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Open Access Publications from the University of California

Scholarly Works (9 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2018)

The collective transmission of forces in multicellular colonies plays an important role in biological processes such as development, endothelial function and wound healing. The quantification of intracellular forces by monolayer stress microscopy (MSM) has improved our understanding of these phenomena, and provides great promise for the functional screening of contractile tissues in the context of drug development. However, current MSM methods present a number of limitations: they assume that the monolayer only deforms laterally, introduce boundary artifacts, and are restricted to low-throughput applications. This dissertation addresses these limitations and provides the first high-throughput quantification of three-dimensional (3D) intracellular stresses in applications relevant to biology and biotechnology, ranging from leukocyte extravasation to high-throughput functional screening of cardiomyocytes.

We introduce a 3D-MSM method that incorporates for the first time monolayer bending. 3D-MSM measures the 3D traction stresses exerted by the cells on a flexible substrate and calculates intracellular stresses by imposing equilibrium of forces and moments in the monolayer. We present 3D-MSM measurements in micropatterned cell islands of different sizes and shapes. We show that intracellular stresses caused by lateral deformation are transmitted across long distances, whereas bending-induced stresses remain confined to 1-2 cell lengths from bending sites. We hypothesize that the localized effect of bending-induced stresses may be important in processes such as cellular extravasation, which are accompanied by large 3D deflections of the endothelium and require localized permeability changes. Overall, these results suggest that bending and lateral stresses can propagate mechanical signals at different length scales, and reveal that the transmission of forces across cell junctions is more three-dimensional and complex than previously believed.

We also present a novel high-throughput functional screening assay that quantifies cardiomyocyte force generation and stiffness changes during the cardiac cycle. The method jointly measures the intracellular stresses within cardiomyocyte monolayers and their elastic modulus. The main novelty of this method is that it utilizes the forces generated by the cells themselves to probe their material properties and is, thus, fully non-invasive. We present the proof-of-concept application of our method with dose-response measurements of a series of benchmark compounds tested on monolayers of hiPSC cardiomyocytes.

    Cover page: Monolayer Stress Microscopy: Developing 3D, Elastographic, and High-Throughput Capabilities
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2016)
    Spotted fever group (SFG) rickettsiae are human pathogens that infect cells in the vasculature. They disseminate through host tissues by a process of cell-to-cell spread that involves protrusion formation, engulfment, and vacuolar escape. Other bacterial pathogens rely on actin-based motility to provide a physical force for spread. Here, we show that SFG species Rickettsia parkeri typically lack actin tails during spread and instead manipulate host intercellular tension and mechanotransduction to promote spread. Using transposon mutagenesis, we identified surface cell antigen 4 (Sca4) as a secreted effector of spread that specifically promotes protrusion engulfment. Sca4 interacts with the cell-adhesion protein vinculin and blocks association with vinculin's binding partner, α-catenin. Using traction and monolayer stress microscopy, we show that Sca4 reduces vinculin-dependent mechanotransduction at cell-cell junctions. Our results suggest that Sca4 relieves intercellular tension to promote protrusion engulfment, which represents a distinctive strategy for manipulating cytoskeletal force generation to enable spread.
      Cover page: Rickettsia Sca4 Reduces Vinculin-Mediated Intercellular Tension to Promote Spread.
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2018)
      Leukocyte transmigration across vessel walls is a critical step in the innate immune response. Upon their activation and firm adhesion to vascular endothelial cells (VECs), leukocytes preferentially extravasate across junctional gaps in the endothelial monolayer (paracellular diapedesis). It has been hypothesized that VECs facilitate paracellular diapedesis by opening their cell-cell junctions in response to the presence of an adhering leukocyte. However, it is unclear how leukocytes interact mechanically with VECs to open the VEC junctions and migrate across the endothelium. In this study, we measured the spatial and temporal evolution of the 3D traction stresses generated by the leukocytes and VECs to elucidate the sequence of mechanical events involved in paracellular diapedesis. Our measurements suggest that the contractile stresses exerted by the leukocytes and the VECs can separately perturb the junctional tensions of VECs to result in the opening of gaps before the initiation of leukocyte transmigration. Decoupling the stresses exerted by the transmigrating leukocytes and the VECs reveals that the leukocytes actively contract the VECs to open a junctional gap and then push themselves across the gap by generating strong stresses that push into the matrix. In addition, we found that diapedesis is facilitated when the tension fluctuations in the VEC monolayer were increased by proinflammatory thrombin treatment. Our findings demonstrate that diapedesis can be mechanically regulated by the transmigrating leukocytes and by proinflammatory signals that increase VEC contractility.
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2019)
        The use of biochemical signaling to derive smooth muscle cells (SMCs) from mesenchymal stem cells (MSCs) has been explored, but the induction of a fully functional SMC phenotype remains to be a major challenge. Cell morphology has been shown to regulate MSC differentiation into various lineages, including SMCs. We engineered substrates with microgrooves to induce cell elongation to study the mechanism underlying the MSC shape modulation in SMC differentiation. In comparison to those on flat substrates, MSCs cultured on engineered substrates were elongated with increased aspect ratios for both cell body and nucleus, as well as augmented cytoskeletal tensions. Biochemical studies indicated that the microgroove-elongated cells expressed significantly higher levels of SMC markers. MicroRNA analyses showed that up-regulation of miR-145 and the consequent repression of KLF4 in these elongated cells promoted MSC-to-SMC differentiation. Rho/ROCK inhibitions, which impair cytoskeletal tension, attenuated cell and nuclear elongations and disrupted the miR-145/KLF4 regulation for SMC differentiation. Furthermore, cell traction force measurements showed that miR-145 is essential for the functional contractility in the microgroove-induced SMC differentiation. Collectively, our findings demonstrate that, through a Rho-ROCK/miR-145/KLF4 pathway, the elongated cell shape serves as a decisive geometric cue to direct MSC differentiation into functional SMCs.
          Cover page: MiR-145 mediates cell morphology-regulated mesenchymal stem cell differentiation to smooth muscle cells
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2015)
          Perturbed biomechanical stimuli are thought to be critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While embryonic cardiomyocytes experience biomechanical stretch every heart beat, their molecular responses to biomechanical stimuli during heart development are poorly understood. We hypothesized that biomechanical stimuli activate specific signaling pathways that impact proliferation, gene expression and myocyte contraction. The objective of this study was to expose embryonic mouse cardiomyocytes (EMCM) to cyclic stretch and examine key molecular and phenotypic responses. Analysis of RNA-Sequencing data demonstrated that gene ontology groups associated with myofibril and cardiac development were significantly modulated. Stretch increased EMCM proliferation, size, cardiac gene expression, and myofibril protein levels. Stretch also repressed several components belonging to the Transforming Growth Factor-β (Tgf-β) signaling pathway. EMCMs undergoing cyclic stretch had decreased Tgf-β expression, protein levels, and signaling. Furthermore, treatment of EMCMs with a Tgf-β inhibitor resulted in increased EMCM size. Functionally, Tgf-β signaling repressed EMCM proliferation and contractile function, as assayed via dynamic monolayer force microscopy (DMFM). Taken together, these data support the hypothesis that biomechanical stimuli play a vital role in normal cardiac development and for cardiac pathology, including HLHS.
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2019)
            Perturbations in biomechanical stimuli during cardiac development contribute to congenital cardiac defects such as hypoplastic left heart syndrome (HLHS). This study sought to identify stretch-responsive pathways involved in cardiac development. miRNA-Seq identified miR-486 as being increased in cardiomyocytes exposed to cyclic stretch in vitro. The right ventricles (RVs) of patients with HLHS experienced increased stretch and had a trend toward higher miR-486 levels. Sheep RVs dilated from excessive pulmonary blood flow had 60% more miR-486 compared with control RVs. The left ventricles of newborn mice treated with miR-486 mimic were 16.9%-24.6% larger and displayed a 2.48-fold increase in cardiomyocyte proliferation. miR-486 treatment decreased FoxO1 and Smad signaling while increasing the protein levels of Stat1. Stat1 associated with Gata-4 and serum response factor (Srf), 2 key cardiac transcription factors with protein levels that increase in response to miR-486. This is the first report to our knowledge of a stretch-responsive miRNA that increases the growth of the ventricle in vivo.
              Cover page: miR-486 is modulated by stretch and increases ventricular growth.
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2015)
              Perturbed biomechanical stimuli are thought to be critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While embryonic cardiomyocytes experience biomechanical stretch every heart beat, their molecular responses to biomechanical stimuli during heart development are poorly understood. We hypothesized that biomechanical stimuli activate specific signaling pathways that impact proliferation, gene expression and myocyte contraction. The objective of this study was to expose embryonic mouse cardiomyocytes (EMCM) to cyclic stretch and examine key molecular and phenotypic responses. Analysis of RNA-Sequencing data demonstrated that gene ontology groups associated with myofibril and cardiac development were significantly modulated. Stretch increased EMCM proliferation, size, cardiac gene expression, and myofibril protein levels. Stretch also repressed several components belonging to the Transforming Growth Factor-β (Tgf-β) signaling pathway. EMCMs undergoing cyclic stretch had decreased Tgf-β expression, protein levels, and signaling. Furthermore, treatment of EMCMs with a Tgf-β inhibitor resulted in increased EMCM size. Functionally, Tgf-β signaling repressed EMCM proliferation and contractile function, as assayed via dynamic monolayer force microscopy (DMFM). Taken together, these data support the hypothesis that biomechanical stimuli play a vital role in normal cardiac development and for cardiac pathology, including HLHS.
                Cover page: Cyclic stretch of embryonic cardiomyocytes increases proliferation, growth, and expression while repressing Tgf-β signaling
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2017)
                Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.
                  Cover page: High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2020)
                  Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs. Compared with conventionally cultured hiPSC-CMs, metabolically matured hiPSC-CMs contract with greater force and show an increased reliance on cardiac sodium (Na+) channels and sarcoplasmic reticulum calcium (Ca2+) cycling. The media enhance the function, long-term survival, and sarcomere structures in engineered heart tissues. Use of the maturation media made it possible to reliably model two genetic cardiac diseases: long QT syndrome type 3 due to a mutation in the cardiac Na+ channel SCN5A and dilated cardiomyopathy due to a mutation in the RNA splicing factor RBM20. The maturation media should increase the fidelity of hiPSC-CMs as disease models.
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