Pharmacokinetics of monoclonal antibodies locally-applied into the middle ear of guinea pigs.

Countless therapeutic antibodies are currently available for the treatment of a broad range of diseases. Some target molecules of therapeutic antibodies are involved in the pathogenesis of sensorineural hearing loss (SNHL), suggesting that SNHL may be a novel target for monoclonal antibody (mAb) therapy. When considering mAb therapy for SNHL, understanding of the pharmacokinetics of mAbs after local application into the middle ear is crucial. To reveal the fundamental characteristics of mAb pharmacokinetics following local application into the middle ear of guinea pigs, we performed pharmacokinetic analyses of mouse monoclonal antibodies to FLAG-tag (FLAG-mAbs), which have no specific binding sites in the middle and inner ear. FLAG-mAbs were rapidly transferred from the middle ear to the cochlear fluid, indicating high permeability of the round window membrane to mAbs. FLAG-mAbs were eliminated from the cochlear fluid 3 h after application, similar to small molecules. Whole-body autoradiography and quantitative assessments of cerebrospinal fluid and serum demonstrated that the biodistribution of FLAG-mAbs was limited to the middle and inner ear. Altogether, the pharmacokinetics of mAbs are similar to those of small molecules when locally applied into the middle ear, suggesting the necessity of drug delivery systems for appropriate mAb delivery to the cochlear fluid after local application into the middle ear.

Mutual interaction between endothelial cells and mural cells enhances BMP9 signaling in endothelial cells.

Hereditary hemorrhagic telangiectasia is characterized by the formation of abnormal vascular networks and caused by the mutation of genes involved in BMP9 signaling. It is also known that the interaction between endothelial cells (ECs) and mural cells (MCs) is critical to maintain vessel integrity. However, it has not yet fully been uncovered whether the EC-MC interaction affects BMP9 signaling or not. To elucidate this point, we analyzed BMP9 signaling in a co-culture of several types of human primary culture ECs and MCs. The co-culture activated the Notch pathway in both types of cells in a co-culture- and BMP9-dependent manner. In HUVECs, the genes induced by BMP9 were significantly and synergistically induced in the presence of pericytes, fibroblasts or mesenchymal stem cells. The synergistic induction was greatly reduced in a non-contact condition. In fibroblasts, PDGFRB expression was potently induced in the presence of HUVECs, and BMP9 additively increased this response. Taken together, these results suggest that the EC-MC interaction potentiates BMP9 signaling both in ECs and MCs and plays a critical role in the maintenance of proper vessel functions.

A novel selective prostaglandin E2 synthesis inhibitor relieves pyrexia and arthritis in Guinea pigs inflammatory models.

Prostaglandin E2 (PGE2), one of the terminal products in the cyclooxygenase pathway, plays an important role in various inflammatory responses. To determine whether selective inhibition of PGE2 may relieve these inflammatory symptoms, we synthesized a selective PGE2 synthesis inhibitor, compound A [1-(6-fluoro-5,7-dimethyl-1,3-benzothiazol-2-yl)-N-[(1S,2R)-2-(hydroxymethyl)cyclohexyl]piperidine-4-carboxamide], then investigated the effects on pyrexia, arthritis and inflammatory pain in guinea pigs. In LPS-stimulated guinea pig macrophages, compound A selectively inhibited inducible PGE2 biosynthesis in a dose-dependent manner whereas enhanced the formation of thromboxane B2 (TXB2). Compound A suppressed yeast-evoked PGE2 production selectively and enhanced the production of TXB2 and 6-keto PGF1αin vivo. In addition, compound A relieved yeast-induced pyrexia and also suppressed paw swelling in an adjuvant-induced arthritis model. The effect on gastrointestinal (GI) ulcer formation was also evaluated and compound A showed a lower GI adverse effect than indomethacin. However, compound A failed to relieve yeast-induced thermal hyperalgesia. These results suggest that selective inhibition of PGE2 synthesis may have anti-pyretic and anti-inflammatory properties without GI side effect, but lack the analgesic efficacy.

Secreted factors from adipose tissue-derived mesenchymal stem cells suppress oxygen/glucose deprivation-induced cardiomyocyte cell death via furin/PCSK-like enzyme activity.

Clinical application of mesenchymal stem cells (MSCs) represents a potential novel therapy for currently intractable deteriorating diseases or traumatic injuries, including myocardial infarction. However, the molecular mechanisms of the therapeutic effects have not been precisely revealed. Herein, we report that conditioned media (CM) from rat adipose tissue-derived MSCs (ASCs) protected adult cardiomyocytes from oxygen/glucose deprivation (OGD)-induced cell death. We focused on furin/PCSK protease activity in ASC-CM because many therapeutic factors of MSCs and soluble cardioprotective factors include the PCSK cleavage site. We found that recombinant furin protected cardiomyocytes from OGD-induced cell death. The ASC-CM had potent furin/PCSK protease activity and the cardioprotective effect of the CM from ASCs in the OGD-assay was abolished by an inhibitor of the furin/PCSK-like enzyme. Microarray analysis and Western blot analysis showed PCSK5A, the secreted type of PCSK5, is the most abundantly secreted PCSK among 7 PCSK family members in ASC. Finally, knockdown of PCSK5A in ASCs decreased both the furin/PCSK protease activity and cardioprotective activity in the CM. These findings indicate an involvement of furin/PCSK-type protease(s) in the anti-ischemic activity of ASCs, and suggest a new mechanism of the therapeutic effect of MSCs.

Elevated expression of periostin in human osteoarthritic cartilage and its potential role in matrix degradation via matrix metalloproteinase-13.

We investigated the role of periostin, an extracellular matrix protein, in the pathophysiology of osteoarthritis (OA). In OA, dysregulated gene expression and phenotypic changes in articular chondrocytes culminate in progressive loss of cartilage from the joint surface. The molecular mechanisms underlying this process are poorly understood. We examined periostin expression by immunohistochemical analysis of lesional and nonlesional cartilage from human and rodent OA knee cartilage. In addition, we used small interfering (si)RNA and adenovirus transduction of chondrocytes to knock down and up-regulate periostin levels, respectively, and analyzed its effect on matrix metalloproteinase (MMP)-13, a disintegrin and MMP with thrombospondin motifs (ADAMTS)-4, and type II collagen expression. We found high periostin levels in human and rodent OA cartilage. Periostin increased MMP-13 expression dose [1-10 µg/ml (EC50 0.5-1 µg/ml)] and time (24-72 h) dependently, significantly enhanced expression of ADAMTS4 mRNA, and promoted cartilage degeneration through collagen and proteoglycan degradation. Periostin induction of MMP-13 expression was inhibited by CCT031374 hydrobromide, an inhibitor of the canonical Wnt/ß-catenin signaling pathway. In addition, siRNA-mediated knockdown of endogenous periostin blocked constitutive MMP-13 expression. These findings implicate periostin as a catabolic protein that promotes cartilage degeneration in OA by up-regulating MMP-13 through canonical Wnt signaling.

Characterization of gene expression profiles for different types of mast cells pooled from mouse stomach subregions by an RNA amplification method.

BACKGROUND: Mast cells (MCs) play pivotal roles in allergy and innate immunity and consist of heterogenous subclasses. However, the molecular basis determining the different characteristics of these multiple MC subclasses remains unclear. RESULTS: To approach this, we developed a method of RNA extraction/amplification for intact in vivo MCs pooled from frozen tissue sections, which enabled us to obtain the global gene expression pattern of pooled MCs belonging to the same subclass. MCs were isolated from the submucosa (sMCs) and mucosa (mMCs) of mouse stomach sections, respectively, 15 cells were pooled, and their RNA was extracted, amplified and subjected to microarray analysis. Known marker genes specific for mMCs and sMCs showed expected expression trends, indicating accuracy of the analysis. We identified 1,272 genes showing significantly different expression levels between sMCs and mMCs, and classified them into clusters on the basis of similarity of their expression profiles compared with bone marrow-derived MCs, which are the cultured MCs with so-called 'immature' properties. Among them, we found that several key genes such as Notch4 had sMC-biased expression and Ptgr1 had mMC-biased expression. Furthermore, there is a difference in the expression of several genes including extracellular matrix protein components, adhesion molecules, and cytoskeletal proteins between the two MC subclasses, which may reflect functional adaptation of each MC to the mucosal or submucosal environment in the stomach. CONCLUSION: By using the method of RNA amplification from pooled intact MCs, we characterized the distinct gene expression profiles of sMCs and mMCs in the mouse stomach. Our findings offer insight into possible unidentified properties specific for each MC subclass.