Biology subjects

Parreno, J.

Publications and source records attributed to Parreno, J..

10 recordsLinked to original sources

bioRxiv2026

Tropomyosin 3.1 Regulates Chondrocyte Cortical F-actin Stability and Homeostasis

Objective: Osteoarthritis is a degenerative joint disease of which the mechanisms and proteins contributing to articular cartilage breakdown have not yet been fully elucidated. In osteoarthritis, chondrocyte F-actin loss coincides with altered chondrocyte homeostasis favoring catabolism. While the loss of F-actin has been implicated as a driver of osteoarthritis progression, the mechanisms by which F-actin is lost and regulates a catabolic shift in gene expression require elucidation. We hypothesized that destabilizing F-actin by targeting inhibition of specific F-actin stabilizer Tropomyosins (Tpms) dysregulates chondrocyte homeostasis through regulation of actin-associated transcription factors, Yes-associated protein (YAP) and Myocardin-related transcription factor A (MRTF). Methods: Using RT-PCR and Sanger sequencing, we determined the expression of Tpm isoforms present in cartilage. We then focused on the regulation of chondrocyte cortical F-actin by the specific Tpm isoform, Tpm3.1. Using confocal imaging for F-actin, we examined the effect of Tpm3.1 knockout in mouse femoral head cartilage and pharmacological inhibition (TR100) of primary bovine chondrocytes. Next, we examined the nuclear translocation of actin-associated regulators, YAP and MRTF, by TPM3.1 inhibition in primary chondrocytes. Finally, we determined the effect of YAP and MRTF inhibition on the regulation of chondrogenic and degradative enzyme gene expression in primary chondrocytes. Results: Our results revealed TPM3.1 is present and localized cortically in mouse and bovine chondrocytes. There was a reduction in cortical F-actin staining in Tpm3.1 knockout mouse femoral head cartilage as well as in primary bovine chondrocytes treated with TR100. In primary chondrocytes, TR100 caused a catabolic shift in mRNA levels. This coincided with a decrease in the nuclear-to-cytoplasmic ratio of both YAP and MRTF in TR100-treated cells. Furthermore, pharmacological inhibition of either YAP or MRTF partially recapitulated the catabolic shift seen in TR100-treated chondrocytes by a reduction of chondrogenic mRNA levels. Conclusion: Collectively, these results provide insight into the regulation of chondrocyte homeostasis by cortical F-actin stability and the downstream mediators of transcription. A greater understanding of the actin-associated molecules that regulate chondrocyte homeostasis in osteoarthritis may lead to novel insights to target disease progression.

cell biology↗

bioRxiv2026

Bioorthogonal Tuning of Hydrogel Stiffness Promotes Zonal Redifferentiation of Passaged Chondrocytes

Generating bioengineered cartilage that recapitulates the depth-dependent phenotype, structure, and function of native articular cartilage remains a challenge. While cartilage is rich in aggrecan and type II collagen, proper function depends on depth-dependent protein expression. Superficial zone chondrocytes (SZCs) secrete proteoglycan-4 (PRG4) to lubricate the cartilage surface. Deep zone chondrocytes produce type X collagen (COLX) to support compressive loading and load transfer to subchondral bone. We previously demonstrated that passaged full-thickness chondrocytes (FTCs) and zonal chondrocytes can re-express cartilage and zone-specific markers following scaffold-free three-dimensional (3D) culture in redifferentiation media. However, in the absence of an instructive matrix, cells expressed low levels of zone-specific proteins and exhibited limited depth-dependent organization. We hypothesize that synthetic extracellular matrix with zone-specific microenvironmental cues will guide zonal differentiation. To this end, passaged primary bovine chondrocytes were encapsulated in a soft, hyaluronan (HA)-based, cell-adhesive, and protease-degradable hydrogel established via bioorthogonal tetrazine (Tz) ligation with norbornene (Nb). When supplemented with TGF{beta}3, FTCs deposited aggrecan and type II collagen with minimal type I collagen. Application of interfacial tetrazine ligation with trans-cyclooctene (TCO) during cell culture resulted in matrix stiffening, leading to upregulation of COLX expression. Conversely, SZCs cultured in soft hydrogels exhibited the greatest PRG4 expression. Establishment of a trilayered construct with region-specific stiffness via the diffusion-controlled reaction promoted PRG4 and COLX expression in defined zones. Together, these findings demonstrate that tunable HA-based hydrogels can enhance zone-specific chondrocyte phenotypes and promote the formation of zonally organized cartilage.

bioengineering↗

bioRxiv2026

Fibronectin Coating of Tissue Culture Polystyrene to Improve Superficial Zone Chondrocyte Expansion

The surface layer of articular cartilage provides for low-friction joint movement and protects the tissue from mechanical wear. The superficial zone chondrocytes (SZCs) of the surface layer produce proteoglycan-4 (PRG4), which is a lubricant that is necessary to reduce friction. Articular cartilage has limited capacity for self-repair and cell-based therapies, such as autologous chondrocyte implantation (ACI), is used to stimulate repair. However, in ACI, cells are expanded on tissue culture polystyrene where SZC poorly attach, proliferate slowly and dedifferentiate. Consequently, expanded SZC produce fibrocartilage tissue with insufficient PRG4. We previously demonstrated that culturing SZC on chondrocyte-derived decellularized extracellular matrix (CM) enhances SZC attachment and preserves phenotype. Since fibronectin (FN) was identified as the most abundant matrix protein within CM, here we tested the hypothesis that FN-coated culture surfaces would partially reproduce the beneficial effects of CM. We found that, similar to CM, SZC on FN-coated polystyrene increased SZC attachment and proliferation. However, unlike CM, SZCs expanded on FN-coated polystyrene remained more dedifferentiated as indicated by spread cells, elevated fibroblastic and contractile mRNA levels, and increased formation of SMA positive stress fibers. Consistent with the dedifferentiated phenotype, SZC on FN-coated polystyrene displayed extensive stress fibers, and higher nuclear myocardin-related-transcription-factor-a (MRTF-A). In contrast, CM reduced stress fiber formation and diminished nuclear MRTF-A in SZC. CM provides matrix cues beyond FN that suppress dedifferentiation and preserve the SZC phenotype. Identifying the matrix cues necessary to improve SZC expansion could lead to the generation of a superior surface in ACI repair tissue.

cell biology↗

bioRxiv2025

The Effects of Hypothermic Storage on Passaged Chondrocyte Viability and Redifferentiation Potential

Cell-based transplantation therapies, such as autologous chondrocyte implantation (ACI), are used to treat focal cartilage defects caused by trauma or degeneration. In ACI, chondrocytes are isolated from non-load-bearing regions of healthy cartilage regions and then sent to a cell manufacturing laboratory, where they are expanded for cell number in monolayer culture. Once a large number of cells are obtained, they are transported to the clinic for reimplantation into the defect site. The storage and transport conditions from cell manufacturing to implantation may be a critical time that could influence cell viability and redifferentiation potential. Although hypothermic storage at sub-physiological temperatures is commonly used to preserve cell viability, long-term storage of cartilage under hypothermic conditions can impair chondrocyte viability and function. However, the impact of short-term, acute hypothermic storage on passaged chondrocytes remains largely unknown. We tested the hypothesis that acute hypothermic storage negatively impacts passaged chondrocyte viability and reduces the capacity for redifferentiation. Passaged chondrocytes were stored either in monolayer culture or in suspension at 36, 19 or 8{degrees}C. In monolayer culture, hypothermic temperatures preserved cell viability with no difference compared to storage at 36{degrees}C for up to three days. Additionally, hypothermic temperatures promoted cell rounding, reduced proliferative capacity, depolymerized filamentous actin, and led to a slight reduction in the mRNA levels of specific matrix molecules compared to 36{degrees}C. Intriguingly, the effects of hypothermia were context-dependent. Exposure of passaged cells in suspension to hypothermia promoted the maintenance of cell viability and reduced aggregation compared to 36{degrees}C. When stored at 8{degrees}C in suspension, passaged cells exhibited enhanced expression of specific matrix molecule mRNA levels compared to cells at 36{degrees}C in suspension. Subsequently, when passaged cells in suspension at 8{degrees}C were seeded in 3D within adherent agarose molds, there was an increase in aggrecan expression 10 days after seeding. The tissues formed by cells stored in suspension at 9{degrees}C were thicker and stained more intensely for aggrecan. Therefore, in contrast to our hypothesis, we found that hypothermic storage did not have a negative impact; when stored for 1 day in suspension, it had lasting effects on matrix deposition. The storage of passaged chondrocytes under hypothermic conditions may be beneficial for ACI, warranting further investigations of cell hypothermic storage for in vivo repair.

bioengineering↗

bioRxiv2024

Actin Polymerization Status Regulates Tendon Homeostasis through Myocardin-Related Transcription Factor-A

The actin cytoskeleton is a potent regulator of tenocyte homeostasis. However, the mechanisms by which actin regulates tendon homeostasis are not entirely known. This study examined the regulation of tenocyte molecule expression by actin polymerization via the globular (G-) actin-binding transcription factor, myocardin-related transcription factor-a (MRTF). We determined that decreasing the proportion of G-actin in tenocytes by treatment with TGF{beta}1 increases nuclear MRTF. These alterations in actin polymerization and MRTF localization coincided with favorable alterations to tenocyte gene expression. In contrast, latrunculin A increases the proportion of G-actin in tenocytes and reduces nuclear MRTF, causing cells to acquire a tendinosis-like phenotype. To parse out the effects of F-actin depolymerization from regulation by MRTF, we treated tenocytes with cytochalasin D. Similar to latrunculin A treatment, exposure of cells to cytochalasin D increases the proportion of G-actin in tenocytes. However, unlike latrunculin A treatment, cytochalasin D increases nuclear MRTF. Compared to latrunculin A treatment, cytochalasin D led to opposing effects on the expression of a subset of genes. The differential regulation of genes by latrunculin A and cytochalasin D suggests that actin signals through MRTF to regulate a specific subset of genes. By targeting the deactivation of MRTF through the inhibitor CCG1423, we verify that MRTF regulates Type I Collagen, Tenascin C, Scleraxis, and -smooth muscle actin in tenocytes. Actin polymerization status is a potent regulator of tenocyte homeostasis through the modulation of several downstream pathways, including MRTF. Understanding the regulation of tenocyte homeostasis by actin may lead to new therapeutic interventions against tendinopathies, such as tendinosis.

cell biology↗

bioRxiv2023

Targeting F-actin stress fibers to suppress the dedifferentiated phenotype in chondrocytes

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.

cell biology↗

bioRxiv2022

Destabilization of F-actin by Mechanical Stress Deprivation or Tpm3.1 Inhibition Promotes a Pathological Phenotype in Tendon Cells

The actin cytoskeleton is a central mediator between mechanical force and cellular phenotype. In tendon, it is speculated that mechanical stress deprivation regulates gene expression by filamentous (F-) actin destabilization. However, the molecular mechanisms that stabilize tenocyte F-actin networks remain unclear. Tropomyosins (Tpms) are master regulators of F-actin networks. There are over 40 mammalian Tpm isoforms, with each isoform having the unique capability to stabilize F-actin sub-populations. Thus, the specific Tpm(s) expressed by a cell defines overall F-actin organization. Here, we investigated F-actin destabilization by stress deprivation of tendon and tested the hypothesis that stress fiber-associated Tpm(s) stabilize tenocyte F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon fascicles downregulated tenocyte genes (collagen-I, tenascin-C, scleraxis, -smooth muscle actin) and upregulated matrix metalloproteinase-3. Concomitant with mRNA modulation were increases in DNAse-I/Phallodin (G/F-actin) staining, confirming F-actin destabilization by tendon stress deprivation. To investigate the molecular regulation of F-actin stabilization, we first identified the Tpms expressed by mouse tendons. Tendon cells from different origins (tail, Achilles, plantaris) express three isoforms in common: Tpm1.6, 3.1, and 4.2. We examined the function of Tpm3.1 since we previously determined that it stabilizes F-actin stress fibers in lens epithelial cells. Tpm3.1 associated with F-actin stress fibers in native and primary tendon cells. Inhibition of Tpm3.1 depolymerized F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin stability in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.

cell biology↗

bioRxiv2021

Microstructural determinants of lens stiffness in rat versus guinea pig lenses

Proper 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 nuclear size. Capsule thickness, epithelial cell area, and fiber cell widths scaled with lens size (i.e., greater in guinea pig lenses than rats), indicating that sizes of these features do not correlate with the stiffness of rat lenses, while suture organization was similar between rats and guinea pigs. However, we found that the hard rat lens nucleus occupies a greater fraction of the lens than the guinea pig lens nucleus, suggesting a role for nuclear size in determining whole lens stiffness. Therefore, while many features contribute to lens biomechanical properties, the size of the lens nucleus with respect to the size of the lens could be a major determinant of lens stiffness in rats versus guinea pigs.

cell biology↗

bioRxiv2019

Tpm3.1 association with actin stress fibers is required for lens epithelial to mesenchymal transition

PurposeEpithelial 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.\n\nMethodsWe 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 TGF{beta}2. 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 sma mRNA (qPCR) and protein (WES immunoassay) analysis.\n\nResultsLens epithelial cells express eight tropomyosin isoforms. Cell culture studies showed that TGF{beta}2 treatment results in an upregulation of Tpm3.1, which associates with actin in stress fibers. TR100 prevents stress fiber formation and reduces sma in TGF{beta}2 treated cells. We confirmed the role of Tpm3.1 in lens epithelial cells in the native lens ex vivo. Culture of whole lenses in the presence of TGF{beta}2 results in stress fiber formation at the basal regions of epithelial cells. Knockout of Tpm3.1 or treatment of lenses with TR100 prevents basal stress fiber formation and reduces epithelial sma levels.\n\nConclusionTargeting specific stress fiber associated tropomyosin isoform, Tpm3.1, is a means to represses lens EMT.

cell biology↗

bioRxiv2018

The effects of mechanical strain on mouse eye lens capsule and cellular microstructure

The understanding of multiscale load transfer within complex soft tissues is incomplete. The eye lens is ideal for multiscale tissue mechanics studies as its principal function is to fine focus light at different distances onto the retina via mechanical shape changes. The biomechanical function, resiliency, and intricate microstructure of the lens make it an excellent non-connective soft tissue model. We hypothesized that compressive strain applied onto whole lens tissue leads to deformation of specific microstructures and that this deformation is reversible following removal of load. For this examination, mouse lenses were compressed by sequential application of increasing load. Using confocal microscopy and quantitative image analysis, we determined that axial strain [≥]10% reduces capsule thickness, expands epithelial cell area, and separates fiber cell tips at the anterior region of the lenses. At the equatorial region, strain [≥]6% increases fiber cell widths. The effects of strain on lens epithelial cell area, capsule thickness, and equatorial fiber cell widths are reversible following the release of lenses from strain. However, although fiber cell tip separation following the removal of low loads is reversible, the separation becomes irreversible with application of higher loads. This irreversible separation between fiber cell tips leads to incomplete bulk lens resiliency. The lens is an accessible biomechanical model system that provides new insights on multiscale transfer of loads in soft tissues.

cell biology↗