Gene-expression changes in knee-joint tissues with aging and menopause: implications for the joint as an organ.

BACKGROUND: When considering the "joint as an organ", the tissues in a joint act as complementary components of an organ, and the "set point" is the cellular activity for homeostasis of the joint tissues. Even in the absence of injury, joint tissues have adaptive responses to processes, like aging and menopause, which result in changes to the set point. PURPOSE: The purpose of this study in a preclinical model was to investigate age-related and menopause-related changes in knee-joint tissues with the hypothesis that tissues will change in unique ways that reflect their differing contributions to maintaining joint function (as measured by joint laxity) and the differing processes of aging and menopause. METHODS: Rabbit knee-joint tissues from three groups were evaluated: young adult (gene expression, n=8; joint laxity, n=7; water content, n=8), aging adult (gene expression, n=6; joint laxity, n=7; water content, n=5), and menopausal adult (gene expression, n=8; joint laxity, n=7; water content, n=8). Surgical menopause was induced with ovariohysterectomy surgery and gene expression was assessed using reverse-transcription quantitative polymerase chain reaction. RESULTS: Aging resulted in changes to 37 of the 150 gene-tissue combinations evaluated, and menopause resulted in changes to 39 of the 150. Despite the similar number of changes, only eleven changes were the same in both aging and menopause. No differences in joint laxity were detected comparing young adult rabbits with aging adult rabbits or with menopausal adult rabbits. CONCLUSION: Aging and menopause affected the gene-expression patterns of the tissues of the knee joint differently, suggesting unique changes to the set point of the knee. Interestingly, aging and menopause did not affect knee-joint laxity, suggesting that joint function was maintained, despite changes in gene expression. Taken together, these findings support the theory of the joint as an organ where the tissues of the joint adapt to maintain joint function.

Diet-induced obesity leads to pro-inflammatory alterations to the vitreous humour of the eye in a rat model.

OBJECTIVE AND DESIGN: The purpose of this study was to investigate if diet-induced obesity (DIO) and subsequent low-level systemic inflammation would result in local increases in pro-inflammatory mediators in the vitreous humour (VH) of the eyes of rats. METHODS: Sixteen male Sprague-Dawley rats were fed a high-fat/high-sucrose (n = 9) or chow control-diet (n = 7) for 12-weeks. RT-qPCR was conducted on RNA from VH cells and a 27-plex Luminex® Assay was conducted on VH fluid and serum. RESULTS: Increased protein levels for IL-1ß, IL-6, and IL-18 in both serum and VH fluid were observed. VH protein levels for IL-13 and IL-17 were also increased. All mediators significantly increased in VH fluid were also positively correlated with percent body fat. Increased mRNA levels in VH cells for an oxidative stress molecule were accompanied by decreased mRNA levels for an antioxidant scavenger, suggesting an antioxidant/oxidant imbalance in the VH with DIO. In addition, decreased mRNA levels for TRAIL, FAS-L and TGF-ß, molecules associated with immune privilege, were also significantly depressed. CONCLUSIONS: DIO-related metabolic disturbances disrupt VH homeostasis in a manner that reflects development of a pro-inflammatory environment. Prolonged exposure to such an environment may lead to overt pathologies with compromised eye function.

Increased lubricin/proteoglycan 4 gene expression and decreased modulus in medial collateral ligaments following ovariohysterectomy in the adult rabbit: Evidence consistent with aging.

This study investigated whether ovariohysterectomy (OVH) surgery to induce menopause resulted in changes to modulus, failure strain and lubricin/proteoglycan 4 (PRG4) gene expression in rabbit medial collateral ligaments (MCLs), similar to aging (Thornton et al., 2015a). The MCLs from adult rabbits that underwent OVH surgery as adolescents (15-week-old) and adults (1-year-old) were compared by evaluating mechanical behaviour (adolescent OVH, n=8; adult OVH, n=7; normal, n=7), gene expression (adolescent OVH, n=9; adult OVH, n=8; normal, n=8), and collagen and glycosaminoglycan (adolescent OVH, n=9; adult OVH, n=8; normal, n=8) and water (adolescent OVH, n=9; adult OVH, n=8; normal, n=8) content. Mechanical behaviour evaluated cyclic, static and total creep strain, and ultimate tensile strength, modulus and failure strain. The RT-qPCR assessed mRNA levels for matrix regulatory genes. Adult OVH MCLs exhibited increased cyclic creep and failure strain, and decreased modulus with increased mRNA levels for lubricin/PRG4 and collagen I compared with normal MCLs. Adolescent OVH MCLs exhibited increased cyclic, static and total creep strain with decreased mRNA levels for the progesterone receptor. Lubricin/PRG4 plays a role in the lubrication of collagen fascicles which is likely related to the decreased modulus and increased failure strain observed in ligaments from adult OVH rabbits. Progesterone and its receptor are thought to play a role in the stretching of ligaments in pelvic organ prolapse and pregnancy which is likely related to the increase in creep strain observed in ligaments from adolescent OVH rabbits. Ovariohysterectomy in adult rabbits resulted in changes that were consistent with the aging MCL.

Aging affects mechanical properties and lubricin/PRG4 gene expression in normal ligaments.

Age-related changes in ligament properties may have clinical implications for injuries in the mature athlete. Previous preclinical models documented mechanical and biochemical changes in ligaments with aging. The purpose of this study was to investigate the effect of aging on ligament properties (mechanical, molecular, biochemical) by comparing medial collateral ligaments (MCLs) from 1-year-old and 3-year-old rabbits. The MCLs underwent mechanical (n=7, 1-year-old; n=7, 3-year-old), molecular (n=8, 1-year-old; n=6, 3-year-old), collagen and glycosaminoglycan (GAG) content (n=8, 1-year-old; n=6, 3-year-old) and water content (n=8, 1-year-old; n=5, 3-year-old) assessments. Mechanical assessments evaluated total creep strain, failure strain, ultimate tensile strength and modulus. Molecular assessments using RT-qPCR evaluated gene expression for collagens, proteoglycans, hormone receptors, and matrix metalloproteinases and their inhibitors. While total creep strain and ultimate tensile strength were not affected by aging, failure strain was increased and modulus was decreased comparing MCLs from 3-year-old rabbits to those from 1-year-old rabbits. The mRNA expression levels for lubricin/proteoglycan 4 (PRG4) and tissue inhibitor of metalloproteinase-3 increased with aging; whereas, the mRNA expression levels for estrogen receptor and matrix metalloproteinase-1 decreased with aging. Collagen and GAG content assays and water content assessments did not demonstrate any age-related changes. The increased failure strain and decreased modulus with aging may have implications for increased susceptibility to ligament damage/injury with aging. Lubricin/PRG4 gene expression was affected by aging and its speculated role in ligament function may be related to interfascicular lubrication, which in turn may lead to altered mechanical function with aging and increases in potential for injury.

Regional molecular and cellular differences in the female rabbit Achilles tendon complex: potential implications for understanding responses to loading.

The aim of this study was: (i) to analyze the morphology and expression of extracellular matrix genes in six different regions of the Achilles tendon complex of intact normal rabbits; and (ii) to assess the effect of ovariohysterectomy (OVH) on the regional expression of these genes. Female New Zealand White rabbits were separated into two groups: (i) intact normal rabbits (n = 4); and (ii) OVH rabbits (n = 8). For each rabbit, the Achilles tendon complex was dissected into six regions: distal gastrocnemius (DG); distal flexor digitorum superficialis; proximal lateral gastrocnemius (PLG); proximal medial gastrocnemius; proximal flexor digitorum superficialis; and paratenon. For each of the regions, hematoxylin and eosin staining was performed for histological evaluation of intact normal rabbit tissues and mRNA levels for proteoglycans, collagens and genes associated with collagen regulation were assessed by real-time reverse transcription-quantitative polymerase chain reaction for both the intact normal and OVH rabbit tissues. The distal regions displayed a more fibrocartilaginous phenotype. For intact normal rabbits, aggrecan mRNA expression was higher in the distal regions of the Achilles tendon complex compared with the proximal regions. Collagen Type I and matrix metalloproteinase-2 expression levels were increased in the PLG compared to the DG in the intact normal rabbit tissues. The tendons from OVH rabbits had lower gene expressions for the proteoglycans aggrecan, biglycan, decorin and versican compared with the intact normal rabbits, although the regional differences of increased aggrecan expression in distal regions compared with proximal regions persisted. The tensile and compressive forces experienced in the examined regions may be related to the regional differences found in gene expression. The lower mRNA expression of the genes examined in the OVH group confirms a potential effect of systemic estrogen on tendon.

Carrageenan-induced transient inflammation in a rabbit knee model: molecular changes consistent with an early osteoarthritis phenotype.

OBJECTIVE: Inflammation following a knee injury is one of the factors associated with initiation of cartilage degeneration leading to osteoarthritis (OA). The hypothesis tested was that inflammation results in elevated expression of proteinases implicated in OA. METHODS: Mature female rabbits received a single carrageenan injection to the right hind knee and the left knee served as the control. Five animals were killed at time points of 1, 2 and 4 weeks. The synovium and cartilage from both knees were collected and analysed for specific mRNA levels. RESULTS: Interleukin (IL)-1ß and IL-6 mRNA levels peaked at 2 weeks and returned to normal levels in tissues by 4 weeks post-carrageenan treatment. Matrix metalloproteinase (MMP)-13, MMP-1, MMP-3 and cathepsin K followed the trend set by the inflammatory cytokines. Both synovium and cartilage tissues exhibited similar patterns of molecular expression, with cartilage from the tibial plateau responding more strongly than the femoral condyles. CONCLUSIONS: The acute inflammatory milieu controls the transient expression of many degradative proteinases in the knee. However, a single acute exposure to inflammation in the rabbit knee is insufficient to create a chronic inflammatory environment and other complementary factors, such as persistent mechanical instability and/or injury, may contribute to the establishment of OA.

Free axial vibrations at 0 to 200 Hz positively affect extracellular matrix messenger ribonucleic acid expression in bovine nucleus pulposi.

STUDY DESIGN: Bovine caudal intervertebral discs (IVDs) were exposed to free axial vibration for 10 to 60 minutes at 0 to 0.5 g and 0 to 200 Hz. Expression of messenger ribonucleic acid for aggrecan, collagen type I, collagen type II, biglycan, decorin, and versican were assayed, as was apoptosis. OBJECTIVE: To determine the vibration conditions which are most effective in altering intervertebral disc IVD gene expression. SUMMARY OF BACKGROUND DATA: Various studies have suggested widely varying effects of vibration in the IVD, ranging from harmful (increased risk of degeneration) to beneficial (increased analgesia) to neutral (no effect). METHODS: Vibration was applied using a custom designed voice coil system, which generated controlled motion in the axial direction. Gene expression in the nucleus pulposus was assessed using RT-PCR and the SYBR green chemistry; apoptosis was assessed using TUNEL staining. RESULTS: Expression of messenger ribonucleic acids for biglycan, collagen type I, collagen type II, decorin, and versican were significantly affected by vibration duration, frequency, and amplitude. Aggrecan was unaffected. Of the 3 factors, amplitude had the largest and widest effect. CONCLUSION: Expression of extracellular matrix genes was significantly upregulated at high amplitudes (>0.4 g) in as little as 10 minutes. This may indicate a potential therapeutic stimulus; periodic application of controlled vibration could positively influence matrix maintenance. Further studies on the protein level and long-term effects are warranted.

Influence of bilateral medial collateral ligament injury on mRNA expression in distal corneal tissues of control and ovariohysterectomized rabbits.

PURPOSE: Corneal tissues are reported to be impacted by physiological changes (eg, menopause), systemic autoimmune diseases, and osteoarthritic-like conditions. In this study, changes in specific mRNA levels in the cornea after a ligament injury in normal and rabbits subjected to surgical menopause were examined. METHODS: Skeletally mature female rabbits were either sham-operated (control) or were subjected to surgical menopause (OVX). Eight weeks post-OVX, subsets of control and OVX animals were subjected to bilateral injuries to their medial collateral ligaments (MCL) of the knee, and 6 and 14 weeks postinjury, corneal tissues were harvested. Using reverse transcriptase-polymerase chain reaction, mRNA levels for several relevant molecules, including matrix molecules, growth factors, cytokines, proteinases, and hormone receptors, were assessed. RESULTS: mRNA levels for estrogen receptor, decorin, collagens, several growth factors, and inflammatory cytokines decreased in central corneal tissue 6 weeks after distal MCL injury in control animals. The central corneal tissues of animals subjected to OVX alone also exhibited decreases in mRNA levels for a similar set of molecules. When OVX animals were further subjected to MCL injury, the mRNA levels for many of these molecules did not vary from those in the uninjured OVX group. Interestingly, mRNA levels for most molecules were still altered 14 weeks post-MCL injury in the control and OVX animals, a time when the MCL has healed. CONCLUSIONS: Corneal tissues respond to changes resulting from OVX and/or injury. OVX combined with a ligament injury does not appear to have an additive impact on corneal mRNA levels for most of the molecules assessed.

Influence of timing (pre-puberty or skeletal maturity) of ovariohysterectomy on mRNA levels in corneal tissues of female rabbits.

PURPOSE: Corneal thickness and curvature are reported to be influenced by hormonal changes associated with menstrual cycle, pregnancy, or menopause. However, the molecular mechanisms leading to these alterations are not clearly understood. The present study focuses on gene expression patterns (mRNA levels) in corneal tissues following surgically induced menopause in an animal model. The impact of lower hormone levels on mRNA levels in corneal tissues after pre-puberty ovariohysterectomy (OVX) was compared to that in skeletally mature adult animals. METHODS: Skeletally mature adult female rabbits were either left unoperated (control) or were subjected to OVX at 54 weeks of age using an approved protocol. The central (approximately 6 mm) and the peripheral corneal tissues were harvested from normal and OVX rabbits eight weeks after surgery. In a second study, young sexually immature rabbits at eight weeks of age were subjected to OVX and corneal tissues were collected when the animals were 22 and 32 weeks of age. In both experiments, RNA was isolated from corneal tissues and RT-PCR was used to assess mRNA levels for several relevant molecules. RESULTS: When mature animals were examined eight weeks after OVX, mRNA levels for molecules such as the estrogen receptor, decorin, collagen I, collagen V, and several growth factors were found to be significantly decreased in central corneal tissues. Interestingly, no corresponding changes in mRNA levels were observed for these same molecules in peripheral corneal tissues. When young, pre-pubertal animals were subjected to OVX, mRNA levels were found to be mainly unchanged for the OVX animals at 22 weeks of age i.e., after 14 weeks of low hormone conditions. However, significant decreases in mRNA levels for a similar subset of molecules were observed when the animals were at least 32 weeks of age, i.e., after 24 weeks of a low hormone environment. Examination of peripheral corneal tissues did not show significant changes in mRNA levels due to OVX at either 22 or 32 weeks of age except for collagens I and V at 32 weeks of age. CONCLUSIONS: These results indicate significant alterations in mRNA levels in the central corneal tissues of rabbits following OVX. Interestingly, peripheral corneal tissues show very little alteration in mRNA levels for the same molecules. Furthermore, OVX had a more rapid impact on mRNA levels in mature animals than in skeletally immature animals. Thus, loss of hormone producing tissues during growth and maturation apparently delayed the impact of hormone removal compared to loss after maturity had been attained and growth stimuli are likely absent. Therefore, specific areas of the cornea are more responsive to hormone levels than others. The impact of the loss of hormones is influenced by the maturation state of the rabbit, but mRNA levels for a similar subset of genes are affected by OVX in both age groups.

Impact of age, systemic glucocorticoids, and progressive knee arthritis on specific mRNA levels in different areas of the rabbit cornea.

PURPOSE: To determine the influence of factors such as age, osteoarthritis (OA), and glucocorticoid treatment on total RNA and mRNA regulation in the cornea and how these factors differ between prepupillary and peripheral areas of the cornea. METHODS: Molecular analyses of corneal tissue were performed using rabbits of different age groups and skeletally mature animals that had undergone anterior cruciate ligament (ACL) transection, an established model of knee OA. Systemic glucocorticoids were administered to cohorts of the osteoarthritic and control animals to determine the influence of distal joint disease on the corneal response. Corneal tissue was analyzed for changes in mRNA levels for several relevant genes: collagen I, collagen III, collagen V, decorin core protein, cyclooxygenase-2 (COX-2), glucocorticoid receptor, and the housekeeping gene beta-actin. RESULTS: The corneal tissue was found to have diminishing total RNA with age, which is consistent with previous studies in the literature. Interestingly, in skeletally mature animals, distal joint OA was found to affect corneal mRNA levels for several important structural and inflammatory genes (collagen I, decorin core protein, and COX-2) in a manner that progressed with OA progression. Although systemic glucocorticoid treatment did not alter mRNA levels in the normal cornea, it did counteract the changes observed early after OA induction (3 weeks) while having less of an effect in later, more established arthritis (6 weeks). CONCLUSIONS: This study reveals that distal joint OA can affect mRNA levels for several structural and inflammatory genes of the cornea, changes that seem to be suppressed by systemic glucocorticoid treatment. These findings indicate that OA has associated systemic factors that influence corneal cell metabolism.

Early inflammatory arthritis in the rabbit: the influence of intraarticular and systemic corticosteroids on mRNA levels in connective tissues of the knee.

OBJECTIVE: Using a rabbit model of inflammatory arthritis, to determine the influence of early disease on expression of specific genes and investigate the influence of intraarticular (IA) and intramuscular (IM) corticosteroids on the regulation of these genes in connective tissues of the rabbit knee. METHODS: Skeletally mature rabbits underwent induction of antigen-induced arthritis or remained untreated as control animals. Four days after disease induction, at an early stage of the disease, animals underwent either IA or IM treatment with glucocorticoids (GC) (5 mg/knee and 10 mg/kg methylprednisolone acetate, respectively). Twenty-four hours following treatment, synovium, menisci, and cartilage of the knee were collected and analyzed for changes in mRNA levels using reverse transcription-polymerase chain reaction for a number of relevant genes: collagen I, collagen II, biglycan, decorin, matrix metalloproteinases-3 and -13 (MMP-3 and MMP-13), cyclooxygenases-1 and -2 (COX-1 and COX-2), tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), inducible nitric oxide synthase (iNOS), hyaluronan synthase-2 (HAS-2), and the housekeeping gene beta-actin. RESULTS: Early inflammatory arthritis led to an overall upregulation of most genes assessed, but a downregulation of some genes (iNOS, HAS-2, COX-1) in some tissues. While genes such as collagen II, MMP-3, and MMP-13 were uniformly downregulated by GC treatment in both normal and arthritic tissues, other genes such as collagen I, biglycan, and decorin differed in their pattern of response depending on the tissue examined, the route of drug administration, and whether normal or arthritic tissue was studied. CONCLUSION: Early mRNA changes in RA-like disease led to alterations in all tissues examined. The changes were uniquely altered by GC treatment. Route of GC administration influenced outcome.

Denervation alters mRNA levels of repair-associated genes in a rabbit medial collateral ligament injury model.

Previous experiments revealed that denervation impairs healing of the MCL. This suggested the hypothesis that denervation would decrease repair-associated mRNA levels in the injured MCL when compared with normally innervated injured MCL. Adult, skeletally mature female rabbits were assigned to one of four groups: unoperated control, femoral nerve transection alone (denervated controls), MCL partial tear or denervated MCL partial tear. At three days, two weeks, six weeks or sixteen weeks post-surgery, cohorts of 6 rabbits from each experimental group were killed. Ligaments were harvested, RNA extracted and RT-PCR was performed using rabbitspecific primers. In the denervated injury group, mRNA levels for the angiogenesis-associated gene MMP-13, matrix components Collagen I and III, growth factor TGF-beta and angiogenesis inhibitors TIMP-3, and TSP-1 had all increased by two-weeks post-injury, in comparison to the non-denervated injury group (p < or = 0.01). An increased level of TSP-1 mRNA was also detected in the denervated injured group at sixteen weeks post injury (p < or = 0.01). Contrary to the initial hypothesis, denervation led to increased mRNA levels for many relevant molecules during the early stages of MCL healing. Thus, inappropriate timing of over-expression of some molecules may potentially contribute to the decreased quality of the scar tissue, particularly molecules such as TSP-1. Neuronal derived factors strongly influence the in vivo metabolic activity of ligament and scar fibroblasts in the initial phases of healing.