Rationale:: We hypothesized that cluster of differentiation 74 (CD74) downregulation on placental macrophages, leading to altered macrophage-trophoblast interaction, is involved in preeclampsia. Objective:: Preeclamptic pregnancies feature hypertension, proteinuria, and placental anomalies. Feto-placental macrophages regulate villous trophoblast differentiation during placental development. Disturbance of this well-balanced regulation can lead to pathological pregnancies. Methods and Results:: We performed whole-genome expression analysis of placental tissue. CD74 was one of the most downregulated genes in placentas from preeclamptic women. By reverse transcriptase-polymerase chain reaction, we confirmed this finding in early-onset (<34 gestational week, n=26) and late-onset (≥34 gestational week, n=24) samples from preeclamptic women, compared with healthy pregnant controls (n=28). CD74 protein levels were analyzed by Western blot and flow cytometry. We identified placental macrophages to express CD74 by immunofluorescence, flow cytometry, and RT-PCR. CD74-positive macrophages were significantly reduced in preeclamptic placentas compared with controls. CD74-silenced macrophages showed that the adhesion molecules ALCAM , ICAM4 , and Syndecan-2, as well as macrophage adhesion to trophoblasts were diminished. Naive and activated macrophages lacking CD74 showed a shift toward a proinflammatory signature with an increased secretion of tumor necrosis factor-α, chemokine (C–C motif) ligand 5, and monocyte chemotactic protein-1, when cocultured with trophoblasts compared with control macrophages. Trophoblasts stimulated by these factors express more CYP2J2 , sFlt1 , TNFα , and IL-8 . CD74-knockout mice showed disturbed placental morphology, reduced junctional zone, smaller placentas, and impaired spiral artery remodeling with fetal growth restriction. Conclusions:: CD74 downregulation in placental macrophages is present in preeclampsia. CD74 downregulation leads to altered macrophage activation toward a proinflammatory signature and a disturbed crosstalk with trophoblasts.

Background: Preeclampsia is a complex and common human-specific pregnancy syndrome associated with placental pathology. The human specificity provides both intellectual and methodological challenges, lacking a robust model system. Given the role of imprinted genes in human placentation and the vulnerability of imprinted genes to loss of imprinting changes, there has been extensive speculation, but no robust evidence, that imprinted genes are involved in preeclampsia. Our study aims to investigate whether disturbed imprinting contributes to preeclampsia. Methods: We first aimed to confirm that preeclampsia is a disease of the placenta by generating and analyzing genome-wide molecular data on well-characterized patient material. We performed high-throughput transcriptome analyses of multiple placenta samples from healthy controls and patients with preeclampsia. Next, we identified differentially expressed genes in preeclamptic placentas and intersected them with the list of human imprinted genes. We used bioinformatics/statistical analyses to confirm association between imprinting and preeclampsia and to predict biological processes affected in preeclampsia. Validation included epigenetic and cellular assays. In terms of human specificity, we established an in vitro invasion-differentiation trophoblast model. Our comparative phylogenetic analysis involved single-cell transcriptome data of human, macaque, and mouse preimplantation embryogenesis. Results: We found disturbed placental imprinting in preeclampsia and revealed potential candidates, including GATA3 and DLX5 , with poorly explored imprinted status and no prior association with preeclampsia. As a result of loss of imprinting, DLX5 was upregulated in 69% of preeclamptic placentas. Levels of DLX5 correlated with classic preeclampsia markers. DLX5 is expressed in human but not in murine trophoblast. The DLX5 ^high phenotype resulted in reduced proliferation, increased metabolism, and endoplasmic reticulum stress-response activation in trophoblasts in vitro. The transcriptional profile of such cells mimics the transcriptome of preeclamptic placentas. Pan-mammalian comparative analysis identified DLX5 as part of the human-specific regulatory network of trophoblast differentiation. Conclusions: Our analysis provides evidence of a true association among disturbed imprinting, gene expression, and preeclampsia. As a result of disturbed imprinting, the upregulated DLX5 affects trophoblast proliferation. Our in vitro model might fill a vital niche in preeclampsia research. Human-specific regulatory circuitry of DLX5 might help explain certain aspects of preeclampsia.

Inflammation plays an important role in host defenses against infectious agents and injury, but it also contributes to the pathophysiology of atherosclerosis. Signal transducer and activated transcription 1 (STAT1) has been identified as a point of convergence for the cross talk between the pro-inflammatory cytokine interferon γ (IFNγ) and the Toll-like receptor-4 (TLR4) ligand LPS in immune cells. However, there is no information available on the role of STAT1 in TLR4-mediated progression of atherosclerosis and on potential synergism between lipopolysaccharides (LPS) and IFNγ signaling in cells from the vasculature. Cultured human microvascular endothelial cells (HMECs) exposed to LPS activated STAT1 in a delayed manner that was inhibited by cycloheximide treatment. Pretreatment of HMECs as well as primary vascular smooth muscle cells (VSMCs) with IFNγ followed by LPS resulted in a significant increase in STAT1 phosphorylation compared with both factors alone. Increased STAT1 protein levels, strictly mediated by IFNγ, correlated with the augmented STAT1 phosphorylation that was absent in TLR4⁻/⁻ cells. As assessed by PCR, Western analysis, and ELISA, this coincided with increased expression of the chemokine interferon gamma-induced protein 10 kDa (IP-10) and the adhesion molecule ICAM-1 in a TLR4-dependent manner.The STAT1-inhibitor fludarabine markedly reduced these effects as well as IFNγ and LPS-dependent adhesion of U937 cells to endothelial cells, emphasizing the potential importance of STAT1 in the integration of both signals. With the established roles of IFNγ and TLRs in atherosclerotic pathology, the STAT1-dependent signal integration between IFNγ and TLR in ECs and VSMCs in response to exogenous and endogenous atherogenic ligands could result in amplification of pro-inflammatory responses in the damaged vessel and be a novel mechanism involved in the initiation and progression of atherosclerosis.

Background: Glioblastoma (GBM) is the deadliest and the most common primary brain tumor in adults. The invasiveness and proliferation of GBM cells can be decreased through the inhibition of Wnt/β-catenin pathway. In this regard, celecoxib is a promising agent, but other COXIBs and 2,5-dimethylcelecoxib (2,5-DMC) await elucidation. Thus, the aim of this study was to analyze the impact of celecoxib, 2,5-DMC, etori-, rofe-, and valdecoxib on GBM cell viability and the activity of Wnt/β-catenin pathway. In addition, the combination of the compounds with temozolomide (TMZ) was also evaluated. Cell cycle distribution and apoptosis, MGMT methylation level, COX-2 and PGE2 EP4 protein levels were also determined in order to better understand the molecular mechanisms exerted by these compounds and to find out which of them can serve best in GBM therapy. Methods: Celecoxib, 2,5-DMC, etori-, rofe- and valdecoxib were evaluated using three commercially available and two patient-derived GBM cell lines. Cell viability was analyzed using MTT assay, whereas alterations in MGMT methylation level were determined using MS-HRM method. The impact of COXIBs, in the presence and absence of TMZ, on Wnt pathway was measured on the basis of the expression of β-catenin target genes. Cell cycle distribution and apoptosis analysis were performed using flow cytometry. COX-2 and PGE2 EP4 receptor expression were evaluated using Western blot analysis. Results: Wnt/β-catenin pathway was attenuated by COXIBs and 2,5-DMC irrespective of the COX-2 expression profile of the treated cells, their MGMT methylation status, or radio/chemoresistance. Celecoxib and 2,5-DMC were the most cytotoxic. Cell cycle distribution was altered, and apoptosis was induced after the treatment with celecoxib, 2,5-DMC, etori- and valdecoxib in T98G cell line. COXIBs and 2,5-DMC did not influence MGMT methylation status, but inhibited COX-2/PGE2/EP4 pathway. Conclusions: Not only celecoxib, but also 2,5-DMC, etori-, rofe- and valdecoxib should be further investigated as potential good anti-GBM therapeutics.

Background: We tested the controversial hypothesis that vitamin D depletion aggravates hypertension and target‐organ damage by influencing renin. Methods and Results: Four‐week‐old double‐transgenic rats ( dTGR ) with excess angiotensin (Ang) II production due to overexpression of the human renin ( hREN ) and angiotensinogen ( hAGT ) genes received vitamin D‐depleted (n=18) or standard chow (n=15) for 3 weeks. The depleted group had very low serum 25‐hydroxyvitamin D levels (mean± SEM ; 3.8±0.29 versus 40.6±1.19 nmol/L) and had higher mean systolic BP at week 5 (158±3.5 versus 134.6±3.7 mm Hg, P <0.001), week 6 (176.6±3.3 versus 162.3±3.8 mm Hg, P <0.01), and week 7 (171.6±5.1 versus 155.9±4.3 mm Hg, P <0.05). Vitamin D depletion led to increased relative heart weights and increased serum creatinine concentrations. Furthermore, the mRNA s of natriuretic peptides, neutrophil gelatinase‐associated lipocalin, hREN , and rR en were increased by vitamin D depletion. Regulatory T cells in the spleen and in the circulation were not affected. Ang metabolites, including Ang II and the counter‐regulatory breakdown product Ang 1 to 7, were significantly up‐regulated in the vitamin D‐depleted groups, while ACE ‐1 and ACE ‐2 activities were not affected. Conclusions: Short‐term severe vitamin D depletion aggravated hypertension and target‐organ damage in dTGR . Our data suggest that even short‐term severe vitamin D deficiency may directly promote hypertension and impacts on renin‐angiotensin system components that could contribute to target‐organ damage. The findings add to the evidence that vitamin D deficiency could also affect human hypertension.

Preeclampsia is a multisystemic syndrome during pregnancy that is often associated with intrauterine growth retardation. Immunologic dysregulation, involving T cells, is implicated in the pathogenesis. The aim of this study was to evaluate the effect of upregulating regulatory T cells in an established transgenic rat model for preeclampsia. Application of superagonistic monoclonal antibody for CD28 has been shown to effectively upregulate regulatory T cells. In the first protocol (treatment protocol), we applied 1 mg of CD28 superagonist or control antibody on days 11 and 15 of pregnancy. In the second protocol (prevention protocol), the superagonist or control antibody was applied on days 1, 5, and 9. Superagonist increased regulatory T cells in circulation and placenta from 8.49±2.09% of CD4-positive T cells to 23.50±3.05% and from 3.85±1.45% to 23.27±7.64%, respectively. Blood pressure and albuminuria (30.6±15.1 versus 14.6±5.5 mg/d) were similar in the superagonist or control antibody–treated preeclamptic group for both protocols. Rats treated with CD28 superagonist showed increased pup weights in the prevention protocol (2.66±0.03 versus 2.37±0.05 g) and in the treatment protocol (3.04±0.04 versus 2.54±0.1 g). Intrauterine growth retardation, calculated by brain:liver weight ratio, was also decreased by the superagonist in both protocols. Further analysis of brain development revealed a 20% increase in brain volume by the superagonist. Induction of regulatory T cells in the circulation and the uteroplacental unit in an established preeclamptic rat model had no influence on maternal hypertension and proteinuria. However, it substantially improved fetal outcome by ameliorating intrauterine growth retardation.