Increased BALF ATX/LPA levels upon LPS-induced ALI.

<p>Mice were administered aerosolized LPS and were sacrificed 6, 12, 24 and 48 hours later (n = 3–5; a representative experiment out of two is shown). A. Hematoxylin & Eosin staining of lung tissue sections following LPS exposure for the indicated times resulted in alveolar wall thickening and leukocyte infiltration into the lung interstitium and alveolar space. B. Increased BALF cellularity upon LPS/ALI. C. Pulmonary microvascular leakage and edema induced by LPS was reflected in BALF protein content. D. Increased ATX activity in LPS/ALI BALFs, as measured with the TOOS assay. E. IHC for ATX in lung tissue showing constitutive expression from the bronchial epithelium, as well as a weak diffuse staining pattern in the lung parenchyma. F-G. BALF total LPC/LPA levels respectively upon LPS/ALI, as measured with HPLC-MS/MS.</p

Hydroxamic Acids Constitute a Novel Class of Autotaxin Inhibitors that Exhibit <i>in Vivo</i> Efficacy in a Pulmonary Fibrosis Model

Autotaxin (ATX) catalyzes the hydrolysis of lysophosphatidylcholine (LPC) generating the lipid mediator lysophosphatidic acid (LPA). Both ATX and LPA are involved in various pathological inflammatory conditions, including fibrosis and cancer, and have attracted great interest as medicinal targets over the past decade. Thus, the development of novel potent ATX inhibitors is of great importance. We have developed a novel class of ATX inhibitors containing the zinc binding functionality of hydroxamic acid. Such novel hydroxamic acids that incorporate a non-natural δ-amino acid residue exhibit high <i>in vitro</i> inhibitory potency over ATX (IC₅₀ values 50–60 nM). Inhibitor <b>32</b>, based on δ-norleucine, was tested for its efficacy in a mouse model of pulmonary inflammation and fibrosis induced by bleomycin and exhibited promising efficacy. The novel hydroxamic ATX inhibitors provide excellent tools for the study of the role of the enzyme and could contribute to the development of novel therapeutic agents for the treatment of fibrosis and other chronic inflammatory diseases

Hydroxamic Acids Constitute a Novel Class of Autotaxin Inhibitors that Exhibit <i>in Vivo</i> Efficacy in a Pulmonary Fibrosis Model

Autotaxin (ATX) catalyzes the hydrolysis of lysophosphatidylcholine (LPC) generating the lipid mediator lysophosphatidic acid (LPA). Both ATX and LPA are involved in various pathological inflammatory conditions, including fibrosis and cancer, and have attracted great interest as medicinal targets over the past decade. Thus, the development of novel potent ATX inhibitors is of great importance. We have developed a novel class of ATX inhibitors containing the zinc binding functionality of hydroxamic acid. Such novel hydroxamic acids that incorporate a non-natural δ-amino acid residue exhibit high <i>in vitro</i> inhibitory potency over ATX (IC₅₀ values 50–60 nM). Inhibitor <b>32</b>, based on δ-norleucine, was tested for its efficacy in a mouse model of pulmonary inflammation and fibrosis induced by bleomycin and exhibited promising efficacy. The novel hydroxamic ATX inhibitors provide excellent tools for the study of the role of the enzyme and could contribute to the development of novel therapeutic agents for the treatment of fibrosis and other chronic inflammatory diseases

Generation of Tg<i>CC10Enpp2</i> mice.

<p>A. Schematic representation of the construct used for the generation of the transgenic mice. B. Genotyping PCR of the 4 offsprings that carried the transgene, out of the 54 that were generated after the injections of the trangene-microinjected zygotes in surrogate mothers. C. All four transgenic lines contained equal copy numbers, as identified with Real-Time PCR. D. Real-Time RT-PCR confirmed the expression of the transgene (L39 is shown). E. Total ATX activity levels in the BALFs of Tg<i>CC10Enpp2</i> mice (L39) were found moderately upregulated with the TOOS assay. F. In the same mice, BALF LPA was also found elevated, as measured with HPLC-MS/MS. (C-F n = 3–8).</p

No effect of Tmx-induced genetic inactivation of ATX in mouse survival rate.

<p>Kaplan–Meier survival curves of R26Cre-ER^T2/<i>Enpp2</i>^n/n and control mice administered with different Tmx concentrations (50, 100 and 180 mg/kg) using two different routes of delivery (IP, PO), as indicated and as described in the text. Presented results are cumulative from 1 (A), 3 (B) and 4 (C and D) experiments. No statistical significant differences were observed, as assessed with the Logrank test. Overlapping curves are indicated by consecutive symbols.</p

Pharmacologic inhibition of ATX has no effects in ALI development.

<p>GWJ-A-23 was injected intraperitoneally before challenging mice with aerosolized LPS. Mice were sacrificed 24 hours later (n = 3–7; a representative experiment out of two is shown). A-C. Histological analysis (A) and indicated BALF measurements (B,C) suggested minor effects in ALI development, despite decreased BALF ATX activity (D) and the corresponding BALF LPA levels (E).</p

Deletion of ATX from myeloid cells had no effects in ALI development.

<p>Aerosolized LPS was administered in mice where ATX was deleted from the myeloid cells (LysMEnpp2^-/-) and wild type littermate mice. Mice were sacrificed 24 hours later. A-C. Histological analysis (A) and BALF measurements (B & C) showed no differences in inflammation and edema between mice lacking ATX expression from myeloid cells and their wild type littermates. D. Conditional deletion of ATX from myeloid cells had no effects in BALF ATX activity.</p

Tmx-induced (180 mg/kg PO) R26Cre-ER^T2-mediated genetic ablation of ATX results in diminished ATX levels in tissues and plasma.

<p>(A) Real-Time RT-PCR analysis of relative ATX mRNA expression levels in different tissues normalized to the expression levels of B2M. (n = 5–10; exp = 2; with the exception of BAT/WAT n = 4, exp = 1). (B) Real-Time RT-PCR analysis of ATX mRNA expression levels, normalized to the expression levels of B2M, in different tissues following Tmx-induced genetic ablation of the <i>Enpp2</i> gene. (n = 5–10; exp = 2; with the exception of BAT/WAT n = 4, exp = 1). (C) Section from a western blot for ATX (4F1 Ab) in the plasma of the indicated mice. The full images, together with a coomassie brilliant blue staining of the same samples as loading control, and an alternate blot with a commercial antibody can be found at <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143083#pone.0143083.s007" target="_blank">S7 Fig</a> (D) Plasma ATX activity in the plasma of the indicated mice as determined with the TOOS assay on natural LPC substrates (n = 13–27; exp = 3). (E) Plasma LPA levels of the indicated mice as determined by HPLC-MS/MS (n = 9–13; exp = 2). (D) Plasma lysophospholipid (LPLs) levels remain unchanged as measured with HPLC-MS/ MS (n = 9–13; exp = 2). All values in every panel are means (± SEM) and are presented (except A) normalised (%) to control values.</p

Pharmacologic inhibition of ATX has no effects in ALI development.

<p>GWJ-A-23 was injected intraperitoneally before challenging mice with aerosolized LPS. Mice were sacrificed 24 hours later (n = 3–7; a representative experiment out of two is shown). A-C. Histological analysis (A) and indicated BALF measurements (B,C) suggested minor effects in ALI development, despite decreased BALF ATX activity (D) and the corresponding BALF LPA levels (E).</p

Genetic deletion of ATX from the bronchial epithelium has minor effects in ALI development.

<p>Aerosolized LPS was administered in mice where ATX was genetically deleted specifically from bronchial epithelial cells (CC10Enpp2^-/-), as well as wild type littermates. Mice were sacrificed 24 hours later (n = 3–8; a representative experiment out of two is shown). A-C. Histological analysis (A) and BALF measurements (B & C) indicated no significant changes in the lungs of mice lacking ATX expression in the bronchial epithelium in comparison to their wild type littermates. D. Conditional deletion of ATX from the cells of the bronchial epithelium led to decreased enzyme activity of ATX in the BALF.</p