Carla Cicala

Novel nonsterodial anti-inflammatory drug derivatives with markedly reduced ulcerogenic properties in the rat

BACKGROUND/AIMS The use of nonsteroidal anti-inflammatory drugs (NSAIDs) is limited by their ability to induce gastrointestinal injury. Two NSAIDs were modified by incorporation of an nitroxybutyl moiety. The short-term ulcerogenic and anti-inflammatory properties of these derivatives were compared with the native NSAIDs. METHODS Rats were given flurbiprofen, ketoprofen, or their respective derivatives, and the extent of gastric damage and effect on gastric prostaglandin E2 synthesis was assessed. The damage-promoting effects of these compounds were also compared following twice-daily administration for 1 week. Anti-inflammatory properties were examined using a carrageenan-induced paw edema model. RESULTS The derivatives of flurbiprofen and ketoprofen caused significantly less short-term gastric mucosal injury at all doses tested, despite producing comparable suppression of prostaglandin synthesis. The NSAID derivatives also showed comparable anti-inflammatory activity to the native compounds. The flurbiprofen derivative inhibited collagen-induced platelet aggregation significantly more than the native NSAID. Plasma nitrate/nitrite levels increased significantly following administration of the flurbiprofen derivative, consistent with release of a nitrogen oxide. CONCLUSIONS Addition of a nitroxybutyl moiety to two NSAIDs markedly reduced the ability of these agents to induce short-term gastric injury but did not interfere with their ability to suppress inflammatory processes, inhibit prostaglandin synthesis, or inhibit platelet aggregation. These NSAID derivatives may therefore represent a novel class of anti-inflammatory drugs with markedly less ulcerogenic effects on the stomach.

Linkage between inflammation and coagulation: An update on the molecular basis of the crosstalk

Inflammation and coagulation cannot be considered as two separate processes, since there are several connecting points making them part of unique, defensive host response. The endothelium can be considered as the first link between inflammation and coagulation, since damaged endothelium during inflammation represents a surface where proteins involved in both coagulation and the development of inflammation are expressed. During inflammation, cytokines modulate the coagulation system by downregulating the expression of thrombomodulin and the activation of protein C pattern but, at the same time, they induce the expression of tissue factor, modifying, in this way, the balance between procoagulant and anticoagulant activities. At the same time, at the site of tissue injury, platelets become activated and release several mediators that modify tissue integrity. Thrombin, formed following activation of the coagulation cascade, is essential to promote haemostasis but also stimulates several cell functions, including chemotaxis and mitogenesis, which are responsible for the spreading of the lesion and the tissue repair process. Therefore, in the study of inflammation the involvement of the coagulation pathway has to be taken into account, since the interaction between coagulation and inflammation pathways is a critical issue.

Factor Xa as an interface between coagulation and inflammation. Molecular mimicry of factor Xa association with effector cell protease receptor-1 induces acute inflammation in vivo.

Coagulation proteases were tested in a rat model of acute inflammation. Subplantar injection of Factor Xa (10-30 microg) produced a time- and dose-dependent edema in the rat paw, and potentiated carrageenin-induced edema. In contrast, the homologous protease Factor IXa was ineffective. This inflammatory response was recapitulated by the Factor Xa sequence L83FTRKL88(G), which mediates ligand binding to effector cell protease receptor-1 (EPR-1), while a control scrambled peptide did not induce edema in vivo. Conversely, injection of the EPR-1-derived peptide S123PGKPGNQNSKNEPP137 (corresponding to the receptor binding site for Factor Xa) inhibited carrageenin-induced rat paw edema, while the adjacent EPR-1 sequence P136PKKRERERSSHCYP150 was without effect. EPR-1-Factor Xa-induced inflammation was characterized by fast onset and prominent perivascular accumulation of activated and degranulated mast cells, was inhibited by the histamine/serotonin antagonists cyproheptadine and methysergide, but was unaffected by the thrombin-specific inhibitor, Hirulog. These findings suggest that through its interaction with EPR-1, Factor Xa may function as a mediator of acute inflammation in vivo. This pathway may amplify both coagulation and inflammatory cascades, thus contributing to the pathogenesis of tissue injury in vivo.

Protease-activated receptor-2 involvement in hypotension in normal and endotoxemic rats in vivo.

BACKGROUND The protease-activated receptor-2 (PAR-2) is expressed by vascular endothelial cells and upregulated by lipopolysaccharide (LPS) in vitro. PAR-2 is activated by a tethered ligand created after proteolytic cleavage by trypsin or experimentally by a synthetic agonist peptide (PAR-2AP) corresponding to the new amino terminus of the tethered ligand. METHODS AND RESULTS Intravenous administration of PAR-2AP (0.1, 0.3, and 1 mg/kg) to rats caused a dose-dependent hypotension. A scrambled peptide was without effect. A specific trypsin inhibitor, biotin-SGKR-chloromethylketone, inhibited trypsin-induced hypotension but not that stimulated by PAR-2AP. In animals treated with LPS 20 hours earlier, we found an increased sensitivity to trypsin and PAR-2AP in the hypotensive response. In particular, PAR-2AP caused hypotension at a low concentration of 30 ng/kg. Moreover, PAR-2 was immunolocalized to endothelial and smooth muscle cells in aorta and jugular vein in LPS-treated rats, and increased levels of PAR-2 mRNA were shown by reverse transcription-polymerase chain reaction analysis. CONCLUSIONS Our findings suggest that PAR-2 is important in the regulation of blood pressure in vivo. A functional upregulation of PAR-2 by LPS was demonstrated by the activity of concentrations of PAR-2AP that were inactive in normal animals. We conclude that PAR-2 may play an important role in the hypotension associated with endotoxic shock and may represent a new therapeutic target.

Inflammation-coagulation network: are serine protease receptors the knot?

Following an injury, the body recruits a mechanism to delimit and repair tissue damage; this phenomenon is known as inflammation. Among the several different pathways that are activated during this process, which is necessary for survival, activation of the coagulation pathway is a key feature. In fact, clinical changes in blood fluidity have been closely related to ongoing inflammation. Recent evidence suggests that serine protease receptors might play a major role in the host defence mechanism at the interface between coagulation and inflammation.

Geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) mediated signal transduction has anti‐inflammatory effects and interacts with glucocorticoid receptor in vivo

Histamine, vascular endothelial growth factor, acetylcholine, oestrogen as well as fluid shear stress activates a mechanism that recruits heat shock protein 90 to the endothelial nitric oxide synthase. The interaction between Hsp90 and eNOS enhances the activation of the enzyme in cells and in intact blood vessels leading to NO production. Intraplantar administration of carrageenan (50 μl paw−1) to mice causes an oedema lasting 72 h. Geldanamycin (0.1, 0.3, 1 mg kg−1), a specific inhibitor of Hsp‐90, that inhibits endothelium‐dependent relaxations of the rat aorta, mesentery and middle artery inhibits carrageenan‐induced mouse paw oedema in a dose dependent manner. Co‐administration to mice of dexamethasone (1 mg kg−1) with geldanamycin (0.3 mg kg−1) at anti‐inflammatory dose causes a loss of the total anti‐inflammatory effect of each agent alone. RU 486 (10 mg kg−1), a well known glucocorticoid receptorial antagonist, does not inhibit oedema formation but prevents the anti‐inflammatory action of dexamethasone (1 mg kg−1). Similarly, RU 486 prevents the anti‐inflammatory action of geldanamycin (0.3 mg kg−1). In conclusion we have described for the first time that geldanamycin, an inhibitor of Hsp90 dependent signal transduction, is anti‐inflammatory in vivo implying that Hsp90 is critical for pathways involved in carrageenan‐induced paw oedema. In addition the ability of GA to block NO release and reduce oedema formation suggests a therapeutic rationale for specific inhibitors of Hsp90 as potential anti‐inflammatory drugs.

Anti-inflammatory actions of an N-terminal peptide from human lipocortin 1

An acetylated polypeptide corresponding to residues 2–26 of human lipocortin 1 was synthesized and the anti‐inflammatory activity assessed in three models of acute inflammation in rat and mouse. In the carrageenin rat paw oedema test, the peptide produced a maximal inhibition of approximately 41% at the 3 h time point with a 10 μg dose. When rat paw oedema was induced by the injection of venom phospholipase A2, the peptide produced a significant inhibition (31%) at the top dose of 20 μg per paw. In the mouse air‐pouch model, systemic treatment with the peptide produced a dramatic reduction in cytokine‐induced leukocyte migration with an ID50 of approximately 40 μg per mouse. The N‐terminal peptide 2–26 shares the actions of lipocortin 1 in these acute models of inflammation.

IL-1β and TNF-α Regulation of the Adenosine Receptor (A2A) Expression: Differential Requirement for NF-κB Binding to the Proximal Promoter

Adenosine is a potent endogenous regulator of airway inflammation that acts through specific receptor subtypes that can either cause constriction (A1R, A2BR, and A3R) or relaxation (A2AR) of the airways. We therefore examined the effects of key inflammatory mediators on the expression of the A2AR in a lung epithelial cell line (A549). IL-1β and TNF-α increased the expression of the A2AR gene at the mRNA and protein levels. In contrast, LPS had no effect on A2AR gene expression. IL-1β and TNF-α rapidly activated p50 and p65, but not C-Rel, RelB, or p52, and both IL-1β- and TNF-α-stimulated A2AR expression was inhibited by the IκB kinase 2 inhibitor AS602868 in a concentration-dependent manner. Using chromatin immunoprecipitation assays, we demonstrate that IL-1β can enhance p65 association with putative κB binding sites in the A2AR promoter in a temporal manner. In contrast, TNF-α failed to enhance p65 binding to these putative sites. Functionally, the two most 5′ κB sites were important for IL-1β-, but not TNF-α-, induced A2AR promoter reporter gene activity. Finally, neither TNF-α nor Il-1β had any effect on A2AR mRNA transcript degradation. These results directly implicate a major role for NF-κB in the regulation of A2AR gene transcription by IL-1β and TNF-α but suggest that the effects of TNF-α on A2AR gene transcription are not mediated through the proximal promoter.

Diabetic Mouse Angiopathy Is Linked to Progressive Sympathetic Receptor Deletion Coupled to an Enhanced Caveolin-1 Expression

Objective—Clinical studies have demonstrated that hyperglycaemia represents a major risk factor in the development of the endothelial impairment in diabetes, which is the first step in vascular dysfunction. Using non-obese diabetic mice, we have evaluated the role of the adrenergic system and eNOS on progression of the disease Methods and Results—When glycosuria is high (20 to 500 mg/dL), there is a selective reduction in the response to &agr;1 and &bgr;2 agonists but not to dopamine or serotonin. When glycosuria is severe (500 to 1000 mg/dL), there is a complete ablation of the contracture response to the &agr;1 receptor agonist stimulation and a marked reduced response to &bgr;2 agonist stimulation. This effect is coupled with a reduced expression of &agr;1 and &bgr;2 receptors, which is caused by an inhibition at transcriptional level as demonstrated by RT-PCR. In the severe glycosuria (500 to 1000 mg/dL), although eNOS expression is unchanged, caveolin-1 expression is significantly enhanced, indicating that high glucose plasma levels cause an upregulation of the eNOS endogenous inhibitory tone. These latter results correlate with functional data showing that in severe glycosuria, there is a significant reduction in acetylcholine-induced vasodilatation. Conclusions—Our results show that in diabetes development, there is a progressive selective downregulation of the &agr;1 and &bgr;2 receptors. At the same time, there is an increased expression of caveolin-1, the endogenous eNOS inhibitory protein. Thus, caveolin-1 could represent a new possible therapeutic target in vascular impairment associated with diabetes.

Inhibition of CD73 Improves B Cell-Mediated Anti-Tumor Immunity in a Mouse Model of Melanoma

CD73 is a cell surface enzyme that suppresses T cell-mediated immune responses by producing extracellular adenosine. Growing evidence suggests that targeting CD73 in cancer may be useful for an effective therapeutic outcome. In this study, we demonstrate that administration of a specific CD73 inhibitor, adenosine 5′-(α,β-methylene)diphosphate (APCP), to melanoma-bearing mice induced a significant tumor regression by promoting the release of Th1- and Th17-associated cytokines in the tumor microenvironment. CD8+ T cells were increased in melanoma tissue of APCP-treated mice. Accordingly, in nude mice APCP failed to reduce tumor growth. Importantly, we observed that after APCP administration, the presence of B cells in the melanoma tissue was greater than that observed in control mice. This was associated with production of IgG2b within the melanoma. Depletion of CD20+ B cells partially blocked the anti-tumor effect of APCP and significantly reduced the production of IgG2b induced by APCP, implying a critical role for B cells in the anti-tumor activity of APCP. Our results also suggest that APCP could influence B cell activity to produce IgG through IL-17A, which significantly increased in the tumor tissue of APCP-treated mice. In support of this, we found that in melanoma-bearing mice receiving anti–IL-17A mAb, the anti-tumor effect of APCP was ablated. This correlated with a reduced capacity of APCP-treated mice to mount an effective immune response against melanoma, as neutralization of this cytokine significantly affected both the CD8+ T cell- and B cell-mediated responses. In conclusion, we demonstrate that both T cells and B cells play a pivotal role in the APCP-induced anti-tumor immune response.