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Murine articular cartilage apoptosis increases with age
The Seventeenth Annual Meeting of the East Coast Connective Tissue Society
Transgenic mice expressing human neutrophil elastase in the lung Seema S. Dalai, Yasunori Okada ~, Kiran Chada b, Jeanine D'Armiento Columbia University, Dept. Medicine, Division of Molecular Medicine; New York, NY; ~'Kanazawa University, Dept. of Mol. Imm. and Path. Cancer Research Inst., Japan; bUMDNJRobert Wood Johnson MedicalSchool, Dept of Biochemistry, Piscataway, NJ. Elastolytic enzymes administered to the lungs of animals results m an emphysemalike phenotype and helped to establish the importance of elastase as the primary enzyme involved m the pathogenesis of emphysema, Howover, these studies were complicated by the hemorrhagic and inflammatory cell infiltrate produced during lntratracheal instillation of elastase. Therefore, transgenic rnice were generated using a lung specific promoter to direct neutrophil elastase expression specifically in the lung parenchyma, The percentage of transgenic mice born was 7% which was lower than the expected transformation frequency of 20-30%. From the eight founder mice (HE mice) born, one died at birth and one died at 1 month of age. The cause of death was not determined, RNA was isolated from the lungs of representative rnice from the remaining surviving founder lines. The transgene message was not detected by both Northern blot and RNAse protection analyses. Histological anslyses on various tissues from the transgenic mice revealed no pathological changes, especially in the lung. To determine if the transgene was lethal, a second series of mlcroinjection experiments were performed and foster mothers were sacrificed for embryos at 13 dpc. A total of 26 masses representing dead embyros were found. 1 l out of 26 (42%) dead embryos were positive for the transgene by PCR analysis indicating that an increased number of transgenic mice were dying in u t e r o . Reverse transcriptasePCR analysis demonstrated endogenous mouse elastase expression in the lungs of normal mice as early as 12 dpc. Therefore we conclude that elastase plays an essential role in the development of the lung, and generic overexpression of neutrophil elastase leads to embryonic lethality.
129
Murine articular cartilage apoptosis increases with age Christopher S. Adams, Stuart Tillman, and Walter E. Horton, Jr. Laboratory of Biological Chemistry, National Institute on Aging, Gerontology Research Center, Baltimore, MD Programmed cell death (apoptosis) has been conclusively demonstrated in cartilage from the sternum and growth plate. This study demonstrates apoptosis occurring in selected cells of the medial and lateral articular cartilages of the C57BL mouse knee. Animals from four age groups (12,18,24 and 30 months old, n = 10 for each) were used. Fragmented DNA were labeled with biotinylated nucleotides by the Klenow enzyme. Biotinylated nucleotides were then detected by strepavidin peroxidase and Trevigen's blue label. Many cells exhibited a morphology which conformed to the classic definition of apoptosis. Fragmented DNA was confined to the nucleus and frequently those nuclei are broken up into condensed masses. In other cells, however, fragmented DNA was observed both in the cytoplasm of the cell as well as against the edges of the lacuna itself. The distribution of cells exhibiting this staining morphology was primarily restricted to the layer of calcified cartilage that lay in direct contact with subarticular bone. For quantification, the number of cells with fragmented DNA was normalized by the area of the articular cartilage. This total articular cartilage area was then divided into calcified cartilage and noncalcified cartilage. No significant differences was observed between the medial and lateral or tibial and femoral cartilages. Percentage of cells demonstrating fragmented DNA was increased in older animals. Between the ages of 18 month and 24 months, there was a statistically significant doubling of the number of apoptotic cells. The oldest animals studied (24 and 30 months old) displayed more apoptotic cells in the upper layers of the cartilage then did the younger animals. This increase in apoptotic articular chondrocytes may be related to an increase in cartilage degeneration with age seen in these animals.
The localization and expression of glycosaminoglycans and growth factors in response to stromal implants Xin Yi Wu, C. T. Brown and V. Trinkaus-Randall Department of Ophthalmology, School of Medicine, Boston, MA
Boston
University
Corneal wounds are associated with changes in matrix proteins. Our goal was to examine the localization and
Transgenic mice expressing human neutrophil elastase in the lung Seema S. Dalai, Yasunori Okada ~, Kiran Chada b, Jeanine D'Armiento Columbia University, Dept. Medicine, Division of Molecular Medicine; New York, NY; ~'Kanazawa University, Dept. of Mol. Imm. and Path. Cancer Research Inst., Japan; bUMDNJRobert Wood Johnson MedicalSchool, Dept of Biochemistry, Piscataway, NJ. Elastolytic enzymes administered to the lungs of animals results m an emphysemalike phenotype and helped to establish the importance of elastase as the primary enzyme involved m the pathogenesis of emphysema, Howover, these studies were complicated by the hemorrhagic and inflammatory cell infiltrate produced during lntratracheal instillation of elastase. Therefore, transgenic rnice were generated using a lung specific promoter to direct neutrophil elastase expression specifically in the lung parenchyma, The percentage of transgenic mice born was 7% which was lower than the expected transformation frequency of 20-30%. From the eight founder mice (HE mice) born, one died at birth and one died at 1 month of age. The cause of death was not determined, RNA was isolated from the lungs of representative rnice from the remaining surviving founder lines. The transgene message was not detected by both Northern blot and RNAse protection analyses. Histological anslyses on various tissues from the transgenic mice revealed no pathological changes, especially in the lung. To determine if the transgene was lethal, a second series of mlcroinjection experiments were performed and foster mothers were sacrificed for embryos at 13 dpc. A total of 26 masses representing dead embyros were found. 1 l out of 26 (42%) dead embryos were positive for the transgene by PCR analysis indicating that an increased number of transgenic mice were dying in u t e r o . Reverse transcriptasePCR analysis demonstrated endogenous mouse elastase expression in the lungs of normal mice as early as 12 dpc. Therefore we conclude that elastase plays an essential role in the development of the lung, and generic overexpression of neutrophil elastase leads to embryonic lethality.
129
Murine articular cartilage apoptosis increases with age Christopher S. Adams, Stuart Tillman, and Walter E. Horton, Jr. Laboratory of Biological Chemistry, National Institute on Aging, Gerontology Research Center, Baltimore, MD Programmed cell death (apoptosis) has been conclusively demonstrated in cartilage from the sternum and growth plate. This study demonstrates apoptosis occurring in selected cells of the medial and lateral articular cartilages of the C57BL mouse knee. Animals from four age groups (12,18,24 and 30 months old, n = 10 for each) were used. Fragmented DNA were labeled with biotinylated nucleotides by the Klenow enzyme. Biotinylated nucleotides were then detected by strepavidin peroxidase and Trevigen's blue label. Many cells exhibited a morphology which conformed to the classic definition of apoptosis. Fragmented DNA was confined to the nucleus and frequently those nuclei are broken up into condensed masses. In other cells, however, fragmented DNA was observed both in the cytoplasm of the cell as well as against the edges of the lacuna itself. The distribution of cells exhibiting this staining morphology was primarily restricted to the layer of calcified cartilage that lay in direct contact with subarticular bone. For quantification, the number of cells with fragmented DNA was normalized by the area of the articular cartilage. This total articular cartilage area was then divided into calcified cartilage and noncalcified cartilage. No significant differences was observed between the medial and lateral or tibial and femoral cartilages. Percentage of cells demonstrating fragmented DNA was increased in older animals. Between the ages of 18 month and 24 months, there was a statistically significant doubling of the number of apoptotic cells. The oldest animals studied (24 and 30 months old) displayed more apoptotic cells in the upper layers of the cartilage then did the younger animals. This increase in apoptotic articular chondrocytes may be related to an increase in cartilage degeneration with age seen in these animals.
The localization and expression of glycosaminoglycans and growth factors in response to stromal implants Xin Yi Wu, C. T. Brown and V. Trinkaus-Randall Department of Ophthalmology, School of Medicine, Boston, MA
Boston
University
Corneal wounds are associated with changes in matrix proteins. Our goal was to examine the localization and