Tissue-specific expression of senescence biomarkers in spontaneously hypertensive rats: evidence of premature aging in hypertension.

Background: Cellular senescence is an age-related physiological process that contributes to tissue dysfunction and accelerated onset of chronic metabolic diseases including hypertension. Indeed, elevation of blood pressure in hypertension coincides with premature vascular aging and dysfunction. In addition, onsets of metabolic disturbance and osteopenia in patients with hypertension have also been reported. It is possible that hypertension enhances premature aging and causes progressive loss of function in multiple organs. However, the landscape of cellular senescence in critical tissues affected by hypertension remains elusive. Materials and Methods: Heart, liver, bone, hypothalamus, and kidney were collected from spontaneously hypertensive rats (SHR) and age- and sex-matched normotensive Wistar rats (WT) at 6, 12, 24 and 36 weeks of age (n = 10 animals/group). Changes in mRNA levels of senescence biomarkers namely cyclin-dependent kinase (CDK) inhibitors (CDKIs), i.e., Cdkn2a (encoding p16Ink4a) and Cdkn1a (encoding p21cip1) as well as senescence-associated secretory phenotypes (SASPs), i.e., Timp1, Mmp12, Il6 and Cxcl1, were determined. Additionally, bone collagen alignment and hydroxy apatite crystal dimensions were determined by synchrotron radiation small- and wide-angle X-ray scattering (SAXS/WAXS) techniques. Results: Real-time PCR revealed that transcript levels of genes encoding CDKIs and SASPs in the heart and liver were upregulated in SHR from 6 to 36 weeks of age. Expression of Timp1 and Cxcl1 was increased in bone tissues isolated from 36-week-old SHR. In contrast, we found that expression levels of Timp1 and Il6 mRNA were decreased in hypothalamus and kidney of SHR in all age groups. Simultaneous SAXS/WAXS analysis also revealed misalignment of bone collagen fibers in SHR as compared to WT. Conclusion: Premature aging was identified in an organ directly affected by high blood pressure (i.e., heart) and those with known functional defects in SHR (i.e., liver and bone). Cellular senescence was not evident in organs with autoregulation of blood pressure (i.e., brain and kidney). Our study suggested that cellular senescence is induced by persistently elevated blood pressure and in part, leading to organ dysfunction. Therefore, interventions that can both lower blood pressure and prevent cellular senescence should provide therapeutic benefits for treatment of cardiovascular and metabolic consequences.

Impact of Storage Conditions and Mold Types on Aflatoxin B1 Concentration in Corn Residue used as Dairy Feed in Small Holder Dairy Farms, Thailand.

The aims of this study were to determine the impact of storage practice and mold types on mold growth and aflatoxin B1 (AFB1) concentration in corn residue from local seed corn plants, the main roughage source of dairy farms in the northern region in Thailand. A total of 223 samples from 2 types of corn residue - dried and wet - were collected. Mold contamination was determined by spread plate technique, and aflatoxin B1 (AFB1) quantification was performed by a commercial enzyme-linked immunosorbent assay. Multivariate linear models were created to determine factors associated with fungal quantity and AFB1 concentration. Results showed that the presence of Cladosporium spp. in the samples was associated with a lower risk of AFB1 contamination (P<0.05). In addition, appropriate storage practices, e.g. keeping feeds under a roof and using floor canvas under feed piles, gave lower risk of mold contamination and decreasing AFB1 contamination.

Culture Conditions that Support Expansion and Chondrogenesis of Middle-Aged Rat Mesenchymal Stem Cells.

OBJECTIVE: Rats are an early preclinical model for cartilage tissue engineering, and a practical species for investigating the effects of aging. However, rats may be a poor aging model for mesenchymal stem cells (MSCs) based on laboratory reports of a severe decline in chondrogenesis beyond young adulthood. Such testing has not been conducted with MSCs seeded in a scaffold, which can improve the propensity of MSCs to undergo chondrogenesis. Therefore, the objective of this study was to evaluate chondrogenesis of middle-aged rat MSCs encapsulated in agarose. DESIGN: MSCs from 14- to 15-month-old rats were expanded, seeded into agarose, and cultured in chondrogenic medium with or without 5% serum for 15 days. Samples were evaluated for cell viability and cartilaginous extracellular matrix (ECM) accumulation. Experiments were repeated using MSCs from 6-week-old rats. RESULTS: During expansion, middle-aged rat MSCs demonstrated a diminishing proliferation rate that was improved ~2-fold in part by transient exposure to chondrogenic medium. In agarose culture in defined medium, middle-aged rat MSCs accumulated ECM to a much greater extent than negative controls. Serum supplementation improved cell survival ~2-fold, and increased ECM accumulation ~3-fold. Histological analysis indicated that defined medium supported chondrogenesis in a subset of cells, while serum-supplementation increased the frequency of chondrogenic cells. In contrast, young rat MSCs experienced robust chondrogenesis in defined medium that was not improved with serum-supplementation. CONCLUSIONS: These data demonstrate a previously-unreported propensity of middle-aged rat MSCs to undergo chondrogenesis, and the potential of serum to enhance chondrogenesis of aging MSCs.

Differential Effects of the Antioxidants N-Acetylcysteine and Pyrrolidine Dithiocarbamate on Mesenchymal Stem Cell Chondrogenesis.

INTRODUCTION: Mesenchymal stem cell (MSC) chondrogenesis is associated with increases in intracellular reactive oxygen species (ROS), which may result in oxidative stress that is detrimental to cartilage regeneration. This study evaluated the ability of the antioxidants N-acetylcysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) to reduce intracellular ROS, and their effect on MSC chondrogenesis and maturation of cartilage-like extracellular matrix. METHODS: Equine bone marrow MSCs were cultured in serum-supplemented chondrogenic medium with or without NAC or PDTC. ROS was quantified in monolayer after 8 and 72 h of culture. MSCs were seeded into agarose, cultured for 15 days, and analyzed for viable cell density, glycosaminoglycan (GAG) and hydroxyproline accumulation, and collagen gene expression. PDTC cultures were evaluated for oxidative damage by protein carbonylation, and mechanical properties via compressive testing. RESULTS: NAC significantly lowered levels of ROS after 8 but not 72 h, and suppressed GAG accumulation (70%). In secondary experiments using serum-free medium, NAC significantly increased levels of ROS at 72 h, and lowered cell viability and extracellular matrix accumulation. PDTC significantly reduced levels of ROS (~ 30%) and protein carbonylation (27%), and enhanced GAG accumulation (20%). However, the compressive modulus for PDTC-treated samples was significantly lower (40%) than controls. Gene expression was largely unaffected by the antioxidants. CONCLUSIONS: NAC demonstrated a limited ability to reduce intracellular ROS in chondrogenic culture, and generally suppressed accumulation of extracellular matrix. Conversely, PDTC was an effective antioxidant that enhanced GAG accumulation, although the concomitant reduction in compressive properties is a significant limitation for cartilage repair.

Effect of culture duration on chondrogenic preconditioning of equine bone marrow mesenchymal stem cells in self-assembling peptide hydrogel.

Ex vivo induction of chondrogenesis is a promising approach to improve upon the use of bone marrow mesenchymal stem cells (MSCs) for cartilage tissue engineering. This study evaluated the potential to induce chondrogenesis with days of culture in chondrogenic medium for MSCs encapsulated in self-assembling peptide hydrogel. To simulate the transition from preconditioning culture to implantation, MSCs were isolated from self-assembling peptide hydrogel into an individual cell suspension. Commitment to chondrogenesis was evaluated by seeding preconditioned MSCs into agarose and culturing in the absence of the chondrogenic cytokine transforming growth factor beta (TGFß). Positive controls consisted of undifferentiated MSCs seeded into agarose and cultured in medium containing TGFß. Three days of preconditioning was sufficient to produce chondrogenic MSCs that accumulated ∼75% more cartilaginous extracellular matrix than positive controls by day 17. However, gene expression of type X collagen was ∼65-fold higher than positive controls, which was attributed to the absence of TGFß. Potential induction of immunogenicity with preconditioning culture was indicated by expression of major histocompatibility complex class II (MHCII), which was nearly absence in undifferentiated MSCs, and ∼7% positive for preconditioned cells. These data demonstrate the potential to generate chondrogenic MSCs with days of self-assembling peptide hydrogel, and the ability to readily recover an individual cell suspension that is suited for injectable therapies. However, continued exposure to TGFß may be necessary to prevent hypertrophy indicated by type X collagen expression, while immunogenicity may be a concern for allogeneic applications. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1368-1375, 2019.

Modulating the oxidative environment during mesenchymal stem cells chondrogenesis with serum increases collagen accumulation in agarose culture.

Chondrogenesis of mesenchymal stem cells (MSCs) is induced in culture conditions that have been associated with oxidative stress, although the extent to which the oxidative environment affects differentiation and extracellular matrix (ECM) accumulation is not known. The objectives of this study were to evaluate the oxidative environment during MSCs chondrogenesis in conventional serum-free medium, and the effect of serum-supplementation on intracellular reactive oxygen species (ROS) and chondrogenesis. Young adult equine MSCs were seeded into agarose and cultured in chondrogenic medium, with or without 5% fetal bovine serum (FBS), for up to 15 days. Samples were evaluated for intracellular ROS, the antioxidant glutathione, ECM and gene expression measures of chondrogenesis, and carbonylation as an indicator of oxidative damage. Intracellular ROS increased with time in culture, and was lower in medium supplemented with FBS. Glutathione decreased ∼12-fold during early chondrogenesis (p < 0.0001), and was not affected by FBS (p = 0.25). After 15 days of culture, FBS supplementation increased hydroxyproline accumulation ∼80% (p = 0.0002); otherwise, measures of chondrogenesis were largely unaffected. Protein carbonylation in chondrogenic MSCs cultures was not significantly different between serum-free and FBS cultures (p = 0.72). Supplementation with adult equine serum increased hydroxyproline accumulation by 45% over serum-free culture (p = 0.0006). In conclusion, this study characterized changes in the oxidative environment during MSC chondrogenesis, and suggested that lowering ROS may be an effective approach to increase collagen accumulation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:506-514, 2018.

Effects of Dexamethasone Concentration and Timing of Exposure on Chondrogenesis of Equine Bone Marrow-Derived Mesenchymal Stem Cells.

OBJECTIVE: Dexamethasone is known to support mesenchymal stem cell (MSC) chondrogenesis, although the effects of dose and timing of exposure are not well understood. The objective of this study was to investigate these variables using a laboratory model of MSC chondrogenesis. DESIGN: Equine MSCs were encapsulated in agarose and cultured in chondrogenic medium with 1 or 100 nM dexamethasone, or without dexamethasone, for 15 days. Samples were analyzed for extracellular matrix (ECM) accumulation, prostaglandin E2 and alkaline phosphatase secretion, and gene expression of selected collagens and catabolic enzymes. Timing of exposure was evaluated by ECM accumulation after dexamethasone was withdrawn over the first 6 days, or withheld for up to 3 or 6 days of culture. RESULTS: ECM accumulation was not significantly different between 1 and 100 nM dexamethasone, but was suppressed ~40% in dexamethasone-free cultures. Prostaglandin E2 secretion, and expression of catabolic enzymes, including matrix metalloproteinase 13, and type X collagen was generally lowest in 100 nM dexamethasone and not significantly different between 1 nM and dexamethasone-free cultures. Dexamethasone could be withheld for at least 2 days without affecting ECM accumulation, while withdrawal studies suggested that dexamethasone supports ECM accumulation beyond day 6. CONCLUSION: One nanomolar dexamethasone supported robust cartilage-like ECM accumulation despite not having an effect on markers of inflammation, although higher concentrations of dexamethasone may be necessary to suppress undesirable hypertrophic differentiation. While early exposure to dexamethasone was not critical, sustained exposure of at least a week appears to be necessary to maximize ECM accumulation.