COVID-19 plasma proteome reveals novel temporal and cell-specific signatures for disease severity and high-precision disease management.

Coronavirus disease 2019 (COVID-19) is a systemic inflammatory condition with high mortality that may benefit from personalized medicine and high-precision approaches. COVID-19 patient plasma was analysed with targeted proteomics of 1161 proteins. Patients were monitored from Days 1 to 10 of their intensive care unit (ICU) stay. Age- and gender-matched COVID-19-negative sepsis ICU patients and healthy subjects were examined as controls. Proteomic data were resolved using both cell-specific annotation and deep-analysis for functional enrichment. COVID-19 caused extensive remodelling of the plasma microenvironment associated with a relative immunosuppressive milieu between ICU Days 3-7, and characterized by extensive organ damage. COVID-19 resulted in (1) reduced antigen presentation and B/T-cell function, (2) increased repurposed neutrophils and M1-type macrophages, (3) relatively immature or disrupted endothelia and fibroblasts with a defined secretome, and (4) reactive myeloid lines. Extracellular matrix changes identified in COVID-19 plasma could represent impaired immune cell homing and programmed cell death. The major functional modules disrupted in COVID-19 were exaggerated in patients with fatal outcome. Taken together, these findings provide systems-level insight into the mechanisms of COVID-19 inflammation and identify potential prognostic biomarkers. Therapeutic strategies could be tailored to the immune response of severely ill patients.

Endothelial-specific loss of IKKß disrupts pulmonary endothelial angiogenesis and impairs postnatal lung growth.

Pulmonary angiogenesis drives alveolarization, but the transcriptional regulators directing pulmonary angiogenesis remain poorly defined. Global, pharmacological inhibition of nuclear factor-kappa B (NF-κB) impairs pulmonary angiogenesis and alveolarization. However, establishing a definitive role for NF-κB in pulmonary vascular development has been hindered by embryonic lethality induced by constitutive deletion of NF-κB family members. We created a mouse model allowing inducible deletion of the NF-κB activator, IKKß, in endothelial cells (ECs) and assessed the effect on lung structure, endothelial angiogenic function, and the lung transcriptome. Embryonic deletion of IKKß permitted lung vascular development but resulted in a disorganized vascular plexus, while postnatal deletion significantly decreased radial alveolar counts, vascular density, and proliferation of both endothelial and nonendothelial lung cells. Loss of IKKß impaired survival, proliferation, migration, and angiogenesis in primary lung ECs in vitro, in association with decreased expression of VEGFR2 and activation of downstream effectors. Loss of endothelial IKKß in vivo induced broad changes in the lung transcriptome with downregulation of genes related to mitotic cell cycle, extracellular matrix (ECM)-receptor interaction, and vascular development, and the upregulation of genes related to inflammation. Computational deconvolution suggested that loss of endothelial IKKß decreased general capillary, aerocyte capillary, and alveolar type I cell abundance. Taken together, these data definitively establish an essential role for endogenous endothelial IKKß signaling during alveolarization. A deeper understanding of the mechanisms directing this developmental, physiological activation of IKKß in the lung vasculature may provide novel targets for the development of strategies to enhance beneficial proangiogenic signaling in lung development and disease.NEW & NOTEWORTHY This study highlights the cell-specific complexity of nuclear factor kappa B signaling in the developing lung by demonstrating that inducible loss of IKKß in endothelial cells impairs alveolarization, disrupts EC angiogenic function, and broadly represses genes important for vascular development.

Plasma proteome of Long-COVID patients indicates HIF-mediated vasculo-proliferative disease with impact on brain and heart function.

AIMS: Long-COVID occurs after SARS-CoV-2 infection and results in diverse, prolonged symptoms. The present study aimed to unveil potential mechanisms, and to inform prognosis and treatment. METHODS: Plasma proteome from Long-COVID outpatients was analyzed in comparison to matched acutely ill COVID-19 (mild and severe) inpatients and healthy control subjects. The expression of 3072 protein biomarkers was determined with proximity extension assays and then deconvoluted with multiple bioinformatics tools into both cell types and signaling mechanisms, as well as organ specificity. RESULTS: Compared to age- and sex-matched acutely ill COVID-19 inpatients and healthy control subjects, Long-COVID outpatients showed natural killer cell redistribution with a dominant resting phenotype, as opposed to active, and neutrophils that formed extracellular traps. This potential resetting of cell phenotypes was reflected in prospective vascular events mediated by both angiopoietin-1 (ANGPT1) and vascular-endothelial growth factor-A (VEGFA). Several markers (ANGPT1, VEGFA, CCR7, CD56, citrullinated histone 3, elastase) were validated by serological methods in additional patient cohorts. Signaling of transforming growth factor-ß1 with probable connections to elevated EP/p300 suggested vascular inflammation and tumor necrosis factor-α driven pathways. In addition, a vascular proliferative state associated with hypoxia inducible factor 1 pathway suggested progression from acute COVID-19 to Long-COVID. The vasculo-proliferative process predicted in Long-COVID might contribute to changes in the organ-specific proteome reflective of neurologic and cardiometabolic dysfunction. CONCLUSIONS: Taken together, our findings point to a vasculo-proliferative process in Long-COVID that is likely initiated either prior hypoxia (localized or systemic) and/or stimulatory factors (i.e., cytokines, chemokines, growth factors, angiotensin, etc). Analyses of the plasma proteome, used as a surrogate for cellular signaling, unveiled potential organ-specific prognostic biomarkers and therapeutic targets.

Transforming Growth Factor-induced Protein Promotes NF-κB-mediated Angiogenesis during Postnatal Lung Development.

Pulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that NF-κB promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NF-κB activation in the pulmonary vasculature are unknown. To identify mechanisms that activate proangiogenic NF-κB signaling in the developing pulmonary vasculature, proteomic analysis of the lung secretome was performed using two-dimensional difference gel electrophoresis. NF-κB activation and angiogenic function was assessed in primary pulmonary endothelial cells (PECs) and TGFBI (transforming growth factor-ß-induced protein)-regulated genes identified using RNA sequencing. Alveolarization and pulmonary angiogenesis was assessed in wild-type and Tgfbi null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support. Secreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PECs. Proteomic analysis identified TGFBI as one protein highly expressed by the early alveolar lung that promoted PEC migration by activating NF-κB via αvß3 integrins. RNA sequencing identified Csf3 as a TGFBI-regulated gene that enhances nitric oxide production in PECs. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization. Our studies identify TGFBI as a developmentally regulated protein that promotes NF-κB-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth.

Single-Cell Transcriptomic Profiling of Vascular Smooth Muscle Cell Phenotype Modulation in Marfan Syndrome Aortic Aneurysm.

OBJECTIVE: To delineate temporal and spatial dynamics of vascular smooth muscle cell (SMC) transcriptomic changes during aortic aneurysm development in Marfan syndrome (MFS). Approach and Results: We performed single-cell RNA sequencing to study aortic root/ascending aneurysm tissue from Fbn1C1041G/+ (MFS) mice and healthy controls, identifying all aortic cell types. A distinct cluster of transcriptomically modulated SMCs (modSMCs) was identified in adult Fbn1C1041G/+ mouse aortic aneurysm tissue only. Comparison with atherosclerotic aortic data (ApoE-/- mice) revealed similar patterns of SMC modulation but identified an MFS-specific gene signature, including plasminogen activator inhibitor-1 (Serpine1) and Kruppel-like factor 4 (Klf4). We identified 481 differentially expressed genes between modSMC and SMC subsets; functional annotation highlighted extracellular matrix modulation, collagen synthesis, adhesion, and proliferation. Pseudotime trajectory analysis of Fbn1C1041G/+ SMC/modSMC transcriptomes identified genes activated differentially throughout the course of phenotype modulation. While modSMCs were not present in young Fbn1C1041G/+ mouse aortas despite small aortic aneurysm, multiple early modSMCs marker genes were enriched, suggesting activation of phenotype modulation. modSMCs were not found in nondilated adult Fbn1C1041G/+ descending thoracic aortas. Single-cell RNA sequencing from human MFS aortic root aneurysm tissue confirmed analogous SMC modulation in clinical disease. Enhanced expression of TGF-ß (transforming growth factor beta)-responsive genes correlated with SMC modulation in mouse and human data sets. CONCLUSIONS: Dynamic SMC phenotype modulation promotes extracellular matrix substrate modulation and aortic aneurysm progression in MFS. We characterize the disease-specific signature of modSMCs and provide temporal, transcriptomic context to the current understanding of the role TGF-ß plays in MFS aortopathy. Collectively, single-cell RNA sequencing implicates TGF-ß signaling and Klf4 overexpression as potential upstream drivers of SMC modulation.

Divergent effects of canonical and non-canonical TGF-ß signalling on mixed contractile-synthetic smooth muscle cell phenotype in human Marfan syndrome aortic root aneurysms.

Aortic root aneurysm formation is a cardinal feature of Marfan syndrome (MFS) and likely TGF-ß driven via Smad (canonical) and ERK (non-canonical) signalling. The current study assesses human MFS vascular smooth muscle cell (SMC) phenotype, focusing on individual contributions by Smad and ERK, with Notch3 signalling identified as a novel compensatory mechanism against TGF-ß-driven pathology. Although significant ERK activation and mixed contractile gene expression patterns were observed by traditional analysis, this did not directly correlate with the anatomic site of the aneurysm. Smooth muscle cell phenotypic changes were TGF-ß-dependent and opposed by ERK in vitro, implicating the canonical Smad pathway. Bulk SMC RNA sequencing after ERK inhibition showed that ERK modulates cell proliferation, apoptosis, inflammation, and Notch signalling via Notch3 in MFS. Reversing Notch3 overexpression with siRNA demonstrated that Notch3 promotes several protective remodelling pathways, including increased SMC proliferation, decreased apoptosis and reduced matrix metalloproteinase activity, in vitro. In conclusion, in human MFS aortic SMCs: (a) ERK activation is enhanced but not specific to the site of aneurysm formation; (b) ERK opposes TGF-ß-dependent negative effects on SMC phenotype; (c) multiple distinct SMC subtypes contribute to a 'mixed' contractile-synthetic phenotype in MFS aortic aneurysm; and (d) ERK drives Notch3 overexpression, a potential pathway for tissue remodelling in response to aneurysm formation.

NF-κB/NKILA signaling modulates the anti-cancerous effects of EZH2 inhibition.

A wealth of evidence supports the broad therapeutic potential of NF-κB and EZH2 inhibitors as adjuvants for breast cancer treatment. We contribute to this knowledge by elucidating, for the first time, unique regulatory crosstalk between EZH2, NF-κB and the NF-κB interacting long non-coding RNA (NKILA). We define a novel signaling loop encompassing canonical and non-canonical actions of EZH2 on the regulation of NF-κB/NKILA homeostasis, with relevance to breast cancer treatment. We applied a respective silencing approach in non-transformed breast epithelial cells, triple negative MDA-MB-231 cells and hormone responsive MCF-7 cells, and measured changes in EZH2/NF-κB/NKILA levels to confirm their interdependence. We demonstrate cell line-specific fluctuations in these factors that functionally contribute to epithelial-to-mesenchymal transition (EMT) remodelling and cell fate response. EZH2 inhibition attenuates MDA-MB-231 cell motility and CDK4-mediated MCF-7 cell cycle regulation, while inducing global H3K27 methylation and an EMT phenotype in non-transformed cells. Notably, these events are mediated by a cell-context dependent gain or loss of NKILA and NF-κB. Depletion of NF-κB in non-transformed cells enhances their sensitivity to growth factor signaling and suggests a role for the host microenvironment milieu in regulating EZH2/NF-κB/NKILA homeostasis. Taken together, this knowledge critically informs the delivery and assessment of EZH2 inhibitors in breast cancer.

Anatomically specific reactive oxygen species production participates in Marfan syndrome aneurysm formation.

Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root aneurysm formation. Reactive oxygen species (ROS) seem to play a role in aortic wall remodelling in MFS, although the mechanism remains unknown. MFS Fbn1C1039G/+ mouse root/ascending (AS) and descending (DES) aortic samples were examined using DHE staining, lucigenin-enhanced chemiluminescence (LGCL), Verhoeff's elastin-Van Gieson staining (elastin breakdown) and in situ zymography for protease activity. Fbn1C1039G/+ AS- or DES-derived smooth muscle cells (SMC) were treated with anti-TGF-ß antibody, angiotensin II (AngII), anti-TGF-ß antibody + AngII, or isotype control. ROS were detected during early aneurysm formation in the Fbn1C1039G/+ AS aorta, but absent in normal-sized DES aorta. Fbn1C1039G/+ mice treated with the unspecific NADPH oxidase inhibitor, apocynin reduced AS aneurysm formation, with attenuated elastin fragmentation. In situ zymography revealed apocynin treatment decreased protease activity. In vitro SMC studies showed Fbn1C1039G/+ -derived AS SMC had increased NADPH activity compared to DES-derived SMC. AS SMC NADPH activity increased with AngII treatment and appeared TGF-ß dependent. In conclusion, ROS play a role in MFS aneurysm development and correspond anatomically with aneurysmal aortic segments. ROS inhibition via apocynin treatment attenuates MFS aneurysm progression. AngII enhances ROS production in MFS AS SMCs and is likely TGF-ß dependent.

Distinct roles for IκB kinases alpha and beta in regulating pulmonary endothelial angiogenic function during late lung development.

Pulmonary angiogenesis is essential for alveolarization, the final stage of lung development that markedly increases gas exchange surface area. We recently demonstrated that activation of the nuclear factor kappa-B (NFκB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFκB in the pulmonary endothelium, and its downstream targets are not known. In this study, we sought to delineate the specific roles for the NFκB activating kinases, IKKα and IKKß, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKKα or IKKß demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKKα induced mild impairments in angiogenic function, silencing IKKß induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKKß regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKKα and IKKß regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKKß as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKKß activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization.

miR-29b participates in early aneurysm development in Marfan syndrome.

RATIONALE: Marfan syndrome (MFS) is a systemic connective tissue disorder notable for the development of aortic root aneurysms and the subsequent life-threatening complications of aortic dissection and rupture. Underlying fibrillin-1 gene mutations cause increased transforming growth factor-ß (TGF-ß) signaling. Although TGF-ß blockade prevents aneurysms in MFS mouse models, the mechanisms through which excessive TGF-ß causes aneurysms remain ill-defined. OBJECTIVE: We investigated the role of microRNA-29b (miR-29b) in aneurysm formation in MFS. METHODS AND RESULTS: Using quantitative polymerase chain reaction, we discovered that miR-29b, a microRNA regulating apoptosis and extracellular matrix synthesis/deposition genes, is increased in the ascending aorta of Marfan (Fbn1(C1039G/+)) mice. Increased apoptosis, assessed by increased cleaved caspase-3 and caspase-9, enhanced caspase-3 activity, and decreased levels of the antiapoptotic proteins, Mcl-1 and Bcl-2, were found in the Fbn1(C1039G/+) aorta. Histological evidence of decreased and fragmented elastin was observed exclusively in the Fbn1(C1039G/+) ascending aorta in association with repressed elastin mRNA and increased matrix metalloproteinase-2 expression and activity, both targets of miR-29b. Evidence of decreased activation of nuclear factor κB, a repressor of miR-29b, and a factor suppressed by TGF-ß, was also observed in Fbn1(C1039G/+) aorta. Furthermore, administration of a nuclear factor κB inhibitor increased miR-29b levels, whereas TGF-ß blockade or losartan effectively decreased miR-29b levels in Fbn1(C1039G/+) mice. Finally, miR-29b blockade by locked nucleic acid antisense oligonucleotides prevented early aneurysm development, aortic wall apoptosis, and extracellular matrix deficiencies. CONCLUSIONS: We identify increased miR-29b expression as key to the pathogenesis of early aneurysm development in MFS by regulating aortic wall apoptosis and extracellular matrix abnormalities.

Inhibiting NF-κB in the developing lung disrupts angiogenesis and alveolarization.

Bronchopulmonary dysplasia (BPD), a chronic lung disease of infancy, is characterized by arrested alveolar development. Pulmonary angiogenesis, mediated by the vascular endothelial growth factor (VEGF) pathway, is essential for alveolarization. However, the transcriptional regulators mediating pulmonary angiogenesis remain unknown. We previously demonstrated that NF-κB, a transcription factor traditionally associated with inflammation, plays a unique protective role in the neonatal lung. Therefore, we hypothesized that constitutive NF-κB activity is essential for postnatal lung development. Blocking NF-κB activity in 6-day-old neonatal mice induced the alveolar simplification similar to that observed in BPD and significantly reduced pulmonary capillary density. Studies to determine the mechanism responsible for this effect identified greater constitutive NF-κB in neonatal lung and in primary pulmonary endothelial cells (PEC) compared with adult. Moreover, inhibiting constitutive NF-κB activity in the neonatal PEC with either pharmacological inhibitors or RNA interference blocked PEC survival, decreased proliferation, and impaired in vitro angiogenesis. Finally, by chromatin immunoprecipitation, NF-κB was found to be a direct regulator of the angiogenic mediator, VEGF-receptor-2, in the neonatal pulmonary vasculature. Taken together, our data identify an entirely novel role for NF-κB in promoting physiological angiogenesis and alveolarization in the developing lung. Our data suggest that disruption of NF-κB signaling may contribute to the pathogenesis of BPD and that enhancement of NF-κB may represent a viable therapeutic strategy to promote lung growth and regeneration in pulmonary diseases marked by impaired angiogenesis.

The spatial transcriptomic landscape of non-small cell lung cancer brain metastasis.

Brain metastases (BrMs) are a common occurrence in lung cancer with a dismal outcome. To understand the mechanism of metastasis to inform prognosis and treatment, here we analyze primary and metastasized tumor specimens from 44 non-small cell lung cancer patients by spatial RNA sequencing, affording a whole transcriptome map of metastasis resolved with morphological markers for the tumor core, tumor immune microenvironment (TIME), and tumor brain microenvironment (TBME). Our data indicate that the tumor microenvironment (TME) in the brain, including the TIME and TBME, undergoes extensive remodeling to create an immunosuppressive and fibrogenic niche for the BrMs. Specifically, the brain TME is characterized with reduced antigen presentation and B/T cell function, increased neutrophils and M2-type macrophages, immature microglia, and reactive astrocytes. Differential gene expression and network analysis identify fibrosis and immune regulation as the major functional modules disrupted in both the lung and brain TME. Besides providing systems-level insights into the mechanism of lung cancer brain metastasis, our study uncovers potential prognostic biomarkers and suggests that therapeutic strategies should be tailored to the immune and fibrosis status of the BrMs.

Androgens Accentuate TGF-ß Dependent Erk/Smad Activation During Thoracic Aortic Aneurysm Formation in Marfan Syndrome Male Mice.

Background Male patients with Marfan syndrome have a higher risk of aortic events and root dilatation compared with females. The role androgens play during Marfan syndrome aneurysm development in males remains unknown. We hypothesized that androgens potentiate transforming growth factor beta induced Erk (extracellular-signal-regulated kinase)/Smad activation, contributing to aneurysm progression in males. Methods and Results Aortic diameters in Fbn1C1039G/+ and littermate wild-type controls were measured at ages 6, 8, 12, and 16 weeks. Fbn1C1039G/+ males were treated with (1) flutamide (androgen receptor blocker) or (2) vehicle control from age 6 to 16 weeks and then euthanized. p-Erk1/2, p-Smad2, and matrix metalloproteinase (MMP) activity were measured in ascending/aortic root and descending aorta specimens. Fbn1C1039G/+ male and female ascending/aortic root-derived smooth muscle cells were utilized in vitro to measure Erk/Smad activation and MMP-2 activity following dihydrotestosterone, flutamide or transforming growth factor beta 1 treatment. Fbn1C1039G/+ males have increased aneurysm growth. p-Erk1/2 and p-Smad2 were elevated in ascending/aortic root specimens at age 16 weeks. Corresponding with enhanced Erk/Smad signaling, MMP-2 activity was higher in Fbn1C1039G/+ males. In vitro smooth muscle cell studies revealed that dihydrotestosterone potentiates transforming growth factor beta-induced Erk/Smad activation and MMP-2 activity, which is reversed by flutamide treatment. Finally, in vivo flutamide treatment reduced aneurysm growth via p-Erk1/2 and p-Smad2 reduction in Fbn1C1039G/+ males. Conclusions Fbn1C1039G/+ males have enhanced aneurysm growth compared with females associated with enhanced p-Erk1/2 and p-Smad2 activation. Mechanistically, in vitro smooth muscle cell studies suggested that dihydrotestosterone potentiates transforming growth factor beta induced Erk/Smad activation. As biological proof of concept, flutamide treatment attenuated aneurysm growth and p-Erk1/2 and p-Smad2 signaling in Fbn1C1039G/+ males.

Quantitative proteomics reveal lineage-specific protein profiles in iPSC-derived Marfan syndrome smooth muscle cells.

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the FBN1 gene that produces wide disease phenotypic variability. The lack of ample genotype-phenotype correlation hinders translational study development aimed at improving disease prognosis. In response to this need, an induced pluripotent stem cell (iPSC) disease model has been used to test patient-specific cells by a proteomic approach. This model has the potential to risk stratify patients to make clinical decisions, including timing for surgical treatment. The regional propensity for aneurysm formation in MFS may be related to distinct smooth muscle cell (SMC) embryologic lineages. Thus, peripheral blood mononuclear cell (PBMC)-derived induced pluripotent stem cells (iPSC) were differentiated into lateral mesoderm (LM, aortic root) and neural crest (NC, ascending aorta/transverse arch) SMC lineages to model MFS aortic pathology. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) proteomic analysis by tandem mass spectrometry was applied to profile LM and NC iPSC SMCs from four MFS patients and two healthy controls. Analysis revealed 45 proteins with lineage-dependent expression in MFS patients, many of which were specific to diseased samples. Single protein-level data from both iPSC SMCs and primary MFS aortic root aneurysm tissue confirmed elevated integrin αV and reduced MRC2 in clinical disease specimens, validating the iPSC iTRAQ findings. Functionally, iPSC SMCs exhibited defective adhesion to a variety of extracellular matrix proteins, especially laminin-1 and fibronectin, suggesting altered cytoskeleton dynamics. This study defines the aortic embryologic origin-specific proteome in a validated iPSC SMC model to identify novel protein markers associated with MFS aneurysm phenotype. Translating iPSC findings into clinical aortic aneurysm tissue samples highlights the potential for iPSC-based methods to model MFS disease for mechanistic studies and therapeutic discovery in vitro.

The X-linked lymphoproliferative syndrome gene product SAP regulates B cell function through the FcgammaRIIB receptor.

X-linked lympho-proliferative (XLP) is an immunodeficiency condition caused by mutation or deletion of the gene encoding the adaptor protein SAP/SH2D1A. Besides defects in T cell and NK cell function and an absence of NKT cells, XLP can also manifest as lymphomas resulting primarily from uncontrolled B cell proliferation upon acute infection by Epstein-Barr virus. While it has been demonstrated that SAP regulates the functions of T cells and NK cells through the SLAM family of immunoreceptors, its role in B cells has not been defined. Here we show that SAP forms a ternary complex with the kinase Lyn and the inhibitory IgG Fc receptor FcgammaRIIB to regulate B cell proliferation and survival. SAP binds directly and simultaneously to the Lyn SH3 domain and an Immuno-receptor Tyrosine-based Inhibitory Motif (ITIM) in FcgammaRIIB, resulting in the activation of the latter. Moreover, SAP associates with FcgammaRIIB in mouse splenic B cells and promotes its tyrosine phosphorylation. Expression of SAP in the A20 B cell line led to a marked reduction in Blnk phosphorylation, a decrease in Akt activation, and a near-complete ablation of phosphorylation of the MAP kinases Erk1/2, p38 and JNK upon colligation of FcgammaRIIB with the B cell receptor (BCR). In contrast, an XLP-causing SAP mutant was much less efficient in eliciting these effects in B cells. Furthermore, compared to A20 cells, SAP transfectants displayed a significantly reduced rate of proliferation and an increased sensitivity to activation-induced cell death. Collectively these data identify an intrinsic function for SAP in inhibitory signaling in B cells and suggests that SAP may play an important role in balancing positive versus negative immune responses.

A functional nuclear localization signal in insulin-like growth factor binding protein-6 mediates its nuclear import.

IGF binding protein (IGFBP)-6 is a member of the IGFBP family that regulates the actions of IGFs. Although IGFBPs exert their functions extracellularly in an autocrine/paracrine manner, several members of the family, such as IGFBP-3 and -5, possess nuclear localization signals (NLS). To date, no NLS has been described for IGFBP-6, an IGFBP that binds preferentially to IGF-II. We report here that both exogenous and endogenous IGFBP-6 could be imported into the nuclei of rhabdomyosarcoma and HEK-293 cells. Nuclear import of IGFBP-6 was mediated by a NLS sequence that bears limited homology to those found in IGFBP-3 and -5. IGFBP-6 nuclear translocation was an active process that required importins. A peptide corresponding to the IGFBP-6 NLS bound preferentially to importin-alpha. A comprehensive peptide array study revealed that, in addition to positively charged residues such as Arg and Lys, amino acids, notably Gly and Pro, within the NLS, played an important part in binding to importins. Overexpression of wild-type IGFBP-6 increased apoptosis, and the addition of IGF-II did not negate this effect. Only the deletion of the NLS segment abolished the apoptosis effect. Taken together, these results suggest that IGFBP-6 is translocated to the nucleus with functional consequences and that different members of the IGFBP family have specific nuclear import mechanisms.

Apoptosis in human embryo development: 1. Cerebral cortex.

We investigated the apoptosis at the beginning of human cerebral cortex development, in the 6th week of embryogenesis, Carnegie stages 16 and 17. Attention was focused on the dorsal wall of the telencephalon to the ventricular zone of proliferation and to the postmitotic zone with beginning of neuronal migration. We identified apoptotic cells in tissue sections by propidium iodide staining, TUNEL and immunohistochemistry for Fas(APO-1/CD95). We determined the distribution and the percentage (reported to the propidium iodide stained nuclei) of apoptotic TUNEL-positive and Fas(APO-1/CD95)-positive cells. TUNEL-positive apoptotic cells in the proliferative zone were 20% in stage 16 and 60% in stage 17. TUNEL-positive apoptotic cells in the postmitotic zone were 8% in stage16 and 30% in stage 17. CD95-positive apoptotic cells in the proliferative zone were 5% in stage 16 and 2% in stage 17. There were no CD95-positive cells in the postmitotic zone. We evidentiated the presence of the suicide receptor Fas(APO-1/CD95) only on a small population of apoptotic neuroblasts in the proliferative zone. The differences between apoptotic distribution and receptors in early corticogenesis suggest that different apoptotic pathways drive the selection of neuronal populations.

Low-oxygen tension and IGF-I promote proliferation and multipotency of placental mesenchymal stem cells (PMSCs) from different gestations via distinct signaling pathways.

The microenvironment of placental mesenchymal stem cells (PMSCs) is dynamic throughout gestation and determines changes in cell fate. In vivo, PMSCs initially develop in low-oxygen tension and low IGF-I concentrations, and both increase gradually with gestation. The impact of varying concentrations of IGF-I and changing oxygen tension on PMSC signaling and multipotency was investigated in PMSCs from early (preterm) and late (term) gestation human placentae. Preterm PMSCs had greater proliferative response to IGF-I, which was further enhanced by low-oxygen tension. Low-oxygen tension alone was sufficient to induce ERK1/2 phosphorylation, whereas IGF-I was required for AKT (protein kinase B) phosphorylation. Low-oxygen tension prolonged ERK1/2 and AKT phosphorylation with a slowed phosphorylation decay even in presence of IGF-I. Low-oxygen tension maintained higher levels of IGF-I receptor and insulin receptor substrate 1 that were otherwise decreased by exposure to IGF-I and induced a differential phosphorylation pattern on IGF-I receptorß and insulin receptor substrate 1. Phosphorylation of ERK1/2 and AKT was different between the preterm and term PMSCs, and phospho-AKT, and not phospho-ERK1/2, was the major determinant of PMSC proliferation and octamer-4 levels. These studies demonstrate that low-oxygen tension regulates the fate of PMSCs from early and late gestations in response to IGF-I, both independently and dependently, via specific signal transduction mechanisms.

Phosphorylation of IGFBP-1 at discrete sites elicits variable effects on IGF-I receptor autophosphorylation.

We previously demonstrated that hypoxia and leucine deprivation cause hyperphosphorylation of IGF-binding protein-1 (IGFBP-1) at discrete sites that markedly enhanced IGF-I affinity and inhibited IGF-I-stimulated cell growth. In this study we investigated the functional role of these phosphorylation sites using mutagenesis. We created three IGFBP-1 mutants in which individual serine (S119/S169/S98) residues were substituted with alanine and S101A was recreated for comparison. The wild-type (WT) and mutant IGFBP-1 were expressed in Chinese hamster ovary cells and IGFBP-1 in cell media was isolated using isoelectric-focusing-free-flow electrophoresis. BIACore analysis indicated that the changes in IGF-I affinity for S98A and S169A were moderate, whereas S119A greatly reduced the affinity of IGFBP-1 for IGF-I (100-fold, P < .0001). Similar results were obtained with S101A. The IGF-I affinity changes of the mutants were reflected in their ability to inhibit IGF-I-induced receptor autophosphorylation. Employing receptor-stimulation assay using IGF-IR-overexpressing P6 cells, we found that WT-IGFBP-1 inhibited IGF-IRß autophosphorylation (~2-fold, P < .001), possibly attributable to sequestration of IGF-I. Relative to WT, S98A and S169A mutants did not inhibit receptor autophosphorylation. S119A, on the other hand, greatly stimulated the receptor (2.3-fold, P < .05). The data with S101A matched S119A. In summary, we show that phosphorylation at S98 and S169 resulted in milder changes in IGF-I action; nonetheless most dramatic inhibitory effects on the biological activity of IGF-I were due to IGFBP-1 phosphorylation at S119. Our results provide novel demonstration that IGFBP-1 phosphorylation at S119 can enhance affinity for IGF-I possibly through stabilization of the IGF-IGFBP-1 complex. These data also propose that the synergistic interaction of distinct phosphorylation sites may be important in eliciting more pronounced effects on IGF-I affinity that needs further investigation.

An oral versus intranasal prime/boost regimen using attenuated human rotavirus or VP2 and VP6 virus-like particles with immunostimulating complexes influences protection and antibody-secreting cell responses to rotavirus in a neonatal gnotobiotic pig model.

We determined the impact of mucosal prime/boost regimens and vaccine type (attenuated Wa human rotavirus [AttHRV] or nonreplicating Wa 2/6 rotavirus-like particles [VLP]) on protection and antibody-secreting cell (ASC) responses to HRV in a neonatal gnotobiotic pig disease model. Comparisons of delivery routes for AttHRV and evaluation of nonreplicating VLP vaccines are important as alternative vaccine approaches to overcome risks associated with live oral vaccines. Groups of neonatal gnotobiotic pigs were vaccinated using combinations of oral (PO) and intranasal (IN) inoculation routes as follows: (i) 3 oral doses of AttHRV (AttHRV3xPO); (ii) AttHRV3xIN; (iii) AttHRVPO, then 2/6VLP2xIN; (iv) AttHRVIN, then 2/6VLP2xIN; and (v) mock-inoculated controls. Subsets of pigs from each group were challenged with virulent Wa HRV [P1A(8) G1] (4 weeks post-primary inoculation) to assess protection. The AttHRVPO+2/6VLP2xIN pigs had the highest protection rates against virus shedding and diarrhea (71% each); however, these rates did not differ statistically among the vaccine groups, except for the AttHRVIN+2/6VLPIN group, which had a significantly lower protection rate (17%) against diarrhea. The isotype, magnitude, and tissue distribution of ASCs were analyzed by enzyme-linked immunospot assay. The highest mean numbers of virus-specific IgG and IgA ASCs were observed pre- and postchallenge in both intestinal and systemic lymphoid tissues of the AttHRVPO+2/6VLPIN group. Thus, the AttHRVPO+2/6VLPIN vaccine regimen using immunostimulating complexes (ISCOM) and multiple mucosal inductive sites, followed by AttHRV3xPO or IN regimens, were the most effective vaccine regimens, suggesting that either AttHRVPO+2/6VLPIN or AttHRV3xIN may be an alternative approach to AttHRV3xPO for inducing protective immunity against rotavirus diarrhea.