Vandetanib Blocks the Cytokine Storm in SARS-CoV-2-Infected Mice.

The portfolio of SARS-CoV-2 small molecule drugs is currently limited to a handful that are either approved (remdesivir), emergency approved (dexamethasone, baricitinib, paxlovid, and molnupiravir), or in advanced clinical trials. Vandetanib is a kinase inhibitor which targets the vascular endothelial growth factor receptor (VEGFR), the epidermal growth factor receptor (EGFR), as well as the RET-tyrosine kinase. In the current study, it was tested in different cell lines and showed promising results on inhibition versus the toxic effect on A549-hACE2 cells (IC50 0.79 µM) while also showing a reduction of >3 log TCID50/mL for HCoV-229E. The in vivo efficacy of vandetanib was assessed in a mouse model of SARS-CoV-2 infection and statistically significantly reduced the levels of IL-6, IL-10, and TNF-α and mitigated inflammatory cell infiltrates in the lungs of infected animals but did not reduce viral load. Vandetanib also decreased CCL2, CCL3, and CCL4 compared to the infected animals. Vandetanib additionally rescued the decreased IFN-1ß caused by SARS-CoV-2 infection in mice to levels similar to that in uninfected animals. Our results indicate that the FDA-approved anticancer drug vandetanib is worthy of further assessment as a potential therapeutic candidate to block the COVID-19 cytokine storm.

Metabolically driven maturation of human-induced-pluripotent-stem-cell-derived cardiac microtissues on microfluidic chips.

The immature physiology of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) limits their utility for drug screening and disease modelling. Here we show that suitable combinations of mechanical stimuli and metabolic cues can enhance the maturation of hiPSC-derived cardiomyocytes, and that the maturation-inducing cues have phenotype-dependent effects on the cells' action-potential morphology and calcium handling. By using microfluidic chips that enhanced the alignment and extracellular-matrix production of cardiac microtissues derived from genetically distinct sources of hiPSC-derived cardiomyocytes, we identified fatty-acid-enriched maturation media that improved the cells' mitochondrial structure and calcium handling, and observed divergent cell-source-dependent effects on action-potential duration (APD). Specifically, in the presence of maturation media, tissues with abnormally prolonged APDs exhibited shorter APDs, and tissues with aberrantly short APDs displayed prolonged APDs. Regardless of cell source, tissue maturation reduced variabilities in spontaneous beat rate and in APD, and led to converging cell phenotypes (with APDs within the 300-450 ms range characteristic of human left ventricular cardiomyocytes) that improved the modelling of the effects of pro-arrhythmic drugs on cardiac tissue.

Integrated Isogenic Human Induced Pluripotent Stem Cell-Based Liver and Heart Microphysiological Systems Predict Unsafe Drug-Drug Interaction.

Three-dimensional (3D) microphysiological systems (MPSs) mimicking human organ function in vitro are an emerging alternative to conventional monolayer cell culture and animal models for drug development. Human induced pluripotent stem cells (hiPSCs) have the potential to capture the diversity of human genetics and provide an unlimited supply of cells. Combining hiPSCs with microfluidics technology in MPSs offers new perspectives for drug development. Here, the integration of a newly developed liver MPS with a cardiac MPS-both created with the same hiPSC line-to study drug-drug interaction (DDI) is reported. As a prominent example of clinically relevant DDI, the interaction of the arrhythmogenic gastroprokinetic cisapride with the fungicide ketoconazole was investigated. As seen in patients, metabolic conversion of cisapride to non-arrhythmogenic norcisapride in the liver MPS by the cytochrome P450 enzyme CYP3A4 was inhibited by ketoconazole, leading to arrhythmia in the cardiac MPS. These results establish integration of hiPSC-based liver and cardiac MPSs to facilitate screening for DDI, and thus drug efficacy and toxicity, isogenic in the same genetic background.

Control of oxygen tension recapitulates zone-specific functions in human liver microphysiology systems.

This article describes our next generation human Liver Acinus MicroPhysiology System (LAMPS). The key demonstration of this study was that Zone 1 and Zone 3 microenvironments can be established by controlling the oxygen tension in individual devices over the range of ca. 3 to 13%. The oxygen tension was computationally modeled using input on the microfluidic device dimensions, numbers of cells, oxygen consumption rates of hepatocytes, the diffusion coefficients of oxygen in different materials and the flow rate of media in the MicroPhysiology System (MPS). In addition, the oxygen tension was measured using a ratiometric imaging method with the oxygen sensitive dye, Tris(2,2'-bipyridyl) dichlororuthenium(II) hexahydrate (RTDP) and the oxygen insensitive dye, Alexa 488. The Zone 1 biased functions of oxidative phosphorylation, albumin and urea secretion and Zone 3 biased functions of glycolysis, α1AT secretion, Cyp2E1 expression and acetaminophen toxicity were demonstrated in the respective Zone 1 and Zone 3 MicroPhysiology System. Further improvements in the Liver Acinus MicroPhysiology System included improved performance of selected nonparenchymal cells, the inclusion of a porcine liver extracellular matrix to model the Space of Disse, as well as an improved media to support both hepatocytes and non-parenchymal cells. In its current form, the Liver Acinus MicroPhysiology System is most amenable to low to medium throughput, acute through chronic studies, including liver disease models, prioritizing compounds for preclinical studies, optimizing chemistry in structure activity relationship (SAR) projects, as well as in rising dose studies for initial dose ranging. Impact statement Oxygen zonation is a critical aspect of liver functions. A human microphysiology system is needed to investigate the impact of zonation on a wide range of liver functions that can be experimentally manipulated. Because oxygen zonation has such diverse physiological effects in the liver, we developed and present a method for computationally modeling and measuring oxygen that can easily be implemented in all MPS models. We have applied this method in a liver MPS in which we are then able to control oxygenation in separate devices and demonstrate that zonation-dependent hepatocyte functions in the MPS recapitulate what is known about in vivo liver physiology. We believe that this advance allows a deep experimental investigation on the role of zonation in liver metabolism and disease. In addition, modeling and measuring oxygen tension will be required as investigators migrate from PDMS to plastic and glass devices.

WAT-on-a-chip: a physiologically relevant microfluidic system incorporating white adipose tissue.

Organ-on-a-chip systems possess a promising future as drug screening assays and as testbeds for disease modeling in the context of both single-organ systems and multi-organ-chips. Although it comprises approximately one fourth of the body weight of a healthy human, an organ frequently overlooked in this context is white adipose tissue (WAT). WAT-on-a-chip systems are required to create safety profiles of a large number of drugs due to their interactions with adipose tissue and other organs via paracrine signals, fatty acid release, and drug levels through sequestration. We report a WAT-on-a-chip system with a footprint of less than 1 mm2 consisting of a separate media channel and WAT chamber connected via small micropores. Analogous to the in vivo blood circulation, convective transport is thereby confined to the vasculature-like structures and the tissues protected from shear stresses. Numerical and analytical modeling revealed that the flow rates in the WAT chambers are less than 1/100 of the input flow rate. Using optimized injection parameters, we were able to inject pre-adipocytes, which subsequently formed adipose tissue featuring fully functional lipid metabolism. The physiologically relevant microfluidic environment of the WAT-chip supported long term culture of the functional adipose tissue for more than two weeks. Due to its physiological, highly controlled, and computationally predictable character, the system has the potential to be a powerful tool for the study of adipose tissue associated diseases such as obesity and type 2 diabetes.

Quantum dot multiplexing for the profiling of cellular receptors.

The profiling of cellular heterogeneity has wide-reaching importance for our understanding of how cells function and react to their environments in healthy and diseased states. Our ability to interpret and model cell behavior has been limited by the difficulties of measuring cell differences, for example, comparing tumor and non-tumor cells, particularly at the individual cell level. This demonstrates a clear need for a generalizable approach to profile fluorophore sites on cells or molecular assemblies on beads. Here, a multiplex immunoassay for simultaneous detection of five different angiogenic markers was developed. We targeted angiogenic receptors in the vascular endothelial growth factor family (VEGFR1, VEGFR2 and VEGFR3) and Neuropilin (NRP) family (NRP1 and NRP2), using multicolor quantum dots (Qdots). Copper-free click based chemistry was used to conjugate the monoclonal antibodies with 525, 565, 605, 655 and 705 nm CdSe/ZnS Qdots. We tested and performed colocalization analysis of our nanoprobes using the Pearson correlation coefficient statistical analysis. Human umbilical vein endothelial cells (HUVEC) were tested. The ability to easily monitor the molecular indicators of angiogenesis that are a precursor to cancer in a fast and cost effective system is an important step towards personalized nanomedicine.

Secondary anchor targeted cell release.

Personalized medicine offers the promise of tailoring therapy to patients, based on their cellular biomarkers. To achieve this goal, cellular profiling systems are needed that can quickly and efficiently isolate specific cell types without disrupting cellular biomarkers. Here we describe the development of a unique platform that facilitates gentle cell capture via a secondary, surface-anchoring moiety, and cell release. The cellular capture system consists of a glass surface functionalized with APTES, d-desthiobiotin, and streptavidin. Biotinylated mCD11b and hIgG antibodies are used to capture mouse macrophages (RAW 264.7) and human breast cancer (MCF7-GFP) cell lines, respectively. The surface functionalization is optimized by altering assay components, such as streptavidin, d-desthiobiotin, and APTES, to achieve cell capture on 80% of the functionalized surface and cell release upon biotin treatment. We also demonstrate an ability to capture 50% of target cells within a dual-cell mixture. This engineering advancement is a critical step towards achieving cell isolation platforms for personalized medicine.

Plasmonic optical trapping in biologically relevant media.

We present plasmonic optical trapping of micron-sized particles in biologically relevant buffer media with varying ionic strength. The media consist of 3 cell-growth solutions and 2 buffers and are specifically chosen due to their widespread use and applicability to breast-cancer and angiogenesis studies. High-precision rheological measurements on the buffer media reveal that, in all cases excluding the 8.0 pH Stain medium, the fluids exhibit Newtonian behavior, thereby enabling straightforward measurements of optical trap stiffness from power-spectral particle displacement data. Using stiffness as a trapping performance metric, we find that for all media under consideration the plasmonic nanotweezers generate optical forces 3-4x a conventional optical trap. Further, plasmonic trap stiffness values are comparable to those of an identical water-only system, indicating that the performance of a plasmonic nanotweezer is not degraded by the biological media. These results pave the way for future biological applications utilizing plasmonic optical traps.

Engineering quantum dot calibration standards for quantitative fluorescent profiling.

Fluorescence-based tools, in particular optimized fluorophores, offer useful approaches to map cellular heterogeneity. Applying quantum dot (Qdot) technology towards heterogeneity profiling would be a novel approach for characterizing cellular dispersion and requires sensitive calibration standards. To this end, we have employed biotin-streptavidin chemistry to design improved quantitative, Qdot calibration beads. These calibration beads include commercially available Innovator's Tool Kit (ITK)-streptavidin Qdots (Qdots) conjugated to biotin coated polystyrene beads, providing a laboratory-accessible approach for quantitative calibration. We have engineered Qdot calibration beads at 525 nm, 565 nm, 605 nm, 655 nm, and 705 nm emission spectra. These beads provide calibration standards within the 0.0072-0.72 nM Qdot range, corresponding to an initial estimation of 800-80 000 Qdots per bead. We measured the proportion of Qdot loss during bead isolation steps and determined an accurate relationship between theoretical Qdot levels and actual Qdot levels using inductively coupled plasma mass spectrometry (ICP-MS). A linear relationship between fluorescence and Qdot number was observed with estimated concentrations of 1.13 × 10-14µg Cd/Qdot 525, 2.34 × 10-14µg Cd/Qdot 565, 1.49 × 10-13µg Cd/Qdot 605, 1.99 × 10-13µg Cd/Qdot 655 and 1.65 × 10-13µg Cd/Qdot 705. We also report the difference in concentration estimates between the fluorescence and ICP-MS methods. This study establishes the optimal conditions for preparing Qdot calibration beads from commercially available Qdots for quantitative, biophotonic applications including measuring cell surface heterogeneity.

Detection and quantification of poliovirus infection using FTIR spectroscopy and cell culture.

BACKGROUND: In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR) spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1) was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles. RESULTS: Buffalo green monkey kidney (BGMK) cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.). A partial least squares (PLS) regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV) of 17 plaque forming units (PFU)/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 103 to 106 PFU/ml, could also be reliably detected. CONCLUSIONS: This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and medical diagnostics.

Polymerized type I collagen reduces chronic cyclosporine nephrotoxicity.

BACKGROUND: Polymerized type I collagen (P-collagen) has been successfully used to reduce human hypertrophic scars due to its anti-fibrotic and anti-inflammatory properties. We therefore carried out a study to determine if P-collagen reduces functional and structural injury in chronic cyclosporine [cyclosporine A (CsA)] nephropathy. METHODS: Four groups of six male Wistar rats fed with a low sodium diet were treated with vehicle, P-collagen (0.8 mg/day, i.p.), CsA (15 mg/kg) or CsA + P-collagen for 15 days. Mean arterial pressure, renal blood flow and glomerular filtration rate were measured in all groups. Structural injury such as arteriolopathy, tubulo-interstitial fibrosis (TI-fibrosis) and positive apoptotic cells were quantified. The mRNA expression levels of transforming growth factor-beta (TGF-beta), kidney injury molecule (Kim-1), alpha-smooth muscle actin (alpha-SMA), glutathione peroxidase, catalase and Cu/Zn superoxide dismutase (SOD) as well as MnSOD were assessed. Antioxidant enzyme activity, renal lipoperoxidation and urinary excretion of oxygen peroxide (UH(2)O(2)V) were determined. RESULTS: Cyclosporine produced renal dysfunction and induced the development of arteriolopathy, TI-fibrosis and tubular apoptosis. These alterations were associated with increases in TGF-beta, Kim-1 and alpha-SMA mRNA levels as well as with a significant increase of oxidative stress and a reduction of SOD activity. P-Collagen partially ameliorated CsA-induced renal dysfunction and structural injury and prevented both tubular apoptosis and increased oxidative stress. This renoprotective effect was found to be associated with a reduction of TGF-beta, Kim-1 and alpha-SMA mRNA levels. CONCLUSIONS: This study has therefore demonstrated that P-collagen appears to have anti-fibrotic and anti-apoptotic properties and highlights the possibility that the compound might be useful in a strategy to reduce chronic CsA nephrotoxicity.