Mechanical Modulation of Cell Migrations by DNA Nanoassemblies

This proposal seeks to test whether mechanical affinity of the complex between RGD, a tripeptide domain found in proteins of extracellular medium, and integrin, a cell membrane receptor protein, is correlated with cell migrations. By attaching the RGD ligand to a magnetic bead via a DNA handle, integrins on cell surface can be evaluated for their binding affinity to the RGD by mechanically pulling away the binding complex using a pair of magnets in magnetic-tweezers instrument. During the pulling, a camera in the magnetic-tweezers setup is employed to record the distance between the magnetic bead and the magnet pair, which is converted to the binding force at the time of bead detachment. The same camera is also used to simultaneously monitor cell morphology changes that are related to cell migrations. Such a setup allows to correlate the mechanical affinity of the RGD-integrin complex to the cell migration. This new technique has also been successful in measuring the extraction force of lipid molecules such as cholesterol away from the cell membrane. Cholesterol is a critical component to increase the rigidity of the lipid rafts in the cell membrane. Cell migration is shown to be inhibited when lipid rafts are dissolved. Cell membrane receptors such as EGFR, HER2, and CD36 are found within lipid rafts. They may be impacted by compromised lipid rafts after cholesterol depletion. All these three receptors play a role in cell migration through cell signaling pathways such as the Src-Fak axis. We propose that these receptors may induce changes in cell migration when the structure and property of lipid rafts are altered by factors such as cholesterol supplement/depletion or overexpression/knock-down of EGFR/HER2/CD36 receptors. Integrin-RGD mechanical affinity will be used as a marker to follow changes in cell migration as integrin binds to the extracellular matrix via peptide domains such as RGD. When lipid rafts change their properties, integrin-RGD mechanical affinity may alter, which leads to variation in cell migration. Previously, mechanical affinity has been shown to correlate with binding affinity; however, it is unknown if mechanical affinity coordinates with intracellular signaling. By depleting cholesterol, migration will be inhibited. We expect that supplementation of cholesterol after depletion may restore cell migration. To investigate this restoration, we will evaluate changes in cell migration at either extracellular or intracellular level. At the extracellular level, we will again use the mechanical affinity of integrin-RGD as a marker for cell migration. At the intracellular level, western blot analysis will be used to follow changes in cell signaling by monitoring expression levels of phosphorylated proteins associated with cell migration. We aim to test if extracellular mechanical binding force corresponds with intercellular signaling as changes in migration occur. In summary, we will evaluate cell migration using integrin-RGD mechanical affinity when changes in lipid raft composition occur (Aim 1) and test if changes in mechanical affinity correspond with cell signaling intensity (Aim 2). 

Exosome derived from NAMPT-Overexpressed Mesenchymal Stem Cell Improves Aortic Valve Stenosis Outcome via Downregulation of miR-146a-3P Expression

Objective: We previously reported that deleting CXCR4 in endothelial cells leads to aortic valve stenosis (AVS). There is no pharmacotherapy available to treat or attenuate AVS. Nicotinamide Phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway. Extracellular NAMPT is exclusively carried by exosomes in the circulation and gets internalized in distant tissues. In this study, we tested the therapeutic effects of exosome derived from NAMPT-overexpressed mouse mesenchymal stem cell (MSC) on AVS in CXCR4 endothelial cell-specific knockout mice (EC CXCR4 KO), and whether microRNA dysregulation plays a role in the stenosis progression.

Methods:  Echocardiography was used to confirm the presence of AVS in 6 weeks old EC CXCR4 KO mice. The mice were then randomly divided into three groups (n=5). AVS mice were injected intraperitoneally with 100 ug of exosomes derived from either NAMPT-overexpressed MSCs, regular MSCs, or PBS once a week for three consecutive weeks. The expression of miRNAs (miR-146a-3P, miR-146a-5P, miR-125b-5P, and miR-142a-5P) was measured by qPCR in the cardiac endothelial cell of control (CXCR4fl/fl), AVS and exosome-treated AVS mice. Aortic valves were stained using Alizarin Red to detect calcification, and the percentage of calcification was compared between different groups.

Results: Treatment with exosome derived from NAMPT-overexpressed MSC significantly improved ejection fraction and reduced aortic valve (AV) peak velocity, AV peak pressure gradient, left ventricular internal dimension (LVID), and microcalcifications in AVS (EC CXCR4 KO) mice compared to those treated with PBS. Regular MSC-derived exosomes also showed a beneficial effect but were not statistically significant. Compared with the CXCR4fl/fl control mice, the expression of miR-146a-3P and miR-142a-5P in cardiac endothelial cells was significantly increased in mice with AVS, and exosome treatment showed a significant reduction of miR-146-3P expression.

Conclusions: Our data suggest that exosomes derived from NAMPT-overexpressing MSCs may improve the outcome of aortic valve stenosis, and miR-146a-3P might play an important role in the disease development and progression.

Using tree sloths as a model to better understand limb bone specializations in suspensory mammals

Primates and tree sloths use below-branch suspension, emphasizing high amounts of tensile limb loading. Prior studies of mammalian bone properties – mostly in upright mammals – have indicated that tensile bone strength is ~25% of compressive strength. However, the loading performance of primate and sloth limb bones has not been well investigated. The goal of the proposed research is to use in vitro bone strain analysis to understand how mammals that frequently load their limbs in tension do so without fear of long bone injury. A variety of mammalian taxa will be sampled to compare limb bone performance across various locomotor strategies.

Mara Voytek