Sara Petrillo

The mitochondrial heme exporter FLVCR1b mediates erythroid differentiation

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.

Hemopexin therapy reverts heme-induced proinflammatory phenotypic switching of macrophages in a mouse model of sickle cell disease

Hemolytic diseases, such as sickle cell anemia and thalassemia, are characterized by enhanced release of hemoglobin and heme into the circulation, heme-iron loading of reticulo-endothelial system macrophages, and chronic inflammation. Here we show that in addition to activating the vascular endothelium, hemoglobin and heme excess alters the macrophage phenotype in sickle cell disease. We demonstrate that exposure of cultured macrophages to hemolytic aged red blood cells, heme, or iron causes their functional phenotypic change toward a proinflammatory state. In addition, hemolysis and macrophage heme/iron accumulation in a mouse model of sickle disease trigger similar proinflammatory phenotypic alterations in hepatic macrophages. On the mechanistic level, this critically depends on reactive oxygen species production and activation of the Toll-like receptor 4 signaling pathway. We further demonstrate that the heme scavenger hemopexin protects reticulo-endothelial macrophages from heme overload in heme-loaded Hx-null mice and reduces production of cytokines and reactive oxygen species. Importantly, in sickle mice, the administration of human exogenous hemopexin attenuates the inflammatory phenotype of macrophages. Taken together, our data suggest that therapeutic administration of hemopexin is beneficial to counteract heme-driven macrophage-mediated inflammation and its pathophysiologic consequences in sickle cell disease.

Glutathione metabolism in cobalamin deficiency type C (cblC).

BackgroundMethylmalonic aciduria with homocystinuria, cblC defect, is the most frequent disorder of vitamin B12 metabolism. CblC patients are commonly treated with a multidrug therapy to reduce metabolite accumulation and to increase deficient substrates. However the long-term outcome is often unsatisfactory especially in patients with early onset, with frequent progression of neurological and ocular impairment. Recent studies, have shown perturbation of cellular redox status in cblC. To evaluate the potential contribution of oxidative stress into the patophysiology of cblC defect, we have analyzed the in vivo glutathione metabolism in a large series of cblC deficient individuals.MethodsLevels of different forms of glutathione were measured in lymphocytes obtained from 18 cblC patients and compared with age-matched controls. Furthermore, we also analyzed plasma cysteine and total homocysteine.ResultsWe found an imbalance of glutathione metabolism in cblC patients with a significant decrease of total and reduced glutathione, along with a significant increase of different oxidized glutathione forms.ConclusionsThese findings show a relevant in vivo disturbance of glutathione metabolism underlining the contribution of glutathione pool depletion to the redox imbalance in treated cblC patients. Our study may be helpful in addressing future research to better understanding the pathogenetic mechanism of the disease and in developing new therapeutic approaches, including the use of novel vitamin B12 derivatives.

Frataxin Deficiency Leads to Reduced Expression and Impaired Translocation of NF-E2-Related Factor (Nrf2) in Cultured Motor Neurons

Oxidative stress has been implicated in the pathogenesis of Friedreich’s Ataxia (FRDA), a neurodegenerative disease caused by the decreased expression of frataxin, a mitochondrial protein responsible of iron homeostasis. Under conditions of oxidative stress, the activation of the transcription factor NF-E2-related factor (Nrf2) triggers the antioxidant cellular response by inducing antioxidant response element (ARE) driven genes. Increasing evidence supports a role for the Nrf2-ARE pathway in neurodegenerative diseases. In this study, we analyzed the expression and the distribution of Nrf2 in silenced neurons for frataxin gene. Decreased Nrf2 mRNA content and a defective activation after treatment with pro-oxidants have been evidenced in frataxin-silenced neurons by RT-PCR and confocal microscopy. The loss of Nrf2 in FRDA may greatly enhance the cellular susceptibility to oxidative stress and make FRDA neurons more vulnerable to injury. Our findings may help to focus on this promising target, especially in its emerging role in the neuroprotective response.

Glutathione imbalance in patients with X-linked adrenoleukodystrophy

Background X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder of X-linked inheritance caused by a mutation in the ABCD1 gene which determines an accumulation of long-chain fatty acids in plasma and tissues. Recent evidence shows that oxidative stress may be a hallmark in the pathogenesis of X-ALD and glutathione plays an important role in the defense against free radicals. In this study we have analyzed glutathione homeostasis in lymphocytes of 14 patients with X-ALD and evaluated the balance between oxidized and reduced forms of glutathione, in order to define the role of this crucial redox marker in this condition. Methods Lymphocytes, plasma and erythrocytes were obtained from the whole blood of 14 subjects with X-ALD and in 30 healthy subjects. Total, reduced and protein-bound glutathione levels were measured in lymphocytes by HPLC analysis. Erythrocyte free glutathione and antioxidant enzyme activities, plasma thiols and carbonyl content were determined by spectrophotometric assays. Results A significant decrease of total and reduced glutathione was found in lymphocytes of patients, associated to high levels of all oxidized glutathione forms. A decline of free glutathione was particularly significant in erythrocytes. The increased oxidative stress in X-ALD was additionally confirmed by the decrease of plasma thiols and the high level of carbonyls. Conclusion Our results strongly support a role for oxidative stress in the pathophysiology of X-ALD and strengthen the importance of the balance among glutathione forms as a hallmark and a potential biomarker of the disease.

Cytokine, chemokine, and growth factor profile of platelet-rich plasma

Abstract During wound healing, biologically active molecules are released from platelets. The rationale of using platelet-rich plasma (PRP) relies on the concentration of bioactive molecules and subsequent delivery to healing sites. These bioactive molecules have been seldom simultaneously quantified within the same PRP preparation. In the present study, the flexible Bio-Plex system was employed to assess the concentration of a large range of cytokines, chemokines, and growth factors in 16 healthy volunteers so as to determine whether significant baseline differences may be found. Besides IL-1b, IL-1ra, IL-4, IL-6, IL-8, IL-12, IL-13, IL-17, INF-γ, TNF-α, MCP-1, MIP-1a, RANTES, bFGF, PDGF, and VEGF that were already quantified elsewhere, the authors reported also on the presence of IL-2, IL-5, IL-7, IL-9, IL-10, IL-15 G-CSF, GM-CSF, Eotaxin, CXCL10 chemokine (IP-10), and MIP 1b. Among the most interesting results, it is convenient to mention the high concentrations of the HIV-suppressive and inflammatory cytokine RANTES and a statistically significant difference between males and females in the content of PDGF-BB. These data are consistent with previous reports pointing out that gender, diet, and test system affect the results of platelet function in healthy subjects, but seem contradictory when compared to other quantification assays in serum and plasma. The inconsistencies affecting the experimental results found in literature, along with the variability found in the content of bioactive molecules, urge further research, hopefully in form of randomized controlled clinical trials, in order to find definitive evidence of the efficacy of PRP treatment in various pathologic and regenerative conditions.

The heme exporter Flvcr1 regulates expansion and differentiation of committed erythroid progenitors by controlling intracellular heme accumulation.

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.

Heme accumulation in endothelial cells impairs angiogenesis by triggering paraptosis

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.

Oxidative stress in Duchenne muscular dystrophy: focus on the NRF2 redox pathway.

Oxidative stress is involved in the pathogenesis of Duchenne muscular dystrophy (DMD), an X-linked genetic disorder caused by mutations in the dystrophin gene and characterized by progressive, lethal muscle degeneration and chronic inflammation. In this study, we explored the expression and signaling pathway of a master player of the anti-oxidant and anti-inflammatory response, namely NF-E2-related Factor 2, in muscle biopsies of DMD patients. We classified DMD patients in two age groups (Class I, 0-2 years and Class II, 2-9 years), in order to evaluate the antioxidant pathway expression during the disease progression. We observed that altered enzymatic antioxidant responses, increased levels of oxidized glutathione and oxidative damage are differently modulated in the two age classes of patients and well correlate with the severity of pathology. Interestingly, we also observed a modulation of relevant markers of the inflammatory response, such as heme oxygenase 1 and Inteleukin-6 (IL-6), suggesting a link between oxidative stress and chronic inflammatory response. Of note, using a transgenic mouse model, we demonstrated that IL-6 overexpression parallels the antioxidant expression profile and the severity of dystrophic muscle observed in DMD patients. This study advances our understanding of the pathogenic mechanisms underlying DMD and defines the critical role of oxidative stress on muscle wasting with clear implications for disease pathogenesis and therapy in human.

DJ-1 modulates mitochondrial response to oxidative stress: Clues from a novel diagnosis of PARK7

DJ‐1 mutations are associated to early‐onset Parkinsons disease and accounts for about 1–2% of the genetic forms. The protein is involved in many biological processes and its role in mitochondrial regulation is gaining great interest, even if its function in mitochondria is still unclear. We describe a 47‐year‐old woman affected by a multisystem disorder characterized by progressive, early‐onset parkinsonism plus distal spinal amyotrophy, cataracts and sensory‐neural deafness associated with a novel homozygous c.461C>A [p.T154K] mutation in DJ‐1. Patients cultured fibroblasts showed low ATP synthesis, high ROS levels and reduced amount of some subunits of mitochondrial complex I; biomarkers of oxidative stress also resulted abnormal in patients blood. The clinical pattern of multisystem involvement and the biochemical findings in our patient highlight the role for DJ‐1 in modulating mitochondrial response against oxidative stress.