Histological Characterization of Class I HLA Molecules in Whole Umbilical Cord Tissue Towards an Inexhaustible Graft Alternative for Reconstructive Surgery.

BACKGROUND: Limited graft availability is a constant clinical concern. Hence, the umbilical cord (UC) is an attractive alternative to autologous grafts. The UC is an inexhaustible tissue source, and its removal is harmless and part of standard of care after the birth of the baby. Minimal information exists regarding the immunological profile of a whole UC when it is considered to be used as a tissue graft. We aimed to characterize the localization and levels of class I human leukocyte antigens (HLAs) to understand the allogenicity of the UC. Additionally, HLA-E and HLA-G are putative immunosuppressive antigens that are abundant in placenta, but their profiles in UC whole tissue are unclear. HYPOTHESIS: The UC as a whole expresses a relatively low but ubiquitous level of HLA-ABC and significant levels of HLA-G and HLA-E. METHODS: Healthy patients with no known pregnancy-related complications were approached for informed consent. UCs at term and between 12 and 19 weeks were collected to compare HLA profiles by gestational age. Formalin-fixed paraffin-embedded tissues were sectioned to 5 µm and immunohistochemically stained with a pan-HLA-ABC, two HLA-G-specific, or an HLA-E-specific antibody. RESULTS: HLA-ABC was consistently found present in UCs. HLA-ABC was most concentrated in the UC vessel walls and amniotic epithelium but more dispersed in the Wharton's Jelly. HLA-E had a similar localization pattern to HLA-ABC in whole UC tissues at both gestational ages, but its protein level was lower. HLA-G localization and intensity were poor in all UC tissues analyzed, but additional analyses by Western immunoblot and mass spectrometry revealed a low level of HLA-G in the UC. CONCLUSION: The UC may address limitations of graft availability. Rather than the presence of HLA-G, the immunosuppressive properties of the UC are more likely due to the abundance of HLA-E and the interaction known to occur between HLA-E and HLA-ABC. The co-localization of HLA-E and HLA-ABC suggests that HLA-E is likely presenting HLA-ABC leader peptides to immune cells, which is known to have a primarily inhibitory effect.

Tumor necrosis factor alpha expression is increased in maternal microvascular endothelial cells in preeclampsia.

Objective: The aim of this study was to explore the expression and effects of tumor necrosis factor alpha (TNFα) in maternal endothelial cells in preeclampsia (PE).Methods: Expression levels in primary microvascular endothelial cells (MVEC) isolated from patients with severe preeclampsia (PE) and normal pregnancies were determined by RT-qPCR with or without treatment of TNFα and inhibitors for downstream signaling.Results: PE MVEC exhibited increased basal TNFα expression. TNFα treatment increased TNFα, VCAM, and endocan expression in MVEC.Conclusion: TNFα expression is increased in PE MVEC and the treatment of these cells with exogenous TNFα modifies their gene expression.

The effect of magnesium sulfate on gene expression in maternal microvascular endothelial cells.

Preeclampsia is a severe complication of pregnancy associated with maternal and fetal morbidity and mortality. To date, magnesium sulfate remains the preferred method of treatment used to reduce the development of eclampsia. Our aim was to investigate the effects of magnesium sulfate on the expression of genes involved with endothelial function in maternal microvascular endothelial cells from both normal and preeclamptic pregnancies. Primary cells from normal pregnancies treated with 80 mg/L magnesium sulfate for 6 h revealed an overall trend of increased expression of angiogenic and vasopressor-related factors by qPCR analyses. Primary cells from preeclamptic pregnancies revealed an overall trend of decreased expression, with significantly lowered levels for vascular endothelial growth factor receptor 2, endothelin, and vascular cell adhesion protein-1. A comparison of treated cells revealed significantly increased levels for endoglin, vascular endothelial growth factor receptor 2, soluble fms-like tyrosine kinase-1, prostacyclin synthase, tumor necrosis factor α, tumor necrosis factor receptor 1, and endothelin in normal versus preeclamptic cells following treatment. These results reveal disparate activation of overall expression activity by magnesium sulfate in maternal endothelial cells from normal pregnancies over preeclamptic pregnancies.

Microvascular endothelial cells from preeclamptic women exhibit altered expression of angiogenic and vasopressor factors.

Preeclampsia (PE) is a severe complication of pregnancy associated with maternal and fetal morbidity and mortality. The underlying pathophysiology involves maternal systemic vascular and endothelial dysfunction associated with circulating antiangiogenic factors, although the specific etiology of the disease remains elusive. Our aim was to investigate the maternal endothelium in PE by exploring the expression of genes involved with endothelial function in a novel platform of maternal primary endothelial cells. Adipose tissue was sampled at the time of caesarean section from both normal and preeclamptic patients. Maternal microvascular endothelial cells were isolated by tissue digestion and CD31 magnetic Dynabeads. Cell purity was confirmed by immunofluorescence microscopy and flow cytometry. Western analyses revealed VEGF activation of VEGF receptor 2 (VEGFR2) and ERK in primary cells. Quantitative PCR analyses revealed significantly altered mRNA levels of various genes involved with angiogenesis and blood pressure control in preeclamptic cells, including soluble fms-like tyrosine kinase-1, endoglin, VEGFR2, angiotensin receptor 1, and endothelin compared with cells isolated from normal pregnancies. Overall, maternal endothelial cells from preeclamptic patients exhibit extensive alteration of expression of factors associated with antiangiogenic and vasoconstrictive phenotypes, shedding light on the underlying mechanisms associated with the vascular dysfunction characteristic of PE.

Attenuation of VEGFR-2 expression by sFlt-1 and low oxygen in human placenta.

Vascular endothelial growth factor receptor 2 (VEGFR-2), the primary receptor for VEGF, is crucial for normal endothelial function. sFlt-1, a truncated and soluble form of VEGFR-1 which binds and inhibits VEGF, is increased in preeclampsia and is positively regulated by low oxygen. Here, we investigated the effects of sFlt-1 and hypoxia on VEGFR-2 expression and signaling in the human placenta. VEGFR-2 transcript and protein levels were significantly decreased in preeclamptic placentae compared to controls (1.82 and 1.85 fold, respectively). An inverse correlation was observed for VEGFR-2 and sFlt-1 levels in both singleton and twin placentae from patients with preeclampsia. Immunofluorescence analyses revealed co-localization of VEGFR-2 and sFlt-1 in placental vasculature and co-immunoprecipitation analyses confirmed VEGFR-2 and sFlt-1 interaction only in preeclamptic placentae compared to age-matched controls. VEGFR-2 transcript and protein levels from explants cultured in 3% O2 were significantly decreased compared to those incubated at 20% O2 (5.9 and 12.47 fold, respectively). Also, VEGFR-2 transcript levels were significantly decreased in early first trimester placentae (low oxygen environment) compared to late first trimester placentae (2.05 fold). We next explored whether sFlt-1 directly affects VEGFR-2 expression. Treatment of first trimester placental explants with sFlt-1 resulted in significantly decreased levels of VEGFR-2 (2.03 fold) and downstream signaling proteins phospho-ERK (1.60 fold) and phospho-Akt (1.64 fold). Our findings show a novel hypoxia-induced and PE-related down-regulation of VEGFR-2 in the human placenta. sFlt-1, which is known to be increased in hypoxic conditions and PE, directly attenuates VEGFR-2 expression and signaling. A direct interaction between sFlt-1 and VEGFR-2 may represent an important mechanism in VEGFR-2 regulation, inhibition of VEGFR-2-mediated processes in placentation and a novel platform to examine the onset of preeclampsia.

The fate of the internalized apelin receptor is determined by different isoforms of apelin mediating differential interaction with beta-arrestin.

Internalization of the apelin receptor by apelin-13 is characterized by dissociation from beta-arrestins and rapid recycling to the cell surface. Paradoxically, the apelin receptor internalized by apelin-36 was sequestered intracellularly. The specific pathways involved in apelin receptor trafficking were resolved using beta-arrestin1 and constitutively active and dominant negative Rab proteins following activation by apelin-13 or apelin-36. beta-Arrestin1 dissociated from the apelin-13-internalized receptor while the apelin-36-internalized receptor was trafficked with beta-arrestin1 to intracellular compartments. The apelin-13-internalized receptor was rapidly recycled to the cell surface through a Rab4-dependent mechanism while Rab7 targeted the receptor to lysosomes. The internalized receptor co-expressed with dominant negative Rab4 were trafficked to lysosomes. These observations revealed a novel ligand-dependent targeting of the apelin receptor to beta-arrestin-associated and -dissociated trafficking pathways and a role for different Rab proteins to direct these pathways.

Unravelling the roles of the apelin system: prospective therapeutic applications in heart failure and obesity.

The apelin receptor was initially classed as an orphan G-protein-coupled receptor, and little was known about its physiological functions until apelin, the endogenous ligand, was identified. Similarities between the structure and anatomical distribution of apelin and its receptor and that of angiotensin II and the angiotensin AT1 receptor provide clues about the physiological functions of this novel signal-transduction system. Now, roles have been established for the apelin system in lowering blood pressure, as a potent cardiac inotrope, in modulating pituitary hormone release and food and water intake, in stress activation, and as a novel adipokine that is excreted from fat cells and regulates insulin. Given its broad array of physiological roles, apelin has attracted much interest as a target for novel therapeutic research and drug design.

Modification of the terminal residue of apelin-13 antagonizes its hypotensive action.

The apelin peptide is the endogenous ligand for the apelin G protein-coupled receptor. The distribution of the apelin peptides and receptor are widespread in the central nervous system and periphery, with reported roles in the hypothalamic-pituitary-adrenal axis, blood pressure regulation and as one of the most potent positive inotropic substances yet identified. In this report, we show that in native tissues preproapelin exists as a dimer. Dimeric preproapelin was reduced to monomers by dithiothreitol treatment, indicating disulfide linkages. To evaluate the role of the carboxyl-terminal phenylalanine in the hypotensive action of apelin-13, analogs were generated and tested for their role on blood pressure regulation. Injections of apelin-13 and apelin-12 (15 microg/kg) into spontaneously hypertensive rats lowered systolic and diastolic blood pressure to result in decreases of approximately 60% and 15% in mean arterial blood pressure, respectively. Apelin-13(13[D-Phe]) treatment did not differ from apelin-13 in either efficacy or duration of effect, whereas apelin-13(F13A) revealed a loss of function. However, concomitant administration of apelin-13(F13A) (30 microg/kg) blocked hypotensive effects of apelin-13 (15 microg/kg), which revealed that apelin-13(F13A) behaved as an apelin-specific antagonist.

Agonist-independent nuclear localization of the Apelin, angiotensin AT1, and bradykinin B2 receptors.

Signaling of the apelin, angiotensin, and bradykinin peptides is mediated by G protein-coupled receptors related through structure and similarities of physiological function. We report nuclear expression as a characteristic of these receptors, including a nuclear localization for the apelin receptor in brain and cerebellum-derived D283 Med cells and the AT(1) and bradykinin B(2) receptors in HEK-293T cells. Immunocytochemical analyses revealed the apelin receptor with localization in neuronal nuclei in cerebellum and hypothalamus, exhibiting expression in neuronal cytoplasm or in both nuclei and cytoplasm. Confocal microscopy of HEK-293T cells revealed the majority of transfected cells displayed constitutive nuclear localization of AT(1) and B(2) receptors, whereas apelin receptors did not show nuclear localization in these cells. The majority of apelin receptor-transfected cerebellum D283 Med cells showed receptor nuclear expression. Immunoblot analyses of subcellular-fractionated D283 Med cells demonstrated endogenous apelin receptor species in nuclear fractions. In addition, an identified nuclear localization signal motif in the third intracellular loop of the apelin receptor was disrupted by a substituted glutamine in place of lysine. This apelin receptor (K242Q) did not exhibit nuclear localization in D283 Med cells. These results demonstrate the following: (i) the apelin receptor exhibits nuclear localization in human brain; (ii) distinct cell-dependent mechanisms for the nuclear transport of apelin, AT(1), and B(2) receptors; and (iii) the disruption of a nuclear localization signal sequence disrupts the nuclear translocation of the apelin receptor. This discovery of apelin, AT(1), and B(2) receptors with agonist-independent nuclear translocation suggests major unanticipated roles for these receptors in cell signaling and function.

Continued discovery of ligands for G protein-coupled receptors.

G protein-coupled receptors are under intense scrutiny as potential targets of drug research, which stems mostly from the sheer size and diversity of this receptor family as well as the recognized high levels of specificity and sensitivity attainable by drugs targeting these receptors. The continued discovery of genes encoding G protein-coupled receptors has provided an extensive reserve of potential therapeutic targets. However, testing experimental therapeutic agents at these receptors requires a high degree of receptor characterization, beginning with the identity of an endogenous ligand. Often, low levels of sequence identity of a newly identified receptor to previously characterized receptors preclude the prompt identification of a ligand. In such cases, innovative techniques commonly referred to as reverse pharmacology have been employed to ascertain the ligand's identity for these "orphan" receptors. To date over 30 endogenous ligands, both novel and previously known, have been paired with orphan G protein-coupled receptors. Here, we briefly summarize the recent identification of neuropeptides W and B and carboxylic acid anions for their respective receptors GPR7, GPR8 and GPR40, GPR41, GPR43.

Novel G-protein-coupled receptor genes expressed in the brain: continued discovery of important therapeutic targets.

The rhodopsin family of G-protein-coupled receptors (GPCRs) is the largest known group of cell-surface mediators of signal transduction. The vast majority of these receptors were discovered by methods based upon shared sequence homologies found throughout this family. While such efforts identified a multitude of receptor subtypes for previously known ligands, numerous receptors have been discovered for which endogenous ligands were unknown. These receptors are commonly referred to as orphan receptors. One of the most important tasks of modern pharmacology lies in elucidating the functions of these receptors. Of particular interest are receptors with recognised expression in the central nervous system, given that many psychiatric and neurodegenerative disorders are mediated by unknown mechanisms. Hence, this collection of putative neurotransmitter and neuromodulator signal mediators represents a substantial and untapped resource for novel drug discovery. Recently, various methodologies have accelerated the discovery of novel ligands for these orphan receptors, identifying the basic components required for further physiological ligand/receptor system characterisation. Equipped with proven ligand identification strategies, the characterisation of all orphan GPCRs and the exploitation of their exciting potential as targets for the discovery of novel drugs is anticipated.