Ana Alcaraz

Classification of Proliferative Pulmonary Lesions of the Mouse Recommendations of the Mouse Models of Human Cancers Consortium

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20–22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

Conditional knockout of focal adhesion kinase in endothelial cells reveals its role in angiogenesis and vascular development in late embryogenesis

Focal adhesion kinase (FAK) is a critical mediator of signal transduction by integrins and growth factor receptors in a variety of cells including endothelial cells (ECs). Here, we describe EC-specific knockout of FAK using a Cre-loxP approach. In contrast to the total FAK knockout, deletion of FAK specifically in ECs did not affect early embryonic development including normal vasculogenesis. However, in late embryogenesis, FAK deletion in the ECs led to defective angiogenesis in the embryos, yolk sac, and placenta, impaired vasculature and associated hemorrhage, edema, and developmental delay, and late embryonic lethal phenotype. Histologically, ECs and blood vessels in the mutant embryos present a disorganized, detached, and apoptotic appearance. Consistent with these phenotypes, deletion of FAK in ECs isolated from the floxed FAK mice led to reduced tubulogenesis, cell survival, proliferation, and migration in vitro. Together, these results strongly suggest a role of FAK in angiogenesis and vascular development due to its essential function in the regulation of multiple EC activities.

A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice

Mcm4 (minichromosome maintenance–deficient 4 homolog) encodes a subunit of the MCM2-7 complex (also known as MCM2–MCM7), the replication licensing factor and presumptive replicative helicase. Here, we report that the mouse chromosome instability mutation Chaos3 (chromosome aberrations occurring spontaneously 3), isolated in a forward genetic screen, is a viable allele of Mcm4. Mcm4Chaos3 encodes a change in an evolutionarily invariant amino acid (F345I), producing an apparently destabilized MCM4. Saccharomyces cerevisiae strains that we engineered to contain a corresponding allele (resulting in an F391I change) showed a classical minichromosome loss phenotype. Whereas homozygosity for a disrupted Mcm4 allele (Mcm4−) caused preimplantation lethality, McmChaos3/− embryos died late in gestation, indicating that Mcm4Chaos3 is hypomorphic. Mutant embryonic fibroblasts were highly susceptible to chromosome breaks induced by the DNA replication inhibitor aphidicolin. Most notably, >80% of Mcm4Chaos3/Chaos3 females succumbed to mammary adenocarcinomas with a mean latency of 12 months. These findings suggest that hypomorphic alleles of the genes encoding the subunits of the MCM2-7 complex may increase breast cancer risk.

Neutrophil Depletion during Toxoplasma gondii Infection Leads to Impaired Immunity and Lethal Systemic Pathology

ABSTRACT The immunomodulatory role of neutrophils during infection withToxoplasma gondii was investigated. Monoclonal antibody-mediated depletion revealed that neutrophils are essential for survival during the first few days of infection. Moreover, neutrophil depletion was associated with a weaker type 1 immune response as measured by decreased levels of gamma interferon, interleukin-12 (IL-12) and tumor necrosis factor alpha. IL-10 was also decreased in depleted animals. Additionally, splenic populations of CD4+T cells, CD8+ T cells, and NK1.1+ cells were decreased in depleted mice. Neutrophil-depleted mice exhibited lesions of greater severity in tissues examined and a greater parasite burden as determined by histopathology and reverse transcription-PCR. We conclude that neutrophils are critical near the time of infection because they influence the character of the immune response and control tachyzoite replication.

Role of FIP200 in cardiac and liver development and its regulation of TNFα and TSC–mTOR signaling pathways

Focal adhesion kinase family interacting protein of 200 kD (FIP200) has been shown to regulate diverse cellular functions such as cell size, proliferation, and migration in vitro. However, the function of FIP200 in vivo has not been investigated. We show that targeted deletion of FIP200 in the mouse led to embryonic death at mid/late gestation associated with heart failure and liver degeneration. We found that FIP200 knockout (KO) embryos show reduced S6 kinase activation and cell size as a result of increased tuberous sclerosis complex function. Furthermore, FIP200 KO embryos exhibited significant apoptosis in heart and liver. Consistent with this, FIP200 KO mouse embryo fibroblasts and liver cells showed increased apoptosis and reduced c-Jun N-terminal kinase phosphorylation in response to tumor necrosis factor (TNF) α stimulation, which might be mediated by FIP200 interaction with apoptosis signal–regulating kinase 1 (ASK1) and TNF receptor–associated factor 2 (TRAF2), regulation of TRAF2–ASK1 interaction, and ASK1 phosphorylation. Together, our results reveal that FIP200 functions as a regulatory node to couple two important signaling pathways to regulate cell growth and survival during mouse embryogenesis.

Inactivation of focal adhesion kinase in cardiomyocytes promotes eccentric cardiac hypertrophy and fibrosis in mice

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a major role in integrin signaling pathways. Although cardiovascular defects were observed in FAK total KO mice, the embryonic lethality prevented investigation of FAK function in the hearts of adult animals. To circumvent these problems, we created mice in which FAK is selectively inactivated in cardiomyocytes (CFKO mice). We found that CFKO mice develop eccentric cardiac hypertrophy (normal LV wall thickness and increased left chamber dimension) upon stimulation with angiotensin II or pressure overload by transverse aortic constriction as measured by echocardiography. We also found increased heart/body weight ratios, elevated markers of cardiac hypertrophy, multifocal interstitial fibrosis, and increased collagen I and VI expression in CFKO mice compared with control littermates. Spontaneous cardiac chamber dilation and increased expression of hypertrophy markers were found in the older CFKO mice. Analysis of cardiomyocytes isolated from CFKO mice showed increased length but not width. The myocardium of CFKO mice exhibited disorganized myofibrils with increased nonmyofibrillar space filled with swelled mitochondria. Last, decreased tyrosine phosphorylation of FAK substrates p130Cas and paxillin were observed in CFKO mice compared with the control littermates. Together, these results provide strong evidence for a role of FAK in the regulation of heart hypertrophy in vivo.

Cardiac developmental defects and eccentric right ventricular hypertrophy in cardiomyocyte focal adhesion kinase (FAK) conditional knockout mice.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that plays an important role in integrin-mediated signal transduction. To explore the role and mechanisms of FAK in cardiac development, we inactivated FAK in embryonic cardiomyocytes by crossing the floxed FAK mice with myosin light chain-2a (MLC2a) Cre mice, which expressed Cre as early as embryonic day 9.5 in the heart. The majority of conditional FAK knockout mice generated from MLC2a-Cre (CFKO-2a) died in the embryonic stage with thin ventricular wall and ventricular septal defects. A small fraction of CFKO-2a mice survived to adulthood with spontaneous eccentric right ventricle hypertrophy. Transmission electron microscopy analysis displayed swelling in the rough endoplasmic reticulum in CFKO-2a embryonic cardiomyocytes. We found that decreased cell proliferation, but not increased cell apoptosis or differentiation, is the reason for the thin ventricular wall in CFKO-2a mice. Microarray analysis suggests that myocyte enhancer factor 2a (MEF2a) can be regulated by FAK and that inactivation of FAK in the embryonic heart compromised MEF2a expression. Last, we found that Src, but not PI3K, is important in mediating signal transduction for the regulation of MEF2a by FAK. Together, these results identified the role and mechanisms of FAK in embryonic cardiac development.

Interleukin-12 promotes pathologic liver changes and death in mice coinfected with Schistosoma mansoni and Toxoplasma gondii.

ABSTRACT We previously demonstrated that mice concurrently infected withSchistosoma mansoni and Toxoplasma gondiiundergo accelerated mortality which is preceded by severe liver damage. Abnormally high levels of serum tumor necrosis factor alpha (TNF-α) in the dually infected mice suggested a role for this and related proinflammatory mediators in the pathologic alterations. In order to evaluate the factors involved in increased inflammatory-mediator production and mortality, interleukin-12−/−(IL-12−/−) mice were coinfected with S. mansoni and T. gondii, and survival and immune responses were monitored. These IL-12−/− mice displayed decreased liver damage and prolonged time to death relative to wild-type animals also coinfected with these parasites. Relative to the response of cells from the coinfected wild-type animals, levels of TNF-α, gamma interferon, and NO produced by splenocytes from coinfected IL-12−/− mice were reduced, and levels of IL-5 and IL-10 were increased, with the net result that the immune response of the dually infected IL-12−/− mice was similar to that of the wild-type mice infected with S. mansoni alone. While dually infected wild-type animals succumb in the absence of overt parasitemia, the delayed death in the absence of IL-12 is associated with relatively uncontrolled T. gondii replication. These data support the view that S. mansoni-infected mice are acutely sensitive to infection with T. gondii as a result of their increased hepatic sensitivity to high levels of proinflammatory cytokines; IL-12 and TNF-α are implicated in this process.

IL-10 Prevents Liver Necrosis During Murine Infection with Trichinella spiralis

Infection with Trichinella spiralis rarely leads to significant morbidity. In this study, we show that IL-10 knockout mice infected with this parasite develop extensive areas of coagulative necrosis in the liver, and newborn larvae are required for lesion formation. Histopathological examination revealed that the hepatic inflammatory infiltrate was mixed but dominated by eosinophils. Accordingly, infected IL-10 knockout mice displayed a marked eosinophilia. IL-10 was expressed during infection in mesenteric lymph node populations and liver tissue. Analysis of cytokine profiles revealed a codominant expression of type 1 and 2 mediators that was enhanced in the absence of IL-10. Additionally, CD11c+ MHC class II+ cells were increased in mesenteric lymph nodes of IL-10 knockout mice, suggesting a possible link between IL-10 and dendritic cell trafficking. Nevertheless, there were no significant differences in mortality or parasite burdens between the strains of mice, indicating that IL-10 is necessary to control the host’s inflammatory response but does not impact establishment of the parasite. Expression of IL-10 appears to be an adaptation used by the liver to protect itself from damage caused by migrating newborn larvae.

IL-10 Regulates Movement of Intestinally Derived CD4+ T Cells to the Liver

Diseases that affect the intestine may have hepatic manifestations, but the mechanisms involved in establishing hepatic disease secondarily remain poorly understood. We previously reported that IL-10 knockout (KO) mice developed severe necrotizing hepatitis following oral infection with Trichinella spiralis. In this study, we used this model of intestinal inflammation to further examine the role of IL-10 in regulating hepatic injury. Hepatic damage was induced by migrating newborn larvae. By delivering the parasite directly into the portal vein, we demonstrated that an ongoing intestinal immune response was necessary for the development of hepatitis. Intestinally derived CD4+ cells increased in the livers of IL-10 KO mice, and Ab-mediated blockade of MAdCAM-1 inhibited the accumulation of CD4+α4β7+ cells in the liver. Moreover, adoptive transfer of intestinally primed CD4+ T cells from IL-10 KO mice caused hepatitis in infected immunodeficient animals. Conversely, transfer of wild-type donor cells reduced the severity of hepatic inflammation in IL-10 KO recipients, demonstrating regulatory activity. Our results revealed that IL-10 prevented migration of intestinal T cells to the liver and inhibited the development of hepatitis.