Dimethyl Sulfoxide Induces Hemolysis and Pulmonary Hypertension.

Vascular and lung injury are well established complications associated with hemolytic disorders, and hemolysis associated pulmonary hypertension (PH) has emerged as the most serious complication of sickle cell disease. The causal relationship between intravascular hemolysis and the development of PH is still under investigation. Previously we have shown that repetitive administration of hemolyzed autologous blood causes PH in rats. Dimethyl sulfoxide (DMSO), a widely used solvent and anti-inflammatory agent, induces hemolysis in vivo. We hypothesized that repetitive administration of DMSO would induce PH in rats. We also examined hemolysis-induced release of adenosine deaminase (ADA) and arginase from red blood cells, which may amplify hemolysis-mediated vascular injury. Acute administration of DMSO (1.5ml/30 min into the right atrium) induced intravascular hemolysis and pulmonary vasoconstriction. DMSO-induced increase in right ventricular peak systolic pressure (RVPSP) was associated with increased release of ADA. Notably, the acute increase in RVPSP was attenuated by administration of an adenosine A2A receptor agonist or by pretreatment of animals with ADA inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). Repetitive administration of DMSO for 10 days produced anemia, hemoglobinuria, hemoglobinemia, splenomegaly, and development of PH. Histopathological analysis revealed pulmonary vascular remodeling. The presented data describe a new model of hemolysis induced PH, suggesting that hemolysis is mechanistically related to pulmonary hypertension, and pointing to a potential pathogenic role that adenosine deaminase and accelerated adenosine metabolism may play in hemolysis associated pulmonary hypertension.

Sex-related differences in clinical outcomes among patients with myocardial infarction with nonobstructive coronary artery disease: A systematic review and meta-analysis.

BACKGROUND: Among patients who present with acute myocardial infarction (MI), 2-6% are found to have non-obstructive coronary arteries (NOCA). Patients with MINOCA are more commonly women and present at a younger age (51-59 years). The influence of sex on adverse event rates remains unclear. METHODS: PubMed, MEDLINE, CENTRAL (Cochrane Central Register of Controlled Trials), EMBASE, EBSCO, Web of Science and CINAHL databases were searched for trials comparing gender differences in clinical outcomes among patients with MINOCA from inception through April 10, 2022. The primary endpoint of the study was composite major adverse clinical events (MACE) including all-cause mortality, non-fatal MI, stroke, and cardiovascular readmissions, and secondary endpoints were the individual components of the MACE. RESULTS: Seven studies with a total of 28,671 MINOCA patients were included (n = 11,249 men and n = 17,422 women) over a mean follow-up of 2 years. Women had more MACE than men (10.1% vs. 9.1%, OR 1.15, 1.04-1.23, I2 = 44.7%). Among secondary endpoints, only the incidence of stroke was higher in women (3.5% vs. 2.2%, OR 1.3, 1.01-1.68, I2 = 0%). All-cause mortality, non-fatal MI, and cardiovascular readmissions were not significantly different between the two groups. CONCLUSIONS: We hypothesize that small vessel disease associated with MINOCA drives MACE in women and the diminishing influence of estrogen, hypercoagulability and underprescribing could contribute to the differences sex-related outcomes.

Does Bentonite Cause Cytotoxic and Whole-Transcriptomic Adverse Effects in Enterocytes When Used to Reduce Aflatoxin B1 Exposure?

Aflatoxin B1 (AFB1) is a major food safety concern, threatening the health of humans and animals. Bentonite (BEN) is an aluminosilicate clay used as a feed additive to reduce AFB1 presence in contaminated feedstuff. So far, few studies have characterized BEN toxicity and efficacy in vitro. In this study, cytotoxicity (WST-1 test), the effects on cell permeability (trans-epithelial electrical resistance and lucifer yellow dye incorporation), and transcriptional changes (RNA-seq) caused by BEN, AFB1 and their combination (AFB1 + BEN) were investigated in Caco-2 cells. Up to 0.1 mg/mL, BEN did not affect cell viability and permeability, but it reduced AFB1 cytotoxicity; however, at higher concentrations, BEN was cytotoxic. As to RNA-seq, 0.1 mg/mL BEN did not show effects on cell transcriptome, confirming that the interaction between BEN and AFB1 occurs in the medium. Data from AFB1 and AFB1 + BEN suggested AFB1 provoked most of the transcriptional changes, whereas BEN was preventive. The most interesting AFB1-targeted pathways for which BEN was effective were cell integrity, xenobiotic metabolism and transporters, basal metabolism, inflammation and immune response, p53 biological network, apoptosis and carcinogenesis. To our knowledge, this is the first study assessing the in vitro toxicity and whole-transcriptomic effects of BEN, alone or in the presence of AFB1.

Defenestrated endothelium delays liver-directed gene transfer in hemophilia A mice.

Hemophilia A is an inherited bleeding disorder caused by defective or deficient coagulation factor VIII (FVIII) activity. Until recently, the only treatment for prevention of bleeding involved IV administration of FVIII. Gene therapy with adeno-associated vectors (AAVs) has shown some efficacy in patients with hemophilia A. However, limitations persist due to AAV-induced cellular stress, immunogenicity, and reduced durability of gene expression. Herein, we examined the efficacy of liver-directed gene transfer in FVIII knock-out mice by AAV8-GFP. Surprisingly, compared with control mice, FVIII knockout (F8TKO) mice showed significant delay in AAV8-GFP transfer in the liver. We found that the delay in liver-directed gene transfer in F8TKO mice was associated with absence of liver sinusoidal endothelial cell (LSEC) fenestration, which led to aberrant expression of several sinusoidal endothelial proteins, causing increased capillarization and decreased permeability of LSECs. This is the first study to link impaired liver-directed gene transfer to liver-endothelium maladaptive structural changes associated with FVIII deficiency in mice.

Bentonite does not affect in vitro ruminal gross fermentations but could modify ruminal metabolome and mineral content. A proof of concept.

Clay minerals, such as bentonite, are used as feed additives capable of adsorbing mycotoxins and heavy metals and have been related to many positive effects on animal health and productivity. However, these compounds seem to induce also side effects and to interact with the intestinal and ruminal microbiota. The present in vitro study is aimed at evaluating the effects of different doses of bentonite on ruminal fermentations, metabolome and mineral content. Five doses of bentonite (0, 2.5, 5, 10 and 50 mg in 150 mL total volume) were incubated (39 °C for 24 h) with a dairy cow Total Mixed Ratio (TMR) and the ruminal fluid obtained from one healthy Holstein lactating cow. The kinetics of gas production (GP) continuously monitored during the incubation evidenced no significant differences in either cumulative GP (mL/g DM) or GP rate (mL/g DM/h) between the treatment groups. After the incubation, metabolome and mineral content of treated ruminal fluids were studied in pooled replicate samples by 1H NMR spectroscopy and Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES), respectively. The NMR analysis led to the identification of 20 metabolites and suggested a clear metabolic differentiation among treatments. The ICP-OES analysis suggested that the addition of bentonite affected the concentration of Al, Ba, Ca, Cr, Mn, Mo and Sr. It is conceivable that bentonite administration does not affect gross ruminal fermentations, while it seems to modify the ruminal metabolome and the concentrations of few minerals in ruminal fluid.

An Iron Shield to Protect Epigallocatehin-3-Gallate from Degradation: Multifunctional Self-Assembled Iron Oxide Nanocarrier Enhances Protein Kinase CK2 Intracellular Targeting and Inhibition.

Protein kinase CK2 is largely involved in cell proliferation and apoptosis and is generally recognized as an Achilles' heel of cancer, being overexpressed in several malignancies. The beneficial effects of (-)-epigallocatechin-3-gallate (EGCG) in the prevention and treatment of several diseases, including cancer, have been widely reported. However, poor stability and limited bioavailability hinder the development of EGCG as an effective therapeutic agent. The combination of innovative nanomaterials and bioactive compounds into nanoparticle-based systems demonstrates the synergistic advantages of nanocomplexes as compared to the individual components. In the present study, we developed a self-assembled core-shell nanohybrid (SAMN@EGCG) combining EGCG and intrinsic dual-signal iron oxide nanoparticles (Surface Active Maghemite Nanoparticles). Interestingly, nano-immobilization on SAMNs protects EGCG from degradation, preventing its auto-oxidation. Most importantly, the nanohybrid was able to successfully deliver EGCG into cancer cells, displaying impressive protein kinase CK2 inhibition comparable to that obtained with the most specific CK2 inhibitor, CX-4945 (5.5 vs. 3 µM), thus promoting the phytochemical exploitation as a valuable alternative for cancer therapy. Finally, to assess the advantages offered by nano-immobilization, we tested SAMN@EGCG against Pseudomonas aeruginosa, a Gram-negative bacterium involved in severe lung infections. An improved antimicrobial effect with a drastic drop of MIC from 500 to 32.7 µM was shown.

Employment of Phenolic Compounds from Olive Vegetation Water in Broiler Chickens: Effects on Gut Microbiota and on the Shelf Life of Breast Fillets.

Olive vegetation water (OVW) is a by-product with a noticeable environmental impact; however, its polyphenols may be reused food and feed manufacture as high-value ingredients with antioxidant/antimicrobial activities. The effect of dietary supplementation with OVW polyphenols on the gut microbiota, carcass and breast quality, shelf life, and lipid oxidation in broiler chickens has been studied. Chicks were fed diets supplemented with crude phenolic concentrate (CPC) obtained from OVW (220 and 440 mg/kg phenols equivalent) until reaching commercial size. Cloacal microbial community (rRNA16S sequencing) was monitored during the growth period. Breasts were submitted to culture-dependent and -independent microbiological analyses during their shelf-life. Composition, fatty acid concentration, and lipid oxidation of raw and cooked thawed breasts were measured. Growth performance and gut microbiota were only slightly affected by the dietary treatments, while animal age influenced the cloacal microbiota. The supplementation was found to reduce the shelf life of breasts due to the growth of spoilers. Chemical composition and lipid oxidation were not affected. The hydroxytyrosol (HT) concentration varied from 178.6 to 292.4 ug/kg in breast muscle at the beginning of the shelf-life period. The identification of HT in meat demonstrates that the absorption and metabolism of these compounds was occurring efficiently in the chickens.

P-selectin deficiency promotes liver senescence in sickle cell disease mice.

Sickle cell disease (SCD) is caused by a homozygous mutation in the ß-globin gene, which leads to erythrocyte sickling, vasoocclusion, and intense hemolysis. P-selectin inhibition has been shown to prevent vasoocclusive events in patients with SCD; however, the chronic effect of P-selectin inhibition in SCD remains to be determined. Here, we used quantitative liver intravital microscopy in our recently generated P-selectin-deficient SCD mice to show that chronic P-selectin deficiency attenuates liver ischemia but fails to prevent hepatobiliary injury. Remarkably, we find that this failure in resolution of hepatobiliary injury in P-selectin-deficient SCD mice is associated with the increase in cellular senescence and reduced epithelial cell proliferation in the liver. These findings highlight the importance of investigating the long-term effects of chronic P-selectin inhibition therapy on liver pathophysiology in patients with SCD.

Effects of honeybush (Cyclopia subternata) extract on physico-chemical, oxidative and sensory traits of typical Italian salami.

Honeybush (Cyclopia subternata Vogel) is an indigenous South African shrub enjoyed as hot brewed tea. "Unfermented" honeybush is also a potential antioxidant bioactive extract for foodstuffs due to its polyphenol content. The effect of "unfermented" honeybush extract (Hob; 0.5%) was evaluated in typical Italian salami and compared with nitrate (Nit; 100 mg/kg) and a control (Ctl; without nitrate or honeybush). After 35 days of ripening, Hob had a higher (p < .01) water activity (0.928), compared with Ctl (0.923) and Nit (0.924). Final pH (5.35-5.24) was not affected by treatments. Lower lipid oxidation was observed in Hob and Nit treatments (p < .001) compared with Ctl. Internal color and odor intensity were similar among treatments. Salami with honeybush extract had less spontaneous outer surface mold growth whereas the Ctl showed intermediate growth (p < .05). Honeybush extract seems a promising natural ingredient with antioxidant action.

Experimental intravascular hemolysis induces hemodynamic and pathological pulmonary hypertension: association with accelerated purine metabolism.

Pulmonary hypertension (PH) is emerging as a serious complication associated with hemolytic disorders, and plexiform lesions (PXL) have been reported in patients with sickle cell disease (SCD). We hypothesized that repetitive hemolysis per se induces PH and angioproliferative vasculopathy and evaluated a new mechanism for hemolysis-associated PH (HA-PH) that involves the release of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) from erythrocytes. In healthy rats, repetitive administration of hemolyzed autologous blood (HAB) for 10 days produced reversible pulmonary parenchymal injury and vascular remodeling and PH. Moreover, the combination of a single dose of Sugen-5416 (SU, 200 mg/kg) and 10-day HAB treatment resulted in severe and progressive obliterative PH and formation of PXL (Day 26, right ventricular peak systolic pressure (mmHg): 26.1 ± 1.1, 41.5 ± 0.5 and 85.1 ± 5.9 in untreated, HAB treated and SU+HAB treated rats, respectively). In rats, repetitive administration of HAB increased plasma ADA activity and reduced urinary adenosine levels. Similarly, SCD patients had higher plasma ADA and PNP activity and accelerated adenosine, inosine, and guanosine metabolism than healthy controls. Our study provides evidence that hemolysis per se leads to the development of angioproliferative PH. We also report the development of a rat model of HA-PH that closely mimics pulmonary vasculopathy seen in patients with HA-PH. Finally, this study suggests that in hemolytic diseases released ADA and PNP may increase the risk of PH, likely by abolishing the vasoprotective effects of adenosine, inosine and guanosine. Further characterization of this new rat model of hemolysis-induced angioproliferative PH and additional studies of the role of purines metabolism in HA-PH are warranted.

Spectrophotometric techniques for the characterization of strains involved in the blue pigmentation of food: Preliminary results.

Near infrared spectroscopy (NIRs) and ultraviolet visible spectroscopy (UV-vis) have been investigated as rapid techniques to characterize foodborne bacteria through the analysis of the spectra of whole cells or microbial suspensions. The use of spectra collected from broth cultures could be used as a fingerprint for strain classification using a combined polyphasic approach. The aim of this study was to evaluate the feasibility of NIRs and UV-vis for the characterization of blue strains belonging to the Pseudomonas fluorescens group. The bacteria were isolated from different food matrices, including some spoiled samples (blue discoloration). Eightyone strains previously identified at the species level were grown in Minimal Bacterial Medium broth under standard conditions at 22°C. Two biological replicates were centrifuged in order to separate the bacterial cells from the extracellular products. Six aliquots per strain were analyzed on a small ring cup in transflectance mode (680-2500 nm, gap 2 nm). A subset of 39 strains was evaluated by UV-vis to determine changes in the spectral characteristics at 48 and 72 hours. Several chemometric approaches were tested to assess the performance of NIRs and UVvis. According to the variable importance in projection (VIP), the 1892-2020 nm spectral region showed the highest level of discrimination between blue strains and others. Additional information was provided in the 680-886 and 1454-1768 nm regions (aromatic C-H bonds) and in the 2036-2134 nm region (fatty acids). Changes in UV-vis spectral data (at 48 and 72 hours) appear to indicate the presence of phenazine and catecholic compounds in extracellular products.

The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

Senescent cells withdraw from the cell cycle and do not proliferate. The prevalence of senescent compared to normally functioning parenchymal cells increases with age, impairing tissue and organ homeostasis. A contentious principle governing this process has been the redox theory of aging. We linked matricellular protein thrombospondin 1 (TSP1) and its receptor CD47 to the activation of NADPH oxidase 1 (Nox1), but not of the other closely related Nox isoforms, and associated oxidative stress, and to senescence in human cells and aged tissue. In human endothelial cells, TSP1 promoted senescence and attenuated cell cycle progression and proliferation. At the molecular level, TSP1 increased Nox1-dependent generation of reactive oxygen species (ROS), leading to the increased abundance of the transcription factor p53. p53 mediated a DNA damage response that led to senescence through Rb and p21cip, both of which inhibit cell cycle progression. Nox1 inhibition blocked the ability of TSP1 to increase p53 nuclear localization and p21cip abundance and its ability to promote senescence. Mice lacking TSP1 showed decreases in ROS production, p21cip expression, p53 activity, and aging-induced senescence. Conversely, lung tissue from aging humans displayed increases in the abundance of vascular TSP1, Nox1, p53, and p21cip Finally, genetic ablation or pharmacological blockade of Nox1 in human endothelial cells attenuated TSP1-mediated ROS generation, restored cell cycle progression, and protected against senescence. Together, our results provide insights into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence and suggest potential targets for controlling the aging process at the molecular level.

TSP1-CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction.

AIMS: Thrombospondin-1 (TSP1) is a ligand for CD47 and TSP1-/- mice are protected from pulmonary hypertension (PH). We hypothesized the TSP1-CD47 axis is upregulated in human PH and promotes pulmonary arterial vasculopathy. METHODS AND RESULTS: We analyzed the molecular signature and functional response of lung tissue and distal pulmonary arteries (PAs) from individuals with (n = 23) and without (n = 16) PH. Compared with controls, lungs and distal PAs from PH patients showed induction of TSP1-CD47 and endothelin-1/endothelin A receptor (ET-1/ETA) protein and mRNA. In control PAs, treatment with exogenous TSP1 inhibited vasodilation and potentiated vasoconstriction to ET-1. Treatment of diseased PAs from PH patients with a CD47 blocking antibody improved sensitivity to vasodilators. Hypoxic wild type (WT) mice developed PH and displayed upregulation of pulmonary TSP1, CD47, and ET-1/ETA concurrent with down regulation of the transcription factor cell homolog of the v-myc oncogene (cMyc). In contrast, PH was attenuated in hypoxic CD47-/- mice while pulmonary TSP1 and ET-1/ETA were unchanged and cMyc was overexpressed. In CD47-/- pulmonary endothelial cells cMyc was increased and ET-1 decreased. In CD47+/+ cells, forced induction of cMyc suppressed ET-1 transcript, whereas suppression of cMyc increased ET-1 signaling. Furthermore, disrupting TSP1-CD47 signaling in pulmonary smooth muscle cells abrogated ET-1-stimulated hypertrophy. Finally, a CD47 antibody given 2 weeks after monocrotaline challenge in rats upregulated pulmonary cMyc and improved aberrations in PH-associated cardiopulmonary parameters. CONCLUSIONS: In pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1-CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.

Cognitive functioning in children from Nigeria with sickle cell anemia.

BACKGROUND: Cognitive impairment is a major neurological complication of sickle cell anemia (SCA) in the United States, but there are limited studies of cognitive impairment in Nigeria, the country with the highest SCA burden. We hypothesized that children from Nigeria with SCA have worse cognitive functioning than comparison children and explored the association between lower cognitive functioning and key laboratory demographic and socioeconomic variables among children with SCA. PROCEDURE: We conducted a cross-sectional survey, supplemented by anthropomorphic and laboratory data, among a convenience sample of children from Nigeria with and without SCA. We administered the Wechsler Intelligence Scale for Children, Version IV. Our primary outcome measures included (1) estimated IQ (Est. IQ), (2) working memory (WM), and (3) processing speed (PS). RESULTS: The sample included 56 children with SCA (mean age 9.20 [SD 2.75], 46.43% girls) and 44 comparison children (mean age 9.41 [SD 2.49], 40.91% girls). Children with SCA performed worse on Est. IQ (84.58 vs. 96.10, P = 0.006) and PS (86.69 vs 96.91, P = 0.009) than comparison children. There was no significant difference in WM between both groups. Factors associated with lower Est. IQ and PS among children with SCA included age, maternal education, weight-for-age Z scores, and height-for age Z scores. CONCLUSION: In this small sample of children from Nigeria, we found worse cognitive functioning in children with SCA than in comparison children, and that sociodemographic and anthropomorphic factors were correlated with cognitive functioning.

Minimum Bactericidal Concentration of Phenols Extracted from Oil Vegetation Water on Spoilers, Starters and Food-Borne Bacteria.

The aim of the study was to assess the in vitro effect of phenols extracted from oil vegetation water (PEOW) on several food-borne strains. Antibacterial activity of PEOW was based on the minimum bactericidal concentration (MBC) on microtitre assay. The taxa tested were: Staphylococcus (n. 5), Listeria (n. 4), Escherichia (n. 2), Salmonella (n. 1), Pseudomonas (n. 3), Lactobacillus (n. 2) and Pediococcus (n. 1). S. aureus and L. monocytogens showed the lowest level of resistance to PEOW (MBC=1.5-3 mg/mL). In contrast, the Gram negative strains (e.g. S. Typhimurium and Pseudomonas spp.) were in some cases unaffected by the tested doses and the MBCs ranged between 6 to 12 mg/mL. Starter cultures were dramatically reduced on growth (e.g. Staphylococcus xylosus; 0.75 mg/mL MBC). The thresholds for pathogenic strains could be considered for further applications of PEOW in food models (e.g. shelf life or challenge test studies).

Addition of Phenols Compounds to Meat Dough Intended for Salami Manufacture and its Antioxidant Effect.

A purified extract of phenols compounds (65% of phenolic content of which decarboxymethyl oleuropein aglycone represented 45% of the wet mass) obtained from vegetation water (a by-product of oil mill) was added to a ground meat dough intended for salami manufacture in two concentration levels: 75 and 150 mg/100 g of dough (F1 and F2, respectively). The control batch was composed of lean and fat cuts of pork in 70:30 ratio, 2.7% salt and a mixed starter culture of staphylococci and pediococci. After stuffing into natural casings, salamis were aged until they reached a total weight loss of 30%. The product was then sliced and packaged in a protective atmosphere (nitrogen:carbon dioxide 80:20) and placed in a refrigerator thermostat (2-4°C) with alternating 12 h of artificial light and darkness. The samples were analysed for the measurement of pH, water activity, organic acidity, peroxide number and secondary products of lipid peroxidation at the time of slicing and after 10, 20 and 30 days of storage into the refrigerated thermostat. The pH and water activity were not substantially different between the control and the two enriched batches. The peroxide number and secondary products of lipid peroxidation values in the two batches with phenols were at least substantially lower than the control sample. In conclusion, the phenol compounds obtained from vegetation water have shown no interference with the ripening process while protecting the dough from oxidation.

Polyphenols from olive mill waste affect biofilm formation and motility in Escherichia coli K-12.

Olive mill wastes are sources of phenolic compounds with a wide array of biological activities, including antimicrobial effects. A potential option for bioremediation to overcome ecological problems is the reutilization of these natural compounds in food production. The aim of this work was to gain a better understanding of the antimicrobial mode of action of a phenols extract from olive vegetation water (PEOVW) at molecular level by studying Escherichia coli as a model microorganism. Genome-wide transcriptional analysis was performed on E. coli K-12 exposed to PEOVW. The repression of genes for flagellar synthesis and the involvement of genes linked to biofilm formation and stress response were observed. Sub-inhibitory concentrations of PEOVW significantly decreased biofilm formation, swarming and swimming motility, thus confirming the gene expression data. This study provides interesting insights on the molecular action of PEOVW on E. coli K-12. Given these anti-biofilm properties and considering that biofilm formation is a serious problem for the food industry and human health, PEOVW has proved to be a high-value natural product.

Platelet bioenergetic screen in sickle cell patients reveals mitochondrial complex V inhibition, which contributes to platelet activation.

Bioenergetic dysfunction, although central to the pathogenesis of numerous diseases, remains uncharacterized in many patient populations because of the invasiveness of obtaining tissue for mitochondrial studies. Although platelets are an accessible source of mitochondria, the role of bioenergetics in regulating platelet function remains unclear. Herein, we validate extracellular flux analysis in human platelets and use this technique to screen for mitochondrial dysfunction in sickle cell disease (SCD) patients, a population with aberrant platelet activation of an unknown mechanism and in which mitochondrial function has never been assessed. We identify a bioenergetic alteration in SCD patients characterized by deficient complex V activity, leading to decreased mitochondrial respiration, membrane hyperpolarization, and augmented oxidant production compared with healthy subjects. This dysfunction correlates with platelet activation and hemolysis in vivo and can be recapitulated in vitro by exposing healthy platelets to hemoglobin or a complex V inhibitor. Further, reproduction of this dysfunction in vitro activates healthy platelets, an effect prevented by attenuation of mitochondrial hyperpolarization or by scavenging mitochondrial oxidants. These data identify bioenergetic dysfunction in SCD patients for the first time and establish mitochondrial hyperpolarization and oxidant generation as potential pathogenic mechanism in SCD as well as a modulator of healthy platelet function.

Thrombospondin-1 activation of signal-regulatory protein-α stimulates reactive oxygen species production and promotes renal ischemia reperfusion injury.

Ischemia reperfusion injury (IRI) causes tissue and organ injury, in part, through alterations in tissue blood flow and the production of reactive oxygen species. The cell surface receptor signal-regulatory protein-α (SIRP-α) is expressed on inflammatory cells and suppresses phagocytosis, but the function of SIRP-α in IRI has not been determined. We reported previously that the matricellular protein thrombospondin-1 is upregulated in IRI. Here, we report a novel interaction between thrombospondin-1 and SIRP-α on nonphagocytic cells. In cell-free experiments, thrombospondin-1 bound SIRP-α. In vascular smooth muscle cells and renal tubular epithelial cells, treatment with thrombospondin-1 led to phosphorylation of SIRP-α and downstream activation of Src homology domain 2-containing phosphatase-1. Thrombospondin-1 also stimulated phosphorylation of p47(phox) (an organizer subunit for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1/2) and increased production of superoxide, both of which were abrogated by knockdown or antibody blockade of SIRP-α. In rodent aortic rings, treatment with thrombospondin-1 increased the production of superoxide and inhibited nitric oxide-mediated vasodilation in a SIRP-α-dependent manner. Renal IRI upregulated the thrombospondin-1-SIRP-α signaling axis and was associated with increased superoxide production and cell death. A SIRP-α antibody that blocks thrombospondin-1 activation of SIRP-α mitigated the effects of renal IRI, increasing blood flow, suppressing production of reactive oxygen species, and preserving cellular architecture. A role for CD47 in SIRP-α activation in these pathways is also described. Overall, these results suggest that thrombospondin-1 binding to SIRP-α on nonphagocytic cells activates NADPH oxidase, limits vasodilation, and promotes renal IRI.

Vasculopathy, inflammation, and blood flow in leg ulcers of patients with sickle cell anemia.

Chronic leg ulcers are frequent and debilitating complications of sickle cell anemia. Inadequate blood supply has been postulated to be an important factor in their occurrence and delayed healing. Little is known about their microcirculatory and histopathological changes. We evaluated the microcirculation of lower extremity ulcers with laser speckle contrast imaging and infrared thermography and obtained clinical and laboratory characteristics in 18 adults with sickle cell anemia and chronic leg ulcers. Skin biopsies were obtained in four subjects. Subjects had markers of severe disease, anemia, high degree of hemolysis, inflammation, and thrombophilia. The highest blood flow was present in the ulcer bed, progressively less in the immediate periwound area, and an unaffected control skin area in the same extremity. Microscopic examination showed evidence of venostasis, inflammation, and vasculopathy. Blood vessels were increased in number, had activated endothelium and evidence of thrombosis/recanalization. High blood flow may be due to chronic inflammation, cutaneous vasodilatation, venostasis, and in situ thrombosis. These changes in skin microcirculation are similar to chronic venous ulcers in the non-sickle cell disease (SCD) population, thus suggesting that leg ulcers may be another end-organ complication with endothelial dysfunction that appears in patients with SCD at a younger age and with higher frequency than in the general population.