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Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2024)

The blood brain barrier (BBB) is a complex, multi-cellular structure that strictly regulates the passage of blood-borne molecules, ions, and cells from the periphery into the central nervous system (CNS). Its constituents include specialized endothelial cells (EC), pericytes, astrocytic end-feet, and the basal lamina. Due to this protective structure, hydrophilic drugs suboptimally accumulate in the brain after systemic administration contributing to the clinical failure of many CNS targeted therapies. Moreover, BBB dysfunction is observed in many neurodegenerative illnesses including Parkinson’s, Alzheimer’s, and Huntington’s disease. Thus, there is considerable interest in elucidating the underpinnings of BBB transport such that endogenous systems may be modulated or coopted to best treat CNS-associated diseases.

While maintained by exogenous factors, BBB properties are localized to and effectuated by the brain endothelium. Unlike ECs of the periphery, brain microvascular ECs (BMECs) maintain specialized tight junctions between single cells that restrict paracellular entry into the CNS, while a limited number of caveolae, reduced pinocytosis, and selective transporters limit transcellular entry. This specialization, induced by the developing neural milieu and maintained by non-endothelial brain constituents, is critical for BMEC identity. Consequently, there is a need for higher-order human-derived BBB in vitro models to replace conventional BMEC monolayer models that are better suited to complement in vivo (mouse) studies.

Based on our lab’s Vascularized Micro Organ (VMO) platform, this body of research aims to develop a novel BBB version – the Vascularized Micro Brain (VMB) – utilizing human-derived endothelial colony forming cells derived endothelial cells (ECFC-ECs), pericytes, astrocytes and iPSC-derived neural progenitor cells (iPSC-NPCs), that mimics the natural process of BBB induction and assembles into a perfused micro-vasculature. Herein, we demonstrate the ability of the VMB to mirror in vivo BBB gene expression and capture complex BBB-like vascular functions including reduced permeability and transcytosis. We believe that the VMB is optimally suited to study the BBB and its functional state during CNS-related pathologies, including neurodegenerative diseases and cancer, with the hope that it can better identify drugs likely to succeed in the clinic. 

    Creative Commons 'BY' version 4.0 license
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2017)
    Physical force has emerged as a key regulator of tissue homeostasis, and plays an important role in embryogenesis, tissue regeneration, and disease progression. Currently, the details of protein interactions under elevated physical stress are largely missing, therefore, preventing the fundamental, molecular understanding of mechano-transduction. This is in part due to the difficulty isolating large quantities of cell lysates exposed to force-bearing conditions for biochemical analysis. We designed a simple, easy-to-fabricate, large-scale cell stretch device for the analysis of force-sensitive cell responses. Using proximal biotinylation (BioID) analysis or phospho-specific antibodies, we detected force-sensitive biochemical changes in cells exposed to prolonged cyclic substrate stretch. For example, using promiscuous biotin ligase BirA* tagged α-catenin, the biotinylation of myosin IIA increased with stretch, suggesting the close proximity of myosin IIA to α-catenin under a force bearing condition. Furthermore, using phospho-specific antibodies, Akt phosphorylation was reduced upon stretch while Src phosphorylation was unchanged. Interestingly, phosphorylation of GSK3β, a downstream effector of Akt pathway, was also reduced with stretch, while the phosphorylation of other Akt effectors was unchanged. These data suggest that the Akt-GSK3β pathway is force-sensitive. This simple cell stretch device enables biochemical analysis of force-sensitive responses and has potential to uncover molecules underlying mechano-transduction.
      Cover page: Biochemical analysis of force-sensitive responses using a large-scale cell stretch device
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2021)
      In recent years, microphysiological system (MPS, also known as, organ-on-a-chip or tissue chip) platforms have emerged with great promise to improve the predictive capacity of preclinical modeling thereby reducing the high attrition rates when drugs move into trials. While their designs can vary quite significantly, in general MPS are bioengineered in vitro microenvironments that recapitulate key functional units of human organs, and that have broad applications in human physiology, pathophysiology, and clinical pharmacology. A critical next step in the evolution of MPS devices is the widespread incorporation of functional vasculature within tissues. The vasculature itself is a major organ that carries nutrients, immune cells, signaling molecules and therapeutics to all other organs. It also plays critical roles in inducing and maintaining tissue identity through expression of angiocrine factors, and in providing tissue-specific milieus (i.e., the vascular niche) that can support the survival and function of stem cells. Thus, organs are patterned, maintained and supported by the vasculature, which in turn receives signals that drive tissue specific gene expression. In this review, we will discuss published vascularized MPS platforms and present considerations for next-generation devices looking to incorporate this critical constituent. Finally, we will highlight the organ-patterning processes governed by the vasculature, and how the incorporation of a vascular niche within MPS platforms will establish a unique opportunity to study stem cell development.
        Cover page: The vascular niche in next generation microphysiological systems
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2016)
        Collective migration of epithelial cells is an integral part of embryonic development, wound healing, tissue renewal and carcinoma invasion. While previous studies have focused on cell-extracellular matrix adhesion as a site of migration-driving, traction force-transmission, cadherin mediated cell-cell adhesion is also capable of force-transmission. Using a soft elastomer coated with purified N-cadherin as a substrate and a Hepatocyte Growth Factor-treated, transformed MDCK epithelial cell line as a model system, we quantified traction transmitted by N-cadherin-mediated contacts. On a substrate coated with purified extracellular domain of N-cadherin, cell surface N-cadherin proteins arranged into puncta. N-cadherin mutants (either the cytoplasmic deletion or actin-binding domain chimera), however, failed to assemble into puncta, suggesting the assembly of focal adhesion like puncta requires the cytoplasmic domain of N-cadherin. Furthermore, the cytoplasmic domain deleted N-cadherin expressing cells exerted lower traction stress than the full-length or the actin binding domain chimeric N-cadherin. Our data demonstrate that N-cadherin junctions exert significant traction stress that requires the cytoplasmic domain of N-cadherin, but the loss of the cytoplasmic domain does not completely eliminate traction force transmission.
          Cover page: Deletion of the cytoplasmic domain of N-cadherin reduces, but does not eliminate, traction force-transmission
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2024)
          BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, has caused nearly 7 million deaths worldwide. Severe cases are marked by an aggressive inflammatory response known as hypercytokinemia, contributing to endothelial damage. Although vaccination has reduced hospitalizations, hypercytokinemia persists in breakthrough infections, emphasizing the need for disease models mimicking this response. Using a 3D microphysiological system (MPS), we explored the vascular role in SARS-CoV-2-induced hypercytokinemia. METHODS: The vascularized micro-organ (VMO) MPS, consisting of human-derived primary endothelial cells (ECs) and stromal cells within an extracellular matrix, was used to model SARS-CoV-2 infection. A non-replicative pseudotyped virus fused to GFP was employed, allowing visualization of viral entry into human ECs under physiologic flow conditions. Expression of ACE2, TMPRSS2, and AGTR1 was analyzed, and the impact of viral infection on ACE2 expression, vascular inflammation, and vascular morphology was assessed. RESULTS: The VMO platform facilitated the study of COVID-19 vasculature infection, revealing that ACE2 expression increased significantly in direct response to shear stress, thereby enhancing susceptibility to infection by pseudotyped SARS-CoV-2. Infected ECs secreted pro-inflammatory cytokines, including IL-6 along with coagulation factors. Cytokines released by infected cells were able to activate downstream, non-infected EC, providing an amplification mechanism for inflammation and coagulopathy. DISCUSSION: Our findings highlight the crucial role of vasculature in COVID-19 pathogenesis, emphasizing the significance of flow-induced ACE2 expression and subsequent inflammatory responses. The VMO provides a valuable tool for studying SARS-CoV-2 infection dynamics and evaluating potential therapeutics.
            Cover page: SARS-CoV-2 infection of endothelial cells, dependent on flow-induced ACE2 expression, drives hypercytokinemia in a vascularized microphysiological system.Creative Commons 'BY' version 4.0 license
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