The Role of Thrombin and Cell Contractility in Regulating Clustering and Collective Migration of Corneal Fibroblasts in Different ECM Environments

Miguel Miron-Mendoza, Eric Graham, Pouriska Kivanany, Jonathan Quiring, W. Matthew Petroll

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

PURPOSE: We previously reported that extracellular matrix composition (fibrin versus collagen) modulates the pattern of corneal fibroblast spreading and migration in 3-D culture. In this study, we investigate the role of thrombin and cell contractility in mediating these differences in cell behavior.

METHODS: To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen and fibrin matrices. To assess 3-dimensional cell migration, compacted collagen matrices seeded with corneal fibroblasts were embedded inside acellular collagen or fibrin matrices. Constructs were cultured in serum-free media containing platelet-derived growth factor (PDGF), with or without thrombin, the Rho kinase inhibitor Y-27632, and/or the myosin II inhibitor blebbistatin. We used 3-dimensional and 4-dimensional imaging to assess cell mechanical behavior, connectivity and cytoskeletal organization.

RESULTS: Thrombin stimulated increased contractility of corneal fibroblasts. Thrombin also induced Rho kinase-dependent clustering of cells plated on top of compliant collagen matrices, but not on rigid substrates. In contrast, cells on fibrin matrices coalesced into clusters even when Rho kinase was inhibited. In nested matrices, cells always migrated independently through collagen, even in the presence of thrombin. In contrast, cells migrating into fibrin formed an interconnected network. Both Y-27632 and blebbistatin reduced the migration rate in fibrin, but cells continued to migrate collectively.

CONCLUSIONS: The results suggest that while thrombin-induced actomyosin contraction can induce clustering of fibroblasts plated on top of compliant collagen matrices, it does not induce collective cell migration inside 3-D collagen constructs. Furthermore, increased contractility is not required for clustering or collective migration of corneal fibroblasts interacting with fibin.

Original languageEnglish (US)
Pages (from-to)2079-2090
Number of pages12
JournalInvestigative ophthalmology & visual science
Volume56
Issue number3
DOIs
StatePublished - Mar 1 2015

Fingerprint

Thrombin
Cluster Analysis
Fibroblasts
Fibrin
Collagen
rho-Associated Kinases
Cell Movement
Fibrillar Collagens
Myosin Type II
Actomyosin
Platelet-Derived Growth Factor
Serum-Free Culture Media
Extracellular Matrix

Keywords

  • 3-D culture
  • cell mechanics
  • corneal keratocytes
  • extracellular matrix
  • thrombin

ASJC Scopus subject areas

  • Medicine(all)

Cite this

The Role of Thrombin and Cell Contractility in Regulating Clustering and Collective Migration of Corneal Fibroblasts in Different ECM Environments. / Miron-Mendoza, Miguel; Graham, Eric; Kivanany, Pouriska; Quiring, Jonathan; Petroll, W. Matthew.

In: Investigative ophthalmology & visual science, Vol. 56, No. 3, 01.03.2015, p. 2079-2090.

Research output: Contribution to journalArticle

@article{9a61dccfe0a046edb6f80af109fcebdd,
title = "The Role of Thrombin and Cell Contractility in Regulating Clustering and Collective Migration of Corneal Fibroblasts in Different ECM Environments",
abstract = "PURPOSE: We previously reported that extracellular matrix composition (fibrin versus collagen) modulates the pattern of corneal fibroblast spreading and migration in 3-D culture. In this study, we investigate the role of thrombin and cell contractility in mediating these differences in cell behavior.METHODS: To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen and fibrin matrices. To assess 3-dimensional cell migration, compacted collagen matrices seeded with corneal fibroblasts were embedded inside acellular collagen or fibrin matrices. Constructs were cultured in serum-free media containing platelet-derived growth factor (PDGF), with or without thrombin, the Rho kinase inhibitor Y-27632, and/or the myosin II inhibitor blebbistatin. We used 3-dimensional and 4-dimensional imaging to assess cell mechanical behavior, connectivity and cytoskeletal organization.RESULTS: Thrombin stimulated increased contractility of corneal fibroblasts. Thrombin also induced Rho kinase-dependent clustering of cells plated on top of compliant collagen matrices, but not on rigid substrates. In contrast, cells on fibrin matrices coalesced into clusters even when Rho kinase was inhibited. In nested matrices, cells always migrated independently through collagen, even in the presence of thrombin. In contrast, cells migrating into fibrin formed an interconnected network. Both Y-27632 and blebbistatin reduced the migration rate in fibrin, but cells continued to migrate collectively.CONCLUSIONS: The results suggest that while thrombin-induced actomyosin contraction can induce clustering of fibroblasts plated on top of compliant collagen matrices, it does not induce collective cell migration inside 3-D collagen constructs. Furthermore, increased contractility is not required for clustering or collective migration of corneal fibroblasts interacting with fibin.",
keywords = "3-D culture, cell mechanics, corneal keratocytes, extracellular matrix, thrombin",
author = "Miguel Miron-Mendoza and Eric Graham and Pouriska Kivanany and Jonathan Quiring and Petroll, {W. Matthew}",
year = "2015",
month = "3",
day = "1",
doi = "10.1167/iovs.15-16388",
language = "English (US)",
volume = "56",
pages = "2079--2090",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "3",

}

TY - JOUR

T1 - The Role of Thrombin and Cell Contractility in Regulating Clustering and Collective Migration of Corneal Fibroblasts in Different ECM Environments

AU - Miron-Mendoza, Miguel

AU - Graham, Eric

AU - Kivanany, Pouriska

AU - Quiring, Jonathan

AU - Petroll, W. Matthew

PY - 2015/3/1

Y1 - 2015/3/1

N2 - PURPOSE: We previously reported that extracellular matrix composition (fibrin versus collagen) modulates the pattern of corneal fibroblast spreading and migration in 3-D culture. In this study, we investigate the role of thrombin and cell contractility in mediating these differences in cell behavior.METHODS: To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen and fibrin matrices. To assess 3-dimensional cell migration, compacted collagen matrices seeded with corneal fibroblasts were embedded inside acellular collagen or fibrin matrices. Constructs were cultured in serum-free media containing platelet-derived growth factor (PDGF), with or without thrombin, the Rho kinase inhibitor Y-27632, and/or the myosin II inhibitor blebbistatin. We used 3-dimensional and 4-dimensional imaging to assess cell mechanical behavior, connectivity and cytoskeletal organization.RESULTS: Thrombin stimulated increased contractility of corneal fibroblasts. Thrombin also induced Rho kinase-dependent clustering of cells plated on top of compliant collagen matrices, but not on rigid substrates. In contrast, cells on fibrin matrices coalesced into clusters even when Rho kinase was inhibited. In nested matrices, cells always migrated independently through collagen, even in the presence of thrombin. In contrast, cells migrating into fibrin formed an interconnected network. Both Y-27632 and blebbistatin reduced the migration rate in fibrin, but cells continued to migrate collectively.CONCLUSIONS: The results suggest that while thrombin-induced actomyosin contraction can induce clustering of fibroblasts plated on top of compliant collagen matrices, it does not induce collective cell migration inside 3-D collagen constructs. Furthermore, increased contractility is not required for clustering or collective migration of corneal fibroblasts interacting with fibin.

AB - PURPOSE: We previously reported that extracellular matrix composition (fibrin versus collagen) modulates the pattern of corneal fibroblast spreading and migration in 3-D culture. In this study, we investigate the role of thrombin and cell contractility in mediating these differences in cell behavior.METHODS: To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen and fibrin matrices. To assess 3-dimensional cell migration, compacted collagen matrices seeded with corneal fibroblasts were embedded inside acellular collagen or fibrin matrices. Constructs were cultured in serum-free media containing platelet-derived growth factor (PDGF), with or without thrombin, the Rho kinase inhibitor Y-27632, and/or the myosin II inhibitor blebbistatin. We used 3-dimensional and 4-dimensional imaging to assess cell mechanical behavior, connectivity and cytoskeletal organization.RESULTS: Thrombin stimulated increased contractility of corneal fibroblasts. Thrombin also induced Rho kinase-dependent clustering of cells plated on top of compliant collagen matrices, but not on rigid substrates. In contrast, cells on fibrin matrices coalesced into clusters even when Rho kinase was inhibited. In nested matrices, cells always migrated independently through collagen, even in the presence of thrombin. In contrast, cells migrating into fibrin formed an interconnected network. Both Y-27632 and blebbistatin reduced the migration rate in fibrin, but cells continued to migrate collectively.CONCLUSIONS: The results suggest that while thrombin-induced actomyosin contraction can induce clustering of fibroblasts plated on top of compliant collagen matrices, it does not induce collective cell migration inside 3-D collagen constructs. Furthermore, increased contractility is not required for clustering or collective migration of corneal fibroblasts interacting with fibin.

KW - 3-D culture

KW - cell mechanics

KW - corneal keratocytes

KW - extracellular matrix

KW - thrombin

UR - http://www.scopus.com/inward/record.url?scp=84961526728&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84961526728&partnerID=8YFLogxK

U2 - 10.1167/iovs.15-16388

DO - 10.1167/iovs.15-16388

M3 - Article

C2 - 25736789

AN - SCOPUS:84961526728

VL - 56

SP - 2079

EP - 2090

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 3

ER -