Safety and feasibility of the gene transfer of hemopexin for conditions with increased free heme.

Heme is a fundamental molecule for several biological processes, but when released in the extracellular space such as in hemolytic diseases, it can be toxic to cells and tissues. Hemopexin (HPX) is a circulating protein responsible for removing free heme from the circulation, whose levels can be severely depleted in conditions such as sickle cell diseases. Accordingly, increasing HPX levels represents an attractive strategy to mitigate the deleterious effects of heme in these conditions. Gene transfer of liver-produced proteins with adeno-associated virus (AAV) has been shown to be an effective and safety strategy in animal and human studies mainly in hemophilia. Here, we report the feasibility of increasing HPX levels using an AAV8 vector expressing human HPX (hHPX). C57Bl mice were injected with escalating doses of our vector, and expression was assessed by enzyme immunoassay (ELISA), Western blot, and quantitative polymerase chain reaction (qPCR). In addition, the biological activity of transgenic hHPX was confirmed using two different models of heme challenge consisting of serial heme injections or phenylhydrazine-induced hemolysis. Sustained expression of hHPX was confirmed for up to 26 weeks in plasma. Expression was dose-dependent and not associated with clinical signs of toxicity. hHPX levels were significantly reduced by heme infusions and phenylhydrazine-induced hemolysis. No clinical toxicity or laboratory signs of liver damage were observed in preliminary short-term heme challenge studies. Our results confirm that long-term expression of hHPX is feasible and safe in mice, even in the presence of heme overload. Additional studies are needed to explore the effect of transgenic HPX protein in animal models of chronic hemolysis.

Association of Ang/Tie2 pathway mediators with endothelial barrier integrity and disease severity in COVID-19.

Endothelial barrier (EB) disruption contributes to acute lung injury in COVID-19, and levels of both VEGF-A and Ang-2, which are mediators of EB integrity, have been associated with COVID-19 severity. Here we explored the participation of additional mediators of barrier integrity in this process, as well as the potential of serum from COVID-19 patients to induce EB disruption in cell monolayers. In a cohort from a clinical trial consisting of thirty patients with COVID-19 that required hospital admission due to hypoxia we demonstrate that i) levels of soluble Tie2 were increase, and of soluble VE-cadherin were decreased when compared to healthy individuals; ii) sera from these patients induce barrier disruption in monolayers of endothelial cells; and iii) that the magnitude of this effect is proportional to disease severity and to circulating levels of VEGF-A and Ang-2. Our study confirms and extends previous findings on the pathogenesis of acute lung injury in COVID-19, reinforcing the concept that EB is a relevant component of this disease. Our results pave the way for future studies that can refine our understanding of the pathogenesis of acute lung injury in viral respiratory disorders, and contribute to the identification of new biomarkers and therapeutic targets for these conditions.

Association of heme-oxygenase 1, hemopexin, and heme levels with markers of disease severity in COVID-19.

Heme-oxygenase 1 (HO-1) is an enzyme with well-known anti-inflammatory and antioxidant properties, whose levels have been previously associated with disease severity in the context of sterile and infectious diseases. Moreover, the heme/HO-1 pathway has been associated with prothrombotic changes in other diseases. Accordingly, the potential of modulating HO-1 levels for the treatment of COVID-19 was extensively speculated during the COVID-19 pandemic, but very few actual data were generated. The aim of our study was to explore the association of HO-1, heme, and hemopexin (HPX) levels with COVID-19 severity and with markers of inflammation and coagulation activation. The study was conducted in 30 consecutive patients with COVID-19 admitted due to hypoxemia, and 30 healthy volunteers matched by sex, age, and geographic region. HO-1 and HPX levels were measured by enzyme immunoassay (ELISA) and heme levels were measured by a colorimetric method. A comprehensive panel of coagulation and fibrinolysis activation was also used. Patients with COVID-19 presented increased levels of HO-1 when compared to controls (5741 ± 2696 vs 1953 ± 612 pg/mL, respectively, P < 0.0001), as well as a trend toward increased levels of HPX (3.724 ± 0.880 vs 3.254 ± 1.022 mg/mL, respectively; P = 0.06). In addition, HO-1 and HPX levels reduced from admission to day + 4. HO-1 levels were associated with duration of intensive care unit stay and with several markers of coagulation activation. In conclusion, modulation of HO-1 could be associated with the prothrombotic state observed in COVID-19, and HO-1 could also represent a relevant biomarker for COVID-19. New independent studies are warranted to explore and expand these findings.

Changes in Heme Levels During Acute Vaso-occlusive Crisis in Sickle Cell Anemia.

OBJECTIVE/BACKGROUND: Sickle cell anemia (SCA) is associated with increased levels of extracellular heme, which is a key mediator of inflammation in this condition. Despite abundant evidence supporting this concept in cell and animal models, few studies addressed the association between heme levels and the development and severity of acute vasoocclusive crises (VOC) in humans. METHODS: A cross-sectional study was conducted in patients with acute VOC. Total extracellular heme levels were measured in both plasma and serum at admission and after convalescence, and correlated with other clinical and laboratory markers of SCA severity. RESULTS: A total of 28 episodes of VOC in 25 patients were included. Heme levels were similar between admission and convalescence, and correlated with the difference between pre and post hemoglobin, and SCA severity estimated by a composite score of clinical and laboratory markers. Heme levels were neither associated with VOC severity nor with markers of hemostasis activation, and were similar to those reported in an independent population of SCA patients at steady state. DISCUSSION: Acute VOC are not characterized by significant increases in total extracellular heme levels. Studies measuring the fraction of free extracellular heme unbound to proteins are warranted to further refine our understanding of the role of heme in acute VOC.

HRT Atlas v1.0 database: redefining human and mouse housekeeping genes and candidate reference transcripts by mining massive RNA-seq datasets.

Housekeeping (HK) genes are constitutively expressed genes that are required for the maintenance of basic cellular functions. Despite their importance in the calibration of gene expression, as well as the understanding of many genomic and evolutionary features, important discrepancies have been observed in studies that previously identified these genes. Here, we present Housekeeping and Reference Transcript Atlas (HRT Atlas v1.0, www.housekeeping.unicamp.br) a web-based database which addresses some of the previously observed limitations in the identification of these genes, and offers a more accurate database of human and mouse HK genes and transcripts. The database was generated by mining massive human and mouse RNA-seq data sets, including 11 281 and 507 high-quality RNA-seq samples from 52 human non-disease tissues/cells and 14 healthy tissues/cells of C57BL/6 wild type mouse, respectively. User can visualize the expression and download lists of 2158 human HK transcripts from 2176 HK genes and 3024 mouse HK transcripts from 3277 mouse HK genes. HRT Atlas also offers the most stable and suitable tissue selective candidate reference transcripts for normalization of qPCR experiments. Specific primers and predicted modifiers of gene expression for some of these HK transcripts are also proposed. HRT Atlas has also been integrated with a regulatory elements resource from Epiregio server.

Endothelial Barrier Integrity Is Disrupted In Vitro by Heme and by Serum From Sickle Cell Disease Patients.

Free extracellular heme has been shown to activate several compartments of innate immunity, acting as a danger-associated molecular pattern (DAMP) in hemolytic diseases. Although localized endothelial barrier (EB) disruption is an important part of inflammation that allows circulating leukocytes to reach inflamed tissues, non-localized/deregulated disruption of the EB can lead to widespread microvascular hyperpermeability and secondary tissue damage. In mouse models of sickle cell disease (SCD), EB disruption has been associated with the development of a form of acute lung injury that closely resembles acute chest syndrome (ACS), and that can be elicited by acute heme infusion. Here we explored the effect of heme on EB integrity using human endothelial cell monolayers, in experimental conditions that include elements that more closely resemble in vivo conditions. EB integrity was assessed by electric cell-substrate impedance sensing in the presence of varying concentrations of heme and sera from SCD patients or healthy volunteers. Heme caused a dose-dependent decrease of the electrical resistance of cell monolayers, consistent with EB disruption, which was confirmed by staining of junction protein VE-cadherin. In addition, sera from SCD patients, but not from healthy volunteers, were also capable to induce EB disruption. Interestingly, these effects were not associated with total heme levels in serum. However, when heme was added to sera from SCD patients, but not from healthy volunteers, EB disruption could be elicited, and this effect was associated with hemopexin serum levels. Together our in vitro studies provide additional support to the concept of heme as a DAMP in hemolytic conditions.

Hypofibrinolysis induced by tranexamic acid does not influence inflammation and mortality in a polymicrobial sepsis model.

The biological relevance of fibrinolysis to the host response to sepsis is illustrated by pathogens such as S. pyogenes and Y. pestis, whose virulence factors are proteins that challenge the balance between pro- and anti-fibrinolytic factors of the host, and by the consistent finding of hypofibrinolysis in the early stages of sepsis. Whether this hypofibrinolytic response is beneficial or detrimental to the host, by containing the spread of pathogens while at the same time limiting the access of immune cell to infectious foci, is still a matter of debate. Tranexamic acid (TnxAc) is an antifibrinolytic agent that is being increasingly used to prevent and control bleeding in conditions such as elective orthopedic surgery, trauma, and post-partum-hemorrhage, which are frequently followed by infection and sepsis. Here we used a model of polymicrobial sepsis to evaluate whether hypofibrinolysis induced by TnxAc influenced survival, tissue injury and pathogen spread. Mice were treated with two doses of TnxAc bid for 48h, and then sepsis was induced by cecal ligation and puncture. Despite the induction of hypofibrinolysis by TnxAc, no difference could be observed in survival, tissue injury (measured by biochemical and histological parameters), cytokine levels or pathogen spread. Our results contribute with a new piece of data to the understanding of the complex interplay between fibrinolysis and innate immunity. While our results do not support the use of TnxAc in sepsis, they also address the thrombotic safety of TnxAc, a low cost and widely used agent to prevent bleeding.

Effects of light emitting diode (LED) therapy at 940 nm on inflammatory root resorption in rats.

The effects of LED therapy at 940 nm on periodontal healing, inflammatory cell infiltration, and root resorption were analyzed in an experimental model of orthodontic tooth movement in rats. Twenty-five male Wistar rats were allocated into four experimental groups: Control animals (Co, n = 5), Controls + LED therapy (CoLED, n = 6), animals submitted to orthodontic force (RR, n = 7) and submitted to orthodontic force + LED therapy (RRLED, n = 7). All procedures were approved by the Committee for Ethics in Animal Experimentation of the Universidade Estadual de Londrina (protocol CEEA 5/2010 37359). A force of 50 g was applied to the right upper molars of RR and RRLED groups. On days 2, 3, and 4 after orthodontic treatment, the CoLED and RRLED groups received LED irradiation (940 nm, 4 J/cm(2)). The animals were killed on day 7 for histological analysis. An increased number of root resorption lacunae was found only in the RR group (p < 0.05). The RR group also presented more osteoclasts (p < 0.005) and inflammatory cell infiltration (p < 0.005) than the control group. The RRLED group presented fewer osteoclasts (p < 0.005) and inflammatory cells (p < 0.005) in the periodontal ligament than the RR group. The CoLED and RRLED groups presented more periodontal fibroblasts (p < 0.005) than non-irradiated groups. RRLED presented more blood vessels (p < 0.01) in the periodontal ligament than the RR group. In conclusion, the results suggest that LED therapy improved periodontal tissue repair and decreased inflammation and root resorption after the application of orthodontic force.

Effects of light emitting diode (LED) therapy and cold water immersion therapy on exercise-induced muscle damage in rats.

The aim of this work is to analyze the effects of LED therapy at 940 nm or cold water immersion therapy (CWI) after an acute bout of exercise on markers of muscle damage and inflammation. Thirty-two male Wistar rats were allocated into four groups: animals kept at rest (control), exercised animals (E), exercised + CWI (CWI), and exercised + LED therapy (LED). The animals swam for 100 min, after which blood samples were collected for lactate analysis. Animals in the E group were returned to their cages without treatment, the CWI group was placed in cold water (10°C) for 10 min and the LED group received LED irradiation on both gastrocnemius muscles (4 J/cm(2) each). After 24 h, the animals were killed and the soleus muscles were submitted to histological analysis. Blood samples were used for hematological and CK analyses. The results demonstrated that the LED group presented fewer areas of muscle damage and inflammatory cell infiltration and lower levels of CK activity than the E group. Fewer areas of damaged muscle fiber were observed in the LED group than in CWI. CWI and LED did not reduce edema areas. Hematological analysis showed no significant effect of either treatment on leukocyte counts. The results suggest that LED therapy is more efficient than CWI in preventing muscle damage and local inflammation after exercise.

Effects of 940 nm light-emitting diode (led) on sciatic nerve regeneration in rats.

The objective of the present study was to evaluate the effect of 940 nm wavelength light emitting diode (LED) phototherapy on nerve regeneration in rats. Forty male Wistar rats weighing approximately 300 g each were divided into four groups: control (C); control submitted to LED phototherapy (CLed); Sciatic Nerve Lesion without LED phototherapy (L); Sciatic Nerve Lesion with LED phototherapy (LLed). The lesion was caused by crushing the right sciatic nerve. A dose of 4 J/cm(2) was used for ten consecutive days beginning on the first postoperative day. Groups C and L were submitted to the same procedure as the LLed group, but the equipment was turned off. The LED phototherapy with 940 nm wavelength reduced the areas of edema, the number of mononuclear cells present in the inflammatory infiltration, and increased functional recovery scores at 7, 14 and 21 days. The results suggest that the use of phototherapy at 940 nm after nerve damage improves morphofunctional recovery and nerve regeneration.