Justin Parreno - Academia.edu (original) (raw)

Papers by Justin Parreno

Wound Repair and Regeneration, 2008

ABSTRACTPrevious studies have shown that the Yorkshire (Y) pig is a model for normal skin wound h... more ABSTRACTPrevious studies have shown that the Yorkshire (Y) pig is a model for normal skin wound healing, while red Duroc (RD) pigs form hypercontracted scars similar to human hypertrophic scars. In order to determine potential intrinsic differences in fibroblast phenotypes, the ability of normal dorsal and ventral dermal fibroblasts from Y and RD pigs to contract collagen gels was assessed. Cells plated in gels were cultured in media supplemented with 2% or 10% FBS ± 1 or 10 ng/mL transfroming growth factor β1. The degree of contraction of the gels was quantified at defined time‐points postrelease. Final contraction levels were dependent on cell density and serum concentration for all cell types. The rates of contraction of RD dorsal fibroblasts were significantly greater than those for Y dorsal fibroblasts. Immunocytochemical analysis revealed the presence of α‐smooth muscle actin in contracted cells. Furthermore, mRNA levels for matrix metalloproteinase‐2 and decorin showed specif...

Molecular and Cellular Biochemistry, 2008

To investigate the molecular aspects of osteoblastic interactions with a type I collagen matrix, ... more To investigate the molecular aspects of osteoblastic interactions with a type I collagen matrix, human osteoblast-like MG-63 cells were cultured in three-dimensional (3D) collagen I gels. MG-63 cells in collagen gels expressed higher osteocalcin mRNA levels than cells in monolayer (2D) on polystyrene surfaces. Gel contraction was assessed via releasing the collagen gels from attachment following 24 h incubation in serum free, TGF-beta1-treated, or 1,25-(OH)(2)D(3)-treated media. 10 ng/ml of TGF-beta1 was optimal for enhancing contraction and led to decreased osteocalcin mRNA levels. In contrast, 50 nM 1,25-(OH)(2)D(3) led to increased osteocalcin mRNA levels, but did not affect contraction. Furthermore, the effect of contraction on gene expression was examined by releasing a subset of gels after 24 h and assessing mRNA levels by RT-PCR. Contracting gels exhibited temporally regulated differential increases in MMP-1, MMP-3, and alpha(2) integrin mRNA levels at specific time points post release. Cytochalasin D treatment immediately following release of gels inhibited contraction in a dose-dependent manner as well as prevented upregulation of MMP-1, MMP-3, and alpha2 integrin mRNA levels in contracting gels. These results suggest that osteoblastic cells generate internal loads that may affect specific gene expression, and these changes can be altered in the presence of biomediators.

Biochemistry and Cell Biology, 2009

Culture of human osteoblast-like MG-63 cells within collagen gels results in the generation of in... more Culture of human osteoblast-like MG-63 cells within collagen gels results in the generation of intrinsic stress. Release of such collagen gels from attachment results in gel contraction and enhanced MMP-1, MMP-3, and α2 integrin mRNA levels. To understand the potential role of microtubules and signaling pathways involved in MG-63 cell-mediated contraction and gene expression, cells were cultured in collagen gels. After 24 h collagen gels were released, then immediately treated with nocodazole or specific protein kinase inhibitors. Contraction was assessed, RNA isolated, and real-time PCR analysis performed. Treatment with high concentrations of a microtubule depolymerization agent, nocodazole, enhanced early contraction and led to elevated mRNA levels for MMP-3, whereas low concentrations inhibited contraction at later time points and did not affect mRNA levels. ROCK inhibitor treatment (Y27632) inhibited collagen gel contraction and led to depressed mRNA levels. The ERK1/2 inhibito...

bioRxiv (Cold Spring Harbor Laboratory), Dec 8, 2023

Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondr... more Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that TPM3.1 inhibition causes F-actin reorganization from stress fibers back to cortical F-actin and also causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition reduces nuclear localization of myocardin related transcription factor, which is known to suppress dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies.

Molecular Biology of the Cell

Mechanical stress deprivation results in actin depolymerization in tendons. Tendon cell F-actin i... more Mechanical stress deprivation results in actin depolymerization in tendons. Tendon cell F-actin is stabilized by tropomyosin isoform 3.1 (Tpm3.1). Inhibition of Tpm3.1 results in pathological changes reminiscent of tendinosis.

Osteoarthritis and Cartilage, 2015

Matrix Biology, 2006

The random orientation of collagen in the resting zone PC concurs with the expected biaxial strai... more The random orientation of collagen in the resting zone PC concurs with the expected biaxial strain field during growth at this surface (zone 3). These findings agree with the hypothesis that the orientation of the PC and PO collagen network follows the growth-induced strain in these tissues.

Osteoarthritis and Cartilage, 2021

In this study the role of the chondrocyte cytoskeleton in regulating cellular mRNA levels in chon... more In this study the role of the chondrocyte cytoskeleton in regulating cellular mRNA levels in chondrocytes was examined. Passaging of chondrocytes in traditional 2D monolayer culture results in chondrocyte dedifferentiation. As compared to primary chondrocytes, passaged cells exhibited a lower g-/f-actin ratio. However tubulin polymerization was not significantly altered by passaging. Pharmacological actin depolymerization partially redifferentiated passaged chondrocytes whereas modulation of tubulin did not. To delineate the mechanism by which actin polymerization regulates gene expression, myocardin related transcription factor A (MRTF) signaling was investigated. Passaging resulted in an increased amount of MRTFa as well as a predominantly nuclear localization of MRTF; these changes occurred within 5 days of culture and were sustained in passaged chondrocytes. Plating passaged cells in high density 3D or in suspension on top of agarose or exposure of cells in monolayer culture to ...

UBC medical journal, 2011

Hutchinson-Gilford Progeria Syndrome (HGPS) is a condition characterized by signs of accelerated ... more Hutchinson-Gilford Progeria Syndrome (HGPS) is a condition characterized by signs of accelerated aging that present within the first year of life. Notable characteristics of children with HGPS include prominent superficial veins, failure to thrive, alopecia, as well as various skeletal and cardiovascular pathologies normally associated with advanced age. The discovery of the lamin A (C to T) gene mutation at position 1824 of the coding sequence has ushered a greater understanding on the essential role of lamin A protein processing. In normal cells, processing prelamin A to mature lamin A is complete following the cleavage of end terminal amino acids. In HGPS, gene mutation results in the deletion of a Zmpste24/FACE1 splice site in prelamin A preventing end terminal cleavage. Thus, prelamin A remains anchored due to c-terminal farnesylation. Lamin A eventually accumulates within the inner nuclear membrane of cells, resulting in disease pathology. The generation of experimental mouse ...

MethodsX

Whole mount imaging of the lens allows for high spatial resolution visualization of lens epitheli... more Whole mount imaging of the lens allows for high spatial resolution visualization of lens epithelial structures by using small molecule fluorescent probes. However, the visualization of specific proteins in lens epithelial cells within whole lenses remains a challenge as the capsule that surrounds the lens does not allow penetration of antibodies. Here we describe a whole mount imaging method that allows us to overcome this challenge by digesting the lens capsules of paraformaldehyde fixed lenses using collagenase. This method enables the penetration of antibodies for effective visualization of proteins in the epithelium of whole lenses.• A limitation to lens whole mount imaging is the ability to visualize specific proteins as the collagen capsule surrounding the lens impedes the penetration of antibodies• This protocol helps overcome this limitation by a light collagenase digestion of the capsule of fixed lenses prior to immunostaining• This method allows for the imaging of specific proteins in the epithelium of the whole lens tissue

ABSTRACTProper ocular lens function requires lens biomechanical flexibility which is lost in pres... more ABSTRACTProper ocular lens function requires lens biomechanical flexibility which is lost in presbyopia during aging. As increasing lens size has been shown previously to correlate with lens biomechanical stiffness in aging, we tested the hypothesis that whole lens size determines gross biomechanical stiffness. We used an allometric approach to evaluate this hypothesis by comparing lenses from three rodent species (mouse, rats and guinea pigs) of varying size. While rat lenses are larger and stiffer than mouse lenses, guinea pig lenses are even larger than rat lenses but are softer than the rat lens. This indicates that lens size is not a sole determinant of lens stiffness and disproves our hypothesis. Therefore, we investigated the scaling of lens microstructural features that could potentially explain the differences in biomechanical stiffness between rat and guinea pig lenses, including lens capsule thickness, epithelial cell area, fiber cell widths, suture organization, and nucl...

Investigative Opthalmology & Visual Science

Purpose Epithelial to mesenchymal transition (EMT) is a cause of anterior and posterior subcapsul... more Purpose Epithelial to mesenchymal transition (EMT) is a cause of anterior and posterior subcapsular cataracts. Central to EMT is the formation of actin stress fibers. Selective targeting of actin stress fiber-associated tropomyosin (Tpm) in epithelial cells may be a means to prevent stress fiber formation and repress lens EMT. Methods We identified Tpm isoforms in mouse immortalized lens epithelial cells and epithelial and fiber cells from whole lenses by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) followed Sanger sequencing. We focused on the role of one particular tropomyosin isoform, Tpm3.1, in EMT. To induce EMT, we treated cells or native lenses with TGFβ2. To test the function of Tpm3.1, we exposed cells or whole lenses to a Tpm3.1-specific chemical inhibitor, TR100, as well as investigated lenses from Tpm3.1 knockout mice. We examined stress fiber formation by confocal microscopy and assessed EMT progression by analysis of alpha-smooth muscle actin (αSMA) mRNA (real-time RT-PCR), and protein (Western immunoassay [WES]). Results Lens epithelial cells express eight Tpm isoforms. Cell culture studies showed that TGFβ2 treatment results in the upregulation of Tpm3.1, which associates with actin in stress fibers. TR100 prevents stress fiber formation and reduces αSMA in TGFβ2-treated cells. Using an ex vivo lens culture model, TGFβ2 treatment results in stress fiber formation at the basal regions of the epithelial cells. Genetic knockout of Tpm3.1 or treatment of lenses with TR100 prevents basal stress fiber formation and reduces epithelial αSMA levels. Conclusions Targeting specific stress fiber associated tropomyosin isoform, Tpm3.1, is a means to repress lens EMT.

$Research paper thumbnail of Age-related changes in eye lens biomechanics, morphology, refractive index and transparency$

Aging

Lifelong eye lens function requires an appropriate gradient refractive index, biomechanical integ... more Lifelong eye lens function requires an appropriate gradient refractive index, biomechanical integrity and transparency. We conducted an extensive study of wild-type mouse lenses 1-30 months of age to define common age-related changes. Biomechanical testing and morphometrics revealed an increase in lens volume and stiffness with age. Lens capsule thickness and peripheral fiber cell widths increased between 2 to 4 months of age but not further, and thus, cannot account for significant age-dependent increases in lens stiffness after 4 months. In lenses from mice older than 12 months, we routinely observed cataracts due to changes in cell structure, with anterior cataracts due to incomplete suture closure and a cortical ring cataract corresponding to a zone of compaction in cortical lens fiber cells. Refractive index measurements showed a rapid growth in peak refractive index between 1 to 6 months of age, and the area of highest refractive index is correlated with increases in lens nucleus size with age. These data provide a comprehensive overview of age-related changes in murine lenses, including lens size, stiffness, nuclear fraction, refractive index, transparency, capsule thickness and cell structure. Our results suggest similarities between murine and primate lenses and provide a baseline for future lens aging studies.

Journal of Orthopaedic Research®

Osteoarthritis (OA) is a degenerative disease that initially manifests as loss of the superficial... more Osteoarthritis (OA) is a degenerative disease that initially manifests as loss of the superficial zone (SZ) of articular cartilage. SZ chondrocytes (SZC) differ in morphology from other chondrocytes as they are elongated and oriented parallel to the tissue surface. Proteoglycan 4 (PRG4) and tenascin C (TNC) are molecules expressed by SZC, which have been shown to be chondroprotective. Identification of the signalling pathway(s) regulating expression of SZ molecules may lead to a therapeutic target that can be used to delay or prevent the onset of OA. The hypothesis of this study is that expression of SZ molecules are regulated in part, by the CDC42actin-myocardin-related transcription factor-A (MRTF-A) signaling pathway. SZC from bovine metacarpal-phalangeal joints were isolated and grown in monolayer culture. Each target in the CDC42-actin-MRTF-A pathway was inhibited and the effect on cell shape, actin cytoskeleton status, and expression of PRG4 and TNC were determined. Treatment with the CDC42 inhibitor ML141 decreased PRG4 and TNC expression, and correlated with increased cell circularity and G-/F-actin ratio. PRG4 and TNC expression were differentially regulated by actin depolymerizing agents, latrunculin B and cytochalasin D. Chemical inhibition of MRTF-A resulted in decreased expression of both PRG4 and TNC; however, specific knockdown by small interfering RNA only decreased expression of TNC indicating that TNC, but not PRG4, is regulated by MRTF-A. Although PRG4 and TNC expression are both regulated by CDC42 and actin, it appears to occur through different downstream signaling pathways. Further study is required to elucidate the pathway regulating PRG4.

Purpose: Epithelial to mesenchymal transition (EMT) is a cause of anterior and posterior subcapsu... more Purpose: Epithelial to mesenchymal transition (EMT) is a cause of anterior and posterior subcapsular cataracts. Central to EMT is the formation of actin stress fibers. Targeting specific, stress fiber associated tropomyosin in epithelial cells may be a means to prevent stress fiber formation and repress lens EMT. Methods: We identified Tpm isoforms in mouse immortalized lens epithelial cells and isolated whole lenses by semi-quantitative PCR followed Sanger sequencing. We focused on the role of one particular tropomyosin isoform, Tpm3.1, in EMT. To stimulate EMT, we cultured cells or native lenses in TGFβ. To test the function of Tpm3.1, we exposed cells or whole lenses to a Tpm3.1-specific chemical inhibitor, TR100, as well as investigated lenses from Tpm3.1 knockout mice. We examined stress fiber formation by confocal microscopy and assessed EMT progression by αsma mRNA (qPCR) and protein (WES immunoassay) analysis. Results: Lens epithelial cells express eight tropomyosin isoforms...

The American Journal of Sports Medicine

Background: Autologous chondrocyte implantation, which uses passaged chondrocytes, commonly leads... more Background: Autologous chondrocyte implantation, which uses passaged chondrocytes, commonly leads to the formation of fibrocartilage. When chondrocytes are passaged to increase cell numbers, they lose their phenotype and ability to form hyaline cartilage. The use of transforming growth factor β (TGFβ) to redifferentiate passaged chondrocytes has been validated in vitro; however, it is unknown if redifferentiated chondrocytes will enhance defect repair when implanted in vivo. Furthermore, fibrin gel is used in orthopaedic surgery as a fixative and scaffold and could be an appropriate carrier to enhance retention of cells in the repair site. Purpose: To investigate if passaged redifferentiated chondrocytes in fibrin gel have the ability to form cartilage tissue and if these redifferentiated cells will enhance the formation of hyaline cartilage in vivo when implanted into critical-size osteochondral defects. Study Design: Controlled laboratory study. Methods: Rabbit and human chondrocy...

Journal of Tissue Engineering and Regenerative Medicine

Chondrocytes dedifferentiate as a result of monolayer culture for cell number expansion. This is ... more Chondrocytes dedifferentiate as a result of monolayer culture for cell number expansion. This is associated with the development of an elongated shape, increased actin polymerization, development of stress fibres, and expression of contractile molecules. Given the changes in actin status with dedifferentiation, the hypothesis of this study was that adseverin, an actin severing and capping protein, plays a role in regulating chondrocyte phenotype and function. This study reports that serial passaging of articular chondrocytes in monolayer culture resulted in loss of adseverin protein expression as early as Day 14 of culture and remained repressed in Passage 2 (P2) cells. Knockdown of adseverin by siRNA in primary chondrocytes promoted an increase in cell size and an elongated shape, actin stress fibres, decreased G‐/F‐actin ratio, and increased number of actin‐free barbed ends. The cells also showed increased expression of the contractile genes and proteins, vinculin and α‐smooth muscle actin, and increased ability to contract collagen gels. These are all features of dedifferentiation. These effects were due to adseverin as adseverin overexpression following transfection of the green fluorescent protein‐adseverin plasmid partially reversed all of these changes in P2 chondrocytes. Furthermore, sox9 and aggrecan chondrogenic gene expression was upregulated, and collagen type I genes expression was downregulated with adseverin overexpression. The change in aggrecan mRNA expression had functional consequence as these cells exhibited increased total proteoglycan synthesis. These findings demonstrate that adseverin regulates features indicative of redifferentiation in passaged articular chondrocytes through modulation of the actin cytoskeleton status and potentially may regulate the maintenance of phenotype in primary chondrocytes.

Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 2018

Generating the best possible bioengineered cartilage from passaged chondrocytes requires culture ... more Generating the best possible bioengineered cartilage from passaged chondrocytes requires culture condition optimization. In this study, the use of adherent agarose mold (adAM) cultures to support redifferentiation of passaged twice (P2) chondrocytes and serve as a scalable platform to assess the effect of growth factor combinations on proteoglycan accumulation by cells was examined. By 2 days in adAM culture, bovine P2 cells were partially redifferentiated as demonstrated by regression of actin-based dedifferentiation signalling and fibroblast matrix and contractile gene expression. By day 10, aggrecan and type II collagen gene expression were significantly increased in adAM cultured cells. At day 20, a continuous layer of cartilage tissue was observed. There was no evidence of tissue contraction by P2 cells in adAM cultures. The matrix properties of the resultant tissue as well as proteoglycan 4 (PRG4) secreted by the cells were dependent on the initial cell seeding density. AdAM c...