Department of PathologyVanderbilt UniversityNashville, Tennessee 37232SummaryInvadopodia are acti... more Department of PathologyVanderbilt UniversityNashville, Tennessee 37232SummaryInvadopodia are actin-rich subcellular protrusions with as-sociatedproteasesusedbycancercellstodegradeextracel-lularmatrix(ECM)[1].Molecularcomponentsofinvadopodiainclude branched actin-assembly proteins, membrane traf-ficking proteins, signaling proteins, and transmembraneproteinases [1]. Similar structures exist in nontransformedcells, such as osteoclasts and dendritic cells, but are gener-ally called podosomes and are thought to be more involvedin cell-matrix adhesion than invadopodia [2–4]. Despite inti-mate contact with their ECM substrates, it is unknownwhether physical or chemical ECM signals regulate invado-podia function. Here, we report that ECM rigidity directly in-creases boththe number andactivity ofinvadopodia. Trans-duction of ECM-rigidity signals depends on the cellularcontractile apparatus [5–7], given that inhibition of non-muscle myosinII,myosinlightchain kinase,andRhokinaseall abrogate in...
Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, Jan 13, 2018
During the dermal wound healing process, the mechanical rigidity of the newly deposited extracell... more During the dermal wound healing process, the mechanical rigidity of the newly deposited extracellular matrix and transforming growth factor-β1 promote the transition of fibroblasts into myofibroblasts. Myofibroblasts generate large cellular forces that contract and remodel the extracellular matrix leading to scar formation. In contrast, myofibroblasts are not detected in fetal dermal wounds which are more compliant and contain less transforming growth factor-β1 than adult wounds. Instead, fetal fibroblasts orchestrate scarless healing of dermal wounds resulting in healed tissues that resemble uninjured dermis. While these biomechanical differences suggest that the fetal wound environment promotes smaller cellular forces which enable regeneration, previous studies indicate that fetal fibroblasts have unique contractile properties that may facilitate scarless dermal repair. Therefore, we tested whether physiologic wound rigidities and transforming growth factor-β1 induce contractile f...
Rho-associated kinase (ROCK) activity drives cell migration via actomyosin contractility. During ... more Rho-associated kinase (ROCK) activity drives cell migration via actomyosin contractility. During invasion, individual cancer cells can transition between 2 modes of migration, mesenchymal and amoeboid. Changes in ROCK activity can cause a switch between these migration phenotypes which are defined by distinct morphologies. However, recent studies have shown that the ROCK isoforms are not functionally redundant as previously thought. Therefore, it is unclear whether the ROCK isoforms play different roles in regulating migration phenotypes. Here, we found that ROCK1 and ROCK2 differentially regulate carcinoma cell morphology resulting in intermediate phenotypes that share some mesenchymal and amoeboid characteristics. These findings suggest that the ROCK isoforms play unique roles in the phenotypic plasticity of mesenchymal carcinoma cells which may have therapeutic implications.
Injured skin in the mammalian fetus can heal regeneratively due to the ability of fetal fibroblas... more Injured skin in the mammalian fetus can heal regeneratively due to the ability of fetal fibroblasts to effectively reorganize the extracellular matrix (ECM). This process occurs without fetal fibroblasts differentiating into highly contractile myofibroblasts which cause scarring and fibrosis in adult wounds. Here, we provide a brief review of fetal wound healing and the evidence supporting a unique contractile phenotype in fetal fibroblasts. Furthermore, we discuss the biomechanical role of the ECM in driving myofibroblast differentiation in wound healing and the implications for new clinical modalities based on the biophysical properties of fetal fibroblasts. We and others have found that fetal fibroblasts are refractory to the environmental stimuli necessary for myofibroblast differentiation in adult wound healing including mechanical stress. Understanding the biomechanical mechanisms that regulate the contractile phenotype of fetal fibroblasts may unlock new avenues for anti-scarring therapies that target myofibroblast differentiation of adult fibroblasts.
Mechanical rigidity in the tumor microenvironment is associated with a high risk of tumor formati... more Mechanical rigidity in the tumor microenvironment is associated with a high risk of tumor formation and aggressiveness. Adhesion-based signaling driven by a rigid microenvironment is thought to facilitate invasion and migration of cancer cells away from primary tumors. Proteolytic degradation of extracellular matrix (ECM) is a key component of this process and is mediated by subcellular actin-rich structures known as invadopodia. Both ECM rigidity and cellular traction stresses promote invadopodia formation and activity, suggesting a role for these structures in mechanosensing. The presence and activity of mechanosensitive adhesive and signaling components at invadopodia further indicates the potential for these structures to utilize myosin-dependent forces to probe and remodel their ECM environments. Here, we provide a brief review of the role of adhesion-based mechanical signaling in controlling invadopodia and invasive cancer behavior.
Keloid formation has been linked to aberrant fibroblast activity, exacerbated by growth factors a... more Keloid formation has been linked to aberrant fibroblast activity, exacerbated by growth factors and inflammatory mediators. Prostaglandin E2 (PGE2), synthesized from arachidonic acid by cyclooxygenases (COX) and synthases (PGES), acts as both an inflammatory mediator and fibroblast modulator. Although PGE2 has known antifibrotic effects in the lower airway, its role in dermal fibrosis in general, and keloid formation in particular, remains unclear. This study focused on: (1) the effects of PGE2 on keloid fibroblast migration, contraction, and collagen synthesis and (2) endogenous PGE2 synthesis in response interleukin-1b. PGE2 decreased keloid fibroblast migration and contraction via an EP2/ EP4-cAMP mechanism that disrupted actin cytoskeletal dynamics and reversed transforming growth factor-b1-induced collagen I and III synthesis. Impaired fibroblast PGE2 production has been linked to lower airway fibrosis and recently to keloid formation. Here, we showed that interleukin-1b stimulation leads to nuclear factor-kB translocation to the nucleus, resulting in up-regulation of COX-2 and microsomal PGE2 synthase 1. Up-regulation of COX-2 in, and secretion of PGE2 by keloid fibroblasts are diminished compared with their normal fibroblast counterparts. We suggest that the antifibrotic effects of PGE2 during keloid formation are potentially diminished due to aberrant paracrine fibroblast signaling. Exogenous PGE2 may supplement decreased endogenous levels and inhibit keloid formation or progression. Wound Rep Reg (2007) 15 122-133 c 2007 by the Wound Healing Society
Cyclooxygenase-2 is up-regulated shortly after dermal injury and it has been shown to have import... more Cyclooxygenase-2 is up-regulated shortly after dermal injury and it has been shown to have important activity during the repair process. Its main product in the skin, prostaglandin E2 (PGE2), modulates both inflammatory and fibrotic processes during wound healing and partially dictates the overall outcome of wound healing. PGE2 signaling has been shown to be altered during fetal wound healing. This study was designed to examine the mechanism(s) by which PGE2 regulates fibroblast migration and contraction and to determine whether these mechanisms are conserved in fetal-derived dermal fibroblasts. Fetal and adult dermal fibroblasts express all four PGE2 receptors. PGE2 inhibits fetal and adult fibroblast migration in a dose-dependent manner through the EP2/EP4-cAMP-protein kinase A pathway. However, fetal fibroblasts appear to be refractory to this effect, requiring a 10-fold higher concentration of PGE2 to achieve a similar degree of inhibition as adult fibroblasts. Inhibition of adult fibroblast migration correlated with disruption of the actin cytoskeleton. In contrast, PGE2 or a cAMP analog did not disrupt the actin cytoskeleton of fetal dermal fibroblasts. These findings were extended using a modified free-floating, fibroblast-populated collagen lattice (FPCL) contraction assay designed to measure fibroblast contraction. PGE2-inhibited FPCL contraction by adult fibroblasts, but fetal fibroblasts exhibited higher rates of FPCL contraction and a blunted response to exogenous modulation by PGE2 or a cyclase activator (forskolin). These findings indicate that fetal dermal fibroblasts are partially refractory to the effects of PGE2, a major inflammatory mediator associated with dermal wound healing. This effect may have significant and specific relevance to the scarless fetal wound-healing phenotype.
In contrast to fetal wound healing, dermal adult wound healing results in imperfect repair and sc... more In contrast to fetal wound healing, dermal adult wound healing results in imperfect repair and scar formation. Fibroblasts are responsible for the contraction and remodeling of the wound matrix, which is influenced by inflammatory mediators including prostaglandin E2 (PGE2). This study addresses the mechanism by which PGE2 regulates contraction of collagen gels by human fetal and adult dermal fibroblasts. We hypothesized that the intrinsic phenotypic properties of the two types of fibroblasts and their responses to PGE2 alter their contraction properties and contribute to different wound healing outcomes. Contraction was evaluated using free-floating fibroblast-populated collagen gels that contract by migratory forces. PGE2 was found to differentially inhibit collagen gel contraction by fetal and adult fibroblasts. This effect was mimicked by a specific PGE2 receptor agonist as well as by two pharmacological agents, indicating a cyclic adenosine monophosphate-dependent signaling pathway mediated through the EP2 receptor. Our results indicate that fetal fibroblast contraction is maintained by a more stable actin cytoskeleton. Therefore, the migratory phenotype may be sufficient for physical remodeling of the wound matrix leading to regenerative repair. Maintenance of this phenotype in the later stages of wound healing could potentially be achieved by targeting cyclic adenosine monophosphate via the EP2 receptor.
Fetal dermal fibroblasts participate in a dramatically different wound healing process compared t... more Fetal dermal fibroblasts participate in a dramatically different wound healing process compared to their adult counterparts, and it is thought that their intrinsic phenotype contributes to the unique properties of fetal repair. In particular, fibroblast migratory and contractile properties have been shown to be important in the development or lack of fibrosis/scarring. Despite extensive study to date, and multiple experimental techniques utilized by various laboratories, the precise differences between fetal and adult dermal fibroblasts remain unclear. We characterized the migratory and contractile dynamics of fetal dermal fibroblasts at the individual cell and population levels under both 2-dimensional (2D) and 3-dimensional (3D) constraints. Data indicate that (1) individual fetal fibroblasts attach and locomote quicker than adult fibroblasts, resulting in faster migration at the population level; (2) use of a 2D bioactive matrix (collagen) dramatically speeds up the transition from attachment to locomotion; and (3) fetal fibroblasts compact 2D collagen matrices faster than adult fibroblasts. These characteristics are maintained inside of a novel 3D construct, which approximates some in vivo tissue repair dynamics. Specifically, fetal fibroblasts invade this construct faster than adult fibroblasts, likely through more dynamic interactions with surrounding collagen fibers. In conclusion, the hyperactive migratory and contractile dynamics of fetal fibroblasts are qualitatively and quantitatively conserved despite transitions from individual cells to whole populations and from 2D to 3D constraints. We conclude that fetal fibroblasts display a robust phenotype, which is only partially altered by changes in substrate and geometric constraints. This phenotype likely is important in dictating the dynamics of fetal tissue repair.
Collagen gels can serve as biomaterials ideal for tissue equivalents, especially if they are remo... more Collagen gels can serve as biomaterials ideal for tissue equivalents, especially if they are remodeled to have fibril anisotropy mimicking native tissue. Type I collagen gel remodeling was studied microscopically to investigate the changes caused by fibroblasts in collagen gel structures, with and without the growth factors PDGF-BB and TGF-beta1. A bidirectional laser trap microrheometry technique was developed that revealed a high degree of local heterogeneity and anisotropy in the structure of the collagen gels during active fibroblast contraction. The use of the growth factors increased not only the gel anisotropy, but the heterogeneity as well, indicating further changes in the collagen fibril orientations. This work shows the ability to influence the remodeling capabilities of fibroblasts by using growth factors in order to begin to elucidate the changes in the local mechanical environment of contracting collagen gels. We present this experimental technique as a method for probing changes in the fibroblast-driven anisotropy of collagen gels as a basis for understanding microstructural tissue organization important in the development of collagen-based tissue equivalents.
Department of PathologyVanderbilt UniversityNashville, Tennessee 37232SummaryInvadopodia are acti... more Department of PathologyVanderbilt UniversityNashville, Tennessee 37232SummaryInvadopodia are actin-rich subcellular protrusions with as-sociatedproteasesusedbycancercellstodegradeextracel-lularmatrix(ECM)[1].Molecularcomponentsofinvadopodiainclude branched actin-assembly proteins, membrane traf-ficking proteins, signaling proteins, and transmembraneproteinases [1]. Similar structures exist in nontransformedcells, such as osteoclasts and dendritic cells, but are gener-ally called podosomes and are thought to be more involvedin cell-matrix adhesion than invadopodia [2–4]. Despite inti-mate contact with their ECM substrates, it is unknownwhether physical or chemical ECM signals regulate invado-podia function. Here, we report that ECM rigidity directly in-creases boththe number andactivity ofinvadopodia. Trans-duction of ECM-rigidity signals depends on the cellularcontractile apparatus [5–7], given that inhibition of non-muscle myosinII,myosinlightchain kinase,andRhokinaseall abrogate in...
Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, Jan 13, 2018
During the dermal wound healing process, the mechanical rigidity of the newly deposited extracell... more During the dermal wound healing process, the mechanical rigidity of the newly deposited extracellular matrix and transforming growth factor-β1 promote the transition of fibroblasts into myofibroblasts. Myofibroblasts generate large cellular forces that contract and remodel the extracellular matrix leading to scar formation. In contrast, myofibroblasts are not detected in fetal dermal wounds which are more compliant and contain less transforming growth factor-β1 than adult wounds. Instead, fetal fibroblasts orchestrate scarless healing of dermal wounds resulting in healed tissues that resemble uninjured dermis. While these biomechanical differences suggest that the fetal wound environment promotes smaller cellular forces which enable regeneration, previous studies indicate that fetal fibroblasts have unique contractile properties that may facilitate scarless dermal repair. Therefore, we tested whether physiologic wound rigidities and transforming growth factor-β1 induce contractile f...
Rho-associated kinase (ROCK) activity drives cell migration via actomyosin contractility. During ... more Rho-associated kinase (ROCK) activity drives cell migration via actomyosin contractility. During invasion, individual cancer cells can transition between 2 modes of migration, mesenchymal and amoeboid. Changes in ROCK activity can cause a switch between these migration phenotypes which are defined by distinct morphologies. However, recent studies have shown that the ROCK isoforms are not functionally redundant as previously thought. Therefore, it is unclear whether the ROCK isoforms play different roles in regulating migration phenotypes. Here, we found that ROCK1 and ROCK2 differentially regulate carcinoma cell morphology resulting in intermediate phenotypes that share some mesenchymal and amoeboid characteristics. These findings suggest that the ROCK isoforms play unique roles in the phenotypic plasticity of mesenchymal carcinoma cells which may have therapeutic implications.
Injured skin in the mammalian fetus can heal regeneratively due to the ability of fetal fibroblas... more Injured skin in the mammalian fetus can heal regeneratively due to the ability of fetal fibroblasts to effectively reorganize the extracellular matrix (ECM). This process occurs without fetal fibroblasts differentiating into highly contractile myofibroblasts which cause scarring and fibrosis in adult wounds. Here, we provide a brief review of fetal wound healing and the evidence supporting a unique contractile phenotype in fetal fibroblasts. Furthermore, we discuss the biomechanical role of the ECM in driving myofibroblast differentiation in wound healing and the implications for new clinical modalities based on the biophysical properties of fetal fibroblasts. We and others have found that fetal fibroblasts are refractory to the environmental stimuli necessary for myofibroblast differentiation in adult wound healing including mechanical stress. Understanding the biomechanical mechanisms that regulate the contractile phenotype of fetal fibroblasts may unlock new avenues for anti-scarring therapies that target myofibroblast differentiation of adult fibroblasts.
Mechanical rigidity in the tumor microenvironment is associated with a high risk of tumor formati... more Mechanical rigidity in the tumor microenvironment is associated with a high risk of tumor formation and aggressiveness. Adhesion-based signaling driven by a rigid microenvironment is thought to facilitate invasion and migration of cancer cells away from primary tumors. Proteolytic degradation of extracellular matrix (ECM) is a key component of this process and is mediated by subcellular actin-rich structures known as invadopodia. Both ECM rigidity and cellular traction stresses promote invadopodia formation and activity, suggesting a role for these structures in mechanosensing. The presence and activity of mechanosensitive adhesive and signaling components at invadopodia further indicates the potential for these structures to utilize myosin-dependent forces to probe and remodel their ECM environments. Here, we provide a brief review of the role of adhesion-based mechanical signaling in controlling invadopodia and invasive cancer behavior.
Keloid formation has been linked to aberrant fibroblast activity, exacerbated by growth factors a... more Keloid formation has been linked to aberrant fibroblast activity, exacerbated by growth factors and inflammatory mediators. Prostaglandin E2 (PGE2), synthesized from arachidonic acid by cyclooxygenases (COX) and synthases (PGES), acts as both an inflammatory mediator and fibroblast modulator. Although PGE2 has known antifibrotic effects in the lower airway, its role in dermal fibrosis in general, and keloid formation in particular, remains unclear. This study focused on: (1) the effects of PGE2 on keloid fibroblast migration, contraction, and collagen synthesis and (2) endogenous PGE2 synthesis in response interleukin-1b. PGE2 decreased keloid fibroblast migration and contraction via an EP2/ EP4-cAMP mechanism that disrupted actin cytoskeletal dynamics and reversed transforming growth factor-b1-induced collagen I and III synthesis. Impaired fibroblast PGE2 production has been linked to lower airway fibrosis and recently to keloid formation. Here, we showed that interleukin-1b stimulation leads to nuclear factor-kB translocation to the nucleus, resulting in up-regulation of COX-2 and microsomal PGE2 synthase 1. Up-regulation of COX-2 in, and secretion of PGE2 by keloid fibroblasts are diminished compared with their normal fibroblast counterparts. We suggest that the antifibrotic effects of PGE2 during keloid formation are potentially diminished due to aberrant paracrine fibroblast signaling. Exogenous PGE2 may supplement decreased endogenous levels and inhibit keloid formation or progression. Wound Rep Reg (2007) 15 122-133 c 2007 by the Wound Healing Society
Cyclooxygenase-2 is up-regulated shortly after dermal injury and it has been shown to have import... more Cyclooxygenase-2 is up-regulated shortly after dermal injury and it has been shown to have important activity during the repair process. Its main product in the skin, prostaglandin E2 (PGE2), modulates both inflammatory and fibrotic processes during wound healing and partially dictates the overall outcome of wound healing. PGE2 signaling has been shown to be altered during fetal wound healing. This study was designed to examine the mechanism(s) by which PGE2 regulates fibroblast migration and contraction and to determine whether these mechanisms are conserved in fetal-derived dermal fibroblasts. Fetal and adult dermal fibroblasts express all four PGE2 receptors. PGE2 inhibits fetal and adult fibroblast migration in a dose-dependent manner through the EP2/EP4-cAMP-protein kinase A pathway. However, fetal fibroblasts appear to be refractory to this effect, requiring a 10-fold higher concentration of PGE2 to achieve a similar degree of inhibition as adult fibroblasts. Inhibition of adult fibroblast migration correlated with disruption of the actin cytoskeleton. In contrast, PGE2 or a cAMP analog did not disrupt the actin cytoskeleton of fetal dermal fibroblasts. These findings were extended using a modified free-floating, fibroblast-populated collagen lattice (FPCL) contraction assay designed to measure fibroblast contraction. PGE2-inhibited FPCL contraction by adult fibroblasts, but fetal fibroblasts exhibited higher rates of FPCL contraction and a blunted response to exogenous modulation by PGE2 or a cyclase activator (forskolin). These findings indicate that fetal dermal fibroblasts are partially refractory to the effects of PGE2, a major inflammatory mediator associated with dermal wound healing. This effect may have significant and specific relevance to the scarless fetal wound-healing phenotype.
In contrast to fetal wound healing, dermal adult wound healing results in imperfect repair and sc... more In contrast to fetal wound healing, dermal adult wound healing results in imperfect repair and scar formation. Fibroblasts are responsible for the contraction and remodeling of the wound matrix, which is influenced by inflammatory mediators including prostaglandin E2 (PGE2). This study addresses the mechanism by which PGE2 regulates contraction of collagen gels by human fetal and adult dermal fibroblasts. We hypothesized that the intrinsic phenotypic properties of the two types of fibroblasts and their responses to PGE2 alter their contraction properties and contribute to different wound healing outcomes. Contraction was evaluated using free-floating fibroblast-populated collagen gels that contract by migratory forces. PGE2 was found to differentially inhibit collagen gel contraction by fetal and adult fibroblasts. This effect was mimicked by a specific PGE2 receptor agonist as well as by two pharmacological agents, indicating a cyclic adenosine monophosphate-dependent signaling pathway mediated through the EP2 receptor. Our results indicate that fetal fibroblast contraction is maintained by a more stable actin cytoskeleton. Therefore, the migratory phenotype may be sufficient for physical remodeling of the wound matrix leading to regenerative repair. Maintenance of this phenotype in the later stages of wound healing could potentially be achieved by targeting cyclic adenosine monophosphate via the EP2 receptor.
Fetal dermal fibroblasts participate in a dramatically different wound healing process compared t... more Fetal dermal fibroblasts participate in a dramatically different wound healing process compared to their adult counterparts, and it is thought that their intrinsic phenotype contributes to the unique properties of fetal repair. In particular, fibroblast migratory and contractile properties have been shown to be important in the development or lack of fibrosis/scarring. Despite extensive study to date, and multiple experimental techniques utilized by various laboratories, the precise differences between fetal and adult dermal fibroblasts remain unclear. We characterized the migratory and contractile dynamics of fetal dermal fibroblasts at the individual cell and population levels under both 2-dimensional (2D) and 3-dimensional (3D) constraints. Data indicate that (1) individual fetal fibroblasts attach and locomote quicker than adult fibroblasts, resulting in faster migration at the population level; (2) use of a 2D bioactive matrix (collagen) dramatically speeds up the transition from attachment to locomotion; and (3) fetal fibroblasts compact 2D collagen matrices faster than adult fibroblasts. These characteristics are maintained inside of a novel 3D construct, which approximates some in vivo tissue repair dynamics. Specifically, fetal fibroblasts invade this construct faster than adult fibroblasts, likely through more dynamic interactions with surrounding collagen fibers. In conclusion, the hyperactive migratory and contractile dynamics of fetal fibroblasts are qualitatively and quantitatively conserved despite transitions from individual cells to whole populations and from 2D to 3D constraints. We conclude that fetal fibroblasts display a robust phenotype, which is only partially altered by changes in substrate and geometric constraints. This phenotype likely is important in dictating the dynamics of fetal tissue repair.
Collagen gels can serve as biomaterials ideal for tissue equivalents, especially if they are remo... more Collagen gels can serve as biomaterials ideal for tissue equivalents, especially if they are remodeled to have fibril anisotropy mimicking native tissue. Type I collagen gel remodeling was studied microscopically to investigate the changes caused by fibroblasts in collagen gel structures, with and without the growth factors PDGF-BB and TGF-beta1. A bidirectional laser trap microrheometry technique was developed that revealed a high degree of local heterogeneity and anisotropy in the structure of the collagen gels during active fibroblast contraction. The use of the growth factors increased not only the gel anisotropy, but the heterogeneity as well, indicating further changes in the collagen fibril orientations. This work shows the ability to influence the remodeling capabilities of fibroblasts by using growth factors in order to begin to elucidate the changes in the local mechanical environment of contracting collagen gels. We present this experimental technique as a method for probing changes in the fibroblast-driven anisotropy of collagen gels as a basis for understanding microstructural tissue organization important in the development of collagen-based tissue equivalents.
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Papers by Aron Parekh