Vascular smooth muscle cell (VSMC) dysfunction is a critical pathological process in postinjury intima hyperplasia. This process is driven by the adherence and accumulation of platelet-derived extracellular vesicles (PEVs) released from activated platelets to VSMCs at the site of injured intima. However, the precise mechanism remains unclear. Thus, the present study aimed to investigate how PEVs adhere to VSMCs and facilitate VSMC dysfunction in postinjury intimal hyperplasia. Morphological results confirmed that PEVs led to VSMC dysfunction and intimal hyperplasia. Integrated single-cell and proteomic analysis revealed that increased secreted phosphoprotein 1 (SPP1) expression in VSMCs played a central role in this process, possibly by mediating PEV adhesion to VSMCs and activating the focal adhesion kinase (FAK)/phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt) axis. In addition, integrin beta 3 (ITGβ3, CD61) on PEVs, with increased expression under pathological conditions, was predicted to interact with SPP1. Co-immunoprecipitation (Co-IP) analysis further confirmed that ITGβ3 interacted with SPP1, thereby activating the FAK/PI3K/AKT phosphorylation and promoting PEV adhesion. Of note, blocking ITGβ3 expression on PEVs reduced PEV adhesion and intimal hyperplasia. Thus, ITGβ3-SPP1-mediated PEV adhesion to VSMCs may be a novel mechanism in intimal hyperplasia, which proposed to be critical for vascular homeostasis.
© The author(s).

Platelet transfusions are essential for managing bleeding and hemostatic dysfunction and could be expanded as a cell therapy due to the multifunctional role of platelets in various diseases. Creating these cell therapies will require modifying transfusable donor platelets to express therapeutic proteins. However, there are currently no appropriate methods for genetically modifying platelets collected from blood donors. Here, we describe an approach using platelet-optimized lipid nanoparticles containing mRNA (mRNA-LNP) to enable exogenous protein expression in human and rat platelets. Within the library of mRNA-LNP tested, exogenous protein expression did not require nor correlate with platelet activation. Transfected platelets retained hemostatic function and accumulated in regions of vascular damage after transfusion into rats with hemorrhagic shock. We expect this technology will expand the therapeutic potential of platelets.

Platelet-neutrophil complexes (PNCs) occur during the inflammatory response to trauma and infections, and their interactions enable cell activation that can lead to tissue destruction. The ability to identify the accumulation and tissue localisation of PNCs is necessary to further understand their role in the organs associated with blast-induced shock wave trauma. Relevant experimental lung injury models often utilise pigs and rats, species for which immunohistochemistry protocols to detect platelets and neutrophils have yet to be established. Therefore, monoplex and multiplex immunohistochemistry protocols were established to evaluate the application of 22 commercially available antibodies to detect platelet (nine rat and five pig) and/or neutrophil (four rat and six pig) antigens identified as having potential selectivity for porcine or rat tissue, using lung and liver sections taken from models of polytrauma, including blast lung injury. Of the antibodies evaluated, one antibody was able to detect rat neutrophil elastase (on frozen and formalin-fixed paraffin embedded (FFPE) sections), and one antibody was successful in detecting rat CD61 (frozen sections only); whilst one antibody was able to detect porcine MPO (frozen and FFPE sections) and antibodies, targeting CD42b or CD49b antigens, were able to detect porcine platelets (frozen and FFPE and frozen, respectively). Staining procedures for platelet and neutrophil antigens were also successful in detecting the presence of PNCs in both rat and porcine tissue. We have, therefore, established protocols to allow for the detection of PNCs in lung and liver sections from porcine and rat models of trauma, which we anticipate should be of value to others interested in investigating these cell types in these species.

Mechanical properties of the tumor microenvironment have emerged as key factors in tumor progression. It has been proposed that increased tissue stiffness can transform stromal fibroblasts into carcinoma-associated fibroblasts. However, it is unclear whether the three to five times increase in stiffness seen in tumor-adjacent stroma is sufficient for fibroblast activation. In this study we developed a three-dimensional (3D) hydrogel model with precisely tunable stiffness and show that a physiologically relevant increase in stiffness is sufficient to lead to fibroblast activation. We found that soluble factors including CC-motif chemokine ligand (CCL) chemokines and fibronectin are necessary for this activation, and the combination of C-C chemokine receptor type 4 (CCR4) chemokine receptors and β1 and β3 integrins are necessary to transduce these chemomechanical signals. We then show that these chemomechanical signals lead to the gene expression changes associated with fibroblast activation via a network of intracellular signaling pathways that include focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K). Finally, we identify the actin-associated protein palladin as a key node in these signaling pathways that result in fibroblast activation.
Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The adhesion receptor β3 integrin regulates diverse cellular functions in various tissues. As β3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of β3 integrin in cardiac fibrosis by using wild type (WT) and β3 integrin null (β3-/-) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, β3-/- mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from β3-/- mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in β3-/- mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and β3-/- mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that β3-/- cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in β3-/- cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that β3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.