Endothelium‐dependent and endothelium‐independent effects of 1‐nitro‐2‐propylbenzene on rat aorta

A group of nitro compounds contains a benzene ring in a short aliphatic chain with the NO₂ group, property that supposedly favors its vasodilator profile. In this study, we evaluated in isolated rat aorta the effects of 1‐nitro‐2‐propylbenzene (NPB), a nitro compound containing the NO₂ in the aromatic ring. In aorta precontracted with KCl, NPB (1‐3000 μm ) induced full endothelium‐independent relaxation. In endothelium‐intact preparations, phenylephrine‐induced contractions were fully relaxed by NPB, effect unaltered by N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME) or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ). In the concentration range of 30–300 μm , NPB slightly but significantly potentiated the phenylephrine‐induced contraction. Such potentiation was unaltered by the thromboxane‐prostanoid receptor antagonist seratrodast, but was abolished by endothelium removal or by preincubation of endothelium‐intact preparations with L‐NAME, ODQ or by ruthenium red and HC‐030031, blockers of subtype 1 of ankyrin transient receptor potential (TRPA₁) channels. Verapamil exacerbated the potentiating effect of NPB. The potentiating effect was undetectable in preparations precontracted by 9,11‐dideoxy‐11α,9α‐epoxymethanoprostaglandin F2α (U‐46619). Relaxation was reduced by ruthenium red while it was enhanced by HC‐030031. In conclusion, NPB has vasodilator properties but with a mechanism of action distinct from its analogues. Contrary to other nitro compounds, its relaxing effects did not involve recruitment of the guanylyl cyclase pathway. NPB has also endothelium‐dependent potentiating properties on phenylephrine‐induced contractions, a phenomenon that putatively required a role of endothelial TRPA₁ channels. The present findings reinforce the notion that the functional group NO₂ in the aliphatic chain of these nitro compounds determines favorably their vasodilator properties.     

Angiostatin K1‐3 induces E‐selectin via AP1 and Ets1: a mediator for anti‐angiogenic action of K1‐3

Summary. Background: Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1‐3 included. We previously showed that K1‐3 was the most potent angiostatin to induce E‐selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1‐3‐induced E‐selectin expression and investigate the role of E‐selectin in the anti‐angiogenic action of K1‐3. Methods and results: Quantitative real time RT‐PCR and Western blotting analyses confirmed a time‐dependent increase of E‐selectin mRNA and protein induced by K1‐3. Subcellular fractionation and immunofluorescence microscopy showed the co‐localization of K1‐3‐induced E‐selectin with caveolin 1 (Cav1) in lipid rafts in which E‐selectin may behave as a signaling receptor. Promoter‐driven reporter assays and site‐directed mutagenesis showed that K1‐3 induced E‐selectin expression via promoter activation and AP1 and Ets‐1 binding sites in the proximal E‐selectin promoter were required for E‐selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1‐3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E‐selectin by K1‐3. A modulatory role of E‐selectin in the anti‐angiogenic action of K1‐3 was manifested by both overexpression and knockdown of E‐selectin followed by cell proliferation assay. Conclusions: We show that K1‐3 induced E‐selectin expression via AP1 and Ets‐1 binding to the proximal E‐selectin promoter (?356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E‐selectin as a novel target for the anti‐angiogenic therapy.     

COX‐1+/−COX‐2−/− genotype in mice is associated with shortened time to carotid artery occlusion through increased PAI‐1

Summary. Background: We found a high incidence of thrombotic deaths in COX‐1^+/?COX‐2^?/? mice and sought to define the mechanism of these events. The cyclooxygenase products thromboxane A₂ and prostacyclin are important in the regulation of coagulation but their role in fibrinolysis is largely unexplored. PAI‐1 blocks fibrinolysis by inhibiting plasminogen activator. Aim: Our objective was to explain the mechanism of increased thrombosis associated with the COX‐1^+/?COX‐2^?/? genotype. Methods: Carotid artery occlusion times were measured after photochemical injury. PAI‐1 levels were measured in the plasma by ELISA. PAI‐1 levels in the aorta were measured by RT‐PCR and Western blotting. Urinary metabolites of Thromboxane A₂ and prostacyclin were measured by ELISA. Results: The COX‐1^+/?COX‐2^?/? genotype is associated with a decreased time to occlusion in the carotid artery thrombosis model (30 ± 5 minutes vs 60 ± minutes in wild type, p<.001). The COX‐1^?/?COX‐2^+/+, COX‐1^+/?COX‐2^+/? and COX‐1^+/? COX‐2^+/+ all had occlusion times similar to wild type. COX‐1^+/+ COX‐2^?/? had a prolonged occlusion time. COX‐1^+/? COX‐2^?/? had increased PAI‐1 levels in the plasma and aorta and with a prolonged euglobulin lysis time (37.4 ± 10.2 hours vs 15.6 ± 9.8 hours in wild type, p<.004). The decreased time to occlusion in the COX‐1^+/?COX2^?/? mice was normalized by an inhibitory antibody to PAI‐1 whereas the antibody had no effect on the time to occlusion in wild type mice. Conclusion: The COX‐1^+/?COX‐2^?/? genotype is associated with a shortened time to occlusion in the carotid thrombosis model and the shortened time to occlusion is mediated through increased PAI‐1 levels resulting in decreased fibrinolysis.     

The PKB inhibitor Akti‐1/2 potentiates PAR‐1‐mediated platelet function independently of its ability to block PKB

Summary. Background: The role of PKB in platelet function is poorly defined due to the lack of genuinely selective small‐molecule inhibitors and limiting genetic models. Recently, a selective, non‐ATP‐competitive PKB inhibitor, Akti‐1/2 has been reported, but the efficacy and specificity against PKB activation in platelet function is unknown. Objective and methods: To determine the effect of the PKB inhibitor Akti‐1/2 on PKB activation and platelet function by Western blotting and aggregometry/flow cytometry, respectively. Results: Akti‐1/2 potently inhibited thrombin‐mediated PKB phosphorylation on Thr³⁰⁸ and Ser⁴⁷³ and phosphorylation of its downstream substrate GSK3β, with a negligible effect on the phosphorylation of pleckstrin, p38, ERK and JNK. Surprisingly, Akti‐1/2 strongly potentiated PAR‐1‐mediated platelet aggregation. This effect persisted in the presence of PI3 kinase inhibitors, indicating a mechanism of action that is independent of PKB. Potentiation of aggregation by Akti‐1/2 was associated with increased [Ca⁺⁺]_i, PKC activation and degranulation and was ablated by agents that antagonized this pathway. Conclusions: The PKB inhibitor Akti‐1/2 increases PAR‐1‐mediated platelet responses in a PKB‐independent, Ca⁺⁺/PKC‐dependent manner. This effect is strong and rapid and may impact on the therapeutic application of Akti‐1/2 and structurally related compounds.     

Mobilization effects of G‐CSF,GM‐CSF,and darbepoetin‐α for allogeneic peripheral blood stem cell transplantation

The effects of GM‐/G‐CSF and darbepoetin‐α on stem cell mobilization were investigated. From February 2005 to March 2007, 30 allogeneic sibling donors were randomly assigned to a G‐CSF group (5 μg/kg/day for 5–7 days) or triple group (GM‐CSF 10 μg/kg/day on 1st and 2nd day, G‐CSF 5 μg/kg/day for 5–7 days, and darbepoetin‐α 40 mg on 1st day). The MNCs and CD34⁺ cells were not different between the two groups, although the doses (×10⁸/kg of recipient body weight) of CD3⁺ cells (3.64 ± 1.75 vs. 2.63 ± 1.36, P = 0.089) and CD8⁺ cells (1.07 ± 0.53 vs. 0.60 ± 0.30, P = 0.006) were lower in the triple group. The engraftments, frequency of RBC transfusions, and hemoglobin recovery were not different between the two groups. The cumulative incidence of overall and Grades II–IV aGVHD was 64.3% vs. 61.1% and 25.9% vs. 27.1% in the G‐CSF and triple regimen group, respectively, whereas the cumulative incidence of cGVHD was 20.8 ± 1.3% and 24.4 ± 1.7%, respectively. In conclusion, the triple regimen did not seem to be superior to G‐CSF alone in terms of the CD34+ cell dose, hemoglobin recovery, and GVHD. However, the CD8+ cell count was significantly lower in the triple regimen group. The role of a lower CD8+ cell count in the graft may need to be elucidated in the future. J. Clin. Apheresis, 2009. © 2009 Wiley‐Liss, Inc.     

Tonsil‐derived mesenchymal stem cells (T‐MSCs) prevent Th17‐mediated autoimmune response via regulation of the programmed death‐1/programmed death ligand‐1 (PD‐1/PD‐L1) pathway

Our knowledge of the immunomodulatory role of mesenchymal stem cells (MSCs) in both the innate and adaptive immune systems has dramatically expanded, providing great promise for treating various autoimmune diseases. However, the contribution of MSCs to Th17‐dominant immune disease, such as psoriasis and its underlying mechanism remains elusive. In this study, we demonstrated that human palatine tonsil‐derived MSCs (T‐MSCs) constitutively express both the membrane‐bound and soluble forms of programmed death‐ligand 1 (PD‐L1), which enables T‐MSCs to be distinguished from MSCs originating from other organs (i.e. bone marrow or adipose tissue). We also found that T‐MSC‐derived PD‐L1 effectively represses Th17 differentiation via both cell‐to‐cell contact and a paracrine effect. Further, T‐MSCs increase programmed death‐1 (PD‐1) expression on T‐cells by secreting IFN‐β, which may enhance engagement with PD‐L1. Finally, transplantation of T‐MSCs into imiquimod‐induced psoriatic skin inflammation in mice significantly abrogated disease symptoms, mainly by blunting the Th17 response in a PD‐L1‐dependent manner. This study suggests that T‐MSCs might be a promising cell source to treat autoimmune diseases such as psoriasis, via its unique immunoregulatory features. Copyright © 2017 John Wiley & Sons, Ltd.     

Impact of polymorphisms of the major histocompatibility complex class II,interleukin‐10, tumor necrosis factor‐α and cytotoxic T‐lymphocyte antigen‐4 genes on inhibitor development in severe hemophilia A

Summary. Background: Approximately 25% of severe hemophilia A (HA) patients develop antibodies to factor VIII protein. Patients: In the present case‐controlled cohort study, 260 severely affected, mutation‐type‐matched HA patients were studied for association of human leukocyte antigen (HLA) class II molecules and polymorphisms in the genes encoding interleukin‐10 (IL‐10), tumor necrosis factor‐α (TNF‐α) and cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) and development of inhibitors. Results: Our results demonstrate a higher frequency of DRB1*15 and DQB1*0602 alleles as well as of the haplotype DRB1*15/DQB1*0602 in inhibitor patients [odds ratio (OR) 1.9; P < 0.05]. In TNF‐α, the A allele of the ?308G>A polymorphism was found with higher frequency in the inhibitor cohort (0.22 vs. 0.13, OR 1.80). This finding was more pronounced for the homozygous A/A genotype (OR 4.7). For IL‐10, the ?1082G allele was observed more frequently in patients with inhibitors (0.55 vs. 0.43; P = 0.008). The functional cytokine phenotype was determined for the first time, on the basis of the genetic background, and this showed that 12% of patients with inhibitors were high‐TNF‐α/high‐IL‐10 producers, as compared with 3% of non‐inhibitor patients (OR 4.4). A trend for a lower frequency of the A allele of the CT60 polymorphism in CTLA‐4 was found in inhibitor patients (0.42 vs. 0.50). Conclusions: In conclusion, the reported data clearly highlighted the participation of HLA molecules in inhibitor formation in a large cohort of patients. The higher frequencies of the ?308G>A polymorphism in TNF‐α and ?1082A>G in IL‐10 in inhibitor patients confirmed the earlier published data. The CT60 single‐nucleotide polymorphism in CTLA‐4 is of apparently less importance.