Browsing by Author "Azhar, Mohamad"

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    Generation of mice carrying a knockout-first and conditional-ready allele of transforming growth factor beta2 gene
    (Wiley, 2014-09) Ahmed, A. S. Ishtiaq; Bose, Gracelyn C.; Huang, Li; Azhar, Mohamad; Department of Pediatrics, Indiana University School of Medicine
    Transforming growth factor beta2 (TGFβ2) is a multifunctional protein which is expressed in several embryonic and adult organs. TGFB2 mutations can cause Loeys Dietz syndrome, and its dysregulation is involved in cardiovascular, skeletal, ocular, and neuromuscular diseases, osteoarthritis, tissue fibrosis, and various forms of cancer. TGFβ2 is involved in cell growth, apoptosis, cell migration, cell differentiation, cell-matrix remodeling, epithelial-mesenchymal transition, and wound healing in a highly context-dependent and tissue-specific manner. Tgfb2(-/-) mice die perinatally from congenital heart disease, precluding functional studies in adults. Here, we have generated mice harboring Tgfb2(βgeo) (knockout-first lacZ-tagged insertion) gene-trap allele and Tgfb2(flox) conditional allele. Tgfb2(βgeo/βgeo) or Tgfb2(βgeo/-) mice died at perinatal stage from the same congenital heart defects as Tgfb2(-/-) mice. β-galactosidase staining successfully detected Tgfb2 expression in the heterozygous Tgfb2(βgeo) fetal tissue sections. Tgfb2(flox) mice were produced by crossing the Tgfb2(+/βgeo) mice with the FLPeR mice. Tgfb2(flox/-) mice were viable. Tgfb2 conditional knockout (Tgfb2(cko/-) ) fetuses were generated by crossing of Tgfb2(flox/-) mice with Tgfb2(+/-) ; EIIaCre mice. Systemic Tgfb2(cko/-) embryos developed cardiac defects which resembled the Tgfb2(βgeo/βgeo) , Tgfb2(βgeo/-) , and Tgfb2(-/-) fetuses. In conclusion, Tgfb2(βgeo) and Tgfb2(flox) mice are novel mouse strains which will be useful for investigating the tissue specific expression and function of TGFβ2 in embryonic development, adult organs, and disease pathogenesis and cancer. genesis
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    Genetic and Developmental Basis of Cardiovascular Malformations
    (Elsevier, 2016-03) Azhar, Mohamad; Ware, Stephanie M.; Department of Pediatrics, IU School of Medicine
    Cardiovascular malformations (CVMs) are the most common birth defect, occurring in 1% to 5% of all live births. Genetic, epigenetic, and environmental factors all influence the development of CVMs, and an improved understanding of the causation of CVMs is a prerequisite for prevention. Cardiac development is a complex, multistep process of morphogenesis that is under genetic regulation. Although the genetic contribution to CVMs is well recognized, the genetic causes of human CVMs are still identified infrequently. This article discusses the key genetic concepts characterizing human CVMs, their developmental basis, and the critical developmental and genetic concepts underlying their pathogenesis.
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    Hyperactive transforming growth factor-β1 signaling potentiates skeletal defects in a neurofibromatosis type 1 mouse model
    (Wiley, 2013-12) Rhodes, Steven D.; Wu, Xiaohua; He, Yongzheng; Chen, Shi; Yang, Hao; Staser, Karl W.; Wang, Jiapeng; Zhang, Ping; Jiang, Chang; Yokota, Hiroki; Dong, Ruizhi; Peng, Xianghong; Yang, Xianlin; Murthy, Sreemala; Azhar, Mohamad; Mohammad, Khalid S.; Xu, Mingjiang; Guise, Theresa A.; Yang, Feng-Chun; Anatomy and Cell Biology, School of Medicine
    Dysregulated transforming growth factor beta (TGF-β) signaling is associated with a spectrum of osseous defects as seen in Loeys-Dietz syndrome, Marfan syndrome, and Camurati-Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF-β1 signaling pivotally underpins osseous defects in Nf1(flox/-) ;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF-β1 levels are fivefold to sixfold increased both in Nf1(flox/-) ;Col2.3Cre mice and in a cohort of NF1 patients. Nf1-deficient osteoblasts, the principal source of TGF-β1 in bone, overexpress TGF-β1 in a gene dosage-dependent fashion. Moreover, Nf1-deficient osteoblasts and osteoclasts are hyperresponsive to TGF-β1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21-Ras-dependent hyperactivation of the canonical TGF-β1-Smad pathway. Reexpression of the human, full-length neurofibromin guanosine triphosphatase (GTPase)-activating protein (GAP)-related domain (NF1 GRD) in primary Nf1-deficient osteoblast progenitors, attenuated TGF-β1 expression levels and reduced Smad phosphorylation in response to TGF-β1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF-β receptor 1 (TβRI) kinase inhibitor, SD-208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1(flox/-) ;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF-β1 signaling in the pathogenesis of NF1-associated osteoporosis and pseudarthrosis, thus implicating the TGF-β signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies
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    Progressive alterations in microstructural organization and biomechanical response in the ApoE mouse model of aneurysm
    (Taylor & Francis, 2013) Haskett, Darren; Azhar, Mohamad; Utzinger, Urs; Vande Geest, Jonathan P.; Pediatrics, School of Medicine
    AAA is a complex disease that leads to a localized dilation of the infrarenal aorta that develops over years. Longitudinal information in humans has been difficult to obtain for this disease, therefore mouse models have become increasingly used to study the development of AAAs. The objective of this study was to determine any changes that occur in the biomechanical response and fiber microstructure in the ApoE(-/-) AngII mouse model of aneurysm during disease progression. Adult ApoE(-/-) AngII infused mice along with wild-type controls were taken at 14 and 28 d. Aortas were excised and tested simultaneously for biaxial mechanical response and ECM organization. Data sets were fit to a Fung-type constitutive model to give peak strains and stiffness values. Images from two photon microscopy were quantified in order to assess the preferred fiber alignment and degree of fiber orientation. Biomechanical results found significant differences that were present at 14 d had returned to normal by 28 d along with significant changes in fiber orientation and dispersion indicating remodeling occurring within the aneurysmal wall. This return of some of the normal biomechanical function, in addition the continuing changes that occur in the microstructure suggest a restorative response that occurs in the ApoE(-/-) AngII infused model after the initial aneurysm formation.
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    Role of Transforming Growth Factor Beta2 in Congenital Heart Disease
    (Office of the Vice Chancellor for Research, 2014-04-11) Bose, Gracelyn C.; Ahmed, Abu; Huang, Li; Sarangdhar, Kirti; Azhar, Mohamad
    Congenital heart disease (CHD) represents the largest class of birth defects in the US and affects about 0.8% of all babies born. As a result of remarkable advances in the medical and surgical management of CHD, more than 75% of children born with CHD now live into adulthood. As such, discovery of the causes for CHD is not only a fundamental research endeavor, but is vital to the health care of this growing community. Inherited genetic mutations in Transforming Growth Factor Beta (TGFB) gene are found in the patients of Loeys-Dietz syndrome. Several cardiac (endocardial, myocardial) and extra-cardiac (second heart field, neural crest) cell lineages that express Tgfb2 contribute to heart development. To study the role of Tgfb2 in different cell types involved in heart development, we have generated Tgfb2 conditional knockout mice. These mice harbor Tgfb2 LacZ-tagged conditional-ready allele (also called tm1a). By using long range PCR (LR-PCR) we have confirmed the germline transmission of Tgfb2tm1a allele. Histological examination shows that Tgfb2tm1a/tm1a embryos develop several congenital heart defects. This indicates that Tgfb2tm1a allele is a knockout-first allele, which is consistent with the original design of our conditional gene targeting scheme. Next, by crossing to Flp recombinase mice we can generate mice with a Tgfb2 conditional-ready allele (also called tm1c). The presence of Tgfb2tm1c allele in the mice is confirmed by genomic PCR. In the future, we plan to use Tgfb2tm1c mice to conditionally delete Tgfb2 in different cardiac or extra-cardiac cell types using well-characterized Cre recombinase transgenic mice. Collectively, we have produced, generated, and validated mice harboring the Tgfb2 LacZ tagged knockout-first and conditional-ready allele. Our results from embryos carrying homozygous Tgfb2tm1a allele indicate that TGFβ2 is required for heart development. Future research will be crucial in expanding knowledge of the unknown cellular etiology of cardiac malformations in patients with TGFB2 mutations.