Browsing by Subject "Regeneration"
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Item Cardiac regeneration: more hope, less hype(2016-02-26) Sadek, Hesham A.Item Expression Analysis of the Regenerating Utricle Sensory Epithelia : From Macroarrays to Parsing Pathways(2005-05-03) Hawkins, Raymond David; Lovett, MichaelI have used a human transcription factor microarray developed in the laboratory of Dr. Michael Lovett to study gene expression differences in the chicken inner ear sensory epithelia. In the initial study, the sensory epithelium from the utricle was compared to that of the cochlea. The purpose of this study was to identify gene expression differences between these two organs. The sensory epithelium from each organ is made up of hair cells and supporting cells. These hair cells are necessary for the detection of sound in the cochlea and for the detection of movement and acceleration in the utricle. The chicken sensory epithelia is of great research interest as it possesses the ability to fully regenerate hair cells that are damaged, whereas mammalian epithelia, once damaged, cannot regenerate. These two organs were compared because the utricle is in a constant state of hair cell turnover, and the cochlea remains quiescent, unless damaged. In order to carry out such microarray expression studies on a small number of cells, between 30,000-50,000 cells, a micro-cDNA amplification method, developed in the lab, was implemented and is described here as well. The experiments were carried out via cross-species hybridizations, and subsequently, a number of genes were validated by quantitative PCR and in situ analysis. Additionally, a library subtraction was used to identify additional genes expressed in the utricle sensory epithelia. In a second microarray expression study, the utricle sensory epithelia was damaged by two independent methods and allowed to recover for various time points for expression profiling on the same transcription factor array. The first method of damage was by laser microbeam to ablate the hair cells such that they die almost instantly. The second method of damage was using ototoxic antibiotics. In each time course, the time points were compared to time matched control epithelia (undamaged). The analysis of this data reveals some very important signaling cascades and developmental pathways involved in hair cell regeneration. Finally, in an effort to functionally validate many of the genes identified during regeneration, gene transcripts were targeted by RNA interference to reduce the expression level and determine the effect on hair cell proliferation. Through this method, several genes were identified to reduce proliferation. Additionally, these experiments were profiled as a means for networking genes into pathways by identifying putative downstream targets in the expression data. An intersection of genes downregulated following inhibitory experiments reveals how several genes potentially lie downstream of one another and form a pathway containing some common regulatory elements.Item Heart Regeneration: An Evolutionary Tale(2013-01-17) Mahmoud, Ahmed Ibrahim; Sadek, Hesham A.Lower vertebrates like urodele amphibians and teleost fish retain a robust cardiac regenerative capacity throughout their life, a phenomenon that is mediated through the proliferation of pre-existing cardiomyocytes. The adult mammalian heart lacks any meaningful endogenous regenerative response following injury. However, embryonic mammalian cardiomyocytes are proliferative and exit the cell cycle shortly after birth. The question of whether the mammalian heart lacks this regenerative potential or is lost early after birth was not clear. We were able to show that the hearts of 1-day-old mice regenerated following partial surgical resection of the neonatal heart, a phenomenon that is lost within a week after birth. Thus, for a brief period after birth, the mammalian heart appears to have the capacity to regenerate due to the proliferative competency of cardiomyocytes. However, one major unresolved question was whether the neonatal mouse heart could also regenerate in response to myocardial ischemia, the most common antecedent of heart failure in humans. To examine this question, we induced myocardial infarction (MI) in 1-day-old mice by ligating the left anterior descending coronary artery, and found that this results in extensive myocardial necrosis and systolic dysfunction. Remarkably, the neonatal mouse heart mounted a robust regenerative response, through proliferation of pre-existing cardiomyocytes, which resulted in full functional recovery within 21 days. Moreover, we were able to demonstrate that the neonatal heart is capable of regeneration following mild, but not severe cryoinjury. Therefore, our work identifies a short period of time after birth where the mammalian heart is capable of regeneration following various types of injury. To unravel the molecular mechanisms that regulate the regenerative capacity of the neonatal mammalian heart, we determined that the miR-15 family regulates neonatal heart regeneration through inducing post-natal cardiomyocyte cell cycle arrest. Moreover, inhibition of the miR-15 family at an early post-natal age until adulthood induces cardiomyocyte proliferation in the adult heart and improves left ventricular systolic function following MI. In conclusion, our findings indicate that the mammalian heart harbors a robust regenerative capacity for a short period of time after birth, mediated by proliferation of pre-existing cardiomyocytes, and that the miR-15 family is an important regulator of post-natal cardiomyocytes cell cycle arrest.Item The hope and hype of cardiac regeneration(2010-08-06) Sadek, Hesham A.Item The Identification and Characterization of MKRP, a Novel Kelch Related Protein(2007-12-17) Embree, Laurence Jonathan; Gurry, Daniel J.The cells of adult myofibers in mammals are terminally differentiated and are incapable of division and self-renewal. Regeneration of damaged skeletal muscle tissue is facilitated through the proliferation and differentiation of resident stem cells known as satellite cells . These satellite cells remain quiescent in uninjured tissue, occupying a sublaminar position between the sarcolemma and basal lamina of adult myofibers. In response to trauma, these cells become activated, and proliferate. The activated cells will reestablish the pool of reside quiescent satellite cells, while others will proliferate, migrate by chemotaxis to the area of injury, withdraw from the cell cycle, and differentiate into new myoblasts. These myoblasts will fuse with and repair the injured fibers, or align with each other and fuse to form new fibers. Genes involved in this process should exhibit an altered pattern of expression in skeletal muscle in response to injury by cardiotoxin, and the changes in expression level can be quantified though the measurement of mRNA levels at specified time points following injury. Utilizing transcriptome analysis, we identified a completely novel transcript that is induced in the myogenic progenitor cells following cardiotoxin injury. The novel transcript contained an open reading frame that coded for a protein belonging to the Kelch superfamily. Expression of the transcript was restricted to skeletal muscle lineages during development, and to myogenic progenitor cells and immature myotubes during injury regeneration. Because of its structural identity and restriction to skeletal muscle, the novel transcript was named the myogenic Kelch related protein (MKRP). Knockdown of MKRP expression using siRNA in C2C12 cells revealed an inhibition of both migration and differentiation in myogenic progenitor cells. A yeast two-hybrid screen identified calsarcin-2 as a potential interacting protein, indicating a possible role for MKRP in the calcineurin pathway during myogenic differentiation.Item Mechanical Signals for Compensatory Lung Growth Assessed by High Resolution Computed Tomography(2008-09-18) Ravikumar, Priya; Hsia, Connie C.W.This dissertation involves the use of high resolution computed tomography (HRCT) to understand the role of intra-thoracic mechanical force and its distribution in regenerative growth in dogs i.e. to quantify lobar lung volumes and density gradients in normal and post-pneumonectomy (following lung resection) lungs. HRCT was used to quantitatively assess regional distribution of lung volume and density gradients among lobes of the lung in order to follow the expansion of remaining lobes following lung resection with a high degree of anatomical precision, and to determine the relationships between lung expansion and alveolar tissue growth. I also extended this work by relating regional lung expansion and growth assessed by radiology to regional alveolar tissue growth assessed by detailed quantitative histology under light and electron microscopy. This study illustrates for the first time a powerful and novel use of in vivo imaging to quantify regional lung distortion and changes in local volume, lung compliance as well as soft tissue density. These changes can be followed non-invasively and serially in a wide range of clinical and investigational applications, such as a) assessing the extent and progression of regional heterogeneity in lung disease or injury; b) assessing local response to treatment or surgical intervention; or c) assessing normal or abnormal patterns of lung growth.Item Muscle Function Improvement in Injured Mice with Combination Treatment(2017-01-17) Kulangara, Rohan G.; Sehat, Alvand, J.; Maredia, Navin; Maxwell, Christian; Liu, Ming-Mei; DeSpain, Kevin; Wolf, Steven E.; Song, JuquanINTRODUCTION: Loss of skeletal muscle from direct injury presents debilitating effects to an individual. Current treatments addressing muscle loss are limited by insufficient reconstitution of functioning muscle. Novel regenerative medicine technologies include the application of Urinary Bladder Matrix (UBM) and mesenchymal stem cells (MSCs) to restore functional muscle tissue. In our previous studies, we found that UBM increased muscle myoblast cell proliferation. Therefore, we examined whether co-treatment with MSCs would further augment regeneration as compared to individual treatments. METHODS: Twenty C57BL/6 male adult mice received bilateral laceration injuries on the gastrocnemius muscle under anesthesia, and were randomly grouped to a designed treatment applied 14 days after injury. Treatment groups were 1) DMEM culture medium, 2) UBM only (150μg), 3) MSCs only (1 million mouse derived cells), and 4) UBM+MSCs. 4 additional mice served as a control baseline not receiving injury. Efficacy of treatment was analyzed through isometric muscle force testing as well as histomorphologic examination at 50 days after injury. Two-way ANOVA was applied for statistical analysis. RESULTS: Isometric muscle force was measured, including twitch (Pt), tetanic (Po), and fatigue isometric functions with the muscle stretched to optimal length (Lo). Muscle twitch (Pt) significantly decreased in the DMEM group compared to the non-injured group at day 50 (p < 0.05). Furthermore, twitch significantly increased with UBM treatment, but not with MSC treatment. Regenerating myofiber nuclei were counted and myofiber cross sectional area was measured with histology. New myotubes were identified as having centrally located nuclei. Further, Ki-67 nuclear immunofluorescence staining was performed to demonstrate proliferating satellite cells. The myofiber cross sectional area and the number of Ki-67/DAPI overlapping stained nuclei significantly increased in the DMEM group compared to the non-injured group (p < 0.05). No differences were observed with other treatments in injured mice at day 50. CONCLUSION: We observed a significant improvement in muscle function with combination treatment and single UBM treatment applied 50 days post-injury. The current animal model provides a tool to study muscle regeneration, and is feasible for clinical translation to address impairment in skeletal muscle function after burn injury.Item Ponce de Leon's fountain: stem cells and the regenerating heart(2004-02-12) Garry, Daniel J.Item Transcriptional Regulation of Neonatal Heart Regeneration and Direct Cardiac Reprogramming(2021-05-01T05:00:00.000Z) Wang, Zhaoning; Sadek, Hesham A.; Olson, Eric N.; Xu, Jian; Kliewer, Steven A.The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. Deciphering the molecular underpinnings of neonatal heart regeneration and the blockade to regeneration in later life may provide novel insights for heart repair. To elucidate the transcriptional responses of the different cellular components of the mouse heart following injury, we performed single cell RNA-sequencing on neonatal hearts at various timepoints following myocardial infarction, and coupled the results with bulk tissue RNA-sequencing and H3K27ac ChIP-sequencing data collected at the same timepoints. This approach provides detailed transcriptional dynamics of heterogeneous cardiac cell types during neonatal heart regeneration. Concomitant single cell ATAC-sequencing exposes underlying dynamics of open chromatin landscapes and regenerative gene regulatory networks of diverse cardiac cell types, and reveals previously unknown extracellular mediators of cardiomyocyte proliferation, angiogenesis, and fibroblast activation. Furthermore, using single-nucleus RNA sequencing, we mapped the dynamic transcriptional landscape of five distinct cardiomyocyte populations in healthy, injured and regenerating mouse hearts. We identified immature cardiomyocytes that enter cell-cycle following injury and disappear as the heart loses the ability to regenerate. These proliferative neonatal cardiomyocytes display a unique transcriptional program dependent on NFYa and NFE2L1 transcription factors, which exert proliferative and protective functions, respectively. Cardiac overexpression of these two factors conferred protection against ischemic injury in mature mouse hearts that were otherwise non-regenerative. Together, these findings provide mechanistic insights into the molecular basis of neonatal heart regeneration, and offer various pathways that can be manipulated to facilitate cardiac repair after injury. Direct reprogramming of fibroblasts into induced cardiac-like myocytes using cardiac transcription factors offers another possible therapeutic approach for cardiac repair. To elucidate the gene regulatory network during direct cardiac reprogramming, we performed a genome-wide analysis of cardiac transcription factors binding sites and active enhancers during reprogramming. We found reprogramming factors cooperatively activate enhancers involved in cardiac development and maturation, and further delineated the regulatory relationships between reprogramming factors and cardiac gene expression. These findings reveal synergistic activation of the cardiac epigenetic landscape by cardiac transcription factors and key signaling pathways that govern direct cardiac reprogramming.Item [UT Southwestern Medical Center News](2013-04-17) Owens, Remekca