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Open Access Publications from the University of California

Scholarly Works (28 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2020)

The humoral immune response is mediated by antigen activated B cells that

have terminally differentiated into antibody secreting cells (ASCs). The ASC pool is

composed of short-lived plasma cells (SLPCs) and long-lived plasma cells (LLPCs) that

secrete antigen-specific antibodies to clear an infection and maintain long-term

protective antibody titers to prevent subsequent reinfections. SLPCs have generally

been viewed to be formed from T cell-independent immune responses and localized in

the spleen. LLPCs have been viewed to be formed from T cell-dependent immune

responses and localized in the bone marrow. However, regardless of the type of

stimulating antigen, ASCs of varying lifespans, both SLPCs and LLPCs are found in the

spleen and bone marrow. Currently, it remains unclear as to what factors and pathways

regulate PC longevity.

Our laboratory previously identified the CREB coactivator CRTC2 as a regulator

of ASC differentiation. DNA double strand breaks associated with class switch

recombination activates a signaling pathway in human germinal center (GC) B cells that

results in the phosphorylation and inactivation of CRTC2. Phosphorylated CRTC2 is relocalized

to the cytoplasm and CRTC2 target genes are down-regulated. Dysregulation

of CRTC2 activity through over-expression of a nucleus-localized and constitutively

active form of CRTC2 (CRTC2-AA) in human tonsillar B cells, prevented GC B cells

from exiting the GC reaction and inhibited ASC differentiation. However, it remained

unclear whether the function of CRTC2 in this in vitro differentiation system would be

recapitulated in vivo and whether CRTC2 played any other roles in an in vivo humoral

immune response.

To evaluate these questions, we generated a transgenic (TG) mouse model

which expresses CRTC2-AA at all stages of B cell development. Using these TG mice,

we demonstrate that Crtc2 repression in PCs is an intrinsic requirement for PC

metabolic fitness. Sustained CRTC2 activity shortened the survival of splenic and bone

marrow PCs which resulted in the reduction of long-lived PCs and antibody deficits in

response to immunizations and acute viral infection. We further demonstrated that TG

PCs adopt characteristics associated with SLPCs which include reduced antibody

secretion, glycolysis, oxidative metabolism, and spare respiratory capacity.

Mechanistically, Crtc2 repression is necessary for the fidelity of PC gene expression

and mRNA alternative-splicing programs, with both programs altered in TG PCs.

Combined, our results show that Crtc2 repression in PCs must occur to support PC

metabolism and extend PC survival and lifespan during a humoral immune response.

We hypothesize that the level of Crtc2 repression in differentiated ASCs determines

metabolic fitness and ultimately PC survival and longevity in the bone marrow.

  • 4 supplemental files
Cover page: CRTC2 Regulates Plasma Cell Metabolism and Survival to Maintain Humoral Immune Responses
  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2021)

Pulmonary arterial hypertension (PAH) is a lung disease characterized by narrowing of the pulmonary arteries causing hemodynamic resistance which eventually leads to right heart failure and death. Current therapies mainly act through vasodilation but none reverse the underlying vascular remodeling characteristic of PAH. A deeper understanding of the molecular and cellular mechanisms of PAH is needed to bridge this translational gap. The goal of this dissertation is to investigate the transcriptional alterations in PAH lungs using integrative multiomics to identify and prioritize candidate genes, pathways, and cell types implicated in PAH.First, we identified reprogramming of genes and pathways in various cell types in the lungs of two commonly used rat models of PAH, namely Sugen-hypoxia (SuHx) and monocrotaline (MCT), using single-cell RNA sequencing (scRNAseq). We found that genes dysregulated in SuHx nonclassical monocytes were significantly enriched for PAH-associated genes and GWAS variants. We further identified candidate drugs predicted to reverse the dysregulated gene programs. This study revealed the distinct and shared reprogramming of genes and pathways in two commonly used PAH models for the first time at single-cell resolution and demonstrated their relevance to human PAH and utility for drug repositioning. Next, we dissected the human PAH lung transcriptome at the tissue level using an innovative network and systems biology methods on a well-powered RNA sequencing dataset of human PAH lungs. We discovered many DEGs and pathways in human PAH lungs at the tissue level, and through integration with clinical data and PAH GWAS, our network analysis revealed co-expressed gene modules that are not only associated with PAH diagnosis and severity, but also risk of PAH implicating their causal role in PAH pathogenesis. Key driver analysis utilizing a comprehensive gene regulatory network of the human lung identified and prioritized candidate genes. Furthermore, we integrated the tissue-level networks with scRNAseq to uncover the specific cell types mediating the tissue-level gene programs. Overall, this integrative multiomics and systems biology study revealed and prioritized the dysregulation of many genes, pathways, and cell types in the lungs of PAH animal models and patients, thereby opening new avenues for therapeutic targeting.

    Cover page: Integrative Multiomics and Systems Biology of Pulmonary Arterial Hypertension
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2020)
    Pulmonary hypertension (PH) developing secondarily in pulmonary fibrosis (PF) patients (PF-PH) is a frequent co-morbidity. The high prevalence of PH in PF patients is very concerning since the presence of PH is a strong predictor of mortality in PF patients. Until recently, PH was thought to arise solely from fibrotic destruction of the lung parenchyma, leading to hypoxic vasoconstriction and loss of vascular bed density. Thus, potential cellular and molecular dysregulation of vascular remodeling as a driver of PF-PH has been under-investigated. The recent demonstrations that there is no correlation between the severity of the fibrosis and development of PH, along with the finding that significant vascular histological and molecular differences exist between patients with and without PH have shifted the etiological paradigm of PF-PH. This review aims to provide a comprehensive translational overview of PH in PF patients from clinical diagnosis and outcome to the latest understanding of the histology and molecular pathophysiology of PF-PH.
      Cover page: Pulmonary hypertension secondary to pulmonary fibrosis: clinical data, histopathology and molecular insightsCreative Commons 'BY' version 4.0 license
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2020)
      The novel 2019 coronavirus disease (COVID-19), resulting from severe acute respiratory syndrome coronarvirus-2 (SARS-CoV-2) infection, typically leads to respiratory failure in severe cases; however, cardiovascular injury is reported to contribute to a substantial proportion of COVID-19 deaths. Preexisting cardiovascular disease (CVD) is among the most common risk factors for hospitalization and death in COVID-19 patients, and the pathogenic mechanisms of COVID-19 disease progression itself may promote the development of cardiovascular injury, increasing risk of in-hospital death. Sex differences in COVID-19 are becoming more apparent as mounting data indicate that males seem to be disproportionately at risk of severe COVID-19 outcome due to preexisting CVD and COVID-19-related cardiovascular injury. In this review, we will provide a basic science perspective on current clinical observations in this rapidly evolving field and discuss the interplay sex differences, preexisting CVD and COVID-19-related cardiac injury.
        Cover page: Sex differences underlying preexisting cardiovascular disease and cardiovascular injury in COVID-19
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2023)
        BACKGROUND: Pulmonary hypertension (PH) is associated with aberrant sympathoexcitation leading to right ventricular failure (RVF), arrhythmias, and death. Microglial activation and neuroinflammation have been implicated in sympathoexcitation in experimental PH. We recently reported the first evidence of thoracic spinal cord (TSC) neuroinflammation in PH rats. Here, we hypothesize that PH is associated with increased cardiopulmonary afferent signaling leading to TSC-specific neuroinflammation and sympathoexcitation. Furthermore, inhibition of TSC neuroinflammation rescues experimental PH and RVF. METHODS: We performed transcriptomic analysis and its validation on the TSC of monocrotaline (n=8) and Sugen hypoxia (n=8) rat models of severe PH-RVF. A group of monocrotaline rats received either daily intrathecal microglial activation inhibitor minocycline (200 μg/kg per day, n=5) or PBS (n=5) from day 14 through 28. Echocardiography and right ventricle-catheterization were performed terminally. Real-time quantitative reverse transcription PCR, immunolocalization, microglia+astrocyte quantification, and terminal deoxynucleotidyl transferase dUTP nick end labeling were assessed. Plasma catecholamines were measured by ELISA. Human spinal cord autopsy samples (Control n=3; pulmonary arterial hypertension n=3) were assessed to validate preclinical findings. RESULTS: Increased cardiopulmonary afferent signaling was demonstrated in preclinical and clinical PH. Our findings delineated common dysregulated genes and pathways highlighting neuroinflammation and apoptosis in the remodeled TSC and highlighted increased sympathoexcitation in both rat models. Moreover, we validated significantly increased microglial and astrocytic activation and CX3CL1 expression in TSC of human pulmonary arterial hypertension. Finally, amelioration of TSC neuroinflammation by minocycline in monocrotaline rats inhibited microglial activation, decreased proinflammatory cytokines, sympathetic nervous system activation and significantly attenuated PH and RVF. CONCLUSIONS: Targeting neuroinflammation and associated molecular pathways and genes in the TSC may yield novel therapeutic strategies for PH and RVF.
          Cover page: Thoracic Spinal Cord Neuroinflammation as a Novel Therapeutic Target in Pulmonary Hypertension.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2023)
          The identification and role of endothelial progenitor cells in pulmonary arterial hypertension (PAH) remain controversial. Single-cell omics analysis can shed light on endothelial progenitor cells and their potential contribution to PAH pathobiology. We aim to identify endothelial cells that may have stem/progenitor potential in rat lungs and assess their relevance to PAH. Differential expression, gene set enrichment, cell-cell communication, and trajectory reconstruction analyses were performed on lung endothelial cells from single-cell RNA sequencing of Sugen-hypoxia, monocrotaline, and control rats. Relevance to human PAH was assessed in multiple independent blood and lung transcriptomic data sets. Rat lung endothelial cells were visualized by immunofluorescence in situ, analyzed by flow cytometry, and assessed for tubulogenesis in vitro. A subpopulation of endothelial cells (endothelial arterial type 2 [EA2]) marked by Tm4sf1 (transmembrane 4 L six family member 1), a gene strongly implicated in cancer, harbored a distinct transcriptomic signature enriched for angiogenesis and CXCL12 signaling. Trajectory analysis predicted that EA2 has a less differentiated state compared with other endothelial subpopulations. Analysis of independent data sets revealed that TM4SF1 is downregulated in lungs and endothelial cells from patients and PAH models, is a marker for hematopoietic stem cells, and is upregulated in PAH circulation. TM4SF1+CD31+ rat lung endothelial cells were visualized in distal pulmonary arteries, expressed hematopoietic marker CD45, and formed tubules in coculture with lung fibroblasts. Our study uncovered a novel Tm4sf1-marked subpopulation of rat lung endothelial cells that may have stem/progenitor potential and demonstrated its relevance to PAH. Future studies are warranted to further elucidate the role of EA2 and Tm4sf1 in PAH.
            Cover page: Tm4sf1-marked Endothelial Subpopulation Is Dysregulated in Pulmonary Arterial Hypertension.
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2021)

            Background

            Right ventricular (RV) dysfunction is a significant prognostic determinant of morbidity and mortality in pulmonary arterial hypertension (PAH). Despite the importance of RV function in PAH, the underlying molecular mechanisms of RV dysfunction secondary to PAH remain unclear. We aim to identify and compare molecular determinants of RV failure using RNA sequencing of RV tissue from 2 clinically relevant animal models of PAH.

            Methods

            We performed RNA sequencing on RV from rats treated with monocrotaline or Sugen with hypoxia/normoxia. PAH and RV failure were confirmed by catheterization and echocardiography. We validated the RV transcriptome results using quantitative real-time polymerase chain reaction, immunofluorescence, and Western blot. Immunohistochemistry and immunofluorescence were performed on human RV tissue from control (n=3) and PAH-induced RV failure patients (n=5).

            Results

            We identified similar transcriptomic profiles of RV from monocrotaline- and Sugen with hypoxia-induced RV failure. Pathway analysis showed genes enriched in epithelial-to-mesenchymal transition, inflammation, and metabolism. Histological staining of human RV tissue from patients with RV failure secondary to PAH revealed significant RV fibrosis and endothelial-to-mesenchymal transition, as well as elevated cellular communication network factor 2 (top gene implicated in epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition) expression in perivascular areas compared with normal RV.

            Conclusions

            Transcriptomic signature of RV failure in monocrotaline and Sugen with hypoxia models showed similar gene expressions and biological pathways. We provide translational relevance of this transcriptomic signature using RV from patients with PAH to demonstrate evidence of epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition and protein expression of cellular communication network factor 2 (CTGF [connective tissue growth factor]). Targeting specific molecular mechanisms responsible for RV failure in monocrotaline and Sugen with hypoxia models may identify novel therapeutic strategies for PAH-associated RV failure.
              Cover page: Transcriptomic Analysis of Right Ventricular Remodeling in Two Rat Models of Pulmonary Hypertension: Identification and Validation of Epithelial-to-Mesenchymal Transition in Human Right Ventricular Failure.
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2020)
              Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons throughout the brain and spinal cord progressively degenerate resulting in muscle atrophy, paralysis and death. Recent studies using animal models of ALS implicate multiple cell-types (e.g., astrocytes and microglia) in ALS pathogenesis in the spinal motor systems. To ascertain cellular vulnerability and cell-type specific mechanisms of ALS in the brainstem that orchestrates oral-motor functions, we conducted parallel single cell RNA sequencing (scRNA-seq) analysis using the high-throughput Drop-seq method. We isolated 1894 and 3199 cells from the brainstem of wildtype and mutant SOD1 symptomatic mice respectively, at postnatal day 100. We recovered major known cell types and neuronal subpopulations, such as interneurons and motor neurons, and trigeminal ganglion (TG) peripheral sensory neurons, as well as, previously uncharacterized interneuron subtypes. We found that the majority of the cell types displayed transcriptomic alterations in ALS mice. Differentially expressed genes (DEGs) of individual cell populations revealed cell-type specific alterations in numerous pathways, including previously known ALS pathways such as inflammation (in microglia), stress response (ependymal and an uncharacterized cell population), neurogenesis (astrocytes, oligodendrocytes, neurons), synapse organization and transmission (microglia, oligodendrocyte precursor cells, and neuronal subtypes), and mitochondrial function (uncharacterized cell populations). Other cell-type specific processes altered in SOD1 mutant brainstem include those from motor neurons (axon regeneration, voltage-gated sodium and potassium channels underlying excitability, potassium ion transport), trigeminal sensory neurons (detection of temperature stimulus involved in sensory perception), and cellular response to toxic substances (uncharacterized cell populations). DEGs consistently altered across cell types (e.g., Malat1), as well as cell-type specific DEGs, were identified. Importantly, DEGs from various cell types overlapped with known ALS genes from the literature and with top hits from an existing human ALS genome-wide association study (GWAS), implicating the potential cell types in which the ALS genes function with ALS pathogenesis. Our molecular investigation at single cell resolution provides comprehensive insights into the cell types, genes and pathways altered in the brainstem in a widely used ALS mouse model.
                Cover page: Single-cell RNA-seq analysis of the brainstem of mutant SOD1 mice reveals perturbed cell types and pathways of amyotrophic lateral sclerosis
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2021)
                Single-cell transcriptomics provide a systematic map of gene expression in different human cell types. The next challenge is to systematically understand cell-type-specific gene function. The integration of CRISPR-based functional genomics and stem cell technology enables the scalable interrogation of gene function in differentiated human cells. Here we present the first genome-wide CRISPR interference and CRISPR activation screens in human neurons. We uncover pathways controlling neuronal response to chronic oxidative stress, which is implicated in neurodegenerative diseases. Unexpectedly, knockdown of the lysosomal protein prosaposin strongly sensitizes neurons, but not other cell types, to oxidative stress by triggering the formation of lipofuscin, a hallmark of aging, which traps iron, generating reactive oxygen species and triggering ferroptosis. We also determine transcriptomic changes in neurons after perturbation of genes linked to neurodegenerative diseases. To enable the systematic comparison of gene function across different human cell types, we establish a data commons named CRISPRbrain.
                  Cover page: Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2023)
                  Myocardial fibrosis and calcification associate with adverse outcomes in nonischemic heart failure. Cardiac fibroblasts (CF) transition into myofibroblasts (MF) and osteogenic fibroblasts (OF) to promote myocardial fibrosis and calcification. However, common upstream mechanisms regulating both CF-to-MF transition and CF-to-OF transition remain unknown. microRNAs are promising targets to modulate CF plasticity. Our bioinformatics revealed downregulation of miR-129-5p and upregulation of its targets small leucine-rich proteoglycan Asporin (ASPN) and transcription factor SOX9 as common in mouse and human heart failure (HF). We experimentally confirmed decreased miR-129-5p and enhanced SOX9 and ASPN expression in CF in human hearts with myocardial fibrosis and calcification. miR-129-5p repressed both CF-to-MF and CF-to-OF transition in primary CF, as did knockdown of SOX9 and ASPN. Sox9 and Aspn are direct targets of miR-129-5p that inhibit downstream β-catenin expression. Chronic Angiotensin II infusion downregulated miR-129-5p in CF in WT and TCF21-lineage CF reporter mice, and it was restored by miR-129-5p mimic. Importantly, miR-129-5p mimic not only attenuated progression of myocardial fibrosis, calcification marker expression, and SOX9 and ASPN expression in CF but also restored diastolic and systolic function. Together, we demonstrate miR-129-5p/ASPN and miR-129-5p/SOX9 as potentially novel dysregulated axes in CF-to-MF and CF-to-OF transition in myocardial fibrosis and calcification and the therapeutic relevance of miR-129-5p.
                    Cover page: Myocardial fibrosis and calcification are attenuated by microRNA–129-5p targeting Asporin and Sox9 in cardiac fibroblastsCreative Commons 'BY' version 4.0 license
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