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Open Access Publications from the University of California

Scholarly Works (25 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2023)

Throughout the vascular tree, endothelial cells function as the interface between circulation and underlying tissues. They act not only as gatekeepers but are also designed to gauge and adapt to circulating factors for regulation of vascular tone, development, and repair. The severity of many diseases, such as cardiovascular disease and diabetes, hinge on endothelial function and the ability to initiate vascularization of hypoxic tissues. Vascular morphogenesis requires a delicate molecular gradient of Notch signaling in endothelial cells to pattern correct angiogenic sprouting. This process is controlled, at least in part, by the distribution of ligands (Dll4 and Jagged1). How Jagged1 (Jag1) expression is compartmentalized in the vascular plexus remains unclear. Understanding the molecular regulators for this process can help our understanding of drivers toward endothelial dysfunction. Here, we showed that mRNA decay protein Zinc-finger protein 36 (ZFP36) is robustly induced in vitro in various human primary endothelial cells (ECs) downstream of canonical vascular endothelial growth factor (VEGF) signaling. This induction, observed at the mRNA and protein level, is uniquely specific to ZFP36 and not shared by other family members ZFP36L1 nor ZFP36L2, which have already been linked to vascular development. This induction was also observed to be primarily facilitated through VEGF receptor 2 (VR2) as receptor inhibition blunted ZFP36 induction. We identified Jag1 mRNA as direct target of zinc finger protein 36 (ZFP36) via crosslinking-immunoprecipitation and verified conserved Jag1 regulation in human primary ECs. Human ECs lacking ZFP36 achieved with CRISPR KO, displayed high levels of JAG1 and increased angiogenic sprouting in vitro. Similarly, in mice with tamoxifen induced endothelial specific vascular endothelial-cadherin Cre-mediated KO of Zfp36, endothelial cells displayed mispatterned and increased levels of JAG1 in the developing retinal vascular plexus. Abnormal levels of JAG1 at the sprouting front altered Notch1 signaling, increasing the number of tip cells; a phenotype that was rescued by crossing the EC specific Zfp36 KO with Jag1 KO mice to achieve haploinsufficiency of Jag1. Additionally, we tested the significance of ZFP36 induction in the broad context of molecular drivers of endothelial plasticity: metabolism, kinase signaling, and shear stress. We were unable to confirm several metabolic targets identified in mouse embryonic fibroblasts (MEFs) in our human primary ECs in vitro. However, conserved sensitivity to changes in kinase activity, confirmed through inhibitor studies, suggests that these discrepancies in targets could be a result of cell specific targets for ZFP36 mRNA binding and regulation. Microenvironment can further play a role in ZFP36 mediated endothelial transcript regulation as we show that shear stress is able to suppress overall ZFP36 expression. Our findings reveal an important feedforward loop, whereby VEGF stimulates ZFP36, consequently suppressing Jag1 to enable adequate levels of Notch signaling during sprouting angiogenesis.

    Cover page: Elucidating the role of Zinc-finger protein 36 in endothelial angiogenic response
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2017)
    Sterol regulatory element-binding protein 1c (SREBP-1c) is a central regulator of lipogenesis whose activity is controlled by proteolytic cleavage. The metabolic factors that affect its processing are incompletely understood. Here, we show that dynamic changes in the acyl chain composition of ER phospholipids affect SREBP-1c maturation in physiology and disease. The abundance of polyunsaturated phosphatidylcholine in liver ER is selectively increased in response to feeding and in the setting of obesity-linked insulin resistance. Exogenous delivery of polyunsaturated phosphatidylcholine to ER accelerated SREBP-1c processing through a mechanism that required an intact SREBP cleavage-activating protein (SCAP) pathway. Furthermore, induction of the phospholipid-remodeling enzyme LPCAT3 in response to liver X receptor (LXR) activation promoted SREBP-1c processing by driving the incorporation of polyunsaturated fatty acids into ER. Conversely, LPCAT3 deficiency increased membrane saturation, reduced nuclear SREBP-1c abundance, and blunted the lipogenic response to feeding, LXR agonist treatment, or obesity-linked insulin resistance. Desaturation of the ER membrane may serve as an auxiliary signal of the fed state that promotes lipid synthesis in response to nutrient availability.
      Cover page: ER phospholipid composition modulates lipogenesis during feeding and in obesity
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2016)
      Cholesterol and fatty acid biosynthesis are regulated by the sterol regulatory element-binding proteins (SREBPs), encoded by Srebf1 and Srebf2. We generated mice that were either deficient or hypomorphic for SREBP-2. SREBP-2 deficiency generally caused death during embryonic development. Analyses of Srebf2(-/-) embryos revealed a requirement for SREBP-2 in limb development and expression of morphogenic genes. We encountered only one viable Srebf2(-/-) mouse, which displayed alopecia, attenuated growth, and reduced adipose tissue stores. Hypomorphic SREBP-2 mice (expressing low levels of SREBP-2) survived development, but the female mice exhibited reduced body weight and died between 8 and 12 weeks of age. Male hypomorphic mice were viable but had reduced cholesterol stores in the liver and lower expression of SREBP target genes. Reduced SREBP-2 expression affected SREBP-1 isoforms in a tissue-specific manner. In the liver, reduced SREBP-2 expression nearly abolished Srebf1c transcripts and reduced Srebf1a mRNA levels. In contrast, adipose tissue displayed normal expression of SREBP target genes, likely due to a compensatory increase in Srebf1a expression. Our results establish that SREBP-2 is critical for survival and limb patterning during development. Reduced expression of SREBP-2 from the hypomorphic allele leads to early death in females and reduced cholesterol content in the liver, but not in adipose tissue.
        Cover page: SREBP-2-deficient and hypomorphic mice reveal roles for SREBP-2 in embryonic development and SREBP-1c expression.
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2024)
        Pulmonary alveolar proteinosis (PAP) is a life-threatening, rare lung syndrome for which there is no cure and no approved therapies. PAP is a disease of lipid accumulation characterized by alveolar macrophage foam cell formation. While much is known about the clinical presentation, there is a paucity of information regarding temporal changes in lipids throughout the course of disease. Our objectives were to define the detailed lipid composition of alveolar macrophages in PAP patients at the time of diagnosis and during treatment. We performed comprehensive mass spectrometry to profile the lipid signature of alveolar macrophages obtained from three independent mouse models of PAP and from PAP and non-PAP patients. Additionally, we quantified changes in macrophage-associated lipids during clinical treatment of PAP patients. We found remarkable variations in lipid composition in PAP patients, which were consistent with data from three independent mouse models. Detailed lipidomic analysis revealed that the overall alveolar macrophage lipid burden inversely correlated with clinical improvement and response to therapy in PAP patients. Specifically, as PAP patients experienced clinical improvement, there was a notable decrease in the total lipid content of alveolar macrophages. This crucial observation suggests that the levels of these macrophage-associated lipids can be utilized to assess the efficacy of treatment. These findings provide valuable insights into the dysregulated lipid metabolism associated with PAP, offering the potential for lipid profiling to serve as a means of monitoring therapeutic interventions in PAP patients.
          Cover page: Alveolar macrophage lipid burden correlates with clinical improvement in patients with pulmonary alveolar proteinosis.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2017)
          Cholesterol homeostasis is maintained through concerted action of the SREBPs and LXRs. Here, we report that RNF145, a previously uncharacterized ER membrane ubiquitin ligase, participates in crosstalk between these critical signaling pathways. RNF145 expression is induced in response to LXR activation and high-cholesterol diet feeding. Transduction of RNF145 into mouse liver inhibits the expression of genes involved in cholesterol biosynthesis and reduces plasma cholesterol levels. Conversely, acute suppression of RNF145 via shRNA-mediated knockdown, or chronic inactivation of RNF145 by genetic deletion, potentiates the expression of cholesterol biosynthetic genes and increases cholesterol levels both in liver and plasma. Mechanistic studies show that RNF145 triggers ubiquitination of SCAP on lysine residues within a cytoplasmic loop essential for COPII binding, potentially inhibiting its transport to Golgi and subsequent processing of SREBP-2. These findings define an additional mechanism linking hepatic sterol levels to the reciprocal actions of the SREBP-2 and LXR pathways.
            Cover page: Inhibition of cholesterol biosynthesis through RNF145-dependent ubiquitination of SCAP
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2018)
            Diet1 modulates intestinal production of the hormone, fibroblast growth factor (FGF)15, which signals in liver to regulate bile acid synthesis. C57BL/6ByJ mice with a spontaneous Diet1-null mutation are resistant to hypercholesterolemia compared with wild-type C57BL/6J mice through enhanced cholesterol conversion to bile acids. To further characterize the role of Diet1 in metabolism, we generated Diet1-/- mice on the C57BL/6J genetic background. C57BL/6J Diet1-/- mice had elevated bile acid levels, reduced Fgf15 expression, and increased gastrointestinal motility and intestinal luminal water content, which are symptoms of bile acid diarrhea (BAD) in humans. Natural genetic variation in Diet1 mRNA expression levels across 76 inbred mouse strains correlated positively with Ffg15 mRNA and negatively with serum bile acid levels. This led us to investigate the role of DIET1 genetic variation in primary BAD patients. We identified a DIET1 coding variant (rs12256835) that had skewed prevalence between BAD cases and controls. This variant causes an H1721Q amino acid substitution that increases the levels of FGF19 protein secreted from cultured cells. We propose that genetic variation in DIET1 may be a determinant of FGF19 secretion levels, and may affect bile acid metabolism in both physiological and pathological conditions.
              Cover page: Diet1, bile acid diarrhea, and FGF15/19: mouse model and human genetic variants.Creative Commons 'BY-ND' version 4.0 license
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2015)
              Specific bile acids are potent signaling molecules that modulate metabolic pathways affecting lipid, glucose and bile acid homeostasis, and the microbiota. Bile acids are synthesized from cholesterol in the liver, and the key enzymes involved in bile acid synthesis (Cyp7a1, Cyp8b1) are regulated transcriptionally by the nuclear receptor FXR. We have identified an FXR-regulated pathway upstream of a transcriptional repressor that controls multiple bile acid metabolism genes. We identify MafG as an FXR target gene and show that hepatic MAFG overexpression represses genes of the bile acid synthetic pathway and modifies the biliary bile acid composition. In contrast, loss-of-function studies using MafG(+/-) mice causes de-repression of the same genes with concordant changes in biliary bile acid levels. Finally, we identify functional MafG response elements in bile acid metabolism genes using ChIP-seq analysis. Our studies identify a molecular mechanism for the complex feedback regulation of bile acid synthesis controlled by FXR.
                Cover page: MAFG Is a Transcriptional Repressor of Bile Acid Synthesis and Metabolism
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2018)
                Diet1 modulates intestinal production of the hormone, fibroblast growth factor (FGF)15, which signals in liver to regulate bile acid synthesis. C57BL/6ByJ mice with a spontaneous Diet1-null mutation are resistant to hypercholesterolemia compared with wild-type C57BL/6J mice through enhanced cholesterol conversion to bile acids. To further characterize the role of Diet1 in metabolism, we generated Diet1-/- mice on the C57BL/6J genetic background. C57BL/6J Diet1-/- mice had elevated bile acid levels, reduced Fgf15 expression, and increased gastrointestinal motility and intestinal luminal water content, which are symptoms of bile acid diarrhea (BAD) in humans. Natural genetic variation in Diet1 mRNA expression levels across 76 inbred mouse strains correlated positively with Ffg15 mRNA and negatively with serum bile acid levels. This led us to investigate the role of DIET1 genetic variation in primary BAD patients. We identified a DIET1 coding variant (rs12256835) that had skewed prevalence between BAD cases and controls. This variant causes an H1721Q amino acid substitution that increases the levels of FGF19 protein secreted from cultured cells. We propose that genetic variation in DIET1 may be a determinant of FGF19 secretion levels, and may affect bile acid metabolism in both physiological and pathological conditions.
                  Cover page: Diet1, bile acid diarrhea, and FGF15/19: mouse model and human genetic variants[S]
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2016)
                  Phospholipids are important determinants of membrane biophysical properties, but the impact of membrane acyl chain composition on dietary-lipid absorption is unknown. Here we demonstrate that the LXR-responsive phospholipid-remodeling enzyme Lpcat3 modulates intestinal fatty acid and cholesterol absorption and is required for survival on a high-fat diet. Mice lacking Lpcat3 in the intestine thrive on carbohydrate-based chow but lose body weight rapidly and become moribund on a triglyceride-rich diet. Lpcat3-dependent incorporation of polyunsaturated fatty acids into phospholipids is required for the efficient transport of dietary lipids into enterocytes. Furthermore, loss of Lpcat3 amplifies the production of gut hormones, including GLP-1 and oleoylethanolamide, in response to high-fat feeding, contributing to the paradoxical cessation of food intake in the setting of starvation. These results reveal that membrane phospholipid composition is a gating factor in passive lipid absorption and implicate LXR-Lpcat3 signaling in a gut-brain feedback loop that couples absorption to food intake.
                    Cover page: Intestinal Phospholipid Remodeling Is Required for Dietary-Lipid Uptake and Survival on a High-Fat DietCreative Commons 'BY' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2017)
                    Idiopathic pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by accumulation of surfactant. Surfactant synthesis and secretion are restricted to epithelial type 2 (T2) pneumocytes (also called T2 cells). Clearance of surfactant is dependent upon T2 cells and macrophages. ABCG1 is highly expressed in both T2 cells and macrophages. ABCG1-deficient mice accumulate surfactant, lamellar body-loaded T2 cells, lipid-loaded macrophages, B-1 lymphocytes, and immunoglobulins, clearly demonstrating that ABCG1 has a critical role in pulmonary homeostasis. We identify a variant in the ABCG1 promoter in patients with PAP that results in impaired activation of ABCG1 by the liver X receptor α, suggesting that ABCG1 basal expression and/or induction in response to sterol/lipid loading is essential for normal lung function. We generated mice lacking ABCG1 specifically in either T2 cells or macrophages to determine the relative contribution of these cell types on surfactant lipid homeostasis. These results establish a critical role for T2 cell ABCG1 in controlling surfactant and overall lipid homeostasis in the lung and in the pathogenesis of human lung disease.
                      Cover page: ABCG1 regulates pulmonary surfactant metabolism in mice and men[S]
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