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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2015)

The purpose of the vascular system is to distribute oxygen rich blood to all organs and extremities, to mediate the transport of nutrients and waste, and to deliver immune cells to sites of infection. It is comprised of the heart and a complex, hierarchically branched network of blood vessels through which the blood is pumped. The principal cell type of the blood vessel is the endothelial cell (EC). They are a subclass of epithelial cells that line the blood vessels and like ceramic tiles, provide a flat-slippery surface for the blood to flow past. Healthy blood circulation is critical for organs to be well oxygenated with the transport of red blood cell and free of infection through the delivery of white blood cells.

Under normal physiological conditions, adult EC are quiescent and do not proliferate. However, in pathological conditions like wound healing and cancer, secreted growth factors and inflammatory cytokines induce endothelial cells to turn on an angiogenesis program. On the other hand, transformed ECs can be the primary cell type of pathology. One such endothelial cell pathology is vascular anomalies (VAs). The emergence of these lesions requires endothelial cells to accumulate mutations causing them to form malformed vessels or solid tumors. It has been predicted that half of the mutations underlying vascular malformations are unknown, especially those that are non-hereditary and triggered by sporadic, somatic mutations. What is known has been determined through genetic linkage analysis of familial forms of the disease. Here we present the novel results of an in vivo forward genetic screen in murine endothelial cells that modeled vascular anomalies. The majority of the approximately 100 disrupted genes identified have not been previously associated with vascular anomalies. Furthermore, we validated a tumor suppressor (Fndc3b) and an oncogene (Pdgfrb) and demonstrated their causation in endothelial dysfunction. The major significant finding of this study is that endothelial cell homeostasis is heavily governed by regulation of the actin cytoskeleton, cytokine and growth factor signaling, and Hippo signaling pathways.

Also a result of the screen was the revelation that a pathological relationship exists between hemogenic endothelial cells and leukemia. Using the same forward genetic approach in a mouse model, we were able to induced mutagenesis at E9.5 in endothelial cells one day before they turned on their hemogenic program at E10.5. Not only were hematopoietic malignancies generated (both myeloid and lymphoid), but novel mutations associated with these cancers were identified. Here we also present data demonstrating the novel role of Pi4ka (identified in association with myeloid leukemia) in hematopoiesis. Together this work takes a look at the critical role of endothelial cells from three different perspectives (tumor angiogenesis, vascular anomalies, and hemogenic endothelial contribution to leukemia), highlighting their importance in physiology and pathology.

    Cover page: Endothelial Mutagenesis Uncovers Key Regulatory Pathways that Maintain Vascular Homeostasis
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2015)
    The past 5 years have witnessed a significant expansion in our understanding of vascular endothelial growth factor (VEGF) signaling. In particular, the process of canonical activation of VEGF receptor tyrosine kinases by homodimeric VEGF molecules has now been broadened by the realization that heterodimeric ligands and receptors are also active participants in the signaling process. Although heterodimer receptors were described 2 decades ago, their impact, along with the effect of additional cell surface partners and novel autocrine VEGF signaling pathways, are only now starting to be clarified. Furthermore, ligand-independent signaling (noncanonical) has been identified through galectin and gremlin binding and upon rise of intracellular levels of reactive oxygen species. Activation of the VEGF receptors in the absence of ligand holds immediate implications for therapeutic approaches that exclusively target VEGF. The present review provides a concise summary of the recent developments in both canonical and noncanonical VEGF signaling and places these findings in perspective to their potential clinical and biological ramifications.
      Cover page: Canonical and Noncanonical Vascular Endothelial Growth Factor Pathways
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2014)
      Although vascular complications are a hallmark of diabetes, the molecular mechanisms that underlie endothelial dysfunction are unclear. We showed that reactive oxygen species generated from hyperglycemia promoted ligand-independent phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). This VEGFR2 signaling occurred within the Golgi compartment and resulted in progressively decreased availability of VEGFR2 at the cell surface. Consequently, the responses of endothelial cells to exogenous VEGF in a mouse model of diabetes were impaired because of a specific deficiency of VEGFR2 at the cell surface, despite a lack of change in transcript abundance. Hyperglycemia-induced phosphorylation of VEGFR2 did not require intrinsic receptor kinase activity and was instead mediated by Src family kinases. The reduced cell surface abundance of VEGFR2 in diabetic mice was reversed by treatment with the antioxidant N-acetyl-L-cysteine, suggesting a causative role for oxidative stress. These findings uncover a mode of ligand-independent VEGFR2 signaling that can progressively lead to continuously muted responses to exogenous VEGF and limit angiogenic events.
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2018)
        Through multiple cell-cell and cell-matrix interactions, epithelial and endothelial sheets form tight barriers. Modulators of the cytoskeleton contribute to barrier stability and act as rheostats of vascular permeability. In this study, we sought to identify cytoskeletal regulators that underlie barrier diversity across vessels. To achieve this, we correlated functional and structural barrier features to gene expression of endothelial cells (ECs) derived from different vascular beds. Within a subset of identified candidates, we found that the guanosine nucleotide exchange factor Vav3 was exclusively expressed by microvascular ECs and was closely associated with a high-resistance barrier phenotype. Ectopic expression of Vav3 in large artery and brain ECs significantly enhanced barrier resistance and cortical rearrangement of the actin cytoskeleton. Mechanistically, we found that the barrier effect of Vav3 is dependent on its Dbl homology domain and downstream activation of Rap1. Importantly, inactivation of Vav3 in vivo resulted in increased vascular leakage, highlighting its function as a key regulator of barrier stability.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2016)

          Objective

          Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions.

          Approach and results

          We combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents.

          Conclusions

          This study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.
            Cover page: Perivascular Macrophages Limit Permeability
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2018)
            Given its role as the source of definitive hematopoietic cells, we sought to determine whether mutations initiated in the hemogenic endothelium would yield hematopoietic abnormalities or malignancies. Here, we find that endothelium-specific transposon mutagenesis in mice promotes hematopoietic pathologies that are both myeloid and lymphoid in nature. Frequently mutated genes included previously recognized cancer drivers and additional candidates, such as Pi4ka, a lipid kinase whose mutation was found to promote myeloid and erythroid dysfunction. Subsequent validation experiments showed that targeted inactivation of the Pi4ka catalytic domain or reduction in mRNA expression inhibited myeloid and erythroid cell differentiation in vitro and promoted anemia in vivo through a mechanism involving deregulation of AKT, MAPK, SRC, and JAK-STAT signaling. Finally, we provide evidence linking PI4KAP2, previously considered a pseudogene, to human myeloid and erythroid leukemia.
              Cover page: A Forward Genetic Screen Targeting the Endothelium Reveals a Regulatory Role for the Lipid Kinase Pi4ka in Myelo- and Erythropoiesis
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2015)
              Autocrine VEGF is necessary for endothelial survival, although the cellular mechanisms supporting this function are unknown. Here, we show that--even after full differentiation and maturation--continuous expression of VEGF by endothelial cells is needed to sustain vascular integrity and cellular viability. Depletion of VEGF from the endothelium results in mitochondria fragmentation and suppression of glucose metabolism, leading to increased autophagy that contributes to cell death. Gene-expression profiling showed that endothelial VEGF contributes to the regulation of cell cycle and mitochondrial gene clusters, as well as several--but not all--targets of the transcription factor FOXO1. Indeed, VEGF-deficient endothelium in vitro and in vivo showed increased levels of FOXO1 protein in the nucleus and cytoplasm. Silencing of FOXO1 in VEGF-depleted cells reversed expression profiles of several of the gene clusters that were de-regulated in VEGF knockdown, and rescued both cell death and autophagy phenotypes. Our data suggest that endothelial VEGF maintains vascular homeostasis through regulation of FOXO1 levels, thereby ensuring physiological metabolism and endothelial cell survival.
                Cover page: Autocrine VEGF maintains endothelial survival through regulation of metabolism and autophagy
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