A Multi-Site Study to Develop and Validate First Trimester, Circulating Microparticle Biomarkers for Tiered Risk Stratification of Spontaneous Preterm Birth in Nulliparas.

BACKGROUND: Despite much research, advances in early prediction of spontaneous preterm birth (sPTB) has been slow. The evolving field of circulating microparticle (CMP) biology may identify novel blood-based, and clinically useful, biomarkers. OBJECTIVE: To test the ability of a previously identified, 7-marker set of CMP-derived proteins from the first trimester of pregnancy, in the form of an in vitro diagnostic multivariate index assay (IVDMIA), to stratify pregnant patients according to their risk for sPTB. STUDY DESIGN: We employed a previously validated set of CMP protein biomarkers, utilizing mass spectrometry assays and a nested case-control design in a subset of participants from the Nulliparous Pregnancy Outcomes Study: Monitoring Mothers-to-be (nuMoM2b). We evaluated these biomarkers in the form of an IVDMIA to predict risk for sPTB at different gestational ages. Plasma samples collected at 9- to 13-weeks' gestation were analyzed. The IVDMIA assigned subjects to one of three sPTB risk categories: low risk (LR), moderate risk (MR), or high risk (HR). Independent validation on a set-aside set confirmed the IVDMIA's performance in risk stratification. RESULTS: Samples from 400 participants from the nuMoM2b cohort were used for the study; of these, 160 delivered <37 weeks and 240 delivered at term. Through Monte Carlo simulation in which the validation results were adjusted based on actual weekly sPTB incidence rates in the nuMoM2b cohort, the IVDMIA stratifications demonstrated statistically significant differences among the risk groups in time-to-event (birth) analysis (p < 0.0001). The incidence-rate adjusted cumulative risks of sPTB at ≤ 32 weeks' gestation were 0.4%, 1.6%, and 7.5%, respectively for the LR, MR, and HR groups, respectively. Compared to the LR group, the corresponding risk ratios (RR) of the IVDMIA assigned MR and HR group were 4.25 (95% CI 2.2 to 7.9) and 19.92 (95% CI 10.4 to 37.4), respectively. CONCLUSION: A first trimester CMP protein biomarker panel can be used to stratify risk for sPTB at different gestational ages. Such a multi-tiered stratification tool could be used to assess risk early in pregnancy to enable timely clinical management and interventions, and, ultimately, to enable the development of tailored care pathways for sPTB prevention.

Circulating microparticle proteins predict pregnancies complicated by placenta accreta spectrum.

Placenta accreta spectrum (PAS) is characterized by abnormal attachment of the placenta to the uterus, and attempts at placental delivery can lead to catastrophic maternal hemorrhage and death. Multidisciplinary delivery planning can significantly improve outcomes; however, current diagnostics are lacking as approximately half of pregnancies with PAS are undiagnosed prior to delivery. This is a nested case-control study of 35 cases and 70 controls with the primary objective of identifying circulating microparticle (CMP) protein panels that identify pregnancies complicated by PAS. Size exclusion chromatography and liquid chromatography with tandem mass spectrometry were used for CMP protein isolation and identification, respectively. A two-step iterative workflow was used to establish putative panels. Using plasma sampled at a median of 26 weeks' gestation, five CMP proteins distinguished PAS from controls with a mean area under the curve (AUC) of 0.83. For a separate sample taken at a median of 35 weeks' gestation, the mean AUC was 0.78. In the second trimester, canonical pathway analyses demonstrate over-representation of processes related to iron homeostasis and erythropoietin signaling. In the third trimester, these analyses revealed abnormal immune function. CMP proteins classify PAS well prior to delivery and have potential to significantly reduce maternal morbidity and mortality.

Vitamin D Assessment Over 48 Weeks in Treatment-Naive HIV Individuals Starting Lopinavir/Ritonavir Monotherapy.

BACKGROUND: Vitamin D deficiency is common in HIV population and has been associated with increased comorbidity risk and poor immunologic status. OBJECTIVE: To evaluate the effect of protease inhibitor lopinavir/ritonavir monotherapy on changes in serum 25-hydroxyvitamin D [25(OH)D] over 48 weeks. METHODS: Thirty-four treatment-naïve HIV individuals initiating lopinavir/ritonavir monotherapy and receiving clinical care from private practice in Houston, Texas, were included. Serum 25-hydroxyvitamin D levels from stored plasma samples collected from IMANI-2 pilot study at both baseline and 48 weeks were analyzed using LC-MS assays. Mean 25(OH)D at baseline and 48 weeks were compared using paired t-tests. Linear regression analysis was used to evaluate factors associated with changes in 25(OH)D. Logistic regression analyses were used to determine the effect of vitamin D status and covariates on CD4 cell count recovery. RESULTS: Mean 25(OH)D was significantly higher at 48 weeks (26.3 ng/mL (SD + 14.9); p=0.0003) compared to baseline (19.8 ng/mL (SD +12.1), with fewer individuals having vitamin D deficiency (41.2%) and severe deficiency (11.8%). Both body mass index and baseline CD4 cell count were significant independent covariates associated with 25(OH)D changes over 48 weeks. Baseline vitamin D status did not affect CD4 cell count recovery. However, in a 24-week multivariate analysis, current tobacco use was significantly associated with a decreased odds of CD4 cell count recovery (AOR 0.106, 95% CI 0.018-0.606; p=0.012). CONCLUSION: Individuals treated with lopinavir/ritonavir monotherapy had significantly higher 25(OH)D after 48 weeks. Current tobacco users had significantly diminished CD4 cell count recovery after starting treatment, warranting further clinical investigation.

Late first trimester circulating microparticle proteins predict the risk of preeclampsia < 35 weeks and suggest phenotypic differences among affected cases.

We hypothesize that first trimester circulating micro particle (CMP) proteins will define preeclampsia risk while identifying clusters of disease subtypes among cases. We performed a nested case-control analysis among women with and without preeclampsia. Cases diagnosed < 34 weeks' gestation were matched to controls. Plasma CMPs were isolated via size exclusion chromatography and analyzed using global proteome profiling based on HRAM mass spectrometry. Logistic models then determined feature selection with best performing models determined by cross-validation. K-means clustering examined cases for phenotypic subtypes and biological pathway enrichment was examined. Our results indicated that the proteins distinguishing cases from controls were enriched in biological pathways involved in blood coagulation, hemostasis and tissue repair. A panel consisting of C1RL, GP1BA, VTNC, and ZA2G demonstrated the best distinguishing performance (AUC of 0.79). Among the cases of preeclampsia, two phenotypic sub clusters distinguished cases; one enriched for platelet degranulation and blood coagulation pathways and the other for complement and immune response-associated pathways (corrected p < 0.001). Significantly, the second of the two clusters demonstrated lower gestational age at delivery (p = 0.049), increased protein excretion (p = 0.01), more extreme laboratory derangement (p < 0.0001) and marginally increased diastolic pressure (p = 0.09). We conclude that CMP-associated proteins at 12 weeks' gestation predict the overall risk of developing early preeclampsia and indicate distinct subtypes of pathophysiology and clinical morbidity.

Validation and Clinical Applications of a Comprehensive Next Generation Sequencing System for Molecular Characterization of Solid Cancer Tissues.

Identification of somatic molecular alterations in primary and metastatic solid tumor specimens can provide critical information regarding tumor biology and its heterogeneity, and enables the detection of molecular markers for clinical personalized treatment assignment. However, the optimal methods and target genes for clinical use are still being in development. Toward this end, we validated a targeted amplification-based NGS panel (Oncomine comprehensive assay v1) on a personal genome machine sequencer for molecular profiling of solid tumors. This panel covers 143 genes, and requires low amounts of DNA (20 ng) and RNA (10 ng). We used 27 FFPE tissue specimens, 10 cell lines, and 24 commercial reference materials to evaluate the performance characteristics of this assay. We also evaluated the performance of the assay on 26 OCT-embedded fresh frozen specimens (OEFF). The assay was found to be highly specific (>99%) and sensitive (>99%), with low false-positive and false-negative rates for single-nucleotide variants, indels, copy number alterations, and gene fusions. Our results indicate that this is a reliable method to determine molecular alterations in both fixed and fresh frozen solid tumor samples, including core needle biopsies.

Circulating microparticle proteins obtained in the late first trimester predict spontaneous preterm birth at less than 35 weeks' gestation: a panel validation with specific characterization by parity.

BACKGROUND: We have previously shown that protein biomarkers associated with circulating microparticles proteins (CMPs) obtained at the end of the first trimester may detect physiologic changes in maternal-fetal interaction such that the risk of spontaneous preterm delivery ≤35 weeks can be stratified. OBJECTIVES: We present here a study extension and validation of the CMP protein multiplex concept using a larger sample set from a multicenter population that allows for model derivation in a training set and characterization in a separate testing set. MATERIALS AND METHODS: Ethylenediaminetetraacetic acid (EDTA) plasma was obtained from 3 established biobanks (Seattle, Boston, and Pittsburgh). Samples were from patients at a median of 10-12 weeks' gestation, and the CMPs were isolated via size-exclusion chromatography followed by protein identification via targeted protein analysis using liquid chromatography-multiple reaction monitoring-mass (LC-MRM) spectrometry. A total of 87 women delivered at ≤35 weeks, and 174 women who delivered at term were matched by maternal age (±2 years) and gestational age at sample draw (±2 weeks). From our prior work, the CMP protein multiplex comprising F13A, FBLN1, IC1, ITIH2, and LCAT was selected for validation. RESULTS: For delivery at ≤35 weeks, the receiver operating characteristic (ROC) curve for a panel of CMP proteins (F13A, FBLN1, IC1, ITIH2, and LCAT) revealed an associated area under the ROC curve (AUC) of 0.74 (95% CI, 0.63-0.81). A separate panel of markers (IC1, LCAT, TRFE, and ITIH4), which stratified risk among mothers with a parity of 0, showed an AUC of 0.77 (95% CI, 0.61-0.90). CONCLUSION: We have identified a set of CMP proteins that provide, at 10-12 weeks gestation, a clinically useful AUC in an independent test population. Furthermore, we determined that parity is pertinent to the diagnostic testing performance of the biomarkers for risk stratification.

Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways.

Klotho is a single-pass transmembrane protein with documented anti-cancer properties. Recent reports have implicated Klotho as an inhibitor of transforming growth factor ß1 induced cell migration in renal fibrosis. Overexpression of epidermal growth factor receptor (EGFR) is known to promote tumor initiation and progression in clear-cell renal cell carcinoma (cRCC). We tested our hypothesis that Klotho inhibits EGF-mediated cell migration in cRCC by interfering with the EGFR signaling complex and mitogen-activated protein kinase (MAPK) pathways. We performed cell adhesion, migration, and biochemical studies in vitro using Caki-1 cell line. In addition, we validated the cell culture studies with expression analysis of six de-identified FFPE tissues from primary and metastatic cRCC patients. Our studies show that Klotho inhibited EGF-induced Caki-1 de-adhesion and decreased spreading on collagen type 1. Klotho also inhibited EGF-induced α2ß1 integrin-dependent cell migration on collagen type 1. To test the involvement of MAPK pathways in EGF-induced Caki-1 cell motility, the cells were pretreated with either SB203580, a specific p38 MAPK inhibitor, or Klotho. SB203580 blocked the EGF-induced Caki-1 cell migration. Klotho had a comparable inhibitory effect. Our FFPE clinical specimens revealed decreased Klotho mRNA expression compared to a control, non-cancer kidney tissue. The decrease in Klotho mRNA levels correlated with increased c-Src expression, while E-Cadherin was relatively reduced in metastatic FFPE specimens where Klotho was least expressed. Taken together, these results suggest that secreted Klotho inhibits EGF-induced pro-migratory cell morphological changes and migration in Caki-1 cells. Our data additionally suggest that decreased Klotho expression may be involved in cRCC metastasis.

Single Nucleotide Polymorphisms and the Central Nervous System: Potential Biomarkers in Identifying Suicide Risk.

The rate of completed suicides continues to rise across nations, cultures, socioeconomic classes, age groups, sexes, military personnel, veterans, and civilians from different backgrounds. Most concerning is the absence of diagnosed mental health disorders in the majority of these cases, per current literature reports. Efforts in the identification and possible prevention of this ultimate, tragic act of self-destruction have been minimally successful. In this article, the authors discuss the possible biological mechanisms including the role for potential markers, single nucleotide polymorphisms (SNPs), and their association with suicidal behaviors.

Morph-X-Select: Morphology-based tissue aptamer selection for ovarian cancer biomarker discovery.

High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering an aptamer affinity reagent and its target biomarker protein. Here, we demonstrate a morphology-based tissue aptamer selection method that enables us to use tissue sections from individual patients and identify high-affinity aptamers and their associated target proteins in a systematic and accurate way. We created a combinatorial DNA aptamer library that has been modified with thiophosphate substitutions of the phosphate ester backbone at selected 5´dA positions for enhanced nuclease resistance and targeting. Based on morphological assessment, we used image-directed laser microdissection (LMD) to dissect regions of interest bound with the thioaptamer (TA) library and further identified target proteins for the selected TAs. We have successfully identified and characterized the lead candidate TA, V5, as a vimentin-specific sequence that has shown specific binding to tumor vasculature of human ovarian tissue and human microvascular endothelial cells. This new Morph-X-Select method allows us to select high-affinity aptamers and their associated target proteins in a specific and accurate way, and could be used for personalized biomarker discovery to improve medical decision-making and to facilitate the development of targeted therapies to achieve more favorable outcomes.

Improving vascular maturation using noncoding RNAs increases antitumor effect of chemotherapy.

Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2-targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.

Acetazolamide sensitive tissue calcification and aging of klotho-hypomorphic mice.

UNLABELLED: Klotho, a protein expressed mainly in the kidney, is required for the inhibitory effect of FGF23 on renal 1,25(OH)2D3 formation. Klotho counteracts vascular calcification and diverse age-related disorders. Klotho-hypomorphic mice (kl/kl) suffer from severe vascular calcification and rapid aging. The calcification is at least in part caused by excessive 1,25(OH)2D3, Ca(2+), and phosphate concentrations in blood, which trigger osteogenic signaling including upregulation of alkaline phosphatase (Alpl). As precipitation of calcium and phosphate is fostered by alkaline pH, extracellular acidosis could counteract tissue calcification. In order to induce acidosis, acetazolamide was added to drinking water (0.8 g/l) of kl/kl and wild-type mice. As a result, acetazolamide treatment of kl/kl mice partially reversed the growth deficit, tripled the life span, almost completely reversed the calcifications in trachea, lung, kidney, stomach, intestine, and vascular tissues, the excessive aortic alkaline phosphatase mRNA levels and the plasma concentrations of osteoprotegerin, osteopontin as well as fetuin-A, without significantly decreasing FGF23, 1,25(OH)2D3, Ca(2+), and phosphate plasma concentrations. In primary human aortic smooth muscle cells, acidotic environment prevented phosphate-induced alkaline phosphatase mRNA expression. The present study reveals a completely novel effect of acetazolamide, i.e., interference with osteoinductive signaling and tissue calcification in kl/kl mice. KEY MESSAGES: Klotho deficient (kl/kl) mice suffer from hyperphosphatemia with dramatic tissue calcification. Acetazolamide (ACM) treatment partially reversed the growth deficit of kl/kl mice. In kl/kl mice, ACM reversed tissue calcification despite continued hyperphosphatemia. ACM tripled the life span of kl/kl mice. In human aortic smooth muscle cells, low extracellular pH prevented osteogenic signaling.

Klotho Regulates 14-3-3ζ Monomerization and Binding to the ASK1 Signaling Complex in Response to Oxidative Stress.

The reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1) signaling complex is a key regulator of p38 MAPK activity, a major modulator of stress-associated with aging disorders. We recently reported that the ratio of free ASK1 to the complex-bound ASK1 is significantly decreased in Klotho-responsive manner and that Klotho-deficient tissues have elevated levels of free ASK1 which coincides with increased oxidative stress. Here, we tested the hypothesis that: 1) covalent interactions exist among three identified proteins constituting the ASK1 signaling complex; 2) in normal unstressed cells the ASK1, 14-3-3ζ and thioredoxin (Trx) proteins simultaneously engage in a tripartite complex formation; 3) Klotho's stabilizing effect on the complex relied solely on 14-3-3ζ expression and its apparent phosphorylation and dimerization changes. To verify the hypothesis, we performed 14-3-3ζ siRNA knock-down experiments in conjunction with cell-based assays to measure ASK1-client protein interactions in the presence and absence of Klotho, and with or without an oxidant such as rotenone. Our results show that Klotho activity induces posttranslational modifications in the complex targeting 14-3-3ζ monomer/dimer changes to effectively protect against ASK1 oxidation and dissociation. This is the first observation implicating all three proteins constituting the ASK1 signaling complex in close proximity.

Klotho Protects Dopaminergic Neuron Oxidant-Induced Degeneration by Modulating ASK1 and p38 MAPK Signaling Pathways.

Klotho transgenic mice exhibit resistance to oxidative stress as measured by their urinal levels of 8-hydroxy-2-deoxyguanosine, albeit this anti-oxidant defense mechanism has not been locally investigated in the brain. Here, we tested the hypothesis that the reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1)/p38 MAPK pathway regulates stress levels in the brain of these mice and showed that: 1) the ratio of free ASK1 to thioredoxin (Trx)-bound ASK1 is relatively lower in the transgenic brain whereas the reverse is true for the Klotho knockout mice; 2) the reduced p38 activation level in the transgene corresponds to higher level of ASK1-bound Trx, while the KO mice showed elevated p38 activation and lower level of-bound Trx; and 3) that 14-3-3ζ is hyper phosphorylated (Ser-58) in the transgene which correlated with increased monomer forms. In addition, we evaluated the in vivo robustness of the protection by challenging the brains of Klotho transgenic mice with a neurotoxin, MPTP and analyzed for residual neuron numbers and integrity in the substantia nigra pars compacta. Our results show that Klotho overexpression significantly protects dopaminergic neurons against oxidative damage, partly by modulating p38 MAPK activation level. Our data highlight the importance of ASK1/p38 MAPK pathway in the brain and identify Klotho as a possible anti-oxidant effector.

CYP2D7 Sequence Variation Interferes with TaqMan CYP2D6 (*) 15 and (*) 35 Genotyping.

TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs) some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false-positive CYP2D6 (*) 15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6 (*) 15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL)-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6 (*) 35) which is also located in exon 1. Although alternative CYP2D6 (*) 15 and (*) 35 assays resolved the issue, we discovered a novel CYP2D6 (*) 15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6 (*) 15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696) SNP of CYP2D6 (*) 43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer and/or probe regions can impact the performance of PCR-based genotype assays, including TaqMan. Regardless of the test platform used, it is prudent to confirm rare allele calls by an independent method.

A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis.

There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.

Klotho ameliorates chemically induced endoplasmic reticulum (ER) stress signaling.

BACKGROUND: Both endoplasmic reticulum (ER) stress, a fundamental cell response associated with stress-initiated unfolded protein response (UPR), and loss of Klotho, an anti-aging hormone linked to NF-κB-induced inflammation, occur in chronic metabolic diseases such as obesity and type 2 diabetes. We investigated if the loss of Klotho is causally linked to increased ER stress. METHODS: We treated human renal epithelial HK-2, alveolar epithelial A549, HEK293, and SH-SH-SY5Y neuroblastoma cells with ER stress-inducing agents, thapsigargin and/or tunicamycin. Effects of overexpression or siRNA-mediated knockdown of Klotho on UPR signaling was investigated by immunoblotting and Real-time PCR. RESULTS: Elevated Klotho levels in HK-2 cells decreased expression of ER stress markers phospho--IRE1, XBP-1s, BiP, CHOP, pJNK, and phospho-p38, all of which were elevated in response to tunicamycin and/or thapsigargin. Similar results were observed using A549 cells for XBP-1s, BiP, and CHOP in response to thapsigargin. Conversely, knockdown of Klotho in HEK 293 cells using siRNA caused further thapsigargin-induced increases in pIRE-1, XBP-1s, and BiP. Klotho overexpression in A549 cells blocked thapsigargin-induced caspase and PARP cleavage and improved cell viability. CONCLUSION: Our data indicate that Klotho has an important role in regulating ER stress and that loss of Klotho is causally linked to ER stress-induced apoptosis.

Long-term alterations in maternal plasma proteome after sFlt1-induced preeclampsia in mice.

OBJECTIVE: Preeclampsia is associated with long-term adverse maternal health, such as cardiovascular and metabolic diseases. The objective of this study was to determine whether preeclampsia in a well-characterized animal model that was induced by overexpression of soluble fms-like tyrosine kinase-1 (sFlt1) results in alterations in the maternal circulating proteome that persist long after delivery. STUDY DESIGN: CD-1 mice at day 8 of gestation were injected with adenovirus that carried sFlt1 or the murine immunoglobulin G2α Fc fragment as control. Depleted maternal plasma was analyzed 6 months after delivery by label-free liquid chromatography-mass spectrometry assay. The tandem mass spectrometry data were searched against a mouse database, and the resultant intensity data were used to compare abundance of proteins across disease/control plasma pool. Results were analyzed with ingenuity pathways analysis. Right-tailed Fisher exact test was used to calculate a probability value. RESULTS: Of 150 proteins that are common for both groups, ingenuity pathways analysis determined 105 proteins that were ready for analysis. Diseases and disorders analysis showed significant enrichment of proteins that are associated with cardiovascular disease. Within this cluster, the most abundant proteins were associated with vascular disease, atherosclerosis, and atherosclerotic lesions. Other top disease clusters were inflammatory response, organismal injury and abnormalities, and hematologic and metabolic disease. CONCLUSION: Exposure to sFlt1-induced preeclampsia alters multiple biologic functions in mothers that persist later in life. Our results suggest that some of the long-term adverse outcomes that are associated with preeclampsia actually may be a consequence rather than a mere unmasking of an underlying predisposition. If similar results are found in humans, the development of preventive strategies for preeclampsia should also improve long-term maternal health.

Spironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice.

Klotho is a potent regulator of 1,25-hydroxyvitamin D3 [1,25(OH)2D3] formation and calcium-phosphate metabolism. Klotho-hypomorphic mice (kl/kl mice) suffer from severe growth deficits, rapid aging, hyperphosphatemia, hyperaldosteronism, and extensive vascular and soft tissue calcification. Sequelae of klotho deficiency are similar to those of end-stage renal disease. We show here that the mineralocorticoid receptor antagonist spironolactone reduced vascular and soft tissue calcification and increased the life span of kl/kl mice, without significant effects on 1,25(OH)2D3, FGF23, calcium, and phosphate plasma concentrations. Spironolactone also reduced the expression of osteoinductive Pit1 and Tnfa mRNA, osteogenic transcription factors, and alkaline phosphatase (Alpl) in calcified tissues of kl/kl mice. In human aortic smooth muscle cells (HAoSMCs), aldosterone dose-dependently increased PIT1 mRNA expression, an effect paralleled by increased expression of osteogenic transcription factors and enhanced ALP activity. The effects of aldosterone were reversed by both spironolactone treatment and PIT1 silencing and were mitigated by FGF23 cotreatment in HAoSMCs. In conclusion, aldosterone contributes to vascular and soft tissue calcification, an effect due, at least in part, to stimulation of spironolactone-sensitive, PIT1-dependent osteoinductive signaling.