Functionalized Biomimetic Hydrogels Enhance Salivary Stem/Progenitor Cell Organization

Complex branched salivary structures remain challenging to replicate within implant ready hydrogels. We showed previously that hyaluronic acid (HA)-based hydrogels enable growth and organization of primary salivary-derived human stem/progenitor cells (hS/PCs) into multicellular spheroids. Here, we systematically functionalized three components of migration-permissive hydrogels to foster salivary tissue morphogenesis. We separately analyzed contributions of an enzymatically degradable crosslinker, a pendant integrin-binding site, and hydrogel porosity to best support high viability, integrin-dependent cell adhesion and migration. Structure size, frequency, and morphology were all affected by hydrogel crosslink density and integration of biofunctional peptides. Viability and proliferation data suggested that integration of integrin binding sites had the greatest effect on hS/PCs behavior. A larger internal matrix space, created by increasing both crosslinker length and PEG content, was needed to form large multicellular hS/PC structures. Peptide-modified hydrogels with more internal space shifted hS/PC organization from spheroidal, surrounded by thick basement membrane, to an asymmetric arrangement with punctate matrix proteins defining a "wrinkled" perimeter. Integrin-binding peptides activated integrin β1, with highest activation observed in hydrogels having both cleavable peptide and integrin ligand. The design parameters we prescribe allowed us to encapsulate hS/PCs in a humanized biomimetic hydrogel matrix able to support morphogenesis needed for salivary restoration.

Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity

Fibrosis is characterized by excessive production of disorganized collagen- and fibronectin-rich extracellular matrices (ECMs) and is driven by the persistence of myofibroblasts within tissues. A key protein contributing to myofibroblast differentiation is extra domain A fibronectin (EDA-FN). We sought to target and interfere with interactions between EDA-FN and its integrin receptors to effectively inhibit profibrotic activity and myofibroblast formation. Molecular docking was used to assist in the design of a blocking polypeptide (antifibrotic 38-amino-acid polypeptide, AF38Pep) for specific inhibition of EDA-FN associations with the fibroblast-expressed integrins α4β1 and α4β7. Blocking peptides were designed and evaluated in silico before synthesis, confirmation of binding specificity, and evaluation in vitro. We identified the high-affinity EDA-FN C-C′ loop binding cleft within integrins α4β1 and α4β7. The polypeptide with the highest predicted binding affinity, AF38Pep, was synthesized and could achieve specific binding to myofibroblast fibronectin-rich ECM and EDA-FN C-C′ loop peptides. AF38Pep demonstrated potent myofibroblast inhibitory activity at 10 µg/mL and was not cytotoxic. Treatment with AF38Pep prevented integrin α4β1-mediated focal adhesion kinase (FAK) activation and early signaling through extracellular-signal-regulated kinases 1 and 2 (ERK1/2), attenuated the expression of pro-matrix metalloproteinase 9 (MMP9) and pro-MMP2, and inhibited collagen synthesis and deposition. Immunocytochemistry staining revealed an inhibition of α-smooth muscle actin (α-SMA) incorporation into actin stress fibers and attenuated cell contraction. Increases in the expression of mRNA associated with fibrosis and downstream from integrin signaling were inhibited by treatment with AF38Pep. Our study suggested that AF38Pep could successfully interfere with EDA-FN C-C′ loop-specific integrin interactions and could act as an effective inhibitor of fibroblast of myofibroblast differentiation.

Structural assessment of SARS-CoV2 accessory protein ORF7a predicts LFA-1 and Mac-1 binding potential

ORF7a is an accessory protein common to SARS-CoV1 and the recently discovered SARS-CoV2, which is causing the COVID19 pandemic. The ORF7a protein has a structural homology with ICAM-1 which binds to the T lymphocyte integrin receptor LFA-1. As COVID19 has a strong immune component as part of the disease, we sought to determine whether SARS-CoV2 would have a similar structural interaction with LFA-1. Using molecular docking simulations, we found that SARS-CoV2 ORF7a has the key structural determinants required to bind LFA-1 but also the related leukocyte integrin Mac-1, which is also known to be expressed by macrophages. Our study shows that SARS-CoV2 ORF7a protein has a conserved Ig immunoglobulin-like fold containing an integrin binding site that provides a mechanistic hypothesis for SARS-CoV2‘s interaction with the human immune system. This suggests that experimental investigation of ORF7a mediated effects on immune cells such as T lymphocytes and macrophages (leukocytes) could help understand the disease further and develop effective treatments.

Fibronectin receptor integrin α5β1 regulates assembly of PP2A complexes through PDE4D: modulation of vascular inflammation and atherosclerosis

Fibronectin in the vascular wall promotes inflammatory activation of the endothelium during vascular remodeling and atherosclerosis. These effects are mediated in part by fibronectin binding to integrin α5, which recruits and activates phosphodiesterase 4D5 (PDE4D5) by inducing its dephosphorylation on an inhibitory site Ser651. Active PDE then hydrolyzes anti-inflammatory cAMP to facilitate inflammatory signaling. To test this model in vivo, we mutated the integrin binding site in PDE4D5 in mice. This mutation reduced endothelial inflammatory activation in athero-prone regions of arteries, and, in a hyperlipidemia model, reduced atherosclerotic plaque size while increasing markers of plaque stability. We then investigated the mechanism of PDE4D5 activation. Proteomics identified the PP2A regulatory subunit B55α as the factor recruiting PP2A to PDE4D5. The B55α-PP2A complex localized to adhesions and directly dephosphorylated PDE4D5. This interaction also unexpectedly stabilized the PP2A-B55α complex. The integrin-regulated, pro-atherosclerotic transcription factor Yap is also dephosphorylated and activated through this pathway. PDE4D5 therefore mediates matrix-specific regulation of EC phenotype via an unconventional adapter role, assembling and anchoring a multifunctional PP2A complex with other targets. These results are likely to have widespread consequences for control of cell function by integrins.

Kindlin-3 Mutation in Mesenchymal Stem Cells Results in Enhanced Chondrogenesis

ABSTRACTIdentifying patient mutations driving skeletal development disorders has driven our understanding of bone development. Integrin adhesion deficiency disease is caused by a Kindlin-3 (fermitin family member 3) mutation and its inactivation results in bleeding disorders and osteopenia. In this study, we uncover a role for Kindlin-3 in the differentiation of bone marrow mesenchymal stem cells (BMSCs) down the chondrogenic lineage. Kindlin-3 expression increased with chondrogenic differentiation similar to RUNX2. BMSCs isolated from a Kindlin-3 deficient patient expressed chondrocyte markers including SOX9 under basal conditions, which were further enhanced with chondrogenic differentiation. Rescue of integrin activation by a constitutively activated β3 integrin construct increased adhesion to multiple extracellular matrices and reduced SOX9 expression to basal levels. Growth plates from mice expressing a mutated Kindlin-3 with the integrin binding site ablated demonstrated alterations in chondrocyte maturation similar to that seen with the human Kindlin-3 deficient BMSCs. These findings suggest that Kindlin-3 expression mirrors RUNX2 during chondrogenesis.SUMMARYThis study by Kerr et al. describes a new role for Kindlin-3 in controlling early chondrocyte differentiation from mesenchymal stem cells and later hypertrophic differentiation of chondrocytes.