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Pain is necessary to alert us to actual or potential tissue damage. Specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. Pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (HSANs). Although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. Using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the FLVCR1 (Feline Leukemia Virus subgroup C Receptor 1) gene, which encodes a broadly expressed heme exporter. Different FLVCR1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations in FLVCR1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to HSAN. Using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the FLVCR1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. Our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans. [ABSTRACT FROM AUTHOR]

We recently described morgana as an essential protein able to regulate centrosome duplication and genomic stability, by inhibiting ROCK. Here we show that morgana +/- mice spontaneously develop a lethal myeloproliferative disease resembling human atypical chronic myeloid leukemia (aCML), preceded by ROCK hyperactivation, centrosome amplification, and cytogenetic abnormalities in the bone marrow (BM). Moreover, we found that morgana is underexpressed in the BM of patients affected by atypical CML, a disorder of poorly understood molecular basis, characterized by nonrecurrent cytogenetic abnormalities. Morgana is also underexpressed in the BM of a portion of patients affected by Philadelphia-positive CML (Ph+, CML) caused by the BCR-ABL oncogene, and in this condition, morgana underexpression predicts a worse response to imatinib, the standard treatment for Ph+ CML. Thus, morgana acts as an oncosuppressor with different modalities: (1) Morgana underexpression induces centrosome amplification and cytogenetic abnormalities, and (2) in Ph+ CML, it synergizes with BCR-ABL signaling, reducing the efficacy of imatinib treatment. Importantly, ROCK inhibition in the BM of patients underexpressing morgana restored the efficacy of imatinib to induce apoptosis, suggesting that ROCK inhibitors, combined with imatinib treatment, pressed. can overcome suboptimal responses in patients in which morgana is underex pressed. [ABSTRACT FROM AUTHOR]

Juvenile idiopathic arthritis (JIA) is a heterogeneous group of diseases characterized by autoimmune arthritis of unknown cause with onset before age of 16 years. Methotrexate provides clinical benefits in JIA. For children who do not respond to methotrexate, treatment with anti-tumor necrosis factor (TNF)-α is an option. However, some patients do not respond or are intolerant to anti-TNF therapy. Induction of peripheral tolerance has long been considered a promising approach to the treatment of chronic autoimmune diseases. We aimed to evaluate the potentialities of two altered peptide ligands (APLs) derived from human heat-shock protein 60, an autoantigen involved in the pathogenesis of autoimmune arthritis, in JIA patients. Interferon (IFN)-γ, TNF-α and interleukin (IL)-10 levels were determined in ex vivo assays using peripheral blood mononuclear cells (PBMC) from these patients. Wild-type peptide and one of these APLs increased IFN-γ and TNF-α levels. Unlike, the other APLs (called APL2) increased the IL-10 level without affecting IFN-γ and TNF-α levels. On the other hand, APL2 induces a marked activation of T cells since it transforms cell cycle phase's distribution of CD4+ T cells from these patients. In addition, we evaluated the therapeutic effect of APL2 in collagen-induced arthritis model. Therapy with APL2 reduced arthritis scores and histological lesions in mice. This effect was associated to a decrease in TNF-α and IL-17 levels. These results indicate a therapeutic potentiality of APL2 for JIA. [ABSTRACT FROM AUTHOR]

A pharmacological therapy aimed at the multiple targets exposed by the complex pathophysiology of heart failure remains an unmet clinical need. One possible solution is the use of growth hormone secretagogue peptides, which have important cardiotropic properties. The aim of this work was to conduct an experimental evaluation of the effect of growth hormone releasing peptide six (GHRP-6) on heart function. Bi-dimensional experimental echocardiography was used to evaluate the cardiotropic effect of GHRP-6 when 400 μg/kg were administered to Balb/c mice, measuring the ventricular ejection fraction. Dose-dependency was studied with dosages of 100, 200 and 400 μg/kg, and the effect of concomitant beta-blocker usage on the effect of this peptide was assessed in animals chronically treated with metoprolol at 30 mg/kg. GHRP-6 increased the left ventricular ejection fraction without changes on heart rate. This inotropic effect was dose-dependent, and was sustained even in animals chronically treated with metoprolol. [ABSTRACT FROM AUTHOR]

Up to date the search of pharmacological approaches to halt the complex pathophysiology of heart failure remains as an unmet clinical need. Growth hormone secretagogue peptides have been endowed with relevant cardiotropic properties. The aim of this work was to evaluate the cardiac effect of the growth hormone releasing peptide six (GHRP-6). Adult healthy Balb/c mice were intraperitoneally injected with GHRP-6 (400 μg/kg). Immediately after, a bidimensional echocardiography was conducted in order to characterize the ventricular ejection fraction. Three doses of GHRP-6 were tested (100, 200 and 400 μg/kg) and the concomitant effect between GHRP-6 and metoprolol (30 mg/kg) was assessed. GHRP-6 increased ventricular ejection fraction without any modification on heart rate. This inotropic effect was dose-dependent, and was sustained even in those animals chronically treated with metropolol. [ABSTRACT FROM AUTHOR]

Summary: Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity. [Copyright &y& Elsevier]

Abstract: Ferroportin (FPN), the sole characterized iron exporter, is mainly controlled by the peptide hormone hepcidin in response to iron, erythroid factors, hypoxia, and inflammation. In addition, intracellular iron level controls FPN translation by modulating the binding of Iron Responsive Proteins at the 5′UTR of FPN mRNA. Recently, hypoxia inducible factor (HIF)2α has been shown to regulate FPN expression in intestinal cells. Here we show that, during experimentally-induced acute anemia in mice, FPN is regulated at transcriptional level in a cell-specific manner. FPN mRNA level increases in duodenum and spleen macrophages, whereas it does not change in liver and is strongly down-regulated in erythroid precursors. These results were confirmed in Caco2, Raw264.7 and K562 cells treated with a hypoxic stimulus. Moreover, we found a differential expression of HIF1α and HIF2α in cells and tissues that might account for the specificity of FPN regulation. Thus, hypoxia, by directly controlling hepcidin and its target FPN, orchestrates a complex regulatory network aimed at ensuring rapid iron recovery from the periphery and efficient iron utilization in the erythroid compartment. [Copyright &y& Elsevier]

Feline leukemia virus subgroup C receptor 1 (FLVCR1) is a cell membrane heme exporter that maintains the balance between heme levels and globin synthesis in erythroid precursors. It was previously shown that Flvcr1- null mice died in utero due to a failure of erythropoiesis. Here, we identify Flvcr1b, a mitochondrial Flvcr1 isoform that promotes heme efflux into the cytoplasm. Flvcr1b overexpression promoted heme synthesis and in vitro erythroid differentiation, whereas silencing of Flvcr1b caused mitochondrial heme accumulation and termination of erythroid differentiation. Furthermore, mice lacking the plasma membrane isoform (Flvcr1a) but expressing Flvcr1b had normal erythropoiesis, but exhibited hemorrhages, edema, and skeletal abnormalities. Thus, FLVCR1b regulates erythropoiesis by controlling mitochondrial heme efflux, whereas FLVCR1a expression is required to prevent hemorrhages and edema. The aberrant expression of Flvcr1 isoforms may play a role in the pathogenesis of disorders characterized by an imbalance between heme and globin synthesis. [ABSTRACT FROM AUTHOR]

Various agents, including chemotherapeutic drugs, can induce cell senescence. However, the mechanisms involved in the aging pathway, particularly the stress that chemotherapy imposes on telomeres, are still undefined. To address these issues, human mesenchymal stem cells (MSCs) were assessed as target cells to investigate the initiation of the aging process by chemotherapy. The MSCs were obtained from bone marrow (BM) cells from normal adults and grown in the presence of platelet lysates. Cultured MSCs were identified for immunophenotype, and for growth and differentiation properties. The MSCs were exposed to 10 nM doxorubicin and 500 ng/mL etoposide, sublethal doses that induce DNA double-stranded breaks. Telomere length (TL) was assessed by flow-fluorescence in situ hybridization and Southern blotting. Initial TL shortening was detectable in MSCs at 5 days after drug exposure, with progressive reduction compared with untreated cells at 7, 14, 21, and 28 days in culture. After a single exposure, MSCs were unable to regain the lost telomere sequences for up to 28 days in culture. The ATM phosphorylation was documented early after drug exposure, while no telomerase activation was observed. Chemotherapy-induced TL shortening was associated with reduced clonogenic activity in vitro and accelerated adipose differentiation. Analogous behavior in the differentiation pattern was observed in naturally aged MSCs. These results indicate that cultured MSCs represent a useful cellular model to investigate novel drugs that may favor or, conversely, might prevent TL loss in human stem cells. The TL shortening is a permanent signature of previous chemotherapy-mediated DNA damage, and predicts impaired proliferative and differentiation potential. [ABSTRACT FROM AUTHOR]

Aims The Raf-MEK1/2-ERK1/2 (ERK1/2—extracellular signal-regulated kinases 1/2) signalling cascade is crucial in triggering cardiac responses to different stress stimuli. Scaffold proteins are key elements in coordinating signalling molecules for their appropriate spatiotemporal activation. Here, we investigated the role of IQ motif-containing GTPase-activating protein 1 (IQGAP1), a scaffold for the ERK1/2 cascade, in heart function and remodelling in response to pressure overload. Methods and results IQGAP1-null mice have unaltered basal heart function. When subjected to pressure overload, IQGAP1-null mice initially develop a compensatory hypertrophy indistinguishable from that of wild-type (WT) mice. However, upon a prolonged stimulus, the hypertrophic response develops towards a thinning of left ventricular walls, chamber dilation, and a decrease in contractility, in an accelerated fashion compared with WT mice. This unfavourable cardiac remodelling is characterized by blunted reactivation of the foetal gene programme, impaired cardiomyocyte hypertrophy, and increased cardiomyocyte apoptosis. Analysis of signalling pathways revealed two temporally distinct waves of both ERK1/2 and AKT phosphorylation peaking, respectively, at 10 min and 4 days after aortic banding in WT hearts. IQGAP1-null mice show strongly impaired phosphorylation of MEK1/2-ERK1/2 and AKT following 4 days of pressure overload, but normal activation of these kinases after 10 min. Pull-down experiments indicated that IQGAP1 is able to bind the three components of the ERK cascade, namely c-Raf, MEK1/2, and ERK1/2, as well as AKT in the heart. Conclusion These data demonstrate, for the first time, a key role for the scaffold protein IQGAP1 in integrating hypertrophy and survival signals in the heart and regulating long-term left ventricle remodelling upon pressure overload. [ABSTRACT FROM PUBLISHER]

Summary: Adrenergic stimulation of the heart engages cAMP and phosphoinositide second messenger signaling cascades. Cardiac phosphoinositide 3-kinase p110γ participates in these processes by sustaining β-adrenergic receptor internalization through its catalytic function and by controlling phosphodiesterase 3B (PDE3B) activity via an unknown kinase-independent mechanism. We have discovered that p110γ anchors protein kinase A (PKA) through a site in its N-terminal region. Anchored PKA activates PDE3B to enhance cAMP degradation and phosphorylates p110γ to inhibit PIP3 production. This provides local feedback control of PIP3 and cAMP signaling events. In congestive heart failure, p110γ is upregulated and escapes PKA-mediated inhibition, contributing to a reduction in β-adrenergic receptor density. Pharmacological inhibition of p110γ normalizes β-adrenergic receptor density and improves contractility in failing hearts. [Copyright &y& Elsevier]

We recently identified p140Cap as a novel adaptor protein, expressed in epithelial-rich tissues and phosphorylated upon cell matrix adhesion and growth factor treatment. Here, we characterise p140Cap as a novel Src-binding protein, which regulates Src activation via C-terminal Src kinase (Csk). p140Cap silencing increases cell spreading, migration rate and Src kinase activity. Accordingly, increased expression of p140Cap activates Csk, leading to inhibition of Src and downstream signalling as well as of cell motility and invasion. Moreover, cell proliferation and ‘in vivo’ breast cancer cell growth are strongly impaired by high levels of p140Cap, providing the first evidence that p140Cap is a novel negative regulator of tumour growth. [ABSTRACT FROM AUTHOR]

The G protein-coupled, receptor-activated phosphoinositide 3-kinase γ (PI3Kγ) mediates inflammatory responses and negatively controls cardiac contractility by reducing cAMP concentration. Here, we report that mice carrying a targeted mutation in the PI3Kγ gene causing loss of kinase activity (PI3KγKD/KD) display reduced inflammatory reactions but no alterations in cardiac contractility. We show that, in PI3KγKD/KD hearts, cAMP levels are normal and that PI3Kγ-deficient mice but not PI3KγKD/KD mice develop dramatic myocardial damage after chronic pressure overload induced by transverse aortic constriction (TAC). Finally, our data indicate that PI3Kγ is an essential component of a complex controlling PDE3B phosphodiesterase-mediated cAMP destruction. Thus, cardiac PI3Kγ participates in two distinct signaling pathways: a kinase-dependent activity that controls PKB/Akt as well as MAPK phosphorylation and contributes to TAC-induced cardiac remodeling, and a kinase-independent activity that relies on protein interactions to regulate PDE3B activity and negatively modulates cardiac contractility. [Copyright &y& Elsevier]

Melusin is a muscle specific protein required for heart hypertrophy in response to mechanical overload. Here we describe a protein 63% homologous to melusin, named chp-1, expressed in all tissues tested, including muscles, and highly conserved from invertebrates to human. Both proteins are characterized in their N-terminal half by a tandemly repeated zinc binding 60 amino acid domain with a motif of uniquely spaced cysteine and histidine residues. These motives are highly conserved from plants to mammals and have been recently named CHORD (for cysteine and histidine rich domain) domains. At the C-terminal end melusin contains a calcium binding stretch of 30 acidic amino acid residues which is absent in chp-1. While invertebrate genome contains only one gene coding for a chp-1 homolog, two genes coding for CHORD containing proteins (chp-1 and melusin) are present in vertebrates. Sequence analysis suggests that the muscle specific CHORD containing protein melusin originated by a gene duplication event during early chordate evolution. [Copyright &y& Elsevier]

Investigates the importance of cell matrix adhesion in cell proliferation and survival following mutation of the integrin cytodomain. Abnormalities of the chorion-allantois fusion; Signaling properties of homozygous murine embryonic fibroblasts; Link between integrin cytoplasmic and adhesion-mediated action of cells.

Examines the role of the integrin cytoplasmic domain-associated protein ICAP-1 in the binding and regulation of Rho family GTPases during cell spreading. Isolation of ICAP-1 by two-hybrid screening and binding specificity; Inhibition of the initial phase of cell spreading; Role of an activated form of Cdc42 in reversing ICAP-1 inhibition of cell spreading.

The Dbl oncogene is a putative exchange factor for the small GTPases RhoA and Cdc42, which are involved in actin polymerization into stress fibers and filopodia, respectively. We report here that, upon adhesion to fibronectin, Dbl-transformed NIH3T3 cells display a contracted, polygonal shape with a high number of short stress fibers. In contrast, untransformed NIH3T3 cells acquire the characteristic fibroblast morphology and organize a regular mesh of long stress fibers. We show that in Dbl-transformed and in untransformed NIH3T3 cells the different shape and actin cytoskeleton organization observed in the early steps of adhesion involves activation of distinct GTPases. Upon adhesion to fibronectin, cell morphology of Dbl-transformed NIH3T3 cells depends on activation of RhoA and not of Cdc42. In contrast Cdc42 activation is necessary to untransfected NIH3T3 cells to acquire their fibroblast shape. In both Dbl-transformed and in untransformed NIH3T3 cells a basal Rac activation is necessary to support stress fiber organization, while constitutive Rac activation promotes ruffles and lamellipodia formation. As a consequence of RhoA activation, Dbl-transformed cells show high activity of ROCK-α and CRIK kinases, two known RhoA effectors. In addition Dbl-transformed and NIH3T3 cells expressing the constitutive active form of RhoA are less motile on fibronectin than cells expressing constitutive active Cdc42. We conclude that in NIH3T3 cells in response to fibronectin the expression of the Dbl oncogene leads to a predominant activation of RhoA which both supports the peculiar cell shape and actin cytoskeleton organization in stress fibers and regulates cell motility. Oncogene (2000) 19, 1428–1436. [ABSTRACT FROM AUTHOR]

Presents information on a study on the roles of the receptor-activated isoforms of phosphoinositide 3-kinase (P13K) in leukocyte function. Assessment on the physiologic role of the P13K gamma isoform; Characterization of the migratory deficiencies observed; Conclusion.

Adhesion of human primary skin fibroblasts and ECV304 endothelial cells to immobilized matrix proteins, β1 or αv integrin antibodies stimulates tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. This tyrosine phosphorylation is transiently induced, reaching maximal levels 30 min after adhesion, and it occurs in the absence of receptor ligands. Similar results were observed with EGF receptor­transfected NIH-3T3 cells. Use of a kinase-negative EGF receptor mutant demonstrates that the integrin-stimulated tyrosine phosphorylation is due to activation of the receptor's intrinsic kinase activity. Integrin­mediated EGF receptor activation leads to Erk-1/MAP kinase induction, as shown by treatment with the specific inhibitor tyrphostin AG1478 and by expression of a dominant-negative EGF receptor mutant. EGF receptor and Erk-1/MAP kinase activation by integrins does not lead per se to cell proliferation, but is important for entry into S phase in response to EGF or serum. EGF receptor activation is also required for extracellular matrix-mediated cell survival. Adhesion-dependent MAP kinase activation and survival are regulated through EGF receptor activation in cells expressing this molecule above a threshold level (5 × 103 receptors per cell). These results demonstrate that integrin­dependent EGF receptor activation is a novel signaling mechanism involved in cell survival and proliferation in response to extra-cellular matrix. [ABSTRACT FROM AUTHOR]

Analyzes the expression of muscle-specific B1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, B1 integrin minus cells. Methodology used; Interaction of talin and a-Actinin with B1A and B1D cytoplasmic domain peptides; Results of the findings.

Interleukin-15 (IL-15) is a pro-inflammatory cytokine that is expressed in several autoimmune and inflammatory diseases. We have identified the 36-45 sequence KVTAMKCFLL on human IL-15 that is recognized by a soluble form of recombinant hIL-15Rα-Fc fusion protein. This sequence synthesized as a 10 aa. peptide binds to the IL-15Rα and was able to block the biological activity of IL-15 in two IL-15 dependent cells lines. Using alanine scan strategy we identified a more active peptide by replacing the second Lys in the sequence for the polar non-charged amino acid threonine. Moreover, soluble IL-15Rα was quantitated by a newly developed enzyme-linked immunosorbent assay (ELISA) using the P8 peptide as capture in samples of synovial fl uid from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). This research won the 2012 Award of the Cuban National Academy of Sciences. [ABSTRACT FROM AUTHOR]

Liver diseases represent a major global health issue, and currently, liver transplantation is the only viable alternative to reduce mortality rates in patients with end-stage liver diseases. However, scarcity of donor organs and risk of recidivism requiring a re-transplantation remain major obstacles. Hence, much hope has turned towards cell-based therapy. Hepatocyte-like cells obtained from embryonic stem cells or adult stem cells bearing multipotent or pluripotent characteristics, as well as cell-based systems, such as organoids, bio-artificial liver devices, bioscaffolds and organ printing are indeed promising. New approaches based on extracellular vesicles are also being investigated as cell substitutes. Extracellular vesicles, through the transfer of bioactive molecules, can modulate liver regeneration and restore hepatic function. This review provides an update on the current state-of-art cell-based and cell-free strategies as alternatives to liver transplantation for patients with end-stage liver diseases. [ABSTRACT FROM AUTHOR]

Defining the molecular mechanisms underlying cardiac resilience is crucial to find effective approaches to protect the heart. A physiologic level of ROS is produced in the heart by fatty acid oxidation, but stressful events can boost ROS and cause mitochondrial dysfunction and cardiac functional impairment. Melusin is a muscle specific chaperone required for myocardial compensatory remodeling during stress. Here we report that Melusin localizes in mitochondria where it binds the mitochondrial trifunctional protein, a key enzyme in fatty acid oxidation, and decreases it activity. Studying both mice and human induced pluripotent stem cell-derived cardiomyocytes, we found that Melusin reduces lipid oxidation in the myocardium and limits ROS generation in steady state and during pressure overload and doxorubicin treatment, preventing mitochondrial dysfunction. Accordingly, the treatment with the lipid oxidation inhibitor Trimetazidine concomitantly with stressful stimuli limits ROS accumulation and prevents long-term heart dysfunction. These findings disclose a physiologic mechanism of metabolic regulation in the heart and demonstrate that a timely restriction of lipid metabolism represents a potential therapeutic strategy to improve cardiac resilience to stress., (© 2024. The Author(s).)

The rise in biological therapies has revolutionized oncology, with immunotherapy leading the charge through breakthroughs such as CAR-T cell therapy for melanoma and B-ALL. Modified bispecific antibodies and CAR-T cells are being developed to enhance their effectiveness further. However, CAR-T cell therapy currently relies on a costly ex vivo manufacturing process, necessitating alternative strategies to overcome this bottleneck. Targeted in vivo viral transduction offers a promising avenue but remains under-optimized. Additionally, novel approaches are emerging, such as in vivo vaccine boosting of CAR-T cells to strengthen the immune response against tumors, and dendritic cell-based vaccines are under investigation. Beyond CAR-T cells, mRNA therapeutics represent another promising avenue. Targeted delivery of DNA/RNA using lipid nanoparticles (LNPs) shows potential, as LNPs can be directed to T cells. Moreover, CRISPR editing has demonstrated the ability to precisely edit the genome, enhancing the effector function and persistence of synthetic T cells. Enveloped delivery vehicles packaging Cas9 directed to modified T cells offer a virus-free method for safe and effective molecule release. While this platform still relies on ex vivo transduction, using cells from healthy donors or induced pluripotent stem cells can reduce costs, simplify manufacturing, and expand treatment to patients with low-quality T cells. The use of allogeneic CAR-T cells in cancer has gained attraction for its potential to lower costs and broaden accessibility. This review emphasizes critical strategies for improving the selectivity and efficacy of immunotherapies, paving the way for a more targeted and successful fight against cancer.

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro . Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system, guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study, we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR, immunofluorescence, and Western blot analysis. Additionally, we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes, including CYP450, urea cycle enzymes, and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes, evident as early as 1 day post-differentiation. Interestingly, HLSCs transiently upregulated stem cell markers during differentiation, followed by downregulation after 7 days. Furthermore, differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24 h, with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications., Competing Interests: GC is a member of the Scientific Advisory Board of Unicyte AG. MT, GS, VF, GC, and MBHS are named inventors in related patents. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tapparo, Saccu, Pasquino, Fonsato, Medana, Schiavo, Mecarelli, Maccagno, Silengo, Bruno, Camussi and Herrera Sanchez.)

Crigler Najjar Syndrome type I (CNSI) is a rare recessive disorder caused by mutations in the Ugt1a1 gene. There is no permanent cure except for liver transplantation, and current therapies present several shortcomings. Since stem cell-based therapy offers a promising alternative for the treatment of this disorder, we evaluated the therapeutic potential of human liver stem cells (HLSC) in immune-compromised NOD SCID Gamma (NSG)/Ugt1 -/- mice, which closely mimic the pathological manifestations in CNSI patients. To assess whether HLSC expressed UGT1A1, decellularised mouse liver scaffolds were repopulated with these cells. After 15 days' culture ex vivo, HLSC differentiated into hepatocyte-like cells showing UGT1A1 expression and activity. For the in vivo human cell engraftment and recovery experiments, DiI-labelled HLSC were injected into the liver of 5 days old NSG/Ugt1 -/- pups which were analysed at postnatal Day 21. HLSC expressed UGT1A1 in vivo, induced a strong decrease in serum unconjugated bilirubin, thus significantly improving phenotype and survival compared to untreated controls. A striking recovery from brain damage was also observed in HLSC-injected mutant mice versus controls. Our proof-of-concept study shows that HLSC express UGT1A1 in vivo and improve the phenotype and survival of NSG/Ugt1 -/- mice, and show promises for the treatment of CNSI.

Heme is required for cell respiration and survival. Nevertheless, its intracellular levels need to be finely regulated to avoid heme excess, which may catalyze the production of reactive oxygen species (ROS) and promote cell death. Here, we show that alteration of heme homeostasis in endothelial cells due to the loss of the heme exporter FLVCR1a, results in impaired angiogenesis. In vitro, FLVCR1a silencing in endothelial cells causes defective tubulogenesis and poor viability due to intracellular heme accumulation. Consistently, endothelial-specific Flvcr1a knockout mice show aberrant angiogenesis responsible for hemorrhages and embryonic lethality. Importantly, we demonstrate that impaired heme export leads to endothelial cell death by paraptosis and provide evidence that endoplasmic reticulum (ER) stress precedes heme-induced paraptosis. These findings highlight a crucial role for the cytosolic heme pool in the control of endothelial cell survival and in the regulation of the angiogenic process. Interfering with endothelial heme export represents a valuable model for a deeper understanding of the molecular mechanisms underlying heme-triggered paraptosis and, in the future, might provide a novel tool for the modulation of angiogenesis in pathophysiologic conditions.

NF-κB is a transcription factor involved in the regulation of multiple physiological and pathological cellular processes, including inflammation, cell survival, proliferation, and cancer cell metastasis. NF-κB is frequently hyperactivated in several cancers, including triple-negative breast cancer. Here we show that NF-κB activation in breast cancer cells depends on the presence of the CHORDC1 gene product Morgana, a previously unknown component of the IKK complex and essential for IκBα substrate recognition. Morgana silencing blocks metastasis formation in breast cancer mouse models and this phenotype is reverted by IκBα downregulation. High Morgana expression levels in cancer cells decrease recruitment of natural killer cells in the first phases of tumor growth and induce the expression of cytokines able to attract neutrophils in the primary tumor, as well as in the pre-metastatic lungs, fueling cancer metastasis. In accordance, high Morgana levels positively correlate with NF-κB target gene expression and poor prognosis in human patients.

The data presented in this article are related to the research article entitled Hemopexin counteracts systolic dysfunction induced by heme-driven oxidative stress (G. Ingoglia, C. M. Sag, N. Rex, L. De Franceschi, F. Vinchi, J. Cimino, S. Petrillo, S. Wagner, K. Kreitmeier, L. Silengo, F. Altruda, L. S. Maier, E. Hirsch, A. Ghigo and E. Tolosano, 2017) [1]. Data show that heme induces reactive oxygen species (ROS) production in primary cardiomyocytes. H9c2 myoblastic cells treated with heme bound to human Hemopexin (Hx) are protected from heme accumulation and oxidative stress. Similarly, the heme-driven oxidative response is reduced in primary cardiomyocytes treated with Hx-heme compared to heme alone. Our in vivo data show that mouse models of hemolytic disorders, β-thalassemic mice and phenylhydrazine-treated mice, have low serum Hx associated to enhanced expression of heme- and oxidative stress responsive genes in the heart. Hx -/- mice do not show signs of heart fibrosis or overt inflammation. For interpretation and discussion of these data, refer to the research article referenced above.

Feline leukemia virus subgroup C receptor 1 (Flvcr1) encodes two heme exporters: FLVCR1a, which localizes to the plasma membrane, and FLVCR1b, which localizes to mitochondria. Here, we investigated the role of the two Flvcr1 isoforms during erythropoiesis. We showed that, in mice and zebrafish, Flvcr1a is required for the expansion of committed erythroid progenitors but cannot drive their terminal differentiation, while Flvcr1b contributes to the expansion phase and is required for differentiation. FLVCR1a-down-regulated K562 cells have defective proliferation, enhanced differentiation, and heme loading in the cytosol, while FLVCR1a/1b-deficient K562 cells show impairment in both proliferation and differentiation, and accumulate heme in mitochondria. These data support a model in which the coordinated expression of Flvcr1a and Flvcr1b contributes to control the size of the cytosolic heme pool required to sustain metabolic activity during the expansion of erythroid progenitors and to allow hemoglobinization during their terminal maturation. Consistently, reduction or increase of the cytosolic heme rescued the erythroid defects in zebrafish deficient in Flvcr1a or Flvcr1b, respectively. Thus, heme export represents a tightly regulated process that controls erythropoiesis., (Copyright© Ferrata Storti Foundation.)

Morgana/CHP-1 is a ubiquitously expressed protein able to inhibit ROCK II kinase activity. We have previously demonstrated that morgana haploinsufficiency leads to multiple centrosomes, genomic instability, and higher susceptibility to tumour development. While a large fraction of human cancers has shown morgana down-regulation, a small subset of tumours was shown to express high morgana levels. Here we demonstrate that high morgana expression in different breast cancer subtypes correlates with high tumour grade, mitosis number, and lymph node positivity. Moreover, morgana overexpression induces transformation in NIH-3T3 cells and strongly protects them from various apoptotic stimuli. From a mechanistic point of view, we demonstrate that morgana causes PTEN destabilization, by inhibiting ROCK activity, hence triggering the PI3K/AKT survival pathway. In turn, morgana down-regulation in breast cancer cells that express high morgana levels increases PTEN expression and leads to sensitization of cells to chemotherapy., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background & Aims: The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice., Methods: We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1a(fl/fl);alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured., Results: Flvcr1a(fl/fl);alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1a(fl/fl);alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450., Conclusions: In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The tissue-specific gene expression changes mediated by the hypoxia inducible factors (HIFs) allow the adaptation of cells to low oxygen tension and control several processes including erythropoiesis, angiogenesis and vasculogenesis. The Feline Leukemia Virus, subgroup C, Receptor 1 (Flvcr1) gene encodes for two isoforms, Flvcr1a and 1b, involved in the export of heme out of the cell and of mitochondria respectively. Studies in mouse models demonstrated a crucial role of Flvcr1 isoforms in erythropoiesis and during embryo development. Here, we showed the modulation of Flvcr1 gene expression in different tissues and cell lines in response to hypoxia. Chromatin immunoprecipitation analysis demonstrated that HIF2α and HIF-dependent transcription factor ETS1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1) bind at the region -318/+39 of the Flvcr1 promoter. Analysis of Caco2 cells in which HIF2α or ETS1 were silenced or overexpressed demonstrated that, both HIF2α and ETS1 are involved in the transcriptional regulation of Flvcr1a and that HIF2α is absolutely required for Flvcr1a induction upon hypoxia. The inclusion of the Flvcr1 gene in the group of HIF2α-responsive genes strengthens its role in hypoxia-stimulated processes like erythropoiesis, vasculogenesis and heme absorption., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The concept that extracellular vesicles may act as paracrine/endocrine effectors is based on the evidence that they are able to transport bioactive molecules between cells, either within a defined microenvironment or remotely, by entering the biologic fluids. Extracellular vesicles, including exosomes and microvesicles, may deliver lipids and various functional transcripts, released from the cell of origin, to target cells. Since extracellular vesicles contain defined patterns of mRNA, microRNA, long non-coding RNA, and occasionally genomic DNA, they may transfer genetic information which induces transient or persistent phenotypic changes in recipient cells. In this review, we will discuss potential physiologic and pathological implications of extracellular vesicles, as well as the diagnostic and therapeutic opportunities that they may provide.

Transferrin receptor 2 (TFR2) is a transmembrane protein that is mutated in hemochromatosis type 3. The TFR2 gene is transcribed in 2 main isoforms: the full-length (alpha) and a shorter form (beta). alpha-Tfr2 is the sensor of diferric transferrin, implicated in the modulation of hepcidin, the main regulator of iron homeostasis. The function of the putative beta-Tfr2 protein is unknown. We have developed a new mouse model (KI) lacking beta-Tfr2 compared with Tfr2 knockout mice (KO). Adult Tfr2 KO mice show liver iron overload and inadequate hepcidin levels relative to body iron stores, even though they increase Bmp6 production. KI mice have normal transferrin saturation, liver iron concentration, hepcidin and Bmp6 levels but show a transient anemia at young age and severe spleen iron accumulation in adult animals. Fpn1 is strikingly decreased in the spleen of these animals. These findings and the expression of beta-Tfr2 in wild-type mice spleen suggest a role for beta-Tfr2 in Fpn1 transcriptional control. Selective inactivation of liver alpha-Tfr2 in KI mice (LCKO-KI) returned the phenotype to liver iron overload. Our results strengthen the function of hepatic alpha-Tfr2 in hepcidin activation, suggest a role for extrahepatic Tfr2 and indicate that beta-Tfr2 may specifically control spleen iron efflux.

Haemopexin (Hx) is an acute phase plasma glycoprotein, mainly produced by the liver and released into plasma where it binds heme with high affinity and delivers it to the liver. This system provides protection against free heme-mediated oxidative stress, limits access by pathogens to heme and contributes to iron homeostasis by recycling heme iron. Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF). Recently, a comparative proteomic analysis has shown an increase of Hx in CSF from patients with Alzheimer's disease, thus suggesting its involvement in heme detoxification in brain. Here, we report that Hx is synthesised in brain by the ventricular ependymal cells. To verify whether Hx is involved in heme scavenging in brain, and consequently, in the control of iron level, iron deposits and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron-loaded oligodendrocytes in the basal ganglia and thalamus of Hx-null mice compared to wild-type controls. Interestingly, there was no increase in H- and L-ferritin expression in these regions. This condition is common to several human neurological disorders such as Alzheimer's disease and Parkinson's disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin-positive cells was observed in the cerebral cortex of Hx-null animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and Cu-Zn superoxide dismutase-1 expression were higher in the brain of Hx-null mice than in that of wild-type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron-related neurodegenerative diseases.

Intravascular hemolysis results in the release of massive amounts of hemoglobin and heme into plasma, where they are rapidly bound by haptoglobin and hemopexin, respectively. Data from haptoglobin and hemopexin knockout mice have shown that both proteins protect from renal damage after phenylhydrazine-induced hemolysis, whereas double-mutant mice were especially prone to liver damage. However, the specific role of hemopexin remains elusive because of the difficulty in discriminating between hemoglobin and heme recovery. To study the specific role of hemopexin in intravascular hemolysis, we established a mouse model of heme overload. Under these conditions, both endothelial activation and vascular permeability were significantly higher in hemopexin-null mice compared with wild-type controls. Vascular permeability was particularly altered in the liver, where congestion in the centrolobular area was believed to be associated with oxidative stress and inflammation. Liver damage in hemopexin- null mice may be prevented by induction of heme oxygenase-1 before heme overload. Furthermore, heme-treated hemopexin-null mice exhibited hyperbilirubinemia, prolonged heme oxygenase-1 expression, excessive heme metabolism, and lack of H-ferritin induction in the liver compared with heme-treated wild-type controls. Moreover, these mutant mice metabolize an excess of heme in the kidney. These studies highlight the importance of hemopexin in heme detoxification, thus suggesting that drugs mimicking hemopexin activity might be useful to prevent endothelial damage in patients suffering from hemolytic disorders.

Background & Aims: Haptoglobin is an acute phase protein responsible for the recovery of free hemoglobin from plasma. Haptoglobin-null mice were previously shown to have an altered heme-iron distribution, thus reproducing what occurs in humans in cases of congenital or acquired anhaptoglobinemia. Here, we report the analysis of iron homeostasis in haptoglobin-null mice., Methods: Iron absorption was measured in tied-off duodenal segments. Iron stores were evaluated on tissue homogenates and sections. The expression of molecules involved in iron homeostasis was analyzed at the protein and messenger RNA levels both in mice and in murine RAW264.7 macrophages stimulated in vitro with hemoglobin., Results: Analysis of intestinal iron transport reveals that haptoglobin-null mice export significantly more iron from the duodenal mucosa to plasma compared with control counterparts. Increased iron export from the duodenum correlates with increased duodenal expression of ferroportin, both at the protein and messenger RNA levels, whereas hepatic hepcidin expression remains unchanged. Up-regulation of the ferroportin transcript, but not of the protein, also occurs in haptoglobin-null spleen macrophages, which accumulate free hemoglobin-derived iron. Finally, we demonstrate that hemoglobin induces ferroportin expression in RAW264.7 cells., Conclusions: Taking together these data, we suggest that haptoglobin, by controlling plasma levels of hemoglobin, participates in the regulation of ferroportin expression, thus contributing to the regulation of iron transfer from duodenal mucosa to plasma.

Targeted imaging requires site-specific accumulation of a contrast agent (CA), and the properties of that agent must be selected according to the abundance of the target to obtain a signal above the detection limit of the instrument. However, numerical estimates of receptors per cell are rarely found in the literature. Integrin receptors would be particularly promising targets because of their accessibility from the blood stream and expression on activated neovascular endothelial cells. We systematically estimated the number of integrin receptors of cell lines and primary cells by flow cytometry analysis. Since integrin receptors are heterodimeric molecules, and alpha(v) forms complexes with various beta subunits, the numbers of alpha(v) and beta(3) subunits are therefore dissimilar. The observed values are 3 . 10(3)-1.4 . 10(4)/cell for alpha(v), and 5.3 . 10(2)-1.1 . 10(4)/cell for beta(3). Despite the low number of exposed receptors, we show that up to single-cell MR visualization can be achieved with the use of iron oxide beads complexed with antibodies as CAs.

Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser967) and revealed constitutive phosphorylation of Thr669, Ser967, Ser1002, and Tyr1045 and stimulation-dependent differential phosphorylation of Tyr1068, Tyr1086, Ser1142, Tyr1148, and Tyr1173. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8-3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide 1137GSHQISLDNPDYQQDFFPK1155; however, only Tyr1148 was phosphorylated upon EGF treatment; in contrast Ser1142 was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes.

Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme-iron recovery. We used haptoglobin-null mice to evaluate the impact of haptoglobin gene inactivation on iron metabolism. Haptoglobin deficiency led to increased deposition of hemoglobin in proximal tubules of the kidney instead of the liver and the spleen as occurred in wild-type mice. This difference in organ distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload.

Background: Public repositories of microarray data contain an incredible amount of information that is potentially relevant to explore functional relationships among genes by meta-analysis of expression profiles. However, the widespread use of this resource by the scientific community is at the moment limited by the limited availability of effective tools of analysis. We here describe CLOE, a simple cDNA microarray data mining strategy based on meta-analysis of datasets from pairs of species. The method consists in ranking EST probes in the datasets of the two species according to the similarity of their expression profiles with that of two EST probes from orthologous genes, and extracting orthologous EST pairs from a given top interval of the ranked lists. The Gene Ontology annotation of the obtained candidate partners is then analyzed for keywords overrepresentation., Results: We demonstrate the capabilities of the approach by testing its predictive power on three proteomically-defined mammalian protein complexes, in comparison with single and multiple species meta-analysis approaches. Our results show that CLOE can find candidate partners for a greater number of genes, if compared to multiple species co-expression analysis, but retains a comparable specificity even when applied to species as close as mouse and human. On the other hand, it is much more specific than single organisms co-expression analysis, strongly reducing the number of potential candidate partners for a given gene of interest., Conclusions: CLOE represents a simple and effective data mining approach that can be easily used for meta-analysis of cDNA microarray experiments characterized by very heterogeneous coverage. Importantly, it produces for genes of interest an average number of high confidence putative partners that is in the range of standard experimental validation techniques.

Background: PIP3, generated by P13-K activates Akt which inactivates AFX/FKHR; with the consequent decrease in p27/Kip.1 expression and enhancement of cyclin D1 expression through FRAP/mTOR. PTEN lipid phosphatase degrades PIP3 and negatively regulates Akt, whereas this is activated by EGFR through PI3. In glioblastomas, PTEN is mutated in 27%-40% and EGFR amplified in 60%-65% of cases., Materials and Methods: PTEN mutation and EGFR amplification by PCRP Akt, p27/Kip.1 and cyclin D1 by immunohistochemistry, apoptosis by TUNEL and MIB.1 LI were studied in a series of 65 operated glioblastomas., Results: EGFR amplification and PTEN mutation were present in 50% and 30% of glioblastomas, respectively. No relationship between EGFR amplification and PTEN mutation, and p27/Kip. 1 and cyclin D1 was found. However, cyclin D1 was positive in 69% of Akt-expressing areas, whereas p27 was positive in 30% only., Conclusion: A direct relationship is more evident between cyclin D1 and p27/Kip.1 and Akt than with PTEN and EGFR.

Aim: Only a minority of patients carrying a defined viral aetiologic agent develop cirrhosis and ultimately hepatocellular carcinoma (HCC), the mechanism underlying the worsening is still undefined. Experimental infection by Helicobacter hepaticus in mice causes chronic hepatitis and HCC and recently, more Helicobacter species (Helicobacter spp.) have been detected in the liver of patients suffering from cholestatic diseases and HCC arising from non-cirrhotic liver. We investigated whether Helicobacter spp. sequences could be detected in the liver of patients with cirrhosis and HCC compared to subjects with metastasis to liver from colon cancer., Methods: Twenty-three liver samples from patients operated upon for HCC superimposed on hepatitis C virus (HCV)-related cirrhosis and 6 from patients with resected metastases from colorectal cancer, were tested by polymerase chain reaction for presence of genomic 16S rRNA of Helicobacter genus using specific primers. DNA sequencing and cag A gene analysis were also performed., Results: Genomic sequences of Helicobacter spp. were found in 17 of 20 (85%) liver samples from patients with HCC and in 2 of 6 samples from patients with liver metastasis. In three samples of the first group the result was uncertain. H pylori was revealed in 16 out of 17 positive samples and Helicobacter pullorum in the other., Conclusion: Helicobacter spp., carcinogenic in mice, were found at a higher frequency in the liver of patients with HCV-related cirrhosis and HCC than those in patients without primary liver disease.

Intravascular hemolysis is associated with several pathologic conditions that include hemoglobinopathies, trauma, malaria, and bacterial infections. Among plasma-protective proteins against oxidative damage caused by red blood cell rupture, haptoglobin and hemopexin are thought to play a crucial role. Haptoglobin and hemopexin, by binding with high-affinity hemoglobin and heme, respectively, exert an antioxidant action by preventing heme-catalyzed free radical production. Moreover, these proteins prevent iron loss by inhibiting glomerular filtration of hemoglobin and heme diffusion through plasma membranes. Analysis of single-null mice demonstrated the antioxidant action of haptoglobin and hemopexin in vivo and suggests that the 2 proteins cooperate in the resolution of hemolytic stress. To evaluate the physiological relevance of the haptoglobin-hemopexin system and the principal targets of its action, we generated haptoglobin-hemopexin double-knockout mice and analyzed them under basal conditions and after acute hemolysis. Whereas haptoglobin-hemopexin double-null mice displayed no obvious alteration in phenotype under basal conditions, nonlethal hemolytic stress in these animals led to pronounced splenomegaly as well as liver inflammation and fibrosis. These data demonstrate that haptoglobin and hemopexin together are essential for protection from splenomegaly and liver fibrosis resulting from intravascular hemolysis.

Integrin-mediated cell adhesion cooperates with growth factor receptors in the control of cell proliferation, cell survival, and cell migration. One mechanism to explain these synergistic effects is the ability of integrins to induce phosphorylation of growth factor receptors, for instance the epidermal growth factor (EGF) receptor. Here we define some aspects of the molecular mechanisms regulating integrin-dependent EGF receptor phosphorylation. We show that in the early phases of cell adhesion integrins associate with EGF receptors on the cell membrane in a macromolecular complex including the adaptor protein p130Cas and the c-Src kinase, the latter being required for adhesion-dependent assembly of the macromolecular complex. We also show that the integrin cytoplasmic tail, c-Src kinase, and the p130Cas adaptor protein are required for phosphorylation of EGF receptor in response to integrin-mediated adhesion. We show that integrins induce phosphorylation of EGF receptor on tyrosine residues 845, 1068, 1086, and 1173, but not on residue 1148, a major site of phosphorylation in response to EGF. In addition we find that integrin-mediated adhesion increases the amount of EGF receptor expressed on the cell surface. Therefore these data indicate that integrin-mediated adhesion induces assembly of a macromolecular complex containing c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosine residues.