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Open Access Publications from the University of California

Scholarly Works (21 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2020)

HEG1 (Heart of glass1), a transmembrane protein, directly binds to and recruits KRIT1 (Krev interaction trapped protein 1) to endothelial junctions to form the HEG1-KRIT1 protein complex that establishes and maintains junctional integrity. Genetic inactivation or knockdown of endothelial HEG1 or KRIT1 leads to the upregulation of transcription factors Krüppel-like Factors 4 and 2 (KLF4 and KLF2), which are factors implicated in endothelial vascular homeostasis. However, the effect of acute inhibition of the HEG1-KRIT1 interaction remains incompletely understood. Through structure-based design, we have established the feasibility of pharmacologically disrupting the KRIT1-HEG1 interaction to uncover acute changes in signaling pathways downstream of the HEG1-KRIT1 protein complex disruption. Previous work in the lab shows that the HEG1-KRIT1 inhibitor 3 (HKi3) is a bona fide interaction inhibitor by competing orthosterically with HEG1 for binding to the KRIT1 band 4.1, ezrin, radixin, and moesin (FERM) domain. In this study, we observed that acute inhibition of the HEG1-KRIT1 interaction by HKi3 triggers PI3K/Akt signaling in endothelial cells. We also observed that inhibition of KRIT1-HEG1 interaction using HKi3 also increase KLF4 and KLF2 mRNA expression levels. In addition, we developed an inducible shRNA expression system to the time- controlled knockdown of genes associated with the HEG1-KRIT1 protein complex that we denominated TRMPV-PDCD10 (tetracycline-responsive element (TRE)-dsRed-miR30-against- PDCD10-PGK-Venus). Using this RNAi system, we generated stable hCMEC/D3 cell lines, each expressing the TRMPV-PDCD10 construct. We observed that TRMPVIR-PDCD10 doxycycline-treated cells show marked disruption of VE-cadherin staining and upregulation in KLF2 and KLF4 mRNA gene expression. Thus, our results demonstrate that acute inhibition of the HEG1-KRIT1 interaction activates PI3K/Akt activity and elevates KLF4 and KLF2 gene expression. Thus, HKi3 provides a new pharmacologic tool to study acute inhibition of the HEG1-KRIT1 protein complex. We also adopted an inducible shRNA expression system to regulate genes in endothelial cells in a time-controlled manner.

    Cover page: Pharmacological inhibition of the HEG1-KRIT1 protein complex upregulate KLF2 and KLF4 expression in endothelial cells
    • Thesis
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2021)

    Over the last decade, the use of microneedle devices has facilitated a painless localized and practical delivery of therapeutic payloads across the skin, with considerable promise in a variety of biomedical applications. However, their efficacy has been limited by the slow diffusion of molecules often requiring external actuation. This dissertation aims to demonstrate the unique advantages of active microneedle platforms. The first theme focuses on the development of an autonomous and degradable, active microneedle platform for deeper and faster intradermal therapeutic delivery, and corresponding in vivo performance in a B16F10 mouse melanoma model. The second theme explores a versatile and effective in situ active microneedle vaccination system for the direct intratumoral delivery of an immunoadjuvant, cowpea mosaic virus nanoparticles, in vivo. The third theme describes a dual-action combinatorial programmable microneedle system by integrating fast and sustained-release compartments with tunable release kinetics. We demonstrate that the fine tuning of microneedle materials allows the device to be tailored to deliver initial payloads in minutes, while simultaneously deliver a second drug over prolonged period of times ranging from weeks to months.

      Cover page: Engineering Active Transdermal Systems with Enhanced Targeted Drug Delivery
      • Thesis
      • Peer Reviewed
      UC San Diego Electronic Theses and Dissertations (2020)

      Cerebral cavernous malformations (CCM) are common vascular lesions made of clusters of endothelia filled with blood that primarily affects the central nervous system. Loss-of-function mutations in the genes; KRIT1 ( Krev1 interaction trapped gene 1, CCM1), CCM2, or PDCD10 (Programmed cell death protein 10, CCM3) propel brain vascular changes marked by loss of endothelial tight and adherens junctions, altered basement membrane composition, increased angiogenesis, and altered number of intercellular signaling pathways (e.g., RhoA/ROCK,

      Angiopoietin-2, reactive oxygen species (ROS), anti-coagulation pathway, and endothelial to mesenchymal transition (EndMT)). Moreover, our group, and others, demonstrated that increased vascular endothelial growth factor (VEGF) signaling and associated vascular permeability are significant contributors to CCM disease. Here we show that genetic inactivation of endothelial Pdcd10 in animals results in normoxic stability of hypoxia-inducible factor 1 α (HIF-1α). Our findings indicate that increase in HIF-1α leads to an upregulation of a hypoxic program with genes implicated in angiogenesis and cell metabolism. Furthermore, we show that an increase in COX-2, a direct HIF-1α target gene, contributes to brain lesion genesis because the administration of COX-2 inhibitor celecoxib significantly prevents CCM lesions in a pre-clinical mice model. Therefore, our findings support the hypothesis that components of the hypoxic program may represent potential therapeutic targets for CCM disease.

        Cover page: Investigating the role of increase in hypoxia-inducible factor-1α expression during cerebral cavernous malformations
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2020)

        Background & aims

        Integrin α4β7 mediates lymphocyte trafficking to the gut and gut-associated lymphoid tissues, a process critical for recruitment of effector lymphocytes from the circulation to the gut mucosa in inflammatory bowel disease (IBD) and murine models of intestinal inflammation. Antibody blockade of β7 integrins generally is efficacious in IBD; however, some patients fail to respond, and a few patients can experience exacerbations. This study examined the effects of loss of β7 integrin function in murine models of IBD.

        Methods

        In a mouse IBD model caused by lack of interleukin 10, a cytokine important in CD25hiFoxP3+ regulatory T cell (Treg) function, genetic deletion of β7 integrin or antibody blockade of α4β7-mucosal addressin cell adhesion molecule-1 interaction paradoxically exacerbated colitis.

        Results

        Loss of β7 impaired the capacity of Tregs homing to the gut and therefore suppress intestinal inflammation in an adoptive T-cell transfer model; however, the intrinsic suppressive function of β7-deficient Tregs remained intact, indicating that the β7 deficiency selectively impacts gut homing. Deletion of β7 integrin did not worsen colitis in an acute dextran sodium sulfate model in which Treg number and function were normal.

        Conclusions

        In Integrin subunit beta (Itgb)7-/-Il10-/- mice, loss of β7-dependent Treg homing to gut-associated lymphoid tissues combined with loss of intrinsic Treg function exacerbated intestinal inflammation. These results suggest that IBD patients with reduced CD25hiFoxP3+ Treg numbers or function or lack of interleukin 10 could be at risk for failure of α4β7 blocking therapy.
          Cover page: β7 Integrin Inhibition Can Increase Intestinal Inflammation by Impairing Homing of CD25hiFoxP3+ Regulatory T Cells
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2016)
          The delayed ignition and propulsion of catalytic tubular microrockets based on fuel-induced chemical dealloying of an inner alloy layer is demonstrated. Such timed delay motor activation process relies on the preferential gradual corrosion of Cu from the inner Pt-Cu alloy layer by the peroxide fuel. The dealloying process exposes the catalytically active Pt surface to the chemical fuel, thus igniting the microrockets propulsion autonomously without external stimuli. The delayed motor activation relies solely on the intrinsic material properties of the micromotor and the surrounding solution. The motor activation time can thus be tailored by controlling the composition of the Cu-Pt alloy layer and the surrounding media, including the fuel and NaCl concentrations and local pH. Speed acceleration in a given fuel solution is also demonstrated and reflects the continuous exposure of the Pt surface. The versatile "blastoff" control of these chemical microrockets holds considerable promise for designing self-regulated chemically-powered nanomachines with a "built-in" activation mechanism for diverse tasks.
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2020)
            β2 integrins are the main adhesion molecules in neutrophils and other leukocytes and are rapidly activated by inside-out signaling, which results in conformational changes that are transmitted through the transmembrane domain (TMD). Here, we investigated the biologic effect of introducing a proline mutation in the β2 integrin TMD to create a flexible kink that uncouples the topology of the inner half of the TMD from the outer half and impairs integrin activation. The β2 integrin alpha chains, αL, αM, αX, and αD, all contain an inserted (I) domain with homology to von Willebrand factor A domain. β2 activation was monitored in a homogenous binding assay of 2 reporter monoclonal antibodies: KIM127 reporting extension (E+ ) and mAb24 reporting the high-affinity (H+ ) conformation of the β2 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of β2 completely inhibited arrest of rolling HL-60 cells in response to the chemokine IL-8. TMD mutant HL-60 cells rolling on P-selectin and ICAM-1 were unable to reduce their rolling velocity in response to IL-8. Quantitative dynamic footprinting live-cell imaging showed that blocking TMD topology transmission impaired the chemokine-induced activation of β2, limiting the appearance of extended high-affinity (E+ H+ ) β2. This also resulted in a defect in early spreading (3 min after arrest), which could be overcome by forced integrin activation using Mn2+ . We conclude that the TMD proline mutation severely impairs β2 integrin extension, cell arrest, and early spreading.
              Cover page: Frontline Science: A flexible kink in the transmembrane domain impairs β2 integrin extension and cell arrest from rolling
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2022)
              Hearing depends on precise synaptic transmission between cochlear inner hair cells and spiral ganglion neurons through afferent ribbon synapses. Neuroligins (Nlgns) facilitate synapse maturation in the brain, but they have gone unstudied in the cochlea. We report Nlgn3 and Nlgn1 knockout (KO) cochleae have fewer ribbon synapses and have impaired hearing. Nlgn3 KO is more vulnerable to noise trauma with limited activity at high frequencies one day after noise. Furthermore, Nlgn3 KO cochleae have a 5-fold reduction in synapse number compared to wild type after two weeks of recovery. Double KO cochlear phenotypes are more prominent than the KOs, for example, 5-fold smaller synapses, 25% reduction in synapse density, and 30% less synaptic output. These observations indicate Nlgn3 and Nlgn1 are essential to cochlear ribbon synapse maturation and function.
                Creative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2020)
                The use of microneedles has facilitated the painless localized delivery of drugs across the skin. However, their efficacy has been limited by slow diffusion of molecules and often requires external triggers. Herein, an autonomous and degradable, active microneedle delivery platform is introduced, employing magnesium microparticles loaded within the microneedle patch, as the built-in engine for deeper and faster intradermal payload delivery. The magnesium particles react with the interstitial fluid, leading to an explosive-like rapid production of H2 bubbles, providing the necessary force to breach dermal barriers and enhance payload delivery. The release kinetics of active microneedles is evaluated in vitro by measuring the amount of IgG antibody (as a model drug) that passed through phantom tissue and a pigskin barrier. In vivo experiments using a B16F10 mouse melanoma model demonstrate that the active delivery of anti-CTLA-4 (a checkpoint inhibitor drug) results in greatly enhanced immune response and significantly longer survival. Moreover, spatially resolved zones of active and passive microneedles allow a combinatorial rapid burst response along with slow, sustained release, respectively. Such versatile and effective autonomous dynamic microneedle delivery technology offers considerable promise for a wide range of therapeutic applications, toward a greatly enhanced outcome, convenience, and cost.
                  Cover page: Built-In Active Microneedle Patch with Enhanced Autonomous Drug Delivery.
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2022)
                  Head and neck squamous cell carcinoma (HNSCC) ranks sixth in cancer incidence worldwide and has a 5-year survival rate of only 63%. Immunotherapies-principally immune checkpoint inhibitors (ICI), such as anti-PD-1 and anti-CTLA-4 antibodies that restore endogenous antitumor T-cell immunity-offer the greatest promise for HNSCC treatment. Anti-PD-1 has been recently approved for first-line treatment of recurrent and metastatic HNSCC; however, less than 20% of patients show clinical benefit and durable responses. In addition, the clinical application of ICI has been limited by immune-related adverse events (irAE) consequent to compromised peripheral immune tolerance. Although irAEs are often reversible, they can become severe, prompting premature therapy termination or becoming life threatening. To address the irAEs inherent to systemic ICI therapy, we developed a novel, local delivery strategy based upon an array of soluble microneedles (MN). Using our recently reported syngeneic, tobacco-signature murine HNSCC model, we found that both systemic and local-MN anti-CTLA-4 therapy lead to >90% tumor response, which is dependent on CD8 T cells and conventional dendritic cell type 1 (cDC1). However, local-MN delivery limited the distribution of anti-CTLA-4 antibody from areas distal to draining lymphatic basins. Employing Foxp3-GFPDTR transgenic mice to interrogate irAEs in vivo, we found that local-MN delivery of anti-CTLA-4 protects animals from irAEs observed with systemic therapy. Taken together, our findings support the exploration of MN-intratumoral ICI delivery as a viable strategy for HNSCC treatment with reduced irAEs, and the opportunity to target cDC1s as part of multimodal treatment options to boost ICI therapy.
                    Cover page: Microneedle-mediated Intratumoral Delivery of Anti-CTLA-4 Promotes cDC1-dependent Eradication of Oral Squamous Cell Carcinoma with Limited irAEs
                    • Article
                    • Peer Reviewed
                    UC San Diego Previously Published Works (2019)
                    Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce integrin activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a limited contribution to integrin activation in CHO cells or platelets. Here, we show that talin1 F1 domain (F1) contains a previously undetected Rap1-binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced integrin activation. The capacity of F1 to mediate Rap1-dependent integrin activation depends on a unique loop in F1 that has a propensity to form a helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical, as charge-reversal mutations led to dramatic suppression of talin1-dependent activation. Thus, a novel Rap1-binding site and a transient lipid-dependent helix in F1 work in tandem to enable a direct Rap1-talin1 interaction to cause integrin activation.
                      Cover page: Rap1 binding and a lipid-dependent helix in talin F1 domain promote integrin activation in tandem
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