Overexpression of Chitinase 3-Like 1/YKL-40 in Lung-Specific IL-18-Transgenic Mice, Smokers and COPD

We analyzed the lung mRNA expression profiles of a murine model of COPD developed using a lung-specific IL-18-transgenic mouse. In this transgenic mouse, the expression of 608 genes was found to vary more than 2-fold in comparison with control WT mice, and was clustered into 4 groups. The expression of 140 genes was constitutively increased at all ages, 215 genes increased gradually with aging, 171 genes decreased gradually with aging, and 82 genes decreased temporarily at 9 weeks of age. Interestingly, the levels of mRNA for the chitinase-related genes chitinase 3-like 1 (Chi3l1), Chi3l3, and acidic mammalian chitinase (AMCase) were significantly higher in the lungs of transgenic mice than in control mice. The level of Chi3l1 protein increased significantly with aging in the lungs and sera of IL-18 transgenic, but not WT mice. Previous studies have suggested Chi3l3 and AMCase are IL-13-driven chitinase-like proteins. However, IL-13 gene deletion did not reduce the level of Chi3l1 protein in the lungs of IL-18 transgenic mice. Based on our murine model gene expression data, we analyzed the protein level of YKL-40, the human homolog of Chi3l1, in sera of smokers and COPD patients. Sixteen COPD patients had undergone high resolution computed tomography (HRCT) examination. Emphysema was assessed by using a density mask with a cutoff of −950 Hounsfield units to calculate the low-attenuation area percentage (LAA%). We observed significantly higher serum levels in samples from 28 smokers and 45 COPD patients compared to 30 non-smokers. In COPD patients, there was a significant negative correlation between serum level of YKL-40 and %FEV1. Moreover, there was a significant positive correlation between the serum levels of YKL-40 and LAA% in COPD patients. Thus our results suggest that chitinase-related genes may play an important role in establishing pulmonary inflammation and emphysematous changes in smokers and COPD patients

Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice.

We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression-not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies

Microarray mRNA expression analysis of phase I and II metabolic enzymes levels in chimeric mice.

<p>mRNA expression levels of phase I and II expressed in human livers in (A and B) uPA/SCID and #1C2 homozygous mice, (C and D) #1C2 homo- and hemizygous mice, and (E and F) uPA/SCID and #1C2 mice were plotted. Phase I metabolic enzymes are CYP1A1, 1A2, 1B1, 2A6, 26A1, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7, 3A43, 39A1, 4A11, 4F2, 4F3, 7A1, 7B1, 8B1. Phase II metabolic enzymes are UGT1A6, 2A1, 2B15, 2B17, 2B4, SULT1A1, 1A3, 1B1, 1E1, 2A1, GSTA1, A4, M1, M2, M3, M4, P1, T1, Z1, and NAT1, 2. The solid and dotted lines represent unity and 4-fold differences, respectively. All values were within the 4-fold difference range.</p

Histopathological findings in chimeric mouse livers and kidneys.

<p>H&E staining of left lateral lobes of (A) uPA/SCID chimeric mice, (B) #1C2 homozygous and (C) #1C2 hemizygous chimeric mice, and magnifications of the rectangular parts (D) on the left side of A and (E) on the right side of A, (F) in B, and (G) in C are shown. h-heps with a clear cytoplasm and lipid droplets occupied most areas of the liver sections (A-G). Arrows show (D) degenerating m-heps and (E) hyperplastic m-hep nodules in uPA/SCID chimeric mice. M-heps with eosinophilic cytoplasm of various sizes are shown by arrows in (F) #1C2 homozygous and (G) hemizygous chimeric mice. (H) The left lateral lobe of a #1C2 hemizygous chimeric mouse was immunostained with anti-hCK8/18 antibodies. H-heps were brown-colored, and (I) an area of rectangle was magnified. m, m-heps, h, h-heps. Kidney sections in (J, M) uPA/SCID, (K, N) #1C2 homozygote and (L, O) hemizygous chimeric mice were stained with H&E. Enlarged glomeruli and glomerulosclerosis were observed in uPA/SCID mice (J, M). No pathological findings were observed in (K, N) #1C2 homozygote and (L, O) hemizygous chimeric mouse kidneys. (M), (N) and (O) were high magnification of (J), (K) and (L), respectively. Bar, 100 μm in D-G, I and J-O. Bar, 1 mm in A-C and H.</p

Information of animals infected with HBV or HCV.

<p>Information of animals infected with HBV or HCV.</p

Production of cDNA-uPA mice.

<p>(A) Construction of the cDNA-uPA transgene. (B) Body weight in 8-week-old mice and (C) serum ALT activity in #1C2, 2C7, 1D3, and non-transgenic control mice. Body weight of #1C2 and #2C7 mice was lower than that of #1D3 and control mice. ALT activity in #1C2 and #2C7 mice increased from the age of 6–8 weeks. The approximate values of ALT are given on the right side of the vertical axis of the graph. These values were calculated as follows: 1 ALT (IU/L) ≈ 1.58 Karmen.</p

Histological findings in uPA/SCID and #1C2 homo- and hemizygous livers.

<p>H&E staining of (A and C) uPA/SCID, (D and F) #1C2 homozygous and (G and I) #1C2 hemizygous mice aged 3 and 14 weeks, respectively. Oil red O staining in 3-week-old (B) uPA/SCID and #1C2 (E) homo and (H) hemizygous mouse livers. Hepatocytes of all 3 groups were degenerating in 3-week-old mice. In 14-week-old mice, degenerating hepatocytes and hyperplastic m-hep colonies were observed in uPA/SCID mice but not in #1C2 homo- and hemizygotes. Varying m-hep sizes were observed in 3-week-old #1C2 homo- and hemizygotes. Lipid droplets were more abundant in uPA/SCID mice and #1C2 homozygotes than #1C2 hemizygotes. De, degenerating hepatocytes. Hy, hyperplastic hepatocyte colony. Bar, 100 μm.</p