Bone Formation in the Context of Growth Retardation Induced by hIGFBP-1 Overexpression in Transgenic Mice

In humans, intrauterine growth retardation (hIUGR) is correlated with an overexpression of insulin-like growthfactor binding protein 1 (IGFBP-1). The affected children also present a delay in bone mineralization. In this study, transgenic 12-day-old mutant mice overexpressing human IGFBP-1 hepatospecifically showed a severe growth retardation. Alcian blue and alizarin red S staining of the skeleton revealed mineralization defects at the posterior level of the skull (delayed suture closure) and in appendicular and axial skeleton. Furthermore, microradiographic analysis showed a reduced bone density in the same areas. Thus, overexpressing of hIGFBP-1 demonstrates early postnatal life growth retardation and a delay in mineralization in transgenic mutant mice. These data show the involvement of the IGF/IGFBP system and more particularly IGFBP-1 in the biomineralization process.     

Islet inflammation and hyperplasia induced by the pancreatic islet-specific overexpression of interleukin-6 in transgenic mice.

Interleukin-6 (IL-6) is thought to be involved in the pathogenesis of autoimmune insulin-dependent diabetes mellitus. To examine this possibility, we developed two lines of transgenic mice (termed RIP-IL6) which overexpressed IL-6 in the pancreatic islet beta cells. RIP-IL6 mice, while showing a modest reduction in body weight, remained normoglycemic throughout their lives. Furthermore, insulin gene expression and glucose tolerance were similar to non-transgenic littermates. Histopathological examination revealed significant changes in the pancreas but not other organs of RIP-IL6 animals, with marked alterations in the architecture of the islets, in the islet cells, and in surrounding tissues. In younger animals these changes included islet hyperplasia with increased mitotic figures, neo-ductular formation, fibrosis, and a scant mononuclear cell infiltration (insulitis). In addition, immunostaining for islet hormones revealed changes in both the topography and density of beta and alpha cells. In older RIP-IL6 mice, a more florid insulitis was observed which was composed predominantly of B220+ B lymphocytes and, to a lesser extent, Mac-1+ macrophages and CD4+ and CD8+ T lymphocytes. Immunostaining for mouse IgG revealed significant numbers of plasma cells in the peri-islet infiltrates, which suggested that IL-6 induced differentiation of the recruited B lymphocytes. Therefore, islet overexpression of IL-6 produces a complex, localized host response implicating this cytokine in not only inflammatory processes that occur in autoimmune diabetes but also cellular neogenesis, which may indicate a role in tissue repair.     

Disruption of anterior segment development by TGF-beta1 overexpression in the eyes of transgenic mice.

Previous experiments showed that transgenic mice expressing a secreted self-activating transforming growth factor (TGF) -beta1 did not show a phenotype in the lens and cornea until postnatal day 21, when anterior subcapsular cataracts, sporadic thickening of the corneal stroma, and thinning of the corneal epithelium were noted (Srinivasan et al., 1998). To examine the effects of higher concentrations of TGF-beta1 on the lens and cornea, we constructed transgenic mice harboring the strong, lens-specific chicken betaB1-crystallin promoter driving an activated porcine TGF-beta1 gene. In contrast to the earlier study, the transgenic mice had microphthalmic eyes with closed eyelids. Already at embryonic day (E) 13.5, the future cornea of the transgenic mice was threefold thicker than that of wild-type littermates due to increased proliferation of corneal stromal mesenchyme cells. Staining of fibronectin and thrombospondin-1 was increased in periocular mesenchyme. At E17.5, the thickened transgenic corneal stroma was vascularized and densely populated by abundant star-shaped, neural cell adhesion molecule-positive cells of mesenchymal appearance surrounded by irregular swirls of collagen and extracellular matrix. The corneal endothelium, anterior chamber, and stroma of iris/ciliary body did not develop, and the transgenic cornea was opaque. Fibronectin, perlecan, and thrombospondin-1 were elevated, whereas type VI collagen decreased in the transgenic corneal stroma. Stromal mesenchyme cells expressed alpha-smooth muscle actin as did lens epithelial cells and cells of the retinal pigmented epithelium. By E17.5, lens fiber cells underwent apoptotic cell death that was followed by apoptosis of the entire anterior lens epithelium between E18.5 and birth. Posteriorly, the vitreous humor was essentially absent; however, the retina appeared relatively normal. Thus, excess TGF-beta1, a mitogen for embryonic corneal mesenchyme, severely disrupts corneal and lens differentiation. Our findings profoundly contrast with the mild eye phenotype observed with presumably lower levels of ectopic TGF-beta and illustrate the complexity of TGF-beta utilization and the importance of dose when assessing the effects of this growth factor.     

Hypertension caused by transgenic overexpression of Rac1

Reactive oxygen species, including superoxide, are important mediators of the pathophysiology of hypertension. In the vasculature, superoxide antagonizes nitric oxide (NO*), resulting in increased vascular tone. The GTP binding protein Rac regulates a wide variety of cellular functions, including the activation of NADPH oxidase, the major source of O2*-in the blood vessel wall. An hypothesis is that Rac1 may act as an important regulator of vascular O2*- production, contributing to the balance between O2*- and NO* and maintaining consequent homeostasis of blood pressure. To alter the activity of vascular NADPH oxidase, the authors developed a transgenic animal model that overexpresses the human cDNA of the constitutively active mutant of Rac1 (RacCA) in smooth muscle cells using the smooth muscle +/--actin promoter. The RacCA transgenic had excessive amounts of O2*- in the vessel wall that, which led to heightened production of peroxynitrite, as detected by increased protein nitration and reduced NO* levels. RacCA mice developed moderate hypertension, which was corrected by N-acetyl-L-cysteine (NAC). RacCA transgenic mice also developed left ventricular hypertrophy as a secondary effect of pressure overload. The data suggest that Rac1 is a critical regulator of the redox state of blood vessels and homeostasis of blood pressure.     

Genetic modification of the phenotypes produced by amyloid precursor protein overexpression in transgenic mice

Overexpression of Alzheimer amyloid precursor protein (APP) produces dramatically different phenotypes in transgenic mice depending on the genetic background. For example, concentrations of APP that produce amyloid plaques in outbred transgenic lines are lethal for inbred FVB/N or C57BL/6J mice. Expression of SOD1 transgenes is protective, suggesting involvement of oxidative damage in premature death, but ablation of Apoe had no significant effect. In contrast, FGF2 transgene overexpression enhances the lethal effects of APP. Differential survival does not appear to reflect genetic differences in APP processing, but rather host responses to APP or its derivatives.      

Neurodevelopmental defects resulting from ATRX overexpression in transgenic mice

Several X-linked mental retardation syndromes are caused by mutations in the ATRX gene. Common clinical features associated with ATRX mutations include severe mental retardation, characteristic facial anomalies and variable degrees of urogenital defects and alpha-thalassemia. Although the ATRX protein is a member of the SWI/SNF family of chromatin remodeling proteins, little is known about the biochemical activity of the ATRX protein or its in vivo function during development. Here we demonstrate that ATRX is part of a large multiprotein complex similar in size to the SWI/SNF complex. Furthermore, we have generated transgenic mice that overexpress ATRX as an initial model for studying the function of this protein during development. Misexpression of ATRX was associated with growth retardation, neural tube defects and a high incidence of embryonic death. Moreover, brains from E10.5 transgenic embryos displayed abnormal growth and organization of the ventricular zone that was highly convoluted in the most severely affected embryos. Transgenic mice that survived to birth exhibited a high incidence of perinatal death, as well as seizures, mild craniofacial anomalies and abnormal behavior. Our findings indicate that ATRX dosage is crucial for normal development and organization of the cortex, and emphasize the relevance of our model for the study of ATRX function and disease pathogenesis.     

Mandibular growth retardation in corticosteroid-treated juvenile mice

Juvenile mice were treated for up to eight weeks with weekly doses of a synthetic analogue of cortisol:triamcinolone hexacetonide. The mandibular condylar cartilage was studied histologically and histochemically at regular intervals. Morphometric measurements were performed along the mandibular posterior vertical dimension (condylar process and ramus). By the second injection significant morphological changes were noted in the condylar cartilage, followed by retardation of bone growth. The most distinctive feature in the cartilage of triamcinolone-treated mice was a marked increase in the dimension of its mineralized zone concomitant with a significant increase in the number of hypertrophic chondrocytes. The role of condylar cartilage in mandibular growth is discussed.     

Liver growth retardation in corticosteroid-treated neonatal mice

This study examined the influence of triamcinolone hexacetonide on the growth and nucleic acid synthesis in the liver of neonatal mice. Following a single dose of the hormone the synthesis of DNA was significantly depressed, a feature that lasted for 72 hours. Thereafter an almost full recovery took place and by 144 hours no significant differences existed between hormone-treated livers and their nontreated controls. The above inhibitory effect was dose-dependent and was specific to steroids with glucocorticoid activity. A markedly different response was noted in regard to RNA synthesis. Hormone-treated livers revealed an initial increase in the uptake of 3H-uridine and RNa synthesis but later on revealed a significant decrease in the incorporation of uridine resulting in a marked reduction in the tissue's content of RNA. Concomitantly, the hormone increased significantly the protein content of the liver. It thus became clear that glucocorticoid hormones possess both a significant inhibitory effect upon the proliferative activity of neonatal hepatocytes and interfere with the latters' protein synthetic machinery, thereby affecting the normal growth of the liver at its critical period of growth and development.     

MMTV promoter-regulated caveolin-1 overexpression yields defective parenchymal epithelia in multiple exocrine organs of transgenic mice

Caveolin-1 (Cav-1) is a major structural protein of caveolae, specialized plasma membrane invaginations that are involved in a cell-specific fashion in diverse cell activities such as molecular transport, cell adhesion, and signal transduction. In normal adult mammals, Cav-1 expression is abundant in mesenchyme-derived cells but relatively low in epithelial parenchyma. However, epithelial Cav-1 overexpression is associated with development and/or progression of many carcinomas. In this study, we generated and characterized a transgenic mouse model of Cav-1 overexpression under the control of a mouse mammary tumor virus (MMTV) long terminal-repeat promoter, which is predominantly expressed in specific epithelial cells. The MMTVcav-1⁺ transgenic mice were fertile, and females bore litters of normal size with no obvious developmental abnormalities. However, by age 11 months, the MMTVcav-1⁺ mice demonstrated overtly different phenotypes in multiple exocrine organs when compared with their nontransgenic MMTVcav-1⁻ littermates. Cav-1 overexpression in MMTVcav-1⁺ mice produced organ-specific abnormalities, including hypotrophy of mammary glandular epithelia, bronchiolar epithelial hyperplasia and atypia, mucous-cell hyperplasia in salivary glands, elongated hair follicles and dermal thickening in the skin, and reduced accumulation of enzymogen granules in pancreatic acinar cells. In addition, the MMTVcav-1⁺ transgenic mice tended to have a greater incidence of malignant tumors, including lung and liver carcinomas and lymphoma, than their MMTVcav-1⁻ littermates. Our results indicate that Cav-1 overexpression causes organ-specific, age-related epithelial disorders and suggest the potential for increased susceptibility to carcinogenesis.     

Mandibular growth retardation in corticosteroid-treated juvenile mice.

Juvenile mice were treated for up to eight weeks with weekly doses of a synthetic analogue of cortisol:triamcinolone hexacetonide. The mandibular condylar cartilage was studied histologically and histochemically at regular intervals. Morphometric measurements were performed along the mandibular posterior vertical dimension (condylar process and ramus). By the second injection significant morphological changes were noted in the condylar cartilage, followed by retardation of bone growth. The most distinctive feature in the cartilage of triamcinolone-treated mice was a marked increase in the dimension of its mineralized zone concomitant with a significant increase in the number of hypertrophic chondrocytes. The role of condylar cartilage in mandibular growth is discussed.     

Inflammation induced by infection potentiates tau pathological features in transgenic mice

Comorbidities that promote the progression of Alzheimer's disease (AD) remain to be uncovered and evaluated in animal models. Because elderly individuals are vulnerable to viral and bacterial infections, these microbial agents may be considered important comorbidities that could potentiate an already existing and tenuous inflammatory condition in the brain, accelerating cognitive decline, particularly if the cellular and molecular mechanisms can be defined. Researchers have recently demonstrated that triggering inflammation in the brain exacerbates tau pathological characteristics in animal models. Herein, we explore whether inflammation induced via viral infection, compared with inflammation induced via bacterial lipopolysaccharide, modulates AD-like pathological features in the 3xTg-AD mouse model and provide evidence to support the hypothesis that infectious agents may act as a comorbidity for AD. Our study shows that infection-induced acute or chronic inflammation significantly exacerbates tau pathological characteristics, with chronic inflammation leading to impairments in spatial memory. Tau phosphorylation was increased via a glycogen synthase kinase-3β-dependent mechanism, and there was a prominent shift of tau from the detergent-soluble to the detergent-insoluble fraction. During chronic inflammation, we found that inhibiting glycogen synthase kinase-3β activity with lithium reduced tau phosphorylation and the accumulation of insoluble tau and reversed memory impairments. Taken together, infectious agents that trigger central nervous system inflammation may serve as a comorbidity for AD, leading to cognitive impairments by a mechanism that involves exacerbation of tau pathological characteristics.     

骨髓基质细胞移植改善APP/PS1转基因小鼠的认知功能

目的探究骨髓基质细胞(Bone marrow stromal cells,BMSCs)移植治疗对APP/PS转基因的阿尔兹海默病(Alzheimer's disease,AD)模型小鼠认知功能障碍的改善作用。方法分离培养C57BL/6小鼠的BMSCs,诱导其分化为神经细胞,通过相差显微镜技术和免疫细胞化学荧光染色技术鉴定其表型与分化。通过显微注射技术,将绿色荧光蛋白(Enhanced green fluorescent protein,eGFP)转基因小鼠(生后3个月)来源的BMSCs注入APP/PS转基因小鼠(生后12个月)侧脑室内后,以荧光显微镜技术检测移植细胞的存活和整合情况,HE染色检测AD模型小鼠海马区萎缩的恢复程度并以Morris水迷宫试验与旷场试验检测移植治疗后小鼠认知功能障碍的改善情况。结果分离自C57BL/6小鼠的BMSCs在增殖培养基中贴壁生长,形态呈纺锤形,免疫表型CD271+/CD45-++,经神经分化诱导培养可形成β-tubulin-III神经元和GFAP星形胶质细胞。GFP转基因小鼠来源的BMSCs移植入APP/PS1转基因小鼠脑内可存活并与宿主海马区神经组织有效整合。移植治疗3周后宿主小鼠海马区面积显著增加(<0.001),水迷宫实验逃生潜伏期明显缩短(<0.01),旷场试验评分显著提高(<0.05)。结论骨髓基质细胞移植治疗可有效改善APP/PS1转基因的AD模型小鼠的认知功能障碍。     

Bone formation in vitro and in nude mice by human osteosarcoma cells

Osteosarcomas contain variable amounts of bony tissue, but the mechanism of bone formation by osteosarcoma is not well understood. While a number of cultured human osteosarcoma cell lines have been established, they are maintained by different media and differ qualitatively with regard to bone formation. We examined different media for their ability to support bone formation in vitro and found that alpha-modification of Eagle's minimal essential medium supplemented with beta glycerophosphate was best for this purpose, because it contained the proper calcium and phosphate concentrations. Subsequently, we compared seven human osteosarcoma cell lines under the same experimental conditions to clarify their ability to induce bone formation. NOS-1 cells most frequently exhibited features of bone formation in vitro and in nude mice. Collagen synthesis by tumour cells themselves seemed to be the most important factor for bone volume. However, even HuO9 cells, which lacked collagen synthesis and failed to form bone in vitro, successfully formed tumours containing bone in nude mice. Histological analysis of HuO9 cells in diffusion chambers implanted in nude mice and the findings of polymerase chain reaction indicated that the phenomenon was probably due to bone morphogenetic protein.     

Enhanced plasminogen activator inhibitor-1 expression in transgenic mice with hepatocyte-specific overexpression of superoxide dismutase or glutathione peroxidase

In this study, we developed a double-transgenic mouse model allowing hepatocyte-specific and regulated expression of the redox-modifying enzymes copper/zinc superoxide dismutase (SOD) and glutathione peroxidase (GPX) by using a tetracycline-regulatable gene expression system. Within this system, the SOD and GPX level can be regulated deliberately by addition or removal of doxycycline hydrochloride to the drinking water. As reactive oxygen species (ROS) have been implicated in a number of pathological conditions, such as atherosclerosis, thrombosis, or liver fibrosis, processes that are also frequently associated with enhanced levels of plasminogen activator inhibitor-1 (PAI-1), it was the aim of the present study to investigate the influence of SOD and GPX overexpression on the regulation of PAI-1. PAI-1 mRNA and protein levels in tetracycline transactivator-dependent SOD-overexpressing double-transgenic mice reached values 2.5- to threefold above the normal mRNA level. By applying doxycycline, a deinduction of the PAI-1 levels was observed. By using the same protocol, PAI-1 mRNA and protein levels were enhanced in GPX double-transgenic mice, and again this response was blunted by the addition of doxycycline. These studies provide some new information regarding the role of ROS within the proteolytic processes in hepatocytes that require PAI-1.     

转基因小鼠高表达的FasL对Sertoli细胞免疫调节功能的影响

目的 :研究转基因小鼠高表达的FasL对Sertoli细胞在睾丸局部感染时的免疫调节作用的影响。方法 :将溶脲脲原体 (UU)分别注入FasL转基因及野生型小鼠膀胱 ,模拟上行性感染的途径。分别在感染后 1、2和 3wk处死小鼠 ,分离睾丸组织观察其病理变化 ,并用免疫组化染色法比较UU感染前后 ,Ser toli细胞上FasL和TGF β、IL 1α、IL 6的表达及分泌格局的差异。从野生型小鼠睾丸组织中分离高纯度的Sertoli细胞 ,与未感染UU的对照组相比 ,观察UU感染后FasL Sertoli细胞介导Fas Jurkat细胞的凋亡能力的变化。结果 :UU感染组的转基因小鼠 ,睾丸组织发生的病理改变比野生型更为明显 ;两种小鼠感染后Sertoli细胞分泌的调节因子变化格局不同 ;感染后Sertoli细胞对Jurkat细胞的杀伤能力增强。结论 :在抗感染免疫中 ,转基因表达的FasL可影响Sertoli细胞分泌细胞因子的格局 ,进而影响睾丸局部的免疫平衡。过高表达的FasL对机体的抗感染应答并非一定有利。     

Chronic overproduction of transforming growth factor-beta1 by astrocytes promotes Alzheimer's disease-like microvascular degeneration in transgenic mice

Cerebrovascular amyloid deposition and microvascular degeneration are frequently associated with Alzheimer's disease (AD), but the etiology and pathogenetic role of these abnormalities are unknown. Recently, transforming growth factor-beta1 (TGF-beta1) was implicated in cerebrovascular amyloid formation in transgenic mice with astroglial overproduction of TGF-beta1 and in AD. We tested whether TGF-beta1 overproduction induces AD-like cerebrovascular degeneration and analyzed how cerebrovascular abnormalities develop over time in TGF-beta1-transgenic mice. In cerebral microvessels from 3- to 4-month-old TGF-beta1-transgenic mice, which display a prominent perivascular astrocytosis, levels of the basement membrane proteins perlecan and fibronectin were severalfold higher than in vessels from nontransgenic mice. Consistent with this increase, cortical capillary basement membranes of TGF-beta1 mice were significantly thickened. These changes preceded amyloid deposition, which began at around 6 months of age. In 9- and 18-month-old TGF-beta1 mice, various degenerative changes in microvascular cells of the brain were observed. Endothelial cells were thinner and displayed abnormal, microvilli-like protrusions as well as occasional condensation of chromatin, and pericytes occupied smaller areas in capillary profiles than in nontransgenic controls. Similar cerebrovascular abnormalities have been reported in AD. We conclude that chronic overproduction of TGF-beta1 triggers an accumulation of basement membrane proteins and results in AD-like cerebrovascular amyloidosis and microvascular degeneration. Closely related processes may induce cerebrovascular pathology in AD.     

Defects in lens fiber differentiation are linked to c-mos overexpression in transgenic mice

We describe three strains of transgenic mice derived by embryo microinjection of DNA consisting of a long terminal repeat (LTR) of Moloney murine sarcoma virus (Mo-MSV) linked to the murine c-mos coding sequences. Southern analysis of the genomic DNA of these strains suggested that in each case the transgene had integrated at a different chromosomal location. The strains were characterized by dominant changes in secondary lens fiber differentiation. Shortly after birth, insufficient elongation of differentiating lens fibers and lack of basement membrane secretion resulted in breakdown of the posterior lens capsule. This, in turn led to posterior protrusion and swelling of lens tissue. In the course of the first 3 weeks after birth, globular lens cells began to fill the entire anterior and posterior chambers of the eye. Concomitantly, there was massive overexpression of c-mos RNA in the lens. Whereas this construct has high transforming activity when transfected into NIH-3T3 cells, no hyperplasia or neoplasia have been observed in the affected lenses. Increased expression of c-mos RNA was not confined to the lens of the eye but has been detected in any of several tissues tested.     

Thermogenic activity of growth hormone transgenic mice

The objective was to determine the effect of a mouse metallothionein/bovine growth hormone transgene on resting metabolic rate (RMR), cold-induced thermogenesis, and beta-agonist stimulated nonshivering thermogenesis in mice. Non-transgenic littermates were used as controls. Open-circuit indirect calorimetry was used to assess RMR and cold-induced thermogenesis in 64 mice. Air temperature in the chamber was set at 31 degrees C for RMR and was decreased to 28, 25, 21, or 17 degrees C to determine cold-induced thermogenesis. Response to the beta-agonist isoproterenol was evaluated by monitoring changes in colonic temperature of 34 mice upon injection of the drug or saline. Despite the fact that RMR tended to be lower in transgenics than in nontransgenics, at 31 degrees C transgenic mice were able to regulate colonic temperature at the same level as nontransgenics, but colonic temperature decreased in transgenics relative to nontransgenics as air temperature was reduced. For each degree decrease in air temperature between 31 and 17 degrees C, nontransgenic mice increased heat production by 1.03 +/- .10 watt/kg, whereas transgenic mice increased it by only .56 +/- .08 watt/kg, indicating that the thermogenic response of transgenics to cold was inferior. The magnitude of the maximal increase in colonic temperature after isoproterenol injection was similar for both groups, but the response was slower in transgenics. We suggest that lean body mass and substrate availability for shivering thermogenesis are reduced in transgenics relative to total body weight, and that they allow colonic temperature to decrease to conserve energy.