Browsing by Subject "Fibroblasts"
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Item Idiopathic pulmonary fibrosis(1988-07-14) Weissler, Jonathan C.Item Investigation of Cell Morphology and Cell-Induced 3-D Matrix Reorganization(2008-05-13) Kim, Areum; Petroll, W. MatthewThe overall goal is to develop and apply quantification techniques for assessing the underlying pattern of cytoskeletal organization and cell-matrix mechanical interactions in corneal fibroblasts at the sub-cellular level. To specifically study how Rho and Rac regulate sub-cellular mechanical behavior, cells were plated inside 3-D matrices and incubated with activators and/or inhibitors of Rho and Rac and 3-D optical section images were collected simultaneously. Cell morphology, collagen density and orientation were quantitatively studied. The first important finding is that Rho kinase-dependent contractile force generation leads to co-alignment of cells. This process contributes to global matrix contraction and thus may play a central role in cell transformation and force generation during wound healing. In contrast, activation of Rac using PDGF induces dramatic cell elongation without significant matrix reorganization. PDGF may play a role in cell migration during wound healing process since migration requires protrusion of cell extensions, but not necessarily large contractile force. In order to obtain more detailed understanding of how cells reorganize matrices over time, 4-D imaging techniques were used. Cells were plated inside 3-D matrices and time-lapse DIC and LSCM imaging was performed while disrupting cytoskeletal proteins in the presence or absence of the Rho kinase inhibitor. Addition of nocodazole induced rapid microtubule disruption which resulted in Rho activation and cellular contraction. The matrix was pulled inward by retracting pseudopodial processes, and focal adhesions appeared to mediate this process. When Rho-kinase was inhibited, disruption of microtubules resulted in retraction of dendritic cell processes, and rapid formation and extension of lamellipodial processes at random locations along the cell body, eventually leading to a convoluted, disorganized cell shape. These data suggest that microtubules modulate both cellular contractility and local collagen matrix reorganization via regulation of Rho/Rho kinase activity. In addition, microtubules appear to play a central role in dynamic regulation of cell spreading mechanics, morphology and polarity in 3-D culture. Taken together, these experiments demonstrate that quantitative static and dynamic imaging of cells in 3-D matrices is capable of providing unique insights into the role of specific signaling pathways on the underlying pattern of cytoskeletal organization and cell-matrix mechanical interactions.Item Large Scale Profiling of Fibroblasts from Pediatric Patients with Inborn Errors of Metabolism Results in the Identification of Siblings with L-2-Hydroxyglutaric Aciduria(2020-01-21) Franklin, Jordan; Gu, Wen; Ni, Min; DeBerardinis, RalphInborn errors of metabolism (IEMs) are caused by germline mutations that interfere with the normal physiological functioning of single metabolic enzymes or nutrient transporters1. Discerning an effective treatment for IEMs requires identifying, validating and understanding the impact of disease-causing mutations that are specific to the patient1. Whole-exome sequencing (WES) is the most common strategy for identifying unknown genetic aberrations1. Metabolomics and metabolic flux analysis (MFA) can help functionally validate whether suspected mutations are causative as well as direct the development of diagnostic and therapeutic approaches1. More specifically, mass spectrometry-based metabolomics enables quantification of metabolite abundance, and isotopically labeled MFA enables assessment of the precise manner in which patient cells utilize nutrients for various catabolic pathways1,2. In this study, we conducted metabolomics and MFA to assess metabolic abnormalities in fibroblasts derived from 27 patients, who possessed both known and unknown IEMs, and to gain mechanistic insight into these diseases. To characterize the metabolic abnormalities, we performed a metabolomics experiment that involved 27 patient-derived fibroblast lines and 4 control lines, which eventually yielded a detailed metabolic profile (681 metabolites) for each sample. Additionally, 13C-labeled glucose and 13C-labeled glutamine were introduced to various cell lines in order to evaluate the flux of glucose and glutamine metabolism. We confirmed the quality of our metabolomics results by conducting unsupervised clustering analysis, which showed close grouping of biological replicates, as well as by validating the metabolic changes in a previously reported patient1. Differential analysis of metabolites for each of the 27 patients provided further insight into their specific metabolic anomalies. Notably, we discovered a pair of siblings with dramatically increased 2-hydroxyglutarate (2-HG) levels. By using a derivatization-based mass spectrometry method, we determined that L-2-HG rather than D-2-HG was elevated in the patient samples. WES of both children identified a homozygous mutation that introduced a stop codon in the L-2-HG dehydrogenase gene. This was consistent with the diagnosis of L-2-hydroxyglutaric aciduria in the children3. Metabolic flux analysis provided mechanistic insight into the disease, showing that L-2-HG was primarily being made from glutamine rather than glucose. The unbiased metabolomics approach enabled the discovery and characterization of an important metabolic deficiency and will likely contribute to the discovery of additional IEMs in the cohort.Item Molecular Dissection of Hand2 During the Formation of Pacemaker-Like Myocytes During Direct Reprogramming(2019-03-06) Fernandez-Perez, Antonio; Zhang, Chun-Li; Cleaver, Ondine; Olson, Eric N.; Munshi, NikhilDirect reprogramming of one cell type into another has great promise for regenerative medicine, disease modeling, and lineage specification. Currently, the conversion of fibroblasts into induced cardiomyocytes (iCM) by Gata4, Mef2c, and Tbx5 (GMT) represents an important avenue for generating de novo cardiac myocytes. Recent evidence has shown that iCM formation and diversity can be enhanced by the addition of Hand2 to GMT (GHMT). These four transcription factors give rise to a heterogenous CM population, consisting of atrial (iAM), ventricular (iVM), and pacemaker myocytes (iPM). However, the molecular mechanisms that drive this plastic fate conversion remain poorly understood. Although chromatin and single-cell studies in GMT-iCM have shown the existence of a set of temporal steps that orchestrate iCM formation, little is known about how Hand2 enhances this process. In the present study, we seek to characterize these Hand2-dependent mechanisms. We hypothesize that Hand2 regulates a discrete pacemaker regulatory network that becomes active during GHMT-iCM reprogramming. To test this, we compared the transcriptional and genomic profiles of fibroblasts, GMT, GHMT, and endogenous mouse Pacemaker cells. We observe similar chromatin landscape and gene expression profiles between Hand2-iPM and endogenous sinoatrial node (SAN), however several known key PM pathways are not active. Activation of these networks further enhances iCM-iPM fo Moreover, we show that Hand2 enhances chromatin accessibility in regions related to sarcomere function and electrical coupling, as well as promoting the closing of regions related to alternative fates. Utilizing integrative genomics between ATAC-seq and RNA-seq datasets, we identify the desmosome machinery as an important feature of iPM formation. In parallel, we define a novel Hand2 domain region that regulates cardiac subtype diversity. Taken together, our results showcase Hand2-dependent mechanisms for iPM formation and gives insight into the improvement of future iPM engineering.Item Molecular Regulation of Direct Cardiac Reprogramming(2017-08-14) Zhou, Huanyu; Zhang, Chun-Li; Hill, Joseph A.; Cleaver, Ondine; Olson, Eric N.A heart attack (also known as myocardial infarction, MI) happens when the flow of blood to the heart is blocked. A massive heart attack can kill billions of cardiomyocytes. The heart has limited regenerative potential because adult mammalian cardiomyocytes cannot proliferate, therefore lost cardiomyocytes cannot be replaced. This causes permanent heart damage and results in decreased contraction properties to a large portion of the heart muscle. Therapeutic treatments for heart attack patients have improved dramatically over the past decades. However, due to the inability to replenish lost cardiomyocytes, heart failure is still the primary cause of death in the world. Cardiac fibroblasts (CFs) constitute ~50% of the cells in the heart and form scar tissue following heart injury. Reprogramming CFs to induced-cardiomyocytes (iCMs) by forced expression of cardiac specific transcription factors holds promise for enhancing cardiac repair by reducing scar tissue while simultaneously generating new cardiomyocytes. However, low efficiency as well as a lack of understanding of molecular mechanism of the reprogramming process have significantly hampered its clinical application. The two goals of my PhD study were 1) to optimize the cardiac reprogramming protocol by increasing the efficiency; and 2) to decipher molecular mechanisms of cardiac reprogramming using the information obtained from the optimization process. To improve the efficiency of reprogramming fibroblasts to iCMs by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of the reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 weeks of induction by AKT1 plus GHMT (AGHMT). Furthermore, AGHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of AKT1 whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3a (Foxo3a) acted downstream of AKT1 to influence fibroblast-to-cardiomyocyte reprogramming. Addition of AGHMT converted 50% of mouse embryo fibroblasts to beating cardiomyocytes. However, only 1% of adult fibroblasts displayed spontaneous beating after three weeks of induction by AGHMT. This indicates that there are "barriers" in adult fibroblasts that hinder cardiac reprogramming. We continued to optimize methods for reprogramming fibroblasts to cardiomyocytes in vitro and in vivo. To identify additional regulators of this reprogramming process, we carried out an unbiased screen of ~1,100 open reading frames (ORFs) encoding transcription factors and cytokines for the ability to enhance reprogramming by AGHMT in adult tail-tip fibroblasts (ATTFs). One of the strongest activators of cardiac reprogramming was Krüppel-Type Zinc-Finger Transcription Factor 281 (ZNF281). Adding ZNF281 in AGHMT converted ~30% of ATTFs to iCMs which is comparable to AGHMT reprogrammed MEFs. We showed that ZNF281 enhanced cardiac reprogramming by associating with GATA4 on cardiac enhancers and by inhibiting inflammatory signaling, which antagonizes cardiac reprogramming. Our findings not only identify AKT1 and ZNF281 as robust and efficient activators of adult cardiac reprogramming, but also provide new insights into the molecular mechanisms underlying direct cardiac reprogramming.Item Small Molecules Modulate Chromatin Accessibility to Promote NEUROG2-Mediated Fibroblast-to-Neuron Reprogramming(2016-07-12) Smith, Derek Kurtis; Johnson, Jane E.; Kim, Tae-Kyung; Olson, Eric N.; Zhang, Chun-LiThe activity of pro-neural signaling molecules and transcription factors is sufficient to induce the transdifferentiation of lineage-restricted fibroblasts into functional neurons; however, a mechanistic model of the immediate-early events that catalyze this conversion has not been well defined. We utilized a high-efficiency reprogramming system of NEUROG2, forskolin (F), and dorsomorphin (D) to characterize the genetic and epigenetic events that initiate an acquisition of neuronal identity in fetal human fibroblasts. NEUROG2 immediately activates a neurogenic program, but is only sufficient to impart a functional identity in the presence of FD. These small molecules promote NEUROG2 and CREB1 co-transcription, induce SOX4 expression, and promote SOX4-dependent chromatin remodeling. Genome-wide occupancy analysis revealed that SOX4 targets numerous SWI/SNF complex subunits and co-binds with NEUROG2 to enhance the expression of diverse neurogenic factors. The overexpression of SWI/SNF chromatin remodeling factors or treatment with small molecules that modify chromatin accessibility enhanced NEUROG2-mediated neuronal reprogramming of adult human skin fibroblasts. This work represents the first comprehensive mechanism for the immediate events that catalyze neuronal transdifferentiation.