Endometrial Cancer Side-Population Cells Show Prominent Migration and Have a Potential to Differentiate into the Mesenchymal Cell Lineage

Cancer stem-like cell subpopulations, referred to as “side-population” (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and, uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, α-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into α-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage.Recently, adult stem cells have been identified in several mature tissues, such as the adult intestine,¹ skin,² muscle,³ blood,⁴ and the nervous system^5–7 A stem cell is an undifferentiated cell that is defined by its ability to both self-renew and to produce mature progeny cells.⁸ Stem cells are classified based on their developmental potential as totipotent, pluripotent, oligopotent, and unipotent. Adult somatic stem cells were originally thought to be tissue specific and only able to give rise to progeny cells corresponding to their tissue of origin. Recent studies, however, have shown that adult mammalian stem cells are able to differentiate across tissue lineage boundaries,^9,10 although this “plasticity” of adult somatic stem cells remains controversial.Stem cell subpopulations (“side-population” (SP) cells) have been identified in many mammals, including humans, based on the ability of these cells to efflux the fluorescent dye Hoechst 33342.¹¹ Recent evidence suggests that the SP phenotype is associated with a high expression level of the ATP-binding cassette transporter protein ABCG2/Bcrp1.¹² Most recently, established malignant cell lines, which have been maintained for many years in culture, have also been shown to contain SP cells as a minor subpopulation.¹³The human endometrium is a highly dynamic tissue undergoing cycles of growth, differentiation, shedding, and regeneration throughout the reproductive life of women. Endometrial adult stem/progenitor cells are likely responsible for endometrial regeneration.¹⁴ Rare populations of human endometrial epithelial and stromal colony-forming cells¹⁵ and SP cells^16,17 have been identified. Although coexpression of CD146 and PDGFRβ isolates a population of mesenchymal stem like cells from human endometrium,¹⁸ specific stem cell markers of endometrium remain unclear. Recently, Gotte et al¹⁹ demonstrated that the adult stem cell marker Musashi-1 was coexpressed with Notch-1 in a subpopulation of endometrial cells. Furthermore, they showed that telomerase and Musashi-1-expressing cells were significantly increased in proliferative endometrium, endometriosis, and endometrial carcinoma tissue, compared with secretary endometrium, suggesting the concept of a stem cell origin of endometriosis and endometrial carcinoma.Recent evidence suggests that cancer stem-like cells exist in several malignant tumors, such as leukemia^20,21 breast cancer,²² and brain tumors,²³ and that these stem cells express surface markers similar to those expressed by normal stem cells in each tissue.^20,24Development of endometrial carcinoma is associated with a variety of genetic alterations. For example, increased expression and activity of telomerase^25,26 and frequent dysregulation of signaling pathways have been observed in endometrial carcinoma. Some of these pathways are important determinants of stem cell activity (Wnt-β-catenin and PTEN).^27–29 These suggest a stem cell contribution to endometrial carcinoma development.Recently, we isolated SP cells from the human endometrium. These SP cells showed long-term proliferating capacity in cultures and produced both gland and stromal-like cells. Additionally, they were able to function as progenitor cells.¹⁶ In this study, we isolated and characterized SP cells from human endometrial cancer cells and from rat endometrial cells expressing oncogenic [¹²Val] human K-Ras protein and demonstrated their cancer stem-like cell phenotypes.     

Cytoprotective Mitochondrial Chaperone TRAP-1 As a Novel Molecular Target in Localized and Metastatic Prostate Cancer

Molecular chaperones of the heat shock protein-90 (Hsp90) family promote cell survival, but the molecular requirements of this pathway in tumor progression are not understood. Here, we show that a mitochondria-localized Hsp90 chaperone, tumor necrosis factor receptor-associated protein-1 (TRAP-1), is abundantly and ubiquitously expressed in human high-grade prostatic intraepithelial neoplasia, Gleason grades 3 through 5 prostatic adenocarcinomas, and metastatic prostate cancer, but largely undetectable in normal prostate or benign prostatic hyperplasia in vivo. Prostate lesions formed in genetic models of the disease, including the transgenic adenocarcinoma of the mouse prostate and mice carrying prostate-specific deletion of the phosphatase tensin homolog tumor suppressor (Pten^pc−/−), also exhibit high levels of TRAP-1. Expression of TRAP-1 in nontransformed prostatic epithelial BPH-1 cells inhibited cell death, whereas silencing of TRAP-1 in androgen-independent PC3 or DU145 prostate cancer cells by small interfering RNA enhanced apoptosis. Targeting TRAP-1 with a novel class of mitochondria-directed Hsp90 inhibitors, ie, Gamitrinibs, caused rapid and complete killing of androgen-dependent or -independent prostate cancer, but not BPH-1 cells, whereas reintroduction of TRAP-1 in BPH-1 cells conferred sensitivity to Gamitrinib-induced cell death. These data identify TRAP-1 as a novel mitochondrial survival factor differentially expressed in localized and metastatic prostate cancer compared with normal prostate. Targeting this pathway with Gamitrinibs could be explored as novel molecular therapy in patients with advanced prostate cancer.Apart from skin tumors, prostate cancer is the most commonly diagnosed malignancy in men in the United States.¹ Despite progress in early diagnosis,² and prolongation of patient survival,³ the disease still carries significant morbidity and mortality, with its advanced and metastatic phase claiming over 30,000 deaths per year in the United States alone. Similar to the genetic heterogeneity of most epithelial malignancies, prostate cancer progresses through a stepwise acquisition of multiple molecular changes,⁴ of which insensitivity to androgen deprivation,⁵ emergence of an ‘osteomimetic’ phenotype responsible for metastatic tropism to the bone,⁶ and deregulated cell proliferation and cell survival,⁷ are pivotal traits.In this context, advanced prostate cancer is almost invariably associated with a heightened anti-apoptotic threshold,⁴ which may contribute to disease progression and resistance to therapy. This process often involves aberrant resistance to mitochondrial cell death,⁸ with reduced organelle permeability to solutes, and attenuated release of mitochondrial apoptogenic proteins in the cytosol.⁹ The regulators of such ‘mitochondrial permeability transition’ normally triggered by cell death stimuli are still largely elusive, but knockout data in mice have identified pro-apoptotic Bcl-2 family proteins and the mitochondrial matrix immunophilin, cyclophilin D, as pivotal effectors of this process, controlling the integrity of the mitochondrial outer membrane,⁸ and the opening a permeability transition pore,^10,11 respectively.Recent data have shown that molecular chaperones of the heat shock protein-90 (Hsp90) family,¹² may function as novel regulators of mitochondrial permeability transition,¹³ especially in tumor cells.¹⁴ Accordingly, Hsp90, and its ortholog, tumor necrosis factor receptor-associated protein-1 (TRAP-1) are abundantly localized to mitochondria of tumor, but not most normal cells, and antagonize cyclophilin D-dependent pore-forming function, potentially via a protein (re)folding mechanism.¹⁴ Consistent with a general role of Hsp90 as a drug target in prostate cancer,¹⁵ this mitochondria-compartmentalized cytoprotective pathway could provide a novel therapeutic target to enhance tumor cell apoptosis.¹⁴In the current study, we demonstrate that TRAP-1 is dramatically expressed in all lesions that comprise the entire natural history of human prostate cancer, as well as genetic disease models in rodents, but undetectable in the normal prostate. Importantly, we show that Gamitrinibs, a novel class of small molecule Hsp90 antagonists selectively engineered to target the pool of these chaperones in mitochondria,¹⁶ cause sudden prostate cancer cell death without affecting nontransformed prostatic epithelium.     

E-Selectin Mediates Stem Cell Adhesion and Formation of Blood Vessels in a Murine Model of Infantile Hemangioma

Hemangioma stem cells (HemSCs) are multipotent cells isolated from infantile hemangioma (IH), which form hemangioma-like lesions when injected subcutaneously into immune-deficient mice. In this murine model, HemSCs are the primary target of corticosteroid, a mainstay therapy for problematic IH. The relationship between HemSCs and endothelial cells that reside in IH is not clearly understood. Adhesive interactions might be critical for the preferential accumulation of HemSCs and/or endothelial cells in the tumor. Therefore, we studied the interactions between HemSCs and endothelial cells (HemECs) isolated from IH surgical specimens. We found that HemECs isolated from proliferating phase IH, but not involuting phase, constitutively express E-selectin, a cell adhesion molecule not present in quiescent endothelial cells. E-selectin was further increased when HemECs were exposed to vascular endothelial growth factor–A or tumor necrosis factor–α. In vitro, HemSC migration and adhesion was enhanced by recombinant E-selectin but not P-selectin; both processes were neutralized by E-selectin–blocking antibodies. E-selectin–positive HemECs also stimulated migration and adhesion of HemSCs. In vivo, neutralizing antibodies to E-selectin strongly inhibited formation of blood vessels when HemSCs and HemECs were co-implanted in Matrigel. These data suggest that endothelial E-selectin could be a major ligand for HemSCs and thereby promote cellular interactions and vasculogenesis in IH. We propose that constitutively expressed E-selectin on endothelial cells in the proliferating phase is one mediator of the stem cell tropism in IH.Infantile hemangioma (IH) is the most common tumor of infancy. A hallmark is its unique life cycle of rapid development in childhood, followed by a slow regression and cessation of growth.¹ Hemangioma endothelial cells (HemECs) in proliferating lesions show X chromosome inactivation patterns, indicative of a clonal origin,^2–4 that is maintained in cultured HemECs.² In comparison to human dermal microvascular endothelial cell (HDMEC), HemECs have constitutively active vascular endothelial growth factor-receptor 2 (VEGFR2) signaling in association with low expression of vascular endothelial growth factor-receptor 1 (VEGFR1/FLT1).⁵ HemECs have a placental microvascular phenotype and may originate from placental endothelial cells.^6,7 Little is known about endothelial cells in the involuting phase of IH.⁸Proliferating hemangiomas express high levels of hypoxia inducible factor 1α (HIF-1α) protein and release factors⁹ that can induce recruitment of bone marrow–derived cells from the circulation into the tumors. These cells could be heterogeneous, composed of endothelial progenitor cells,^9,10 myeloid cells,¹¹ and possibly CD133-positive cells that include hemangioma stem cells (HemSCs). HemSCs isolated from specimens of human proliferating IH are multipotent, and exhibit a mesenchymal morphology and robust proliferation in vitro.¹² In contrast to HemECs, HemSCs can form human blood vessels with the immunophenotype and dynamics of IH when injected subcutaneously into nude mice.¹² A central function of HemSCs in IH is supported by our recent study in which we showed that corticosteroid act specifically on the HemSCs to down-regulate vascular endothelial growth factor–A (VEGF-A) expression.¹³ However, it is undetermined whether HemSCs arise in the tumors or whether they are recruited to tumor site in response to pathological endothelial cells in a specific microenvironment.E-selectin has been detected in proliferating phase specimens of IH and has been shown to decrease in involuting phase specimens.^14,15 Here, we analyzed E-selectin expression in endothelial cells expanded from proliferating and involuting IH tumors, and its potential role in functional interactions between HemSCs and ECs. Our findings implicate E-selectin in hemangioma blood vessel development, and suggest that E-selectin on HemECs may engage stem cells in vasculogenesis.     

Dendritic Cells Express Hematopoietic Prostaglandin D Synthase and Function as a Source of Prostaglandin D2 in the Skin

Prostaglandin D2 (PGD2), an arachidonic acid metabolite, has been implicated in allergic responses. A major source of PGD2 in the skin is mast cells that express hematopoietic PGD synthase (H-PGDS). In this study, we show the expression of H-PGDS in human dendritic cells (DCs) and the regulatory mechanisms by which DCs produce PGD2. We detected H-PGDS in epidermal Langerhans cells, dermal DCs, plasmacytoid DCs, and myeloid DCs. Monocyte-derived DCs rapidly secreted PGD2 when stimulated with the calcium ionophore A23187. More importantly, pretreatment of monocyte-derived DCs with PMA (phorbol 12-myrisate 13-acetate) synergistically enhanced the rapid PGD2 secretion induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, whereas PMA alone did not induce PGD2 secretion. Lipopolysaccharide (LPS) reduced H-PGDS expression, but interferon-γ followed by LPS induced significant PGD2 production in a delayed time course at 6 hours. This effect was associated with inhibition of LPS-induced H-PGDS reduction. Interestingly, an irritant compound, SDS, also induced a rapid PGD2 release. PGD2 synergistically enhanced CCL22/macrophage-derived chemokine synthesis in interferon-γ-treated human keratinocytes. In addition, bone marrow-derived DCs from wild-type mice stimulated lymph node cells to produce higher amounts of interleukin-17 than did DCs from mice lacking the H-PGDS gene. Thus, DCs could be an important source of skin PGD2 and may mediate or regulate skin inflammation by releasing PGD2 in response to various stimuli, contributing to the innate and/or acquired immune responses.Prostaglandin D2 (PGD2) is one of the arachidonic acid metabolites and exerts a range of biological activities, including vasodilatation, bronchoconstriction, and inhibition of platelet aggregation.^1–4 PGD2 is also implicated in allergic diseases. PGD2 production is observed in bronchoalveolar lavage fluid from asthmatic patients.⁵ Mice that overproduce PGD2 exhibit an enhanced allergic lung response, eosinophilia, and increased Th2-type cytokine production.⁶ We have demonstrated that PGD2 plays an essential role in IgE-mediated skin responses in mice.⁷ A possible anti-pruritic potential of PGD2 in the scratching behavior of mice was recently proposed.^8,9PGD2 exerts its effect through D prostanoid (DP) and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) receptors. DP and CRTH2 are members of the G protein-coupled, seven transmembrane receptor family. DP is coupled with Gs protein, whereas Gi protein is associated with CRTH2.¹⁰ DP-mediated signals inhibit dendritic cell (DC) migration.^11–13 Effects of PGD2 on DC maturation and interleukin-12 production are also mediated by the DP receptor.¹⁴ On the other hand, CRTH2 signals induce calcium mobilization and chemotaxis in eosinophils and basophils.¹⁰ In addition, CRTH2 signals enhance interleukin-4, -5, and -13 production from Th2 cells.¹⁵PGD2 synthesis is mediated by the isomerization of prostaglandin H2 (PGH2) into PGD2 through the enzymatic activity of PGD synthase (PGDS).¹⁶ Two types of PGDS have been identified: lipocalin-type PGDS and hematopoietic PGDS (H-PGDS).^16,17 Lipocalin-type PGDS is present in meningeal cells, epithelial cells of the choroids plexus, and oligodendrocytes in the brain and is involved in the sleep-wake cycle.¹⁸ H-PGDS was originally isolated from rat spleen as a cytosolic glutathione (GSH)-requiring enzyme.^19,20 Mast cells express H-PGDS and rapidly secrete PGD2 in response to antigen stimulation.^21,22 Thus, mast cells are a major source of PGD2 in the skin and contribute to inflammation,^23,24 although a small population of Th2-type cells contains H-PGDS.²⁵H-PGDS is also detected in antigen-presenting cells, such as histiocytes and/or DCs in rat spleen, thymus, and skin.^26,27 We have revealed that epidermal Langerhans cells in mouse skin express H-PGDS.⁷ Thus, it can be postulated that DCs could be a source of PGD2 in skin tissues and may affect various immune cells and effector cells, including DCs themselves, through DP and/or CRTH2 receptors. However, H-PGDS in human DCs and their capability for PGD2 secretion have not been fully characterized. In the present study, we analyzed H-PGDS expression in human DCs and identified regulatory mechanisms of PGD2 production with a variety of stimuli. Moreover, the biological significance of DC-derived PGD2 in terms of chemokine synthesis from keratinocytes and cytokine production from lymphocytes were also assessed.     

Interaction of MCM7 and RACK1 for Activation of MCM7 and Cell Growth

MCM7 is one of the pivotal DNA replication licensing factors in controlling DNA synthesis and cell entry into S phase. Its expression and DNA copy number are some of the most predictive factors for the growth and behavior of human malignancies. In this study, we identified that MCM7 interacts with the receptor for activated protein kinase C 1 (RACK1), a protein kinase C (PKC) adaptor, in vivo and in vitro. The RACK1 binding motif in MCM7 is located at the amino acid 221-248. Knocking down RACK1 significantly reduced MCM7 chromatin association, DNA synthesis, and cell cycle entry into S phase. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate dramatically decreased MCM7 DNA replication licensing and induced cell growth arrest. Activation of PKC induced redistribution of RACK1 from nucleus to cytoplasm and decreased RACK1-chromatin association. The MCM7 mutant that does not bind RACK1 has no DNA replication licensing or oncogenic transformation activity. As a result, this study demonstrates a novel signaling mechanism that critically controls DNA synthesis and cell cycle progression.Miniature chromosome maintenance (MCM) proteins were initially identified from autonomously replicating sequence in Saccharomyces cerevisiae. Mutations of some of these proteins, such as MCM7 or MCM3 result in loss of the large chunk of yeast chromosomes in yeast. MCM7 cDNA encodes a 543-amino acid protein and is ubiquitously expressed in all tissues. A large body of studies indicate that MCM7 is a critical component of DNA replication licensing complex in the yeast and xenopus.^1–4 Some studies suggest that MCM4, MCM6, and MCM7 complex contains DNA helicase activity.^5,6 DNA replication licensing complex is multimeric and phase specific. In yeast, DNA replication licensing proteins, such as MCM2-7 and several replication origin binding proteins, such as Cdc6, germinin, and Cdt1, form DNA replication licensing complex in G1 phase to enable DNA replication and to promote cell cycle entry into S phase. Initial implication of MCM7 involvement in human malignancies came from positive immunostaining of MCM7 in several human malignancies, including endometrial carcinoma,⁷ melanoma,⁸ esophageal adenocarcinoma,⁹ colorectal adenocarcinoma,¹⁰ oral squamous cell carcinoma,¹¹ glioblastoma,¹² and thyroid cancer.¹³ The first study addressing the oncogenic role of MCM7 in prostate cancer came from genome analysis of prostate cancer by performing a genome wide copy number analysis using biotin-labeled genome DNA on Affymetrix U95av2 chip.¹⁴ The DNA copy number of MCM7 was found to increase severalfold accompanied with a concomitant increase of MCM7 mRNA level. Subsequent validation analyses suggest that either copy number and/or protein level increase of MCM7 are associated with prostate cancer relapse and metastasis. Amplification of MCM7 was also found in esophageal carcinoma.⁹ The magnitude of MCM7 amplification correlates with the expression of MCM7, tumor grades, and the aggressiveness of esophageal cancer.⁹ It is presumed that amplification of MCM7 is the driving force of MCM7 overexpression in primary human malignancies. MCM7 is probably the primary target of Rb, the tumor suppressor that controls cell entry into S phase.¹⁵ There is growing evidence that other signaling pathways also regulate MCM7 activity.Receptor for activated protein kinase C 1 (RACK1), was initially identified as an adaptor of several protein kinase C (PKC) isoforms.¹⁶ The binding of RACK1 and PKC anchor PKC to its substrate to initiate second messenger signaling. It is suggested, according to recent studies that RACK1 interacts with a variety of other signaling molecules, including ras-GTPase activating protein,¹⁷ dynamin-1,¹⁸ src,¹⁹ integrins,²⁰ PTPμ,²¹ phosphodiesterase,²² hypoxia induced factor-1,²³ and so forth, that play an important role in several physiological processes, including, growth, hypoxia response, migration, adhesion, and cell differentiation. RACK1 only binds PKC activated by diacylglycerol or phorbol ester, but not quiescent PKC. In this study, we showed that RACK1 binds with MCM7 N-terminus. The MCM7/RACK1 interaction appears essential for DNA replication activity of MCM7.     

PTEN,PIK3CA,p-AKT,and p-p70S6K Status: Association with Trastuzumab Response and Survival in Patients with HER2-Positive Metastatic Breast Cancer

Phosphatase and tensin homolog (PTEN) is a key modulator of trastuzumab sensitivity in HER2-overexpressing breast cancer. Because PTEN opposes the downstream signaling of phosphoinositide 3-kinase (PI3K), we investigated the role of PTEN and other components of the PI3K pathway in trastuzumab resistance. We analyzed the status of PTEN, p-AKT-Ser473, and p-p70S6K-Thr389 using immunohistochemistry. PIK3CA mutation status was analyzed by direct sequencing. Primary tumor tissue was available from 137 patients with HER2-overexpressing metastatic breast cancer who had received trastuzumab-based chemotherapy. We observed that each of the four biomarkers alone did not significantly correlate with trastuzumab response, whereas PTEN loss alone significantly correlated with shorter survival times (P = 0.023). PI3K pathway activation, defined as PTEN loss and/or PIK3CA mutation, was associated with a poor response to trastuzumab (P = 0.047) and a shorter survival time (P = 0.015). PTEN loss was significantly associated with a poor response to trastuzumab (P = 0.028) and shorter survival time (P = 0.008) in patients who had received first-line trastuzumab and in patients with estrogen receptor- (P = 0.029) and progesterone receptor-negative tumors (P = 0.033). p-AKT-Ser473 and p-p70S6K-Thr389 each had a limited correlation with trastuzumab response. When these markers were combined with PTEN loss, an increased correlation with patient outcome was observed. In conclusion, PI3K pathway activation plays a pivotal role in trastuzumab resistance. Our findings may facilitate the evaluation of tumor response to trastuzumab-based and targeted therapies.Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20% to 25% of invasive breast cancers. Patients with HER2-overexpressing tumors experience a shorter time to relapse and shorter overall survival than patients with tumors of normal HER2 levels.^1,2 HER2 overexpression can lead to activation of many downstream signaling molecules, including Ras-Gap, Src, phosphoinositide 3-kinase (PI3K)/AKT, and many other signaling events.^3,4 Trastuzumab (Herceptin; Genentech, CA), a humanized monoclonal antibody that directly targets the extracellular domain of HER2, has a remarkable therapeutic efficacy in treating patients with HER2-expressing metastatic breast cancer (MBC)⁵ and patients with HER2-positive early-stage disease in adjuvant settings.^6,7 Trastuzumab treatment, when combined with chemotherapy, resulted in a significant improvement in patients'' response rate, time to progression, and duration of response.⁸ The underlying mechanisms of trastuzumab''s antitumor activities include, but are not limited to, inducing antibody-dependent cellular cytotoxicity,⁹ inhibiting HER2 extracellular domain cleavage,¹⁰ activating phosphatase and tensin homolog (PTEN),¹¹ and inhibiting PI3K/AKT survival signaling.¹² However, the overall response rate to trastuzumab is low, and almost half of patients with HER2-positive breast cancer exhibit an initial resistance to trastuzumab-based therapy.^11,13 Despite the large amounts of preclinical and clinical data, the causes of trastuzumab resistance are still poorly understood.¹⁴The PI3K pathway, downstream of HER2, plays a central role in regulating a number of cellular processes, such as apoptosis, migration, angiogenesis, cell proliferation, and glucose metabolism, and it is involved in trastuzumab resistance.^15,16 PI3K phosphorylates phosphatidylinositols on the cell membrane, generating phosphatidylinositol-3,4,5-trisphosphate (PIP3) from phosphatidylinositol-4,5-bisphosphate (PIP2). Then, at the cell membrane, PIP3 recruits protein kinases and activates protein kinase B (PKB)/AKT.¹⁷ In breast cancer cells, HER2 overexpression can lead to activation of the PI3K/AKT pathway.¹⁸ The activation of AKT and its downstream signaling have been demonstrated to inhibit cell cycle arrest and block trastuzumab-mediated apoptosis.¹² AKT phosphorylation and AKT kinase activities were found to be increased in trastuzumab-resistant cells, derived from BT474 HER2-overexpressing breast cancer cells, when compared with parental cells.¹⁹ These data provide insight into the trastuzumab-resistance mechanism of PI3K/AKT signaling.¹⁵Aberrations in the components of the PI3K pathway have been reported in most solid tumors, including breast cancer.¹⁶ PTEN is a tumor suppressor that dephosphorylates the D3 position of PIP3 and inhibits the PI3K/AKT pathway.²⁰ Loss of PTEN function as a result of mutation, deletion, or promoter methylation has been reported in nearly 50% of breast cancers.¹¹ In addition, the gene encoding one of the PI3K catalytic subunits, p110α (PIK3CA), has been found to be mutated in about 25% of breast cancers.^21,22,23 Most of the reported mutations are localized to hotspots in exons 9 and 20 of the PIK3CA gene, which result in increased PI3K pathway signaling.^22,24 We previously discovered that PTEN activation is a novel mechanism of trastuzumab antitumor function, and PTEN loss confers trastuzumab resistance in HER2-overexpressing breast cancer cells.¹¹ PTEN loss significantly predicted poor response to trastuzumab-based therapy in a small cohort of HER2-positive patients with MBC.¹¹ Later, it was reported that both low PTEN levels and PI3K-activating PIK3CA mutations contribute to trastuzumab resistance in HER2-overexpressing breast cancer.^25,26 PTEN loss or PIK3CA mutations, which indicate activation of the PI3K pathway, are considered as markers for poor response to trastuzumab in patients with HER2-overexpressing breast cancer.²⁵ On the other hand, some studies found no correlation between PTEN expression and trastuzumab response or survival in patients with HER2-positive breast cancer.^27,28 These contradictory findings prompted us to further investigate the association between PTEN status and clinical outcomes in a large cohort of patients with MBC who were treated with trastuzumab-based therapy. We tested the hypothesis that a comprehensive assessment of PI3K pathway activation status provides biomarkers that can identify patients who may not benefit from trastuzumab-based therapy.     

Murine Cerebral Malaria Is Associated with a Vasospasm-Like Microcirculatory Dysfunction,and Survival upon Rescue Treatment Is Markedly Increased by Nimodipine

Brain hemodynamics in cerebral malaria (CM) is poorly understood, with apparently conflicting data showing microcirculatory hypoperfusion and normal or even increased blood flow in large arteries. Using intravital microscopy to assess the pial microvasculature through a closed cranial window in the murine model of CM by Plasmodium berghei ANKA, we show that murine CM is associated with marked decreases (mean: 60%) of pial arteriolar blood flow attributable to vasoconstriction and decreased blood velocity. Leukocyte sequestration further decreased perfusion by narrowing luminal diameters in the affected vessels and blocking capillaries. Remarkably, vascular collapse at various degrees was observed in 44% of mice with CM, which also presented more severe vasoconstriction. Coadministration of artemether and nimodipine, a calcium channel blocker used to treat postsubarachnoid hemorrhage vasospasm, to mice presenting CM markedly increased survival compared with artemether plus vehicle only. Administration of nimodipine induced vasodilation and increased pial blood flow. We conclude that vasoconstriction and vascular collapse play a role in murine CM pathogenesis and nimodipine holds potential as adjunctive therapy for CM.Cerebral malaria (CM) caused by Plasmodium falciparum claims the lives of nearly 1 million children every year.¹ Despite antimalarial treatment, 10% to 20% of patients die, and one in every four survivors develops neurological sequelae,^2,3 therefore adjunctive therapies are urgently needed. A number of clinical trials addressing potential adjunctive therapies for CM showed no proven benefits and some interventions were even deleterious,⁴ stressing the need for a better understanding of CM pathogenesis to develop effective therapies.An unresolved issue of CM pathogenesis regards the role of brain hemodynamic perturbations and ischemia. Sequestration of parasitized red blood cells (pRBCs) containing mature forms of the parasite in the brain microvasculature is a characteristic postmortem finding in human CM cases⁵ and together with rosetting⁶ and reduced RBC deformability⁷ may result in the obstruction of blood flow potentially leading to ischemia and hypoxia. In vivo studies of the microcirculation in human CM support this mechanism, with direct observation of retinal microvasculature showing impaired perfusion, retinal whitening, vascular occlusion, and ischemia.⁸ Accordingly, microvascular obstruction observed in the rectal mucosa of CM patients was proportional to the severity of the disease.⁹ In addition, hypovolemia, shock and intracranial hypertension, commonly associated with poor outcomes in CM,⁴ reduce tissue perfusion, and tissue hypoxia is one of the likely explanations for the acidosis frequently observed in severe malaria.^7,10 Ischemic damage has also been shown in children with CM and was associated with severe neurological sequelae.¹¹ On the other hand, transcranial Doppler sonography studies showed normal or even increased cerebral blood flow (CBF) velocities^12–15 in large arteries during CM, which associated with microcirculatory obstruction has been suggested to increase cerebral blood volume leading to intracranial hypertension.¹⁶ Alternatively, collateral flow has been proposed as a mechanism to reconcile the findings of normal or increased CBF velocities and impaired perfusion,¹⁷ an interpretation supported by findings of hyperdynamic flow in capillaries adjacent to obstructed vessels.⁹ Interventions that improve cerebral perfusion have been proposed to be beneficial in CM.^8,18The murine model of CM by Plasmodium berghei ANKA (PbA) shares many features with the human pathology,¹⁹ including the presence of multiple brain microhemorrhages and vascular obstruction, although the nature of the sequestered cell (leukocytes) differs. In murine CM, magnetic resonance imaging (MRI) and spectroscopy studies showed the presence of brain edema, decreased CBF, and ischemia.^20,21 Lack of resolution in MRI, however, precludes detailed studies of the microcirculation, which is a major target and player in CM pathogenesis. A few studies have addressed the in vivo microcirculatory changes in murine models of severe malaria,^22–24 however in sites other than the brain (cremaster muscle or skin). In the present work, we used for the first time brain intravital microscopy to follow the dynamic changes in the pial microcirculation during the course of PbA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow, and eventually vascular collapse, events similar to postsubarachnoid hemorrhage (SAH) vasospasm.²⁵ We also show that nimodipine, a calcium channel blocker used to treat post-SAH vasospasm,^25,26 markedly increased survival when given off-label to mice with CM as adjunctive therapy to artemether.     

Genetic testing in asymptomatic minors Background considerations towards ESHG Recommendations

Although various guidelines and position papers have discussed, in the past, the ethical aspects of genetic testing in asymptomatic minors, the European Society of Human Genetics had not earlier endorsed any set of guidelines exclusively focused on this issue. This paper has served as a background document in preparation of the development of the policy recommendations of the Public and Professional Committee of the European Society of Human Genetics. This background paper first discusses some general considerations with regard to the provision of genetic tests to minors. It discusses the concept of best interests, participation of minors in health-care decisions, parents'' responsibilities to share genetic information, the role of clinical genetics and the health-care system in communication within the family. Second, it discusses, respectively, the presymptomatic and predictive genetic testing for adult-onset disorders, childhood-onset disorders and carrier testing.Although various guidelines and position papers have discussed, in the past, the ethical aspects of genetic testing in asymptomatic minors,^{1, 2} the European Society of Human Genetics had not earlier endorsed any set of guidelines exclusively focused on this issue. This background paper was preceded by an in-depth research on the topic by Eurogentest.³ Eurogentest (http://www.eurogentest.org aims to develop the necessary infrastructure, tools, resources, guidelines and procedures that will structure, harmonize and improve the overall quality of all the EU genetic services at the molecular, cytogenetic, biochemical and clinical level.⁴ Attention has also been paid to the provision of appropriate counselling related to genetic testing, the education of patients and professionals, as well as to the ethical, legal and social issues surrounding testing. The focus of the ethics unit of Eurogentest was oriented towards the study of the ethical issues related to genetic testing in minors. This work was the starting point for this background paper, which has been prepared and supported by different types of evidence. First, research has been performed on the existing recommendations regarding predictive genetic testing in minors¹ and carrier testing,² with the intention of identifying areas of agreement and disagreement. Second, the literature on medico–ethical and medico–legal aspects of predictive genetic testing in minors,⁵ carrier testing,^{6, 7} the position of minors⁸ and patient rights⁹ was studied. Third, a systematic literature review was performed to gather information regarding the attitudes of the different stakeholders (minors, health-care professionals, parents and relatives of the affected individuals) towards genetic testing in asymptomatic minors.^{10, 11} Fourth, the attitudes of European clinical geneticists regarding genetic testing in asymptomatic minors were gathered.^{12, 13, 14}In 2007, contacts were made with the Public and Professional Policy Committee of the European Society of Human Genetics with the aim of developing policy recommendations on the issue. On the basis of a decision of the PPPC meeting during the ESHG conference in Nice (June 2007), an ad hoc committee, consisting of Pascal Borry (Eurogentest), Kris Dierickx (Eurogentest), Angus Clarke, Gerry Evers-Kiebooms (PPPC) and Martina Cornel (PPPC), was created. This ad hoc committee met on 15 November 2007 to discuss a first draft of a background paper and recommendations that were prepared by Pascal Borry under the supervision of Kris Dierickx. A revised version was discussed during a PPPC meeting in Amsterdam (April 2008) and Barcelona (June 2008). In order not to repeat issues that have been discussed elsewhere, reference will often be made to the above-referenced publications.     

Non-invasive prenatal testing: ethical issues explored

This paper explores the ethical implications of introducing non-invasive prenatal diagnostic tests (NIPD tests) in prenatal screening for foetal abnormalities. NIPD tests are easy and safe and can be performed early in pregnancy. Precisely because of these features, it is feared that informed consent may become more difficult, that both testing and selective abortion will become ‘normalized'', and that there will be a trend towards accepting testing for minor abnormalities and non-medical traits as well. In our view, however, the real moral challenge of NIPD testing consists in the possibility of linking up a technique with these features (easy, safe and early) with new genomic technologies that allow prenatal diagnostic testing for a much broader range of abnormalities than is the case in current procedures. An increase in uptake and more selective abortions need not in itself be taken to signal a thoughtless acceptance of these procedures. However, combining this with considerably enlarging the scope of NIPD testing will indeed make informed consent more difficult and challenge the notion of prenatal screening as serving reproductive autonomy. If broad NIPD testing includes later-onset diseases, the ‘right not to know'' of the future child will become a new issue in the debate about prenatal screening. With regard to the controversial issue of selective abortion, it may make a morally relevant difference that after NIPD testing, abortion can be done early. A lower moral status may be attributed to the foetus at that moment, given the dominant opinion that the moral status of the foetus progressively increases with its development.Since the discovery of cell-free foetal DNA/RNA (cffDNA/RNA) in maternal plasma in 1997,¹ the possibility to use this cffDNA/RNA for non-invasive prenatal diagnosis (NIPD) has been investigated many times.^{2, 3, 4, 5, 6} cffDNA/RNA can be obtained from a maternal blood sample, as early as 4 weeks of gestation,⁷ but currently only reliably so from 7 weeks of gestation.⁴ This development holds the promise of NIPD testing early in pregnancy and without the small, but significant risk of foetal loss that the current invasive procedures of chorionic villus sampling (CVS) and amniocentesis (AP) carry. NIPD testing for the determination of a Y-signal for pregnancies at risk of X-linked disorders and for diagnosis of Rhesus factor status in RhD-negative women is now being translated into clinical practice.⁴ In many European countries, discussion about broader applications of NIPD testing can be expected in the coming years.^{8, 9} The feasibility of NIPD for trisomy 21, 13 and 18 has already been shown,² but large-scale independent studies are still needed. Sex-chromosomal abnormalities (eg, Turner syndrome (X0) and triple X syndrome (XXX)) could in principle be diagnosed by NIPD testing as well,⁴ if reliable quantitative tests become available in the future and the maternal ‘background'' can be excluded from testing. Even if accurate NIPD testing for the mentioned abnormalities becomes possible, the clinical utility of the test remains to be assessed. This includes balancing the benefits to the harms also with regard to its psychological, ethical, legal, social and economic implications.^{10, 11} The possible ethical implications of NIPD as a new approach to prenatal testing have so far been reviewed in a few publications.^{4, 8, 9, 12, 13, 14, 15, 16, 17} Apart from clear benefits related to avoiding the miscarriage risk of present invasive methods, important potential drawbacks have been mentioned as well. For one thing, proper counselling and informed consent is argued to become more challenging when offering NIPD testing. Moreover, there is a concern that the ease and safety of NIPD may lead to prenatal screening being increasingly conceived as a matter of course, both by those making the offer and by the women undergoing the test. Related to this is the concern that selective abortion of foetuses with minor abnormalities, the wrong sex or unwanted paternity, will become normalized.This paper aims to expand and refine these ethical evaluations and will add some new ethical perspectives with regard to possible implications of NIPD at present and in the future.In our view, it is not so much the fact that foetal material used for prenatal testing can be obtained early and non-invasively (allowing easy and safe testing) that would lead to moral challenges. Rather, it is the fact that a technology with these features would be open to being used for testing a potentially much broader range of abnormalities than those included in the presently used method of microscopic chromosome analysis (karyotyping).Although NIPD testing can also be applied in high genetic-risk families and for the management of pregnancy, the focus of this paper will primarily be on the application of NIPD testing in the screening context. The reason for this focus on prenatal screening is that in the near future, the question if, and if so, in what way NIPD testing is to be applied within prenatal screening strategies should be considered and discussed by policy makers, health care professionals and society at large.To avoid confusion, a preliminary remark is needed on terminology. In medicine, ‘screening'' is often used as referring to a kind of test for risk assessment or disease discovery. However, after the convention in normative and regulatory discourse, we will use ‘screening'' as referring to any systematic and unsolicited offer of predictive testing (using whatever types of test) involving individuals who themselves have no reason (yet) to seek medical help for the condition in question.¹⁸ In this broader sense, screening stands in contrast to ‘diagnosis'' as testing on indication.     

Neutrophil Elastase Is Produced by Pulmonary Artery Smooth Muscle Cells and Is Linked to Neointimal Lesions

Previously, we reported that murine gammaherpesvirus-68 (M1-MHV-68) induces pulmonary artery (PA) neointimal lesions in S100A4-overexpressing, but not in wild-type (C57), mice. Lesions were associated with heightened lung elastase activity and PA elastin degradation. We now investigate a direct relationship between elastase and PA neointimal lesions, the nature and source of the enzyme, and its presence in clinical disease. We found an association exists between the percentage of PAs with neointimal lesions and elastin fragmentation in S100A4 mice 6 months after viral infection. Confocal microscopy documented the heightened susceptibility of S100A4 versus C57 PA elastin to degradation by elastase. A transient increase in lung elastase activity occurs in S100A4 mice, 7 days after M1-MHV-68, unrelated to inflammation or viral load and before neointimal lesions. Administration of recombinant elafin, an elastase-specific inhibitor, ameliorates early increases in serine elastase and attenuates later development of neointimal lesions. Neutrophils are the source of elevated elastase (NE) in the S100A4 lung, and NE mRNA and protein levels are greater in PA smooth muscle cells (SMC) from S100A4 mice than from C57 mice. Furthermore, elevated NE is observed in cultured PA SMC from idiopathic PA hypertension versus that in control lungs and localizes to neointimal lesions. Thus, PA SMC produce NE, and heightened production and activity of NE is linked to experimental and clinical pulmonary vascular disease.Pulmonary arterial (PA) neointimal lesions are observed in patients with PA hypertension that is idiopathic (IPAH) or associated with other medical conditions. These vascular abnormalities cause narrowing and even obliteration of the vessel lumen and contribute to the progressive increase in pulmonary vascular resistance that can lead to right ventricular failure (reviewed in Ref. ¹). Only a few murine or rodent models recapitulate this pathological feature, eg, mice exposed to ovalbumin or aspergillus² or to schistosomiasis,³ rats treated with the vascular endothelial receptor blocker Sugen 5416, exposed to chronic hypoxia and recovered in room air,⁴ mice that overexpress IL-6 and are subjected to chronic hypoxia,⁵ or mice that overexpress S100A4.⁶ The latter mice, when over 1 year of age, can on rare occasions “spontaneously” develop severe neointimal lesions.⁶ However, these lesions are observed consistently following infection with murine gammaherpesvirus-68 (MHV-68).⁷ S100A4 is also known as metastasin-1 (mts-1), and is a member of the calcium binding family of proteins that clusters on chromosome 1 and that has been related to cancer and inflammation.⁸The S100A4-overexpressing mouse infected with MHV-68 is relevant to clinical PAH. Increased immunoreactivity for S100A4 is observed in vascular lesions in patients with advanced PAH,⁶ and human immunodeficiency virus (HIV) and HHV-8/Kaposi''s sarcoma virus, the human homologue of MHV-68, have been implicated in clinical PAH. Specifically, the viral protein for HHV-8 has been detected in neointimal and plexiform lesions in lung tissues from some,⁹ albeit not all,¹⁰ series of PAH patients.Pulmonary vascular neointimal and plexiform lesions in S100A4 mice are associated with fragmentation of elastic laminae and with heightened activity of a serine elastase.⁷ Fragmentation of PA elastin has been observed in PAs of PAH patients,¹¹ and heightened activity of a serine elastase has been identified in the PA in a variety of experimental forms of PAH^12–15 and in cultured PA smooth muscle cells (SMC).^16–19 Moreover, inhibition of this elastase can attenuate or prevent^12–14 and even reverse¹⁵ experimental pulmonary vascular disease in rodents. In all rodent models where elastase inhibitors were used, the pulmonary vascular lesions were characterized by loss or increased muscularization of normally nonmuscular peripheral arteries at the alveolar wall and duct level, and medial hypertrophy of proximal muscular arteries. Neointimal lesion formation, however, was not present. Elastase inhibition should, however, attenuate these lesions since proliferation and migration of SMC in the neointima are likely the consequences of elastase-mediated release of growth factors from the extracellular matrix^16,17 and activation of growth factor receptors.²⁰We therefore hypothesized, and subsequently demonstrated in this study, that in the S100A4 overexpressing versus C57 mouse, heightened susceptibility of elastin to fragmentation, coupled to elevated serine elastase activity following M1-MHV-68 infection, can contribute to the development of neointimal lesions. We identified the elastase involved as neutrophil elastase (NE) produced by PA SMC, suggesting that it is the endogenous vascular elastase previously related to PAH in other experimental models.^12,16,18,19 Moreover, we showed that NE is produced in significantly greater amounts by cultured murine S100A4 versus C57 PA SMC, and by human PA SMC from IPAH versus control lungs, and we localized NE to neointimal and plexiform lesions in human lung specimens.     

Aberrant CD8+ T-cell responses and memory differentiation upon viral infection of an ataxia-telangiectasia mouse model driven by hyper-activated Akt and mTORC1 signaling

Immune system-related pathology is common in ataxia-telangiectasia (A-T) patients and mice that lack the protein kinase, A-T mutated (ATM). However, it has not been studied how ATM influences immune responses to a viral infection. Using the lymphocytic choriomeningitis virus (LCMV) infection model, we show that ATM^−/− mice, despite having fewer naïve CD8⁺ T cells, effectively clear the virus. However, aberrant CD8⁺ T-cell responses are observed, including defective expansion and contraction, effector-to-memory differentiation, and a switch in viral-epitope immunodominance. T-cell receptor-activated, but not naïve, ATM^−/− splenic CD8⁺ T cells have increased ribosomal protein S6 and Akt phosphorylation and do not proliferate well in response to IL-15, a cytokine important for memory T-cell development. Accordingly, pharmacological Akt or mammalian target of rapamycin complex 1 (mTORC1) inhibition during T-cell receptor activation alone rescues the IL-15 proliferation defect. Finally, rapamycin treatment during LCMV infection in vivo increases the number of memory T cells in ATM^−/− mice. Altogether, these results show that CD8⁺ T cells lacking ATM have hyperactive Akt and mTORC1 signaling in response to T-cell receptor activation, which results in aberrant cytokine responses and memory T-cell development. We speculate that similar signaling defects contribute to the immune system pathology of A-T, and that inhibition of Akt and/or mTORC1 may be of therapeutic value.Ataxia-telangiectasia (A-T) is a human disease caused by mutations in the gene encoding the PI3-kinase-like protein kinase A-T mutated (ATM).¹ A-T is a multifaceted disease with complex pathology. Cerebellar degeneration underlies the hallmark ataxia symptoms, but another prominent issue is immune system-related pathology, including immunodeficiency and lymphoid cancers.² A-T patients commonly acquire hematological malignancies (eg, leukemia and lymphoma) that together with recurrent bronchial infections account for most of the mortality from the disease.³ ATM gene knock-out mouse models of A-T exhibit many features of the human disease,^4–6 including sexual immaturity, immune system defects, hematopoietic stem cell defects, and thymic lymphoma, the latter of which is the most common cause of death in these animals.^4,7Immunodeficiency associated with decreased production of immunoglobulins A, E, and G2, and thymic hypoplasia has been documented in A-T patients.^8,9 The latter involves decreased peripheral CD4⁺ and CD8⁺ T-lymphocyte pools resulting from developmental defects in the thymic microenvironment.⁹ Because ATM is recruited to double-strand breaks, it is likely that defects in the V(D)J recombination process, which results in a block in differentiation at the CD4⁺/CD8⁺ double-positive stage in the thymus, cause lower thymic output of mature CD4⁺ and CD8⁺ cells. This is corroborated by the ability of a functional T-cell receptor (TCR)-αβ transgene to rescue the deficit in peripheral T cells in ATM^−/− mice.¹⁰ Despite the defective thymic development of T cells in A-T patients, the immune function of mature T cells has been reported to be essentially normal.¹¹ However, to date, there have been no studies of how deficiency of ATM affects the response to an infection in A-T patients or in the mouse models of the disease.The best-defined role for ATM is in the nuclear DNA damage response^2,12; however, other functions for ATM have been described.^12,13 For example, ATM is important for mitochondrial homeostasis,^14,15 insulin signaling,^13,16 phosphorylation of 5''-AMP-activated protein kinase (AMPK),^17–19 and activation of Akt.^16,20 In addition, ATM signals to TSC2 in response to reactive oxygen species,²¹ to inhibit mammalian target of rapamycin complex 1 (mTORC1) that itself is regulated by AMPK and Akt.^22–24 Finally, treatment of mice with the mTORC1 inhibitor rapamycin significantly increases the life span of ATM^−/− mice by delaying development of thymic lymphoma.²⁵ Altogether, these results highlight how the loss of ATM might disrupt the integration of signals that feed into the nutrient-sensing mTORC1 pathway.The CD8⁺ T-cell response is a crucial arm of the adaptive immune system. In response to an infection, these cells are activated through the TCR, proliferate, and differentiate into cytotoxic effector cells that kill infected cells. Most of these cells die after clearance of the pathogen, but a subpopulation survives, loses effector cell properties, and become memory T cells.^26,27 Memory T cells are important for fighting recurrent infections, as they are programmed to respond faster and more effectively to the pathogen. The CD8⁺ T-cell response to infection involves differentiation into short-lived effector cells and memory-precursor cells^26,28 that can be monitored based on surface expression of KLRG1 and CD127 markers. Effector CD8⁺ T cells are preferentially represented in the KLRG1^hi population,^29,30 whereas cells that are CD127^hi, which is the receptor for IL-7, and KLRG1^lo preferentially become long-lived memory T cells.^31–33The response of CD8⁺ T cells to TCR activation and the pathways involved in effector and memory cell differentiation are well-documented.³⁴ These include roles of the AMPK and mTORC1 pathways at several levels. For example, TCR activation leads to rapid activation of AMPK in response to Ca²⁺ signaling, presumably in anticipation of the enormous energy demand required for T-cell expansion.³⁵ In addition, we have shown that AMPK/mTORC1 signaling dynamically regulates mitochondrial biogenesis during TCR activation.³⁶ Finally, treatment of mice with the AMPK activator metformin or the mTORC1 inhibitor rapamycin enhances memory T-cell differentiation by boosting fatty acid oxidation.^33,37 Similarly, mTORC1 regulates differentially effector and memory T-cell commitment,³⁸ and it is a negative regulator of memory T-cell differentiation in mice.^33,37,39The goal of this study was to determine how loss of ATM affects normal CD8⁺ T-cell activation and differentiation upon viral infection and to understand how alterations in mTORC1 and related pathways due to lack of ATM might contribute to the immune-related pathology of A-T using ATM^−/− mice as a model of the disease and the well-characterized murine lymphocytic choriomeningitis virus (LCMV) infection paradigm.     

Cytomegalovirus Impairs Cytotrophoblast-Induced Lymphangiogenesis and Vascular Remodeling in an in Vivo Human Placentation Model

We investigated human cytomegalovirus pathogenesis by comparing infection with the low-passage, endotheliotropic strain VR1814 and the attenuated laboratory strain AD169 in human placental villi as explants in vitro and xenografts transplanted into kidney capsules of SCID mice (ie, mice with severe combined immunodeficiency). In this in vivo human placentation model, human cytotrophoblasts invade the renal parenchyma, remodel resident arteries, and induce a robust lymphangiogenic response. VR1814 replicated in villous and cell column cytotrophoblasts and reduced formation of anchoring villi in vitro. In xenografts, infected cytotrophoblasts had a severely diminished capacity to invade and remodel resident arteries. Infiltrating lymphatic endothelial cells proliferated, aggregated, and failed to form lymphatic vessels. In contrast, AD169 grew poorly in cytotrophoblasts in explants, and anchoring villi formed normally in vitro. Likewise, viral replication was impaired in xenografts, and cytotrophoblasts retained invasive capacity, but some partially remodeled blood vessels incorporated lymphatic endothelial cells and were permeable to blood. The expression of both vascular endothelial growth factor (VEGF)-C and basic fibroblast growth factor increased in VR1814-infected explants, whereas VEGF-A and soluble VEGF receptor-3 increased in those infected with AD169. Our results suggest that viral replication and paracrine factors could undermine vascular remodeling and cytotrophoblast-induced lymphangiogenesis, contributing to bleeding, hypoxia, and edema in pregnancies complicated by congenital human cytomegalovirus infection.Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, with an incidence in the United States of approximately 1% to 3% of live births.¹ Primary maternal HCMV infection during gestation poses a 40% to 50% risk of intrauterine transmission, whereas recurrent infection in seropositive mothers rarely causes disease.^2,3 Symptomatic infants (25%) have intrauterine growth restriction (IUGR) and permanent birth defects, including neurological deficiencies, retinopathy, and sensorineuronal deafness.^4–6 Congenital disease is more severe when primary maternal infection occurs in the first trimester.⁷ IUGR and spontaneous abortion in the absence of fetal HCMV infection can result from placental pathology.^8–10 Placentas infected in early gestation show long-standing damage and fibrosis at the uterine-placental interface, which impairs critical functions and results in a hypoxic intrauterine environment.^10–15 Despite the prevalence and the medical and societal impact of congenital HCMV infection, the mechanisms of virus replication, pathogenesis, and transplacental transmission are still unresolved because of the complex nature of placental development and extreme species specificity of HCMV, which replicates only in human tissues.Differentiating/invading cytotrophoblasts switch to an endothelial phenotype in a process that is similar to vasculogenesis.¹⁶ The cells up-regulate novel adhesion molecules and proteinases that enable their attachment to and invasion of the uterus. Interstitial invasion requires down-regulation of integrins characteristic of epithelial cells and novel expression of the integrins α1β1, α5β1, and αvβ3.¹⁷ Endovascular cytotrophoblasts that remodel uterine blood vessels transform their adhesion receptor phenotype to resemble that of endothelial cells, expressing vascular-endothelial cadherin, platelet-endothelial adhesion molecule-1, and vascular endothelial adhesion molecule-1.^16,18 Like endothelial cells, cytotrophoblasts express substances that influence vasculogenesis and angiogenesis, including the vascular endothelial growth factor (VEGF) family ligands VEGF-A and VEGF-C and receptors VEGFR-1 [fms-like tyrosine kinase 1 (Flt-1)] and VEGFR-3.^19–21 Expression of these molecules changes as the cells differentiate/invade, and they regulate cytotrophoblast survival in the remodeled uterine vasculature. Finally, as hemiallogeneic embryonic/fetal cells, invasive cytotrophoblasts must avoid maternal immune responses. Their expression of the nonclassical major histocompatibility complex (MHC) class I molecules HLA-G^22,23 and HLA-C, which have limited polymorphisms,^24,25 contributes to their lack of immunogenicity.Previous studies led to a rudimentary understanding of HCMV infection of the human placenta and identified several molecular mechanisms that impair functions of differentiating/invading cytotrophoblasts. HCMV infection dysregulates the expression of key integrins required for cell invasiveness,^26,27 reduces the expression of matrix metalloproteinase-9,²⁷ and down-regulates cell-cell and cell-matrix adhesion molecules,²⁸ including those required for pseudovasculogenesis¹⁶ and vascular remodeling.¹⁸ The immunosuppressive viral cytokine cmv IL-10 further reduces cytotrophoblast invasion through paracrine effects that increase IL-10 expression.^27,28 Peroxisome proliferator-activated receptor γ activation by infection also compromises cytotrophoblast functions.^29,30 In chorionic villi, the neonatal Fc receptor for IgG, expressed in syncytiotrophoblasts that contact maternal blood, transcytoses circulating maternal antibodies.^31–33 In conjunction with neutralizing titers, developmental expression of HCMV receptors, EGFR, and integrins^34–37 determines susceptibility to infection.^33,38–40 HCMV infects spatially distinct populations of cytotrophoblasts that express α1β1 and αvβ3 integrins used as surface receptors.⁴¹How HCMV disseminates to the placenta and the early stages of pathogenesis in pregnancy are still unresolved because of the virus'' extreme host range restriction. A successful approach to overcome the obstacle to studies of HCMV in vivo has been to infect SCID mice (ie, mice with severe combined immunodeficiency) that have received xenografts of human tissues. Infection of human fetal thymus/liver under the mouse kidney capsule showed that medullary epithelial cells are prominent targets of HCMV replication.⁴² Thus, dramatic interstrain differences were evident in replication of low-passage clinical isolates and laboratory strains in thymus/liver xenografts in vivo.^42,43 The strain Toledo replicates to high titers in implants, whereas the laboratory strains AD169 and Towne, serially passaged in fibroblasts, are attenuated and fail to propagate in tissues in vivo.⁴³ AD169 lacks a 15-kb segment of viral genome that encodes at least 19 open reading frames present in the genomes of all pathogenic clinical strains.⁴⁴ A deletion mutant of Toledo lacking these sequences, although exhibiting only a minor growth defect in fibroblasts, fails to replicate in thymus/liver implants in SCID mice, evidence that genes in this region are central to infection in vivo.⁴⁵ HCMV replication in endothelial and epithelial cells correlates with determinants specified by ORFs UL128-131A,^46,47 which are highly conserved in clinical isolates⁴⁸ and which elicit neutralizing antibodies in humans.^49,50Herein, we investigated HCMV pathogenesis in infected human placental villous explants and in xenografts maintained in vivo. We used a model of human placentation to investigate the vascular effects of fetal cytotrophoblasts in human placental villi transplanted beneath the kidney capsules of SCID mice.²¹ VR1814, a clinical isolate, infected cell column cytotrophoblasts in placental explants and impaired the formation of anchoring villi in vitro. In xenografts, VR1814-infected placental cells had a severely diminished capacity to invade and form lymphatic vessels. In striking contrast, AD169 replicated poorly in villous explants. In SCID mice, AD169-infected cytotrophoblasts remodeled the resident arteries, but these were faulty. Our results show, for the first time to our knowledge, that HCMV genes dispensable for growth in culture function as determinants of pathogenesis that could contribute to vascular anomalies that originate in early placentation.     

Therapeutic Administration of the Direct Thrombin Inhibitor Argatroban Reduces Hepatic Inflammation in Mice with Established Fatty Liver Disease

Thrombin generation is increased in patients with nonalcoholic fatty liver disease (NAFLD) and in mouse models of diet-induced obesity. Deficiency in the thrombin receptor protease activated receptor-1 reduces hepatic inflammation and steatosis in mice fed a Western diet. However, it is currently unclear whether thrombin inhibitors can modify the pathogenesis of established NAFLD. We tested the hypothesis that thrombin inhibition could reverse hepatic steatosis and inflammation in mice with established diet-induced NAFLD. Low-density lipoprotein receptor–deficient LDLr^−/− mice were fed a control diet or a Western diet for 19 weeks. Mice were given the direct thrombin inhibitor argatroban ∼15 mg/kg/day or its vehicle via a miniosmotic pump for the final 4 weeks of the study. Argatroban administration significantly reduced hepatic proinflammatory cytokine expression and reduced macrophage and neutrophil accumulation in livers of mice fed a Western diet. Argatroban did not significantly impact hepatic steatosis, as indicated by histopathology, Oil Red O staining, and hepatic triglyceride levels. Argatroban reduced serum triglyceride and cholesterol levels in mice fed a Western diet. Argatroban reduced both α-smooth muscle actin expression and Type 1 collagen mRNA levels in livers of mice fed a Western diet, indicating reduced activation of hepatic stellate cells. This study indicates that therapeutic intervention with a thrombin inhibitor attenuates hepatic inflammation and several profibrogenic changes in mice fed a Western diet.More than 70% of patients with abdominal obesity develop concurrent nonalcoholic fatty liver disease (NAFLD).¹ NAFLD, the hepatic manifestation of metabolic syndrome, is characterized by excess accumulation of lipids in the liver (ie, hepatic steatosis)^2,3 and affects approximately 25% of the Western population.⁴ Steatosis accompanied by marked histological inflammation is termed nonalcoholic steatohepatitis (NASH), which is the most severe form of NAFLD and a major cause of liver fibrosis and cirrhosis.^5,6 Progression from simple steatosis to NASH is indicative of a poor clinical outcome and currently has no effective pharmacological treatment options. In addition, both obesity and NAFLD are associated with an increased risk of developing type 2 diabetes mellitus⁷ and cardiovascular disease.^8,9 Therefore, there is an immediate need to identify novel pharmacological approaches to treat NAFLD.A significant commonality among obesity-related diseases is inflammation. Obesity and hepatic steatosis are associated with increased expression of many inflammatory mediators in the liver.¹⁰ The expression of several of these mediators, particularly those involved in leukocyte recruitment, is further increased in patients with NASH.¹⁰ Several compelling studies have demonstrated that inflammatory chemokines such as monocyte chemoattractant protein-1 (MCP-1) and the subsequent recruitment and activation of hepatic macrophages (ie, Kupffer cells) are essential components of NAFLD pathogenesis.^11–14 A systemic proinflammatory state, driven in part by hepatic inflammation, is associated with an increased risk of type 2 diabetes^15,16 and adverse cardiovascular outcomes.¹⁷ In particular, systemic levels of high sensitivity C-reactive protein (hs-CRP), a biomarker of risk for acute cardiovascular events,¹⁸ are primarily dictated by the proinflammatory environment in the liver. Indeed, hs-CRP levels are independently associated with hepatic steatosis in patients with metabolic syndrome.⁸ These studies indicate that increased hepatic inflammation is a focal point of multiple diseases stemming from the metabolic syndrome. Of importance, the molecular triggers of hepatic inflammation in metabolic diseases such as obesity are not completely understood. To this end, understanding the cellular and molecular pathways coordinating hepatic inflammation in metabolic disease could lead to the development of clinical therapies that target inflammation as an underlying cause of multiple interrelated diseases.Because the liver is the primary site of coagulation factor synthesis, liver diseases are often accompanied by a rebalancing of the hemostatic profile.¹⁹ Indeed, abdominal obesity, metabolic syndrome, and NAFLD are each associated with activation of the blood coagulation cascade, including increased generation of the serine protease thrombin.^20–23 Moreover, thrombin generation is increased in mouse models of diet-induced obesity and hypercholesterolemia.^24,25 Previous studies have shown that the induction of tissue factor on monocytes is essential for thrombin generation in mice fed a Western diet.²⁶ Various hepatic manifestations of diet-induced obesity, including hepatic steatosis, are reduced in tissue factor–deficient mice.²⁴ Moreover, we found previously that mice lacking a thrombin receptor, protease activated receptor-1 (PAR-1), did not develop hepatic steatosis when fed a Western diet.²⁴ Although compelling, these genetic approaches do not directly address the question of whether intervention with pharmacological agents, perhaps anticoagulants, can reduce established liver disease. Indeed, it is currently unclear whether pharmacological inhibition of thrombin alters the course of established diet-induced fatty liver disease in mice.To this end, we tested the hypothesis that pharmacological inhibition of thrombin could therapeutically reverse diet-induced hepatic inflammation and steatosis in hypercholesterolemic low density lipoprotein receptor–deficient (LDLr^−/−) mice.     

Organic Cation Transporter 2 Mediates Cisplatin-Induced Oto- and Nephrotoxicity and Is a Target for Protective Interventions

The use of the effective antineoplastic agent cisplatin is limited by its serious side effects, such as oto- and nephrotoxicity. Ototoxicity is a problem of special importance in children, because deafness hampers their language and psychosocial development. Recently, organic cation transporters (OCTs) were identified in vitro as cellular uptake mechanisms for cisplatin. In the present study, we investigated in an in vivo model the role of OCTs in the development of cisplatin oto- and nephrotoxicity. The functional effects of cisplatin treatment on kidney (24 hours excretion of glucose, water, and protein) and hearing (auditory brainstem response) were studied in wild-type and OCT1/2 double-knockout (KO) mice. No sign of ototoxicity and only mild nephrotoxicity were observed after cisplatin treatment of knockout mice. Comedication of wild-type mice with cisplatin and the organic cation cimetidine protected from ototoxicity and partly from nephrotoxicity. For the first time we showed that OCT2 is expressed in hair cells of the cochlea. Furthermore, cisplatin-sensitive cell lines from pediatric tumors showed no expression of mRNA for OCTs, indicating the feasibility of therapeutic approaches aimed to reduce cisplatin toxicities by competing OCT2-mediated cisplatin uptake in renal proximal tubular and cochlear hair cells. These findings are very important to establish chemotherapeutical protocols aimed to maximize the antineoplastic effect of cisplatin while reducing the risk of toxicities.Cisplatin, one of the most effective and potent anticancer drugs, is used in the treatment of a wide variety of both pediatric and adult malignancies.¹ When combined with bleomycin and etoposide, cisplatin is considered to be curative treatment for testicular cancer.² However, the chemotherapeutic use of cisplatin is limited by serious side effects, such as nephrotoxicity and ototoxicity, sometimes requiring a reduction in dose or discontinuation of treatment. Even though nephrotoxicity can be managed with some success by concomitant use of i.v. hydration, it is still a major factor that limits the administration and efficacy of cisplatin in cancer therapy.³ Ototoxicity remains an unresolved clinical problem, especially in infants and younger children, where it leads to a considerable risk of delayed language development because of impaired perception of higher frequency consonant sounds that is of great importance in the presence of background noise. This may have devastating consequences for a young child''s social and educational development.⁴The molecular mechanisms of cellular cisplatin toxicity have been investigated intensively.² The ototoxic effects of cisplatin include loss of outer hair cells, blebbing of outer and inner hair cells, degeneration of the stria vascularis, and a decrease in the number of spiral ganglion cells.⁵ In the kidney, cisplatin highly accumulates in cells of the terminal proximal tubule and of the distal nephron, where it causes either apoptosis or necrosis, depending on exposure time and concentration.² Recently, some attention has been paid to the role of specific cellular uptake processes in cisplatin toxicity. The copper transporter (Ctr)1 seems to be involved in cisplatin accumulation in tumor cells⁶ as well as in the renal epithelial cells.⁷ However, the cisplatin uptake into renal tubular cells is also mediated by organic cation transporter (OCT)2 and the function of OCT2 is critically involved in the development of toxicity.^8,9 In humans, OCT2 (hOCT2) is highly expressed at the basolateral side of all three segments of the proximal tubule.¹⁰ It represents a subtype of three OCTs that belong to the SLC22 transporter family. OCTs are highly expressed in intestine, liver, and/or kidney and play a pivotal role in drug absorption and excretion.¹¹ They are polyspecific, electrogenic uniporters that may operate in both directions. Because the membrane potential provides part of the driving force, they preferably mediate organic cation uptake into cells at normal membrane potential.¹² OCT2 has been proposed as target for protective therapeutic interventions accompanying cisplatin treatment. In vitro studies showed that administration of cisplatin together with a second substrate of organic cation transporters decreases cellular cisplatin accumulation protecting renal cells from cisplatin toxicity.^8,9In the present study, we demonstrated in an in vivo model that OCT2 plays a pivotal role in the development of cisplatin induced oto- and nephrotoxicity. For the first time we showed that cisplatin ototoxicity is linked to the expression of OCT2 in hair cells of the cochlea. Furthermore, the feasibility of a therapeutic approach aimed to reduce cisplatin toxicities has been demonstrated in vivo. These findings are very important to establish chemotherapeutic protocols aimed to maximize the antineoplastic effect of cisplatin while reducing the risk of toxicities.