in immunodeficient mice, were present in the chorion from 15-24 weeks gestation, but were absent at term. The second trimester cells also engrafted secondary recipients in serial transplantation experiments. Thus, the human chorion contains functionally mature hematopoietic stem cells at mid-gestation.

Preeclampsia: novel insights from global RNA profiling of trophoblast subpopulations.

BACKGROUND: The maternal signs of preeclampsia, which include the new onset of high blood pressure, can occur because of faulty placentation. We theorized that transcriptomic analyses of trophoblast subpopulations in situ would lend new insights into the role of these cells in preeclampsia pathogenesis. OBJECTIVE: Our goal was to enrich syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts from the placentas of severe preeclampsia cases. Total RNA was subjected to global transcriptional profiling to identify RNAs that were misexpressed compared with controls. STUDY DESIGN: This was a cross-sectional analysis of placentas from women who had been diagnosed with severe preeclampsia. Gestational age-matched controls were placentas from women who had a preterm birth with no signs of infection. Laser microdissection enabled enrichment of syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts. After RNA isolation, a microarray approach was used for global transcriptional profiling. Immunolocalization identified changes in messenger RNA expression that carried over to the protein level. Differential expression of non-protein-coding RNAs was confirmed by in situ hybridization. A 2-way analysis of variance of non-coding RNA expression identified particular classes that distinguished trophoblasts in cases vs controls. Cajal body foci were visualized by coilin immunolocalization. RESULTS: Comparison of the trophoblast subtype data within each group (severe preeclampsia or noninfected preterm birth) identified many highly differentially expressed genes. They included molecules that are known to be expressed by each subpopulation, which is evidence that the method worked. Genes that were expressed differentially between the 2 groups, in a cell-type-specific manner, encoded a combination of molecules that previous studies associated with severe preeclampsia and those that were not known to be dysregulated in this pregnancy complication. Gene ontology analysis of the syncytiotrophoblast data highlighted the dysregulation of immune functions, morphogenesis, transport, and responses to vascular endothelial growth factor and progesterone. The invasive cytotrophoblast data provided evidence of alterations in cellular movement, which is consistent with the shallow invasion often associated with severe preeclampsia. Other dysregulated pathways included immune, lipid, oxygen, and transforming growth factor-beta responses. The data for endovascular cytotrophoblasts showed disordered metabolism, signaling, and vascular development. Additionally, the transcriptional data revealed the differential expression in severe preeclampsia of 2 classes of non-coding RNAs: long non-coding RNAs and small nucleolar RNAs. The long non-coding RNA, urothelial cancer associated 1, was the most highly up-regulated in this class. In situ hybridization confirmed severe preeclampsia-associated expression in syncytiotrophoblasts. The small nucleolar RNAs, which chemically modify RNA structure, also correlated with severe preeclampsia. Thus, we enumerated Cajal body foci, sites of small nucleolar RNA activity, in primary cytotrophoblasts that were isolated from control and severe preeclampsia placentas. In severe preeclampsia, cytotrophoblasts had approximately double the number of these foci as the control samples. CONCLUSION: A laser microdissection approach enabled the identification of novel messenger RNAs and non-coding RNAs that were misexpressed by various trophoblast subpopulations in severe preeclampsia. The results suggested new avenues of investigation, in particular, the roles of PRG2, Kell blood group determinants, and urothelial cancer associated 1 in syncytiotrophoblast diseases. Additionally, many of the newly identified dysregulated molecules might have clinical utility as biomarkers of severe preeclampsia.

Preeclampsia and Inflammatory Preterm Labor Alter the Human Placental Hematopoietic Niche.

BACKGROUND: The human placenta is a source of hematopoietic stem and progenitor cells (HSPCs). The RUNX1 transcription factor is required for the formation of functional HSPCs. The impact of preeclampsia (PE) and preterm labor (PTL, spontaneous preterm labor [sPTL] and inflammatory preterm labor [iPTL]) on HSPC localization and RUNX1 expression in the human placenta is unknown. METHODS: We compared the frequency and density of HSPC in control samples from sPTL (n = 6) versus PE (n = 6) and iPTL (n = 6). We examined RUNX1 protein and RNA expression in placentas from normal pregnancies (5-22 weeks, n = 8 total) and in placentas from the aforementioned pregnancy complications (n = 5/group). RESULTS: Hematopoietic stem and progenitor cells were rare cell types, associated predominantly with the vasculature of placental villi. The HSPC density was greater in the chorionic plate (CP) compared to the villi (P < .001) and greater in PE and iPTL samples as compared to controls within the CP (not significant) and overall (P < .05). During the fetal period, RUNX1 was expressed in the mesenchyme of the CP and villi. Inflammatory PTL samples were more likely to exhibit intraluminal RUNX1(+) cell populations (P < .001) and RUNX1(+) cell clusters attached to arterial endothelial cells. CONCLUSION: Placental HSPCs likely arise from hematopoietic niches comprised RUNX1(+) mesenchyme and vascular endothelium. Pregnancy complications that result in preterm birth differentially affect placental HSPC localization and RUNX1 expression. Our results support previous findings that inflammation positively regulates hematopoiesis. We present new evidence that hemogenic endothelium may be active at later stages of human fetal development in the context of inflammation.

Quantitative proteomic analyses of mammary organoids reveals distinct signatures after exposure to environmental chemicals.

Common environmental contaminants such as bisphenols and phthalates and persistent contaminants such as polychlorinated biphenyls are thought to influence tissue homeostasis and carcinogenesis by acting as disrupters of endocrine function. In this study we investigated the direct effects of exposure to bisphenol A (BPA), mono-n-butyl phthalate (Pht), and polychlorinated biphenyl 153 (PCB153) on the proteome of primary organotypic cultures of the mouse mammary gland. At low-nanomolar doses each of these agents induced distinct effects on the proteomes of these cultures. Although BPA treatment produced effects that were similar to those induced by estradiol, there were some notable differences, including a reduction in the abundance of retinoblastoma-associated protein and increases in the Rho GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle protein CDC42. Both Pht and PCB153 induced changes that were distinct from those induced by estrogen, including decreased levels of the transcriptional corepressor C-terminal binding protein 1. Interestingly, the three chemicals appeared to alter the abundance of distinct splice forms of many proteins as well as the abundance of several proteins that regulate RNA splicing. Our combined results indicate that the three classes of chemical have distinct effects on the proteome of normal mouse mammary cultures, some estrogen-like but most estrogen independent, that influence diverse biological processes including apoptosis, cell adhesion, and proliferation.

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma.

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Stromal cell derived factor-2 (Sdf2): a novel protein expressed in mouse.

The stromal derived factor (SDFs) family comprises a group of molecules generated by stromal cells. SDF1 and SDF4 are chemokines; SDF2 and SDF5 are not yet functionally and structurally defined. In human and mouse, Sdf2 has a paralogous gene, Sdf2l1, whose protein sequences are 78% similar and 68% identical. Human SDF2L1 is an endoplasmic reticulum-stress inducible-gene. In Arabidopsis thaliana, SDF2-like (39% and 37% amino acid sequence identity with Mus musculus Sdf2 and Sdf2l1) has also been implicated in activating the UPR in ER-stress. Here we have cloned, expressed and purified recombinant Sdf2 and raised an anti-Sdf2 antibody. We demonstrated that the protein is expressed in several tissues and is localized in the endoplasmic reticulum. We suggest that Sdf2, initially predicted as a secretory protein because it lacks the canonical ER retention signals in its C-terminal, could be ER-resident through accessory binding proteins or other amino acid sequence motifs, as suggested for the homolog protein SDF2-like. Furthermore, the crystal structure of SDF2-like from Arabidopsis thaliana is a typical ß-trefoil containing three MIR motifs; all hydrophobic residues considered important for maintaining the bottom layer of the ß-trefoil barrel seem to be conserved in the Sdf2 family. Multiple alignment using 43 sequences for SDF2 and 38 for SDF2L1 paralogous families also revealed a very similar residue conservation profile. Comparing the amino acid sequence and predicted 3D structure with other Sdf2-like proteins we suggest a role of mouse Sdf2 in the Unfolded Protein Response and ER-stress, similar to that of Sdf2l1 from human and mouse and SDF2-like from Arabidopsis thaliana. Chronic ER stress has been associated with many human diseases including cancer and diabetes. Identification of new factors associated with the ER stress pathway can help to identify and define key targets of this response.

Variation and quantification among a target set of phosphopeptides in human plasma by multiple reaction monitoring and SWATH-MS2 data-independent acquisition.

Human plasma contains proteins that reflect overall health and represents a rich source of proteins for identifying and understanding disease pathophysiology. However, few studies have investigated changes in plasma phosphoproteins. In addition, little is known about the normal variations in these phosphoproteins, especially with respect to specific sites of modification. To address these questions, we evaluated variability in plasma protein phosphorylation in healthy individuals using multiple reaction monitoring (MRM) and SWATH-MS2 data-independent acquisition. First, we developed a discovery workflow for phosphopeptide enrichment from plasma and identified targets for MRM assays. Next, we analyzed plasma from healthy donors using an analytical workflow consisting of MRM and SWATH-MS2 that targeted phosphopeptides from 58 and 68 phosphoproteins, respectively. These two methods produced similar results showing low variability in 13 phosphosites from 10 phosphoproteins (CVinter < 30%) and high interpersonal variation of 16 phosphosites from 14 phosphoproteins (CVinter > 30%). Moreover, these phosphopeptides originate from phosphoproteins involved in cellular processes governing homeostasis, immune response, cell-extracellular matrix interactions, lipid and sugar metabolism, and cell signaling. This limited assessment of technical and biological variability in phosphopeptides generated from plasma phosphoproteins among healthy volunteers constitutes a reference for future studies that target protein phosphorylation as biomarkers.

Phosphoprotein secretome of tumor cells as a source of candidates for breast cancer biomarkers in plasma.

Breast cancer is a heterogeneous disease whose molecular diversity is not well reflected in clinical and pathological markers used for prognosis and treatment selection. As tumor cells secrete proteins into the extracellular environment, some of these proteins reach circulation and could become suitable biomarkers for improving diagnosis or monitoring response to treatment. As many signaling pathways and interaction networks are altered in cancerous tissues by protein phosphorylation, changes in the secretory phosphoproteome of cancer tissues could reflect both disease progression and subtype. To test this hypothesis, we compared the phosphopeptide-enriched fractions obtained from proteins secreted into conditioned media (CM) derived from five luminal and five basal type breast cancer cell lines using label-free quantitative mass spectrometry. Altogether over 5000 phosphosites derived from 1756 phosphoproteins were identified, several of which have the potential to qualify as phosphopeptide plasma biomarker candidates for the more aggressive basal and also the luminal-type breast cancers. The analysis of phosphopeptides from breast cancer patient plasma and controls allowed us to construct a discovery list of phosphosites under rigorous collection conditions, and second to qualify discovery candidates generated from the CM studies. Indeed, a set of basal-specific phosphorylation CM site candidates derived from IBP3, CD44, OPN, FSTL3, LAMB1, and STC2, and luminal-specific candidates derived from CYTC and IBP5 were selected and, based on their presence in plasma, quantified across all cell line CM samples using Skyline MS1 intensity data. Together, this approach allowed us to assemble a set of novel cancer subtype specific phosphopeptide candidates for subsequent biomarker verification and clinical validation.

Reversal of gene dysregulation in cultured cytotrophoblasts reveals possible causes of preeclampsia.

During human pregnancy, a subset of placental cytotrophoblasts (CTBs) differentiates into cells that aggressively invade the uterus and its vasculature, anchoring the progeny and rerouting maternal blood to the placenta. In preeclampsia (PE), CTB invasion is limited, reducing placental perfusion and/or creating intermittent flow. This syndrome, affecting 4%-8% of pregnancies, entails maternal vascular alterations (e.g., high blood pressure, proteinuria, and edema) and, in some patients, fetal growth restriction. The only cure is removal of the faulty placenta, i.e., delivery. Previously, we showed that defective CTB differentiation contributes to the placental component of PE, but the causes were unknown. Here, we cultured CTBs isolated from PE and control placentas for 48 hours, enabling differentiation and invasion. In various severe forms of PE, transcriptomics revealed common aberrations in CTB gene expression immediately after isolation, including upregulation of SEMA3B, which resolved in culture. The addition of SEMA3B to normal CTBs inhibited invasion and recreated aspects of the PE phenotype. Additionally, SEMA3B downregulated VEGF signaling through the PI3K/AKT and GSK3 pathways, effects that were observed in PE CTBs. We propose that, in severe PE, the in vivo environment dysregulates CTB gene expression; the autocrine actions of the upregulated molecules (including SEMA3B) impair CTB differentiation, invasion and signaling; and patient-specific factors determine the signs.

Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS).

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

Protein composition of bronchoalveolar lavage fluid and airway surface liquid from newborn pigs.

The airway mucosa and the alveolar surface form dynamic interfaces between the lung and the external environment. The epithelial cells lining these barriers elaborate a thin liquid layer containing secreted peptides and proteins that contribute to host defense and other functions. The goal of this study was to develop and apply methods to define the proteome of porcine lung lining liquid, in part, by leveraging the wealth of information in the Sus scrofa database of Ensembl gene, transcript, and protein model predictions. We developed an optimized workflow for detection of secreted proteins in porcine bronchoalveolar lavage (BAL) fluid and in methacholine-induced tracheal secretions [airway surface liquid (ASL)]. We detected 674 and 3,858 unique porcine-specific proteins in BAL and ASL, respectively. This proteome was composed of proteins representing a diverse range of molecular classes and biological processes, including host defense, molecular transport, cell communication, cytoskeletal, and metabolic functions. Specifically, we detected a significant number of secreted proteins with known or predicted roles in innate and adaptive immunity, microbial killing, or other aspects of host defense. In greatly expanding the known proteome of the lung lining fluid in the pig, this study provides a valuable resource for future studies using this important animal model of pulmonary physiology and disease.

Maternal decidual macrophages inhibit NK cell killing of invasive cytotrophoblasts during human pregnancy.

Human pregnancy is an immunological paradox. Semiallogeneic (fetal) placental cells (extravillous cytotrophoblasts [CTBs]) invade the uterine lining (decidua), which contains a unique decidual natural killer (dNK) cell population, identified by the cell surface phenotype CD56(bright) CD16(-) CD3(-) and CD14(+) CD206(+) macrophages (dMac). Previous reports suggested that human dNK cells are not a threat to the fetoplacental unit because they are anergic. In contrast, here we showed that purified and exogenously stimulated dNK cells are capable killers of cellular targets, including semiallogeneic CTBs. However, dMacs in the decidual leukocyte (DL) population restrained dNK killing through a transforming growth factor beta1 (TGF-beta1)-dependent mechanism. Our findings support a new model whereby dNK cells, capable of killing CTBs, are prevented from doing so by neighboring macrophages, thus protecting the fetal cells from NK cell attack. We speculate that this mechanism would inhibit dNK cell-mediated killing, even under conditions where high levels of cytokines may stimulate dNK cells, which could pose a threat to the developing placenta.