Synthesis and Biological Evaluation of Derivatives of 2-{2-Fluoro-4-[(2-oxocyclopentyl)methyl]phenyl}propanoic Acid: Nonsteroidal Anti-Inflammatory Drugs with Low Gastric Ulcerogenic Activity

We previously reported that 2-fluoroloxoprofen has lower gastric ulcerogenic activity than loxoprofen, a nonsteroidal anti-inflammatory drug (NSAID) without selectivity for COX-2. We synthesized derivatives of 2-fluoroloxoprofen and studied their properties. Compared to 2-fluoroloxoprofen, one derivative, <b>11a</b>, exhibited higher anti-inflammatory activity and an equivalent ulcerogenic effect. These results suggest that <b>11a</b> could be therapeutically beneficial for use as an NSAID

Macrophage-Derived Angiopoietin-Like Protein 2 Exacerbates Brain Damage by Accelerating Acute Inflammation after Ischemia-Reperfusion

<div><p>Ischemic stroke is a leading cause of death and disability worldwide. Several reports suggest that acute inflammation after ischemia-reperfusion exacerbates brain damage; however, molecular mechanisms underlying this effect remain unclear. Here, we report that MAC-3-positive immune cells, including infiltrating bone marrow-derived macrophages and activated microglia, express abundant angiopoietin-like protein (ANGPTL) 2 in ischemic mouse brain in a transient middle cerebral artery occlusion (MCAO) model. Both neurological deficits and infarct volume decreased in transient MCAO model mice established in <i>Angptl2</i> knockout (KO) relative to wild-type mice. Acute brain inflammation after ischemia-reperfusion, as estimated by expression levels of pro-inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor alpha (TNF)-α, was significantly suppressed in <i>Angptl2</i> KO compared to control mice. Moreover, analysis employing bone marrow chimeric models using <i>Angptl2</i> KO and wild-type mice revealed that infiltrated bone marrow-derived macrophages secreting ANGPTL2 significantly contribute to acute brain injury seen after ischemia-reperfusion. These studies demonstrate that infiltrating bone marrow-derived macrophages promote inflammation and injury in affected brain areas after ischemia-reperfusion, likely via ANGPTL2 secretion in the acute phase of ischemic stroke.</p></div

Acute brain inflammation after ischemic stroke is attenuated in <i>Angptl2</i>-deficient mice.

<p>(<b>A</b> and <b>B</b>) Relative expression of <i>Il1b</i>, <i>Tnfa</i>, and <i>Il10</i> mRNAs in cortex (sham: WT = 7 and KO = 7; day 1: WT = 6 and KO = 7; day 3: WT = 5 and KO = 6) (<b>A</b>) and ipsilateral caudate putamen (sham: WT = 5 and KO = 8; day 1: WT = 7 and KO = 6; day 3: WT = 7 and KO = 5) (<b>B</b>). mRNA levels were normalized to those of T<i>bp</i>. Values are expressed as fold-increases relative to values in WT, which were set at “1” in each case. (<b>C</b>) Representative images showing MAC-3 immunostaining in the ipsilateral cerebral hemisphere 24 hours after sham or transient MCAO treatment. (Panel 1) Sham-treated wild-type (WT) mice; (Panel 2) transient MCAO-treated wild-type mice; (Panel 3) transient MCAO-treated <i>Angptl2</i> KO mice. Scale bar: 100μm. (<b>D</b>) (1) Number of MAC-3-positive cells in the ipsilateral hemisphere of transient MCAO mice 24 hours after transient MCAO (sham = 7; WT = 7; KO = 6). Results are expressed as means ± s.e.m. (2, 3) Relative <i>Cd68</i> mRNA expression normalized to <i>Tbp</i> mRNA in cortex (sham = 6; WT = 8; KO = 8) (2) and ipsilateral caudate putamen (sham = 6; WT = 8; KO = 8) (3) 24 hours after transient MCAO. Values are expressed as fold-increases relative to values in sham-treated WT mice, which were set to “1” in each case. Results are expressed as means ± s.e.m. ns: not significant, ^*<i>P</i><0.05, ^**<i>P</i><0.01.</p

Macrophage-derived ANGPTL2 exacerbates neuronal injury in the acute phase of ischemic stroke.

<p>Schematic diagram showing ANGPTL2 function in ischemic stroke. In normal conditions, brain resident cells express little ANGPTL2. However, following ischemic stroke, bone marrow-derived macrophages infiltrate the brain interstitium and secrete abundant ANGPTL2. Subsequently, ANGPTL2 from activated macrophages induces those same macrophages to increase expression of pro-inflammatory cytokines, including TNF-α or IL-1, in an autocrine or paracrine manner. Pro-inflammatory cytokines damage neurons or supporting cells and worsen functional neurological deficits induced by brain infarction.</p

ANGPTL2 increases transcription of genes encoding pro-inflammatory cytokines in macrophages.

<p>mRNA expression levels of (left) <i>Il1b</i>, (middle) <i>Tnfa</i>, and (right) <i>Il10</i> following either vehicle or recombinant ANGPTL2 (rANGPTL2) treatment of RAW 264.7 cells (n = 5). mRNAs were harvested at 0, 3, 6, 12, 24 hours after cells were treated with 5 μg/mL rANGPTL2. Data are normalized to <i>Actb</i> levels. mRNA expression in vehicle-treated cells was set to 1 for each transcript assessed. Results are expressed as means ± s.e.m. ^*<i>P</i>< 0.01.</p

Bone marrow-derived ANGPTL2 functions in progression of neuronal damage following brain ischemia.

<p>(<b>A</b>) Schema showing relationships between donors and recipients in the bone marrow transplantation (BMT) experiment. (<b>B</b>) Schema showing the time course of BMT, surgery and sacrifice in the mouse transient MCAO model. (<b>C</b>) Graph showing neurological scores assessed by the 18-point neurological severity scale 24 and 72 hours after MCAO treatment (Day1: WT→WT = 6; KO→WT = 5; WT→KO = 7; KO→KO = 7. Day3: WT→WT = 8; KO→WT = 8; WT→KO = 12; KO→KO = 11). Results are expressed as means ± s.e.m. (<b>D</b>) Representative images showing TTC-stained brain sections 24 hours after transient MCAO in bone marrow chimeric mice. Scale bar: 2mm. (<b>E</b>) Infarct volumes as assessed by TTC-staining of brain sections 24 and 72 hours after stroke onset in bone marrow chimeric mice (Day1: WT→WT = 6; KO→WT = 5; WT→KO = 7; KO→KO = 7. Day3: WT→WT = 8; KO→WT = 8; WT→KO = 12; KO→KO = 11). Results are expressed as means ± s.e.m. ^*<i>P</i><0.05, and ^**<i>P</i><0.01.</p

Tissue Inhibitor of Metalloproteinase-3 Knockout Mice Exhibit Enhanced Energy Expenditure through Thermogenesis

<div><p>Tissue inhibitors of metalloproteinases (TIMPs) regulate matrix metalloproteinase activity and maintain extracellular matrix homeostasis. Although TIMP-3 has multiple functions (e.g., apoptosis, inhibition of VEGF binding to VEGF receptor, and inhibition of TNFα converting enzyme), its roles in thermogenesis and metabolism, which influence energy expenditure and can lead to the development of metabolic disorders when dysregulated, are poorly understood. This study aimed to determine whether TIMP-3 is implicated in metabolism by analyzing TIMP-3 knockout (KO) mice. TIMP-3 KO mice had higher body temperature, oxygen consumption, and carbon dioxide production than wild-type (WT) mice, although there were no differences in food intake and locomotor activity. These results suggest that metabolism is enhanced in TIMP-3 KO mice. Real-time PCR analysis showed that the expression of PPAR-δ, UCP-2, NRF-1 and NRF-2 in soleus muscle, and PGC-1α and UCP-2 in gastrocnemius muscle, was higher in TIMP-3 KO mice than in WT mice, suggesting that TIMP-3 deficiency may increase mitochondrial activity. When exposed to cold for 8 hours to induce thermogenesis, TIMP-3 KO mice had a higher body temperature than WT mice. In the treadmill test, oxygen consumption and carbon dioxide production were higher in TIMP-3 KO mice both before and after starting exercise, and the difference was more pronounced after starting exercise. Our findings suggest that TIMP-3 KO mice exhibit enhanced metabolism, as reflected by a higher body temperature than WT mice, possibly due to increased mitochondrial activity. Given that TIMP-3 deficiency increases energy expenditure, TIMP-3 may present a novel therapeutic target for preventing metabolic disorders.</p></div

A Molecular Clock Regulates Angiopoietin-Like Protein 2 Expression

<div><p>Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of <i>ANGPTL2</i> expression are poorly understood. Here, we assess circadian rhythmicity of <i>ANGPTL2</i> expression in various mouse tissues. We observed that <i>ANGPTL2</i> rhythmicity was similar to that of the <i>PER2</i> gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human <i>ANGPTL2</i> gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the <i>ANGPTL2</i> promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, <i>Cry</i>-deficient mice exhibited arrhythmic <i>Angptl2</i> expression. Taken together, these data suggest that periodic expression of <i>ANGPTL2</i> is regulated by a molecular clock.</p> </div

Metabolic parameters in TIMP-3 KO and wild type mice.

<p>Body temperature (A), body weight (B), food intake per body weight (C) and locomotor activity (D) in TIMP-3 knockout (KO) and wild type (WT) mice at 15 weeks or 8 months of age are presented as mean ± SD (n = 5–7/group). ^†p<0.01, ^‡p<0.001.</p

Real-time PCR-based analysis of mitochondrial activity in soleus muscle of TIMP-3 KO and wild type mice.

<p>Expression of PGC-1α (A), PPAR-δ (B), PPAR-γ (C), UCP-2 (D), NRF-1 (E) and NRF-2 (F) in TIMP-3 knockout (KO) and wild type (WT) mice is presented as mean ± SD (n = 6–7/group). *p<0.05 ^†p<0.01.</p