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Proteomic Analysis of the Pericyte Derived Extracellular Matrix

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Abstract

Pericytes (PC) line the post capillary venules and deposit extracellular matrix (ECM) proteins that provide vascular integrity during angiogenesis and regulatory signals during inflammation. Here we describe the characterization of soluble and structural ECM proteins that are deposited by PC. ECM components in PC-derived ECM were qualitatively and quantitatively analyzed via liquid chromatography/tandem mass spectrometry LC/MS–MS, while Western blot analysis was used to confirm the presence of commonly investigated matrix proteins. Functional annotation was used to categorize specific types of proteins, including structural collagens, glycoproteins, proteoglycans, ancillary angiogenic and inflammatory proteins. Further, the functional ability of PC derived ECM to support endothelial cell tubule formation, neutrophil adhesion, polarization and chemotaxis were evaluated. Our findings indicate that PC ECM is compositionally distinct to smooth muscle cell (SMC) ECM, and functionally supports vasculogenesis and neutrophil adhesion as well as SMC ECM. While differences in neutrophil polarization and cell ruffling were seen between PC ECM and SMC ECM adherent neutrophils, no difference in migratory velocities were observed. This first characterization of PC derived ECM provides insight into its potential use as a component of implantable engineered scaffolds and for investigations of basic cellular response to the microvascular basement membrane.

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Abbreviations

ECM:

Extracellular matrix

PC:

Pericytes

SMC:

Smooth muscle cells

TEVG:

Tissue engineered vascular grafts

EGF:

Epidermal growth factor

PDGF-B:

Platelet-derived growth factor subunit B

MMP:

Matrix metalloproteinase

fMLP:

Formyl-Met-Leu-Phe

ICAM-1:

Intercellular adhesion molecule-1

VCAM-1:

Vascular cell adhesion molecule-1

PECAM:

Platelet endothelial cell adhesion molecule

VE cadherin:

Vascular endothelial cadherin

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Acknowledgments

We would like to thank TuKiet Lam and Jean Kanyo at the W. M. Keck Mass Spectrometry and Proteomics Facility at Yale for their assistance with performing the mass spectrometry and protein identification. Support for L. A. B. is from NIH Grant 5T32DK101019-02. We extend our gratitude to Laura Niklason (Yale School of Medicine) for the kind gift of the SMC. Lastly, we acknowledge Yue Hou for use of the Matlab code to evaluate neutrophil ruffling.

Conflict of interest

Lola A. Brown, Parid Sava, Cesar Garcia, and Anjelica L. Gonzalez declare that they have no conflict of interest.

Ethical Standards

All human subject research was carried out in accordance with Yale University Human Investigation Committee (HIC) of the Internal Review Board (IRB) as part of the Human Research Protection Program guidelines and approved as HIC protocol #0902004786. No animal studies were performed for this article. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all donors who were included in the study.

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    Authors and Affiliations

    1. Biomedical Engineering, Yale University, 55 Prospect Street, New Haven, CT, 06520, USA

      Lola A. Brown, Parid Sava, Cesar Garcia & Anjelica L. Gonzalez

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    1. Lola A. Brown
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    2. Parid Sava
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    Correspondence to Anjelica L. Gonzalez.

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    Associate Editor Christine Schmidt oversaw the review of this article.

    Lola A. Brown and Parid Sava contributed equally to this work.

    This article is part of the 2015 Young Innovators Issue.

    Anjelica L. Gonzalez is the Donna L. Dubinsky Assistant Professor of Biomedical Engineering at Yale University. Prior to her arrival at Yale, she obtained a B.S. in Biological Engineering from Utah State University and a Ph.D. in Computational Biology from Baylor College of Medicine. Her graduate research in Larry V. McIntire’s lab was focused on the development of synthetic biomaterials for investigation of integrin dependent cell migration. This work translated directly into her current research, which is focused on the development of experimental models of human microvasculature for investigation of immunological processes. This research has demonstrated usefulness in advancing the current paradigm of the leukocyte adhesion cascade, identifying biochemical and mechanical regulators of leukocyte recruitment during inflammation. Anjelica’s work in synthetic extracellular matrices and regulation of leukocyte function has also been translated to the advancement and creation of therapeutic devices for treatment of lymphomas and dermal wounds.

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    Supplemental Fig. 1: Flow cytometric characterization of cultured PC isolated from human placental microvessels. Representative flow cytometry plots demonstrating that PC uniformly express the cell surface molecules NG2, CD90, PDGFR-β, and CD146 and the intracellular protein α-SMA. PC do not express the intracellular proteins SMMHC, a marker expressed by SMCs, CD31 or CD34, markers expressed by endothelial cells, or CD45, a marker expressed by leukocytes. Specific staining is shown by the bolded black line whereas isotype control is shown in grey.

    Supplemental Table 1: Full mass spectrometry protein Identification of pericyte and smooth muscle cell derived proteins from each donor

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    Supplementary material 2 (DOCX 171 kb)

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    Brown, L.A., Sava, P., Garcia, C. et al. Proteomic Analysis of the Pericyte Derived Extracellular Matrix. Cel. Mol. Bioeng. 8, 349–363 (2015). https://doi.org/10.1007/s12195-015-0408-5

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