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The short release half-life of carbon monoxide (CO) is a major obstacle to the effective therapeutic use of carbon monoxide-releasing molecule-2 (CORM-2). The potential of CORM-2-entrapped ultradeformable liposomes (CORM-2-UDLs) to enhance the release half-life of CO and alleviate skin inflammation was investigated in the present study. CORM-2-UDLs were prepared by using soy phosphatidylcholine to form lipid bilayers and Tween 80 as an edge activator. The deformability of CORM-2-UDLs was measured and compared with that of conventional liposomes by passing formulations through a filter device at a constant pressure. The release profile of CO from CORM-2-UDLs was evaluated by myoglobin assay. In vitro and in vivo anti-inflammatory effects of CORM-2-UDLs were assessed in lipopolysaccharide-stimulated macrophages and TPA-induced ear edema model, respectively. The deformability of the optimized CORM-2-UDLs was 2.3 times higher than conventional liposomes. CORM-2-UDLs significantly prolonged the release half-life of CO from 30 s in a CORM-2 solution to 21.6 min. CORM-2-UDLs demonstrated in vitro anti-inflammatory activity by decreasing nitrite production and pro-inflammatory cytokine levels. Furthermore, CORM-2-UDLs successfully ameliorated skin inflammation by reducing ear edema, pathological scores, neutrophil accumulation, and inflammatory cytokines expression. The results demonstrate that CORM-2-UDLs could be used as promising therapeutics against acute skin inflammation., Graphical abstract The short release half-life of carbon monoxide is a major obstacle to the effective therapeutic use of carbon monoxide-releasing molecule-2 (CORM-2). Herein, CORM-2-entrapped ultradeformable liposomes (CORM-2-UDLs) were designed to prolong carbon monoxide release half-life and enhance skin permeation of CORM-2 for better anti-inflammatory effects in skin inflammation model.Image 1

Toxic carbon monoxide in small concentrations has pro-apoptotic, anti-allergic, vasodilator effects, and stimulates angiogenesis. The problem with the therapeutic use of low doses of carbon monoxide is that it is difficult to dose. To control the amount and gradual release of carbon monoxide, non-toxic preparation is used - CO donor based on carbonyl compound of ruthenium (CORM-2). The aim – is to identify the effect of CORM-2 on the level of immunoglobulins in the blood serum and bone marrow of mice under conditions of inducing an immune response. Materials and methods. 3 groups of 15 white laboratory mice each were formed. Induction of the immune response was due to the intraperitoneal administration of xenogenic red blood cells. The first experimental group on the first day of immunization received CORM-2 (20 mg/kg), group No. 2 – on the 5th day after immunization (period of the productive phase). The control group consisted of immunized animals that did not receive CORM-2. The amount of IgA, IgM, and IgG in blood serum was determined by ELISA on the 2nd and 6th day after immunization. At the end of the experiment, bone marrow cell populations were counted. Results. After the injection of CORM-2 during the induction phase of the immune response, it inhibits the production of immunoglobulins. In comparison with the control, the level of IgA and IgG is reduced, but the amount of IgM remains unchanged. In the bone marrow, the number of monocytes, erythroblasts, and normoblasts, as well as lymphoblasts and plasma cells, increased. At the same time, the number of myeloblasts, myelocytes, basophilic normoblasts, and megakaryocytes decreased. The use of CORM-2 during the productive phase caused a decrease in the level of IgM and IgG with a simultaneous increase in the level of IgA. The number of neutrophils, eosinophils, monocytes, polychromophilic and oxyphilic normoblasts, lymphocytes, and plasma cells in the bone marrow increased. However, the number of myeloblasts, promyelocytes, myelocytes, metamyelocytes, basophilic normoblasts, and megakaryocytes decreased. Conclusions. The impact of the CORM-2 on the inductive phase of the immune response leads to inhibition of the production of immunoglobulins. The injection of CORM-2 during the productive phase of the immune response decreased the level of IgM and IgG, but at the same time, an increase in the level of IgA was observed. After the injection of CORM-2, in the bone marrow, the number of monocytes, lymphocytes, and plasma cells increased. The results indicate that CORM-2 is able to modulate the immune response.

Carbon monoxide (CO)-releasing molecules (CORMs) are used to deliver CO, a biological ‘gasotransmitter’, in biological chemistry and biomedicine. CORMs kill bacteria in culture and in animal models, but are reportedly benign towards mammalian cells. CORM-2 (tricarbonyldichlororuthenium(II) dimer, Ru2Cl4(CO)6), the first widely used and commercially available CORM, displays numerous pharmacological, biochemical and microbiological activities, generally attributed to CO release. Here, we investigate the basis of its potent antibacterial activity against Escherichia coli and demonstrate, using three globin CO sensors, that CORM-2 releases negligible CO (&lt, 0.1 mol CO per mol CORM-2). A strong negative correlation between viability and cellular ruthenium accumulation implies that ruthenium toxicity underlies biocidal activity. Exogenous amino acids and thiols (especially cysteine, glutathione and N-acetyl cysteine) protected bacteria against inhibition of growth by CORM-2. Bacteria treated with 30 μM CORM-2, with added cysteine and histidine, exhibited no significant loss of viability, but were killed in the absence of these amino acids. Their prevention of toxicity correlates with their CORM-2-binding affinities (Cys, Kd 3 μM, His, Kd 130 μM) as determined by 1H-NMR. Glutathione is proposed to be an important intracellular target of CORM-2, with CORM-2 having a much higher affinity for reduced glutathione (GSH) than oxidised glutathione (GSSG) (GSH, Kd 2 μM, GSSG, Kd 25,000 μM). The toxicity of low, but potent, levels (15 μM) of CORM-2 was accompanied by cell lysis, as judged by the release of cytoplasmic ATP pools. The biological effects of CORM-2 and related CORMs, and the design of biological experiments, must be re-examined in the light of these data.

Toxic carbon monoxide in small concentrations has pro-apoptotic, anti-allergic, vasodilator effects, and stimulates angiogenesis. The problem with the therapeutic use of low doses of carbon monoxide is that it is difficult to dose. To control the amount and gradual release of carbon monoxide, non-toxic preparation is used - CO donor based on carbonyl compound of ruthenium (CORM-2).The aim – is to identify the effect of CORM-2 on the level of immunoglobulins in the blood serum and bone marrow of mice under conditions of inducing an immune response.Materials and methods. 3 groups of 15 white laboratory mice each were formed. Induction of the immune response was due to the intraperitoneal administration of xenogenic red blood cells. The first experimental group on the first day of immunization received CORM-2 (20 mg/kg), group No. 2 – on the 5th day after immunization (period of the productive phase). The control group consisted of immunized animals that did not receive CORM-2. The amount of IgA, IgM, and IgG in blood serum was determined by ELISA on the 2nd and 6th day after immunization. At the end of the experiment, bone marrow cell populations were counted.Results. After the injection of CORM-2 during the induction phase of the immune response, it inhibits the production of immunoglobulins. In comparison with the control, the level of IgA and IgG is reduced, but the amount of IgM remains unchanged. In the bone marrow, the number of monocytes, erythroblasts, and normoblasts, as well as lymphoblasts and plasma cells, increased. At the same time, the number of myeloblasts, myelocytes, basophilic normoblasts, and megakaryocytes decreased. The use of CORM-2 during the productive phase caused a decrease in the level of IgM and IgG with a simultaneous increase in the level of IgA. The number of neutrophils, eosinophils, monocytes, polychromophilic and oxyphilic normoblasts, lymphocytes, and plasma cells in the bone marrow increased. However, the number of myeloblasts, promyelocytes, myelocytes, metamyelocytes, basophilic normoblasts, and megakaryocytes decreased.Conclusions. The impact of the CORM-2 on the inductive phase of the immune response leads to inhibition of the production of immunoglobulins. The injection of CORM-2 during the productive phase of the immune response decreased the level of IgM and IgG, but at the same time, an increase in the level of IgA was observed. After the injection of CORM-2, in the bone marrow, the number of monocytes, lymphocytes, and plasma cells increased. The results indicate that CORM-2 is able to modulate the immune response., Токсический монооксид углерода в незначительных количествах обладает проапоптическим, противоаллергическим действием, имеет вазодилататорное влияние, стимулирует ангиогенез. Проблема его терапевтического использования заключается в сложности точной дозировки. Для контроля количества и постепенного высвобождения монооксида углерода используют нетоксический препарат – донор СО на основе карбонильного соединения рутения (CORM-2).Цель работы - выявить влияние CORM-2 на уровень иммуноглобулинов сыворотки крови и костного мозга мышей в условиях индукции иммунного ответа.Материалы и методы. Сформировано 3 группы по 15 белых лабораторных мышей. Индукция иммунного ответа была получена путем внутрибрюшинного введения ксеногенных эритроцитов. Первая экспериментальная группа в первый день иммунизации получила CORM-2 (20 мг/кг), группа №2 – на 5 день после иммунизации (период продуктивной фазы). Контрольная группа состояла из иммунизированных мышей, которые не получали CORM-2. Определяли количество IgA, IgM и IgG в сыворотке крови методом ИФА анализа на 2 и 6 дни после иммунизации соответственно. В конце эксперимента подсчитывали популяции клеток костного мозга.Результаты. Введение CORM-2 в индукционную фазу иммунного ответа сдерживает продукцию иммуноглобулинов. В сравнении с контролем, уровень IgА и IgG является сниженным, но количество IgМ остается неизменным. В костном мозге увеличилось количество моноцитов, эритробластов и нормобластов, а также лимфобластов и плазматических клеток. Одновременно снизилось количество миелобластов, миелоцитов, базофильных нормобластов и мегакариоцитов. Введение CORM-2 в период продуктивной фазы обуславливал снижение уровня IgМ и IgG с одновременным повышением уровня IgА. Количество нейтрофилов, эозинофилов, моноцитов, полихроматофильных и оксифильных нормобластов, лимфоцитов и плазматических клеток в костном мозге увеличилось. Вместе с тем количество миелобластов, промиелоцитов, миелоцитов, метамиелоцитов, базофильных нормобластов и мегакариоцитов уменьшилось.Выводы. Введение CORM-2 в период продуктивной фазы иммунного ответа снизило уровень IgМ и IgG, но одновременно обуславливает повышение уровня IgА. После введения CORM-2 в костном мозге увеличивалось количество моноцитов, лимфоцитов и плазматических клеток. Полученные результаты указывают на то, что CORM-2 способен модулировать иммунный ответ., Токсичний монооксид вуглецю в незначних концентраціях володіє проапоптичною, протиалергійною дією, має вазодилататорний вплив, стимулює ангіогенез. Проблема його терапевтичного застосування полягає у складності точного дозування. Для контролю кількості та поступового вивільнення монооксиду карбону застосовують нетоксичний препарат – донор СО на основі карбонільної сполуки рутенію (CORM-2).Мета роботи - виявити вплив CORM-2 на рівень імуноглобулінів сироватки крові та кісткового мозку мишей в умовах індукції імунної відповіді.Матеріали та методи. Сформували 3 групи по 15 білих лабораторних мишей, з них – дві експериментальні та одна контрольна. Індукцію імунної відповіді спричиняли шляхом внутрішньоочеревинного уведення ксеногенних еритроцитів. Перша експериментальна група в перший день імунізації отримала CORM-2 (20 мг/кг), група №2 – на 5 день після імунізації (період продуктивної фази). Контрольну групу становили імунізовані миші, які не отримували CORM-2. Визначали кількість IgA, IgM, IgG у сироватці крові методом ІФА аналізу на 2 і 6 дні після імунізації. Наприкінці експерименту підраховували популяції клітин кісткового мозку.Результати. Введення CORM-2 в індукційну фазу імунної відповіді стримує продукцію імуноглобулінів. Порівнюючи з контролем, рівень IgА та IgG – знижений, але кількість IgМ залишалася незмінною. У кістковому мозку збільшилася кількість моноцитів, еритробластів і нормобластів, а також лімфобластів і плазматичних клітин. Одночасно знизилася кількість мієлобластів, мієлоцитів, базофільних нормобластів, мегакаріоцитів. Уведення CORM-2 в період продуктивної фази спричиняв зниження рівня IgМ та IgG з одночасним підвищенням рівня IgА. Кількість нейтрофілів, еозинофілів, моноцитів, поліхромнофільних та оксифільних нормобластів, лімфоцитів і плазматичних клітин у кістковому мозку збільшилася. Поряд з тим кількість мієлобластів, промієлоцитів, мієлоцитів, метамієлоцитів, базофільних нормобластів і мегакаріоцитів зменшилася.Висновки. CORM-2 в індукційну фазу імунної відповіді спричиняє пригнічення продукції імуноглобулінів. Уведення CORM-2 в період продуктивної фази імунної відповіді знижує рівень IgМ та IgG, але одночасно спостерігається підвищення рівня IgА. Після застосування CORM-2 у кістковому мозку збільшується кількість моноцитів, лімфоцитів, плазматичних клітин. Результати, що отримали, вказують: CORM-2 здатен модулювати імунну відповідь.

During the process of human islet isolation a cascade of stressful events are triggered and negatively influence islet yield, viability, and function, including the production of proinflammatory cytokines and activation of apoptosis. Carbon monoxide-releasing molecule 2 (CORM-2) is a donor of carbon monoxide (CO) and can release CO spontaneously. Accumulating studies suggest that CORM-2 exerts cytoprotective and anti-inflammatory properties. However, the effect of CORM-2 on islet isolation is still unclear. In this study, we found that CORM-2 pretreatment significantly decreased the expression of critical inflammatory genes, including tissue factor, intercellular adhesion molecule-1, chemokine ( C-C motif) ligand 2, C-X-C motif chemokine 10, Toll-like receptor 4, interleukin-1β, interleukin-6, and tumor necrosis factor-α ( TNF-α). The isolated islets of the CORM-2 pretreatment group showed reduced apoptotic rate, improved viability, and higher glucose-stimulated insulin secretion, and functional gene expression in comparison to control group. Importantly, CORM-2 pretreatment prevented the impairment caused by TNF-α, evidenced by the improved glucose-stimulated index and transplantation outcomes. The present study demonstrated the anti-inflammatory property of CORM-2 during human islet isolation, and we suggest that CORM-2 pretreatment is an appealing treatment to mitigate inflammation-mediated islet dysfunction during isolation and culture ex vivo and to preserve long-term islet survival and function.

The up-regulation of heme oxygenase-1 (HO-1) is mediated through nicotinamaide adenine dinucleotide phosphate (NADPH) oxidases (Nox) and reactive oxygen species (ROS) generation, which could provide cytoprotection against inflammation. However, the molecular mechanisms of carbon monoxide-releasing molecule (CORM)-2-induced HO-1 expression in human tracheal smooth muscle cells (HTSMCs) remain unknown. Here, we found that pretreatment with CORM-2 attenuated the lipopolysaccharide (LPS)-induced intercellular adhesion molecule (ICAM-1) expression and leukocyte count through the up-regulation of HO-1 in mice, which was revealed by immunohistochemistrical staining, Western blot, real-time PCR, and cell count. The inhibitory effects of HO-1 by CORM-2 were reversed by transfection with HO-1 siRNA. Next, Western blot, real-time PCR, and promoter activity assay were performed to examine the HO-1 induction in HTSMCs. We found that CORM-2 induced HO-1 expression via the activation of protein kinase C (PKC)&alpha, and proline-rich tyrosine kinase (Pyk2), which was mediated through Nox-derived ROS generation using pharmacological inhibitors or small interfering ribonucleic acids (siRNAs). CORM-2-induced HO-1 expression was mediated through Nox-(1, 2, 4) or p47phox, which was confirmed by transfection with their own siRNAs. The Nox-derived ROS signals promoted the activities of extracellular signal-regulated kinase 1/2 (ERK1/2). Subsequently, c-Fos and c-Jun&mdash, activator protein-1 (AP-1) subunits&mdash, were up-regulated by activated ERK1/2, which turned on transcription of the HO-1 gene by regulating the HO-1 promoter. These results suggested that in HTSMCs, CORM-2 activates PKC&alpha, /Pyk2-dependent Nox/ROS/ERK1/2/AP-1, leading to HO-1 up-regulation, which suppresses the lipopolysaccharide (LPS)-induced airway inflammation.

The upregulation of heme oxygenase-1 (HO-1) by the carbon monoxide-releasing molecule (CORM)-2 may be mediated through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [Nox] and reactive oxygen species (ROS) generation, which could provide cytoprotection against various cellular injuries. However, the detailed mechanisms of CORM-2-induced HO-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain largely unknown. Therefore, we dissected the mechanisms underlying CORM-2-induced HO-1 expression in HPAEpiCs. We found that the administration of mice with CORM-2 attenuated the tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule-1 (ICAM-1) expression and leukocyte count as revealed by immunohistochemical staining, western blot, real-time polymerase chain reaction (PCR), and cell count. Furthermore, TNF-α-induced ICAM-1 expression associated with monocyte adhesion to HPAEpiCs was attenuated by infection with adenovirus (adv)-HO-1 or incubation with CORM-2. These inhibitory effects of HO-1 were reversed by pretreatment with hemoglobin (Hb). Moreover, CORM-2-induced HO-1 expression was mediated via the phosphorylation of p47phox, c-Src, epidermal growth factor receptor (EGFR), Akt, and NF-E2-related factor 2 (Nrf2), which were inhibited by their pharmacological inhibitors, including diphenyleneiodonium (DPI) or apocynin (APO), ROS [N-acetyl-L-cysteine (NAC)], PP1, AG1478, PI3K (LY294002), or Akt (SH-5), and small interfering RNAs (siRNAs). CORM-2-enhanced Nrf2 expression, and anti-oxidant response element (ARE) promoter activity was also inhibited by these pharmacological inhibitors. The interaction between Nrf2 and AREs was confirmed with a chromatin immunoprecipitation (ChIP) assay. These findings suggest that CORM-2 increases the formation of the Nrf2 and AREs complex and binds with ARE-binding sites via Src, EGFR, and PI3K/Akt, which further induces HO-1 expression in HPAEpiCs. Thus, the HO-1/CO system might suppress TNF-α-mediated inflammatory responses and exert a potential therapeutic strategy in pulmonary diseases.

Amaç: Erektik disfonksiyon temelinde endotel bozukluğun geliştirdiği çok boyutlu bir hastalık olmakla birlikte hipertansiyon, diyabet gibi kardiyvasküler hastalıkların erken belirtisi olarak düşünülmektedir. Son yıllarda kardiyovasküler hastalıklarda gazotransmitterlerin önemi giderek artmıştır. Gazotransmitter olarak bilinen NO, H2S ve CO arasındaki etkileşim birçok çalışmaya konu olmuştur. Bu çalışmadaki amaç CO donörü olan CORM-2'nin corpus cavernosumda gevşeme yanıtları üzerine etkilerinin ve diğer gazotransmiterler olan NO ve H2S aracılı yanıtlarla ilişkisinin araştırılmasıdır. Gereç ve Yöntem: CO'nun H2S ve NO ile ilişkisinin araştırılması için fare korpus kavernosum dokularında NO inhibitörü olan L-NAME ve H2S inhibitörü olan AOAA varlığında kümülatif CORM-2 yanıtları alınmıştır. Endojen H2S' e CORM-2 etkisini değerlendirebilmek amacıyla CORM-2 inkübasyonu yapılmış dokularda kümülatif L-sistein yanıtları alınmıştır. Bir diğer deney setinde ise CORM-2'nin yukarıda bahsedilen inhibitörler varlığında H2S' e etkisinin değerlendirilmesi amacı ile metilen mavisi analizleri gerçekleştirilmiştir. Bulgular: H2S inhibitörü olan AOAA varlığında alınan CORM-2 yanıtları endotelli ve endotelsiz dokularda farklılık göstermektedir. L-NAME varlığında alınan CORM2 yanıtlarında kontrol grubuna kıyasla Emaks değerleri anlamlı olarak azalmaktadır. CORM-2 inkübasyonu varlığında L-sistein yanıtlarında ise Emaks'da anlamlılık bulunmamıştır. Metilen mavisi deneyinde elde edilen sonuçlara göre, L-sistein ile indüklenen CORM-2'nin H2S düzeylerini artırdığı bu artışın ise AOAA ile geri döndüğü gösterilmiştir. Sonuç: Bütün çalışmaların doğrultusunda CORM-2 yanıtlarının endotelli ve endotelsiz dokularda farklı olması CO'nun H2S ve NO ile etkileşimine ışık tutacak yeni bir bulgu içermektedir., Objective: Although it is a multidimensional disease developed by endothelial disorder on the basis of erectic dysfunction, it is considered as an early symptom of cardiovascular diseases such as hypertension and diabetes. In recent years, the importance of gazo transmitters has increased in cardiovascular diseases. The interaction between NO, H2S and CO, known as gasotransmitters, has been the subject of many studies. The aim of this study was to investigate the effects of CO donor CORM-2 on relaxation responses in corpus cavernosum and its relationship with other gasotransmitters, NO and H2S mediated responses. Materials and Methods: In order to investigate the relationship between CO and H2S and NO, cumulative CORM-2 responses were obtained in the presence of LNAME which is NO inhibitor and AOAA is H2S inhibitor in mouse corpus cavernosum tissues. In order to evaluate the effect of CORM-2 on endogenous H2S, cumulative L-cysteine responses were obtained in CORM-2 incubated tissues. In another experimental set, methylene blue analyzes were performed to evaluate the effect of CORM-2 on H2S in the presence of the above-mentioned inhibitors. Results: In the presence of H2S inhibitor AOAA, CORM-2 responses differ in endothelial and non-endothelial tissues. In the presence of L-NAME, the CORM-2 responses significantly decreased the Emax values compared to the control group. In the presence of CORM-2 incubation, L-cysteine responses were not significant in Emax. According to the results obtained in the methylene blue experiment, it was shown that L-cysteine-induced CORM-2 increased H2S levels and this increase was returned by AOAA. Conclusion: According to all studies, the differentiation of CORM-2 responses in endothelial and non-endothelial tissues contains a new finding to shed light on the interaction of CO with H2S and NO.

Neuropathic pain is still an extremely important problem in today's medicine because opioids, which are commonly used to reduce pain, have limited efficacy in this type of pathology. Therefore, complementary therapy is needed. Our experiments were performed in rats to evaluate the contribution of the purinergic system, especially P2X4 receptor (P2X4R), in the modulation of glia activation and, consequently, the levels of nociceptive interleukins after chronic constriction injury (CCI) of the right sciatic nerve, a rat model of neuropathic pain. Moreover, we studied how intrathecal (ith.) injection of a P2X4R antagonist Tricarbonyldichlororuthenium (II) dimer (CORM-2) modulates nociceptive transmission and opioid effectiveness in the CCI model. Our results demonstrate that repeated ith. administration of CORM-2 once daily (20 μg/5 μl, 16 and 1 h before CCI and then daily) for eight consecutive days significantly reduced pain-related behavior and activation of both spinal microglia and/or astroglia induced by CCI. Moreover, even a single administration of CORM-2 on day 7 after CCI attenuated mechanical and thermal hypersensitivity as efficiently as morphine and buprenorphine. In addition, using Western blot, we have shown that repeated ith. administration of CORM-2 lowers the CCI-elevated level of MMP-9 and pronociceptive interleukins (IL-1β, IL-18, IL-6) in the dorsal L4-L6 spinal cord and/or DRG. Furthermore, in parallel, CORM-2 upregulates spinal IL-1Ra; however, it does not influence other antinociceptive factors, IL-10 and IL-18BP. Additionally, based on our biochemical results, we hypothesize that p38MAPK, ERK1/2 and PI3K/Akt but not the NLRP3/Caspase-1 pathway are partly involved in the CORM-2 analgesic effects in rat neuropathic pain. Our data provide new evidence that P2X4R may indeed play a significant role in neuropathic pain development by modulating neuroimmune interactions in the spinal cord and DRG, suggesting that its blockade may have potential therapeutic utility.

Introduction: Heme oxygenase-1 (HO-1) is the rate limiting enzyme that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). The first human case of HO-1 deficiency showed abnormalities in blood coagulation and the fibrinolytic system. Thus, HO-1 or HO-1 products, such as CO, might regulate coagulation and the fibrinolytic system. This study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberates CO, modulates the expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVECs), and TF expression in peripheral blood mononuclear cells (PBMCs). Additionally, we examined the mechanism by which CO exerts its effects. Materials and Methods: HUVECs were pretreated with 50 μM CORM-2 for 3 hours, and stimulated with tumor necrosis factor-α (TNF-α, 10 ng/ml) for an additional 0-5 hours. PBMCs were pretreated with 50-100 μM CORM-2 for 1hour followed by stimulating with lipopolysaccharid (LPS, 10 ng/ml) for additional 0-9 hours. The mRNA and protein levels were determined by RT-PCR and western blotting, respectively. Results: Pretreatment with CORM-2 significantly inhibited TNF-α-induced TF and PAI-1 up-regulation in HUVECs, and LPS-induced TF expression in PBMCs. CORM-2 inhibited TNF-α-induced activation of p38 MAPK, ERK1/2, JNK, and NF-κB signaling pathways in HUVECs. Conclusions: CORM-2 suppresses TNF-α-induced TF and PAI-1 up-regulation, and MAPKs and NF-κB signaling pathways activation by TNF-α in HUVECs. CORM-2 suppresses LPS-induced TF up-regulation in PBMCs. Therefore, we envision that the antithrombotic activity of CORM-2 might be used as a pharmaceutical agent for the treatment of various inflammatory conditions. © 2012 Elsevier Ltd., Thesis of Keiko Maruyama / 丸山 慶子 博士論文 金沢大学医薬保健学総合研究科（保健学専攻）

Folic Acid (FA)-tagged protein nanoemulsions were found to be preferentially internalized on B-cell lymphoma cell line (A20 cell line), which, for the first time, are reported to express folate receptor (FR)-alpha. Carbon monoxide releasing molecule-2 (CORM-2) was incorporated in the oil phase of the initial formulation. FA-functionalized nanoemulsions loaded with CORM-2 exhibited a considerable antitumor effect and an increased survival of BALB/c mice bearing subcutaneous A20 lymphoma tumors. The developed nanoemulsions also demonstrated to be well tolerated by these immunocompetent mice. Thus, the results obtained in this study demonstrate that FA-tagged protein nanoemulsions can be successfully used in cancer therapy, with the important ability to delivery drugs intracellularly., SFRH/BD/81479/2011 and SFRH/BD/81269/2011 scholarships from Fundação para a Ciência e a Tecnologia (FCT). This work has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC-COMPETE and by Portuguese funds from FCT through the project PEst-OE/BIA/UI4050/2014.

Background Increased resistance to antimicrobial agents is a characteristic of many bacteria growing in biofilms on for example indwelling urinary catheters or in intracellular bacterial reservoirs. Biofilm-related infections caused by multidrug-resistant bacteria, such as extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, are a major challenge. The aim of this study was to investigate if a carbon monoxide-releasing molecule (CORM-2) has antibacterial effects against ESBL-producing uropathogenic E. coli (UPEC) in the biofilm mode of growth and following colonization of host bladder epithelial cells. Results The effect of CORM-2 was examined on bacteria grown within an established biofilm (biofilm formed for 24 h on plastic surface) by a live/dead viability staining assay. CORM-2 (500 μM) exposure for 24 h killed approximately 60 % of the ESBL-producing UPEC isolate. A non-ESBL-producing UPEC isolate and the E. coli K-12 strain TG1 were also sensitive to CORM-2 exposure when grown in biofilms. The antibacterial effect of CORM-2 on planktonic bacteria was reduced and delayed in the stationary growth phase compared to the exponential growth phase. In human bladder epithelial cell colonization experiments, CORM-2 exposure for 4 h significantly reduced the bacterial counts of an ESBL-producing UPEC isolate. Conclusion This study shows that CORM-2 has antibacterial properties against multidrug-resistant UPEC under biofilm-like conditions and following host cell colonization, which motivate further studies of its therapeutic potential.

Objective: The aim of this study was to observe the counter-effect of carbon monoxide-releasing molecule-2 (CORM-2) on lipopolysaccharide (LPS)-suppressed thrombomodulin (TM) and endothelial protein C receptor (EPCR) expressions from human umbilical vein endothelial cell (HUVEC), and to reveal its mechanisms. Methods: HUVECs were divided into 5 treatment groups, wherein reagents were added simultaneously. TM and EPCR proteins of the cells and the culture medium levels of soluble TM, soluble EPCR, and matrix metalloproteinase-2 (MMP-2) were detected after administration, whereas mRNA levels of TM and EPCR, as well as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity among groups, were also evaluated. Results: No significant difference was observed in any indicator between CORM-2 and sham groups. Addition of LPS produced drastic increase in MMP-2 expression, NF-κB activity, shedding of TM and EPCR (into the culture medium), as well as remarkable decrease in both mRNA and protein expressions of TM and EPCR, and cell viability. LPS + CORM-2 treatment significantly reduced the increase in MMP-2, NF-κB activity, and TM/EPCR shedding, whereas maintained both mRNA and protein levels of TM and EPCR, and preserved cell viability. Conclusions: CORM-2 protects HUVEC from LPS-induced injury, by way of suppressing NF-κB activity, which downregulates TM and EPCR mRNAs. It also decreases MMP-2 expression and prevents the shedding of TM and EPCR from the surface of endothelial cells, so as to preserve their protective effect.

Background/Aims: Carbon monoxide (CO) interferes with cytochrome-dependent cellular functions and acts as gaseous transmitter. CO is released from CO-releasing molecules (CORM) including tricarbonyl-dichlororuthenium (II) dimer (CORM-2), molecules considered for the treatment of several disorders including vascular dysfunction, inflammation, tissue ischemia and organ rejection. Cytochrome P450-sensitive function include formation of 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) by renal 25-hydroxyvitamin D-3 1-alpha-hydroxylase (Cyp27b1). The enzyme is regulated by PTH, FGF23 and klotho. 1,25(OH)(2)D-3 regulates Ca2+ and phosphate transport as well as klotho expression. The present study explored, whether CORM-2 influences 1,25(OH)(2)D-3 formation and klotho expression. Methods: Mice were treated with intravenous CORM-2 (20 mg/kg body weight). Plasma 1,25(OH)(2)D-3 and FGF23 concentrations were determined by ELISA, phosphate, calcium and creatinine concentrations by colorimetric methods, transcript levels by quantitative RT-PCR and protein expression by western blotting. Fgf23 mRNA transcript levels were further determined in rat osteosarcoma UMR106 cells without or with prior treatment for 24 hours with 20 mu M CORM-2. Results: CORM-2 injection within 24 hours significantly increased FGF23 plasma levels and decreased 1,25(OH)(2)D-3 plasma levels, renal Cyp27b1 gene expression as well as renal klotho protein abundance and transcript levels. Moreover, treatment of UMR106 cells with CORM-2 significantly increased Fgf23 transcript levels. Conclusion: CO-releasing molecule CORM-2 enhances FGF23 expression and release and decreases klotho expression and 1,25(OH)(2)D-3 synthesis.

Background: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO – a critical gasotransmitter – in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. Methods: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli — cytochrome bd-I, cytochrome bd-II and cytochrome bo′, to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (Km 0.24μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. Results: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo′. Cytochromes bo′ and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. Conclusions: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. General significance: Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.