Elsevier

Journal of Surgical Research

Volume 253, September 2020, Pages 53-62
Journal of Surgical Research

Vascular
Inhibitory Effects of PRG4 on Migration and Proliferation of Human Venous Cells

https://doi.org/10.1016/j.jss.2020.03.028Get rights and content

Abstract

Background

Proteoglycan 4 (PRG4; lubricin) is a member of two gene co-expression network modules associated with human vein graft failure. However, little is known about PRG4 and the vascular system. Therefore, we have investigated the effects of recombinant human PRG4 (rhPRG4) on cell migration and proliferation in human veins.

Methods

Effects of rhPRG4 on cell migration, proliferation, and neointima formation were determined in human venous tissue and cultured venous smooth muscle cells (SMCs), adventitial cells, and endothelial cells. Expression of PRG4 by cultured human saphenous veins, failed vein grafts, and varicose veins was determined by immunostaining or Western blotting.

Results

Limited expression of PRG4 in fresh saphenous veins was dramatically increased around medial SMCs after culture ex vivo. rhPRG4 inhibited the migration of cultured SMCs, adventitial cells, and endothelial cells, as well as the proliferation of endothelial cells. rhPRG4 also inhibited the migration of SMCs and adventitial cells from tissue explants, but there was no effect on cell proliferation or neointima formation in ex vivo whole veins. Finally, PRG4 was largely absent in two examples of venous pathology, that is, failed human vein grafts and varicose veins.

Conclusions

Although rhPRG4 can inhibit the migration of venous SMCs, endothelial cells, and adventitial cells, and the proliferation of endothelial cells, PRG4 was only increased around medial SMCs in veins after ex vivo culture. PRG4 was not observed around medial SMCs in failed human vein grafts and varicose veins, suggesting the possibility that a failure of PRG4 upregulation may promote these pathologies.

Introduction

Saphenous vein grafts are commonly used to bypass atherosclerotic arteries of the heart and limbs. Unfortunately, up to 30% of these grafts fail midterm, that is, during the first 18 mo1, 2, 3 because of intimal hyperplasia and adverse remodeling by mechanisms that remain largely unknown.4 This contrasts with late-term failure (>2 y), which results from atherosclerosis that can be treated with drugs such as statins,5 and early failure (<1 mo), which results from thrombosis from technical problems. In contrast to late-term failure, atherosclerotic risk factors, such as plasma cholesterol and diabetes, do not predict midterm vein graft failure.6

We have recently reported that proteoglycan 4 (PRG4, lubricin) is a member of two novel weighted gene co-expression network modules that are associated with midterm human vein graft failure.7 PRG4 was first discovered as an N-terminal fragment that increased proliferative activity of cells of endothelial and hematopoietic lineage.8,9 It has anti-inflammatory and lubricating properties10,11 that protect against both age- and injury-induced osteoarthritis, surgical adhesions, and epithelial dysfunction in a model of cystitis.12, 13, 14, 15, 16, 17 PRG4 inhibits inflammation and fibrosis,10,13,14,18,19 which may play a role in vein graft failure.4,20 However, nothing is known about the potential effects on cell proliferation and migration, which play a central role in intimal hyperplasia. Therefore, we have investigated the effects of recombinant human PRG4 (rhPRG4) on cell migration and proliferation using ex vivo and in vitro human vein models.

Section snippets

Materials

Recombinant rhPRG4 was produced as an endotoxin-free full-length product in CHO-M cells by Lubris BioPharma (Framingham, MA, USA).21

Veins

Two different sets of veins from patients with normal kidney function were obtained for study. In the first, paired human saphenous vein remnants obtained at the time of peripheral vascular bypass and at the time of reoperation for vein graft stenosis were fixed for histology with patient consent under protocols approved by Asahikawa Medical University (Asahikawa,

PRG4 is increased in the media of veins after ex vivo culture

In human vein specimens left over from bypass surgery, PRG4 was seen associated with elastic fibers in the adventitia, endothelial cells of the vasa vasorum, and with remaining luminal endothelial cells (arrows, Fig. 1A). In regard to the latter, about half of the luminal endothelial cells are lost during surgical preparation of the grafts in the operating room (data not presented).29 There was little or no PRG4 immunostaining in the media or intima (Fig. 1A, the medial-adventitial boundary is

Discussion

We observed that rhPRG4 inhibits major aspects of the vascular response to injury—namely, cell migration and proliferation. In the case of migration, PRG4 inhibits SMCs, adventitial cells, and endothelial cells, whereas in the case of proliferation, it only inhibits endothelial cells. The integrated effect of inhibiting these three distinct cell types on intimal hyperplasia in a vein graft is not clear. The inhibitory effect on SMCs, which have been considered the major culprit cell of intimal

Acknowledgment

The authors thank Dr Nam Tran, Dr Niten Singh, Dr Ben Starnes, Dr Elina Quiroga, Dr Mark Meissner, Mike Crandall, and Wendy Hamar (Department of Surgery, University of Washington, Seattle, WA) for help with procurement of vein specimens. The authors also thank Alyssa Morin for help with Western blots.

This work was supported by an unrestricted gift from Lubris Biopharma, Inc (RDK); an American Heart Association Grant-in-Aid (RDK); and a Liaoning Province science and technology research project

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    1

    Current address: Department of Surgery, Ohio State University Wexner Medical Center, 452 W 10th Ave, Columbus, OH 43210, USA.

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