Abstract Background Plasma gelsolin (pGSN) is an important part of the blood actin buffer that prevents negative consequences of possible F-actin deposition in the microcirculation and has various functions during host immune response. Recent reports reveal that severe COVID-19 correlates with reduced levels of pGSN. Therefore, using an in vitro system, we investigated whether pGSN could attenuate increased permeability of the blood–brain barrier (BBB) during its exposure to the portion of the SARS-CoV-2 spike protein containing the receptor binding domain (S1 subunit). Materials and methods Two- and three-dimensional models of the human BBB were constructed using the human cerebral microvascular endothelial cell line hCMEC/D3 and exposed to physiologically relevant shear stress to mimic perfusion in the central nervous system (CNS). Trans-endothelial electrical resistance (TEER) as well as immunostaining and Western blotting of tight junction (TJ) proteins assessed barrier integrity in the presence of the SARS-CoV-2 spike protein and pGSN. The IncuCyte Live Imaging system evaluated the motility of the endothelial cells. Magnetic bead-based ELISA was used to determine cytokine secretion. Additionally, quantitative real-time PCR (qRT-PCR) revealed gene expression of proteins from signaling pathways that are associated with the immune response. Results pGSN reversed S1-induced BBB permeability in both 2D and 3D BBB models in the presence of shear stress. BBB models exposed to pGSN also exhibited attenuated pro-inflammatory signaling pathways (PI3K, AKT, MAPK, NF-κB), reduced cytokine secretion (IL-6, IL-8, TNF-α), and increased expression of proteins that form intercellular TJ (ZO-1, occludin, claudin-5). Conclusion Due to its anti-inflammatory and protective effects on the brain endothelium, pGSN has the potential to be an alternative therapeutic target for patients with severe SARS-CoV-2 infection, especially those suffering neurological complications of COVID-19.
more »
« less
Variable fluid flow regimes alter human brain microvascular endothelial c ell–cell junctions and cytoskeletal structure
The human brain microvasculature is constantly exposed to variable fluid flow regimes and their influence on the endothelium depends in part on the synchronous cooperative behavior between cell–cell junctions and the cytoskeleton. In this study, we exposed human cerebral microvascular endothelial cells to a low laminar flow (1 dyne⋅cm−2), high laminar flow (10 dyne⋅cm−2), low oscillatory flow (±1 dyne⋅cm−2), or high oscillatory flow (±10 dyne⋅cm−2) for 24 hr. After this time, endothelial cell–cell junction and cytoskeletal structural response was characterized through observation of zonula occludens‐1 (ZO‐1), claudin‐5, junctional adhesion molecule‐A (JAM‐A), vascular endothelial cadherin (VE‐Cad), and F‐actin. In addition, we also characterized cell morphology through measurement of cell area and cell eccentricity. Our results revealed the greatest change in junctional structure reorganization for ZO‐1 and JAM‐A to be observed under low laminar flow conditions while claudin‐5 exhibited the greatest change in structural reorganization under both low and high laminar flow conditions. However, VE‐Cad displayed the greatest structural response under a high laminar flow, reflecting the unique responses each cell–cell junction protein had to each fluid flow regime. In addition, cell area and cell eccentricity displayed most significant changes under the high laminar flow and low oscillatory flow, respectively. We believe this study will be useful to the field of cell mechanics and mechanobiology.
more » « less- Award ID(s):
- 2045750
- NSF-PAR ID:
- 10305615
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Cytoskeleton
- Volume:
- 78
- Issue:
- 6
- ISSN:
- 1949-3584
- Page Range / eLocation ID:
- p. 323-334
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Long‐term daily oral administration of intestinal permeation enhancers is safe and effective in micemore » « less
Abstract Although protein drugs are powerful biologic therapeutics, they cannot be delivered orally because their large size and hydrophilicity limit their absorption across the intestinal epithelium. One potential solution is the incorporation of permeation enhancers into oral protein formulations; however, few have advanced clinically due to toxicity concerns surrounding chronic use. To better understand these concerns, we conducted a 30‐day longitudinal study of daily oral permeation enhancer use in mice and resultant effects on intestinal health. Specifically, we investigated three permeation enhancers: sodium caprate (C10), an industry standard, as well as 1‐phenylpiperazine (PPZ) and sodium deoxycholate (SDC). Over 30 days of treatment, all mice gained weight, and none required removal from the study due to poor health. Furthermore, intestinal permeability did not increase following chronic use. We also quantified the gene expression of four tight junction proteins (claudin 2, claudin 3, ZO‐1, and JAM‐A). Significant differences in gene expression between untreated and permeation enhancer‐treated mice were found, but these varied between treatment groups, with most differences resolving after a 1‐week washout period. Immunofluorescence microscopy revealed no observable differences in protein localization or villus architecture between treated and untreated mice. Overall, PPZ and SDC performed comparably to C10, one of the most clinically advanced enhancers, and results suggest that the chronic use of some permeation enhancers may be therapeutically viable from a safety standpoint.
-
Endothelial mechanobiology is a key consideration in the progression of vascular dysfunction, including atherosclerosis. However mechanistic connections between the clinically associated physical stimuli, vessel stiffness and shear stress, and how they interact to modulate plaque progression remain incompletely characterized. Vessel-chip systems are excellent candidates for modeling vascular mechanobiology as they may be engineered from the ground up, guided by the mechanical parameters present in human arteries and veins, to recapitulate key features of the vasculature. Here, we report extensive validation of a vessel-chip model of endothelial yes-associated protein (YAP) mechanobiology, a protein sensitive to both matrix stiffness and shearing forces and, importantly, implicated in atherosclerotic progression. Our model captures the established endothelial mechanoresponse, with endothelial alignment, elongation, reduction of adhesion molecules, and YAP cytoplasmic retention under high laminar shear. Conversely, we observed disturbed morphology, inflammation, and nuclear partitioning under low, high, and high oscillatory shear. Examining targets of YAP transcriptional co-activation, connective tissue growth factor (CTGF) is strongly downregulated by high laminar shear, whereas it is strongly upregulated by low shear or oscillatory flow. Ankyrin repeat domain 1 (ANKRD1) is only upregulated by high oscillatory shear. Verteporfin inhibition of YAP reduced the expression of CTGF but did not affect ANKRD1. Lastly, substrate stiffness modulated the endothelial shear mechanoresponse. Under high shear, softer substrates showed the lowest nuclear localization of YAP whereas stiffer substrates increased nuclear localization. Low shear strongly increased nuclear localization of YAP across stiffnesses. Together, we have validated a model of endothelial mechanobiology and describe a clinically relevant biological connection between matrix stiffness, shear stress, and endothelial activation via YAP mechanobiology.more » « less
-
Normal fibroblasts are present within the extracellular matrix (ECM). They can become activated, leading to increased proliferation and ECM protein secretion such as collagen type I to promote tissue remodeling. These cells are also involved in adult pathologies including cancer metastasis and cardiac and renal fibrosis. One source of activated fibroblasts is endothelial to mesenchymal transformation (EndMT), in which endothelial cells lose their cell–cell and cell–ECM adhesions, gain invasive properties, and become mesenchymal cells. While EndMT is well characterized in developmental biology, the mechanisms and functional role of EndMT in adult physiology and pathology have not been fully investigated. A microfluidic device with an incorporated three-dimensional ECM chamber was developed to study the role of combined steady fluid shear stress magnitudes and transforming growth factor-βeta 1 (TGF-β1) on EndMT. Low (1 dyne per cm 2 ) steady shear stress and TGF-β1 exposure induced EndMT in endothelial cells, including upregulation of mesenchymal protein and gene expression markers. Cells exposed to TGF-β1 and high (20 dynes per cm 2 ) steady shear stress did not undergo EndMT, and protein and gene expression of mesenchymal markers was significantly downregulated. Mesenchymally transformed cells under static conditions with and without TGF-β1 showed significantly more collagen production when compared to fluidic conditions. These results confirm that both low shear stress and TGF-β1 induce EndMT in endothelial cells, but this process can be prevented by exposure to physiologically relevant high shear stress. These results also show conditions most likely to cause tissue pathology.more » « less
-
more » « less
Abstract Recent developments in digital light processing (DLP) can advance the structural and biochemical complexity of perfusable
in vitro models of the blood–brain barrier. Here, we describe a strategy to functionalize complex, DLP-printed vascular models with multiple peptide motifs in a single hydrogel. Different peptides can be clicked into the walls of distinct topologies, or the peptide motifs lining channel walls can differ from those in the bulk of the hydrogel. The flexibility of this approach is used to both characterize the effects of various bioactive domains on endothelial coverage and tight junction formation, in addition to facilitating astrocyte attachment in the hydrogel surrounding the endothelialized vessel to mimic endothelial–astrocyte interaction. Peptides derived from proteins mediating cell-extracellular matrix (e.g. RGD and IKVAV) and cell–cell (e.g. HAVDI) adhesions are used to mediate endothelial cell attachment and coverage. HAVDI and IKVAV-lined channels exhibit significantly greater endothelialization and increased zonula-occluden-1 (ZO-1) localization to cell–cell junctions of endothelial cells, indicative of tight junction formation. RGD is then used in the bulk hydrogel to create an endothelial–astrocyte co-culture model of the blood–brain barrier that overcomes the limitations of previous platforms incapable of complex topology or tunable bioactive domains. This approach yields an adjustable, biofabricated platform to interrogate the effects of cell-matrix interaction on blood–brain barrier mechanobiology.
