Carol Reno

POSTMORTEM STABILITY OF TOTAL RNA ISOLATED FROM RABBIT LIGAMENT, TENDON AND CARTILAGE

The stability of RNA, particularly mRNA, in tissues is under complex regulation. Most studies to date have focused on very cellular tissues and not connective tissues such as ligaments, tendons and cartilage. As the availability of such tissues for transplantation or research purposes is frequently delayed following death, it is important to determine whether RNA stability in such tissues is influenced by time postmortem. To approach this question, skeletally mature NZW rabbits were used to investigate RNA integrity over time in dense, hypocellular connective tissues and in several hypercellular organ tissues such as brain, kidney, liver and lung. Samples were analyzed at varying intervals postmortem with respect to rRNA integrity by agarose gel electrophoresis and ethidium bromide staining and mRNA integrity by Northern blot analysis and RT-PCR. No degradation of rRNA or loss in integrity of mRNA for genes of low and high copy number was observed up to 96 h postmortem. These findings confirm that it is likely appropriate to use properly stored postmortem dense connective tissues for molecular biological investigations.

Altered levels of extracellular matrix molecule mRNA in healing rabbit ligaments

RT-PCR methods were used to amplify, semi-quantify, clone and sequence cDNA fragments specific for rabbit extracellular matrix molecules biglycan, collagen I, collagen III, decorin, lumican, versican, fibromodulin, and also glyceraldehyde-3-phosphate dehydrogenase (G3PDH), using RNA isolated from rabbit ligaments. Sequence analysis of two independent clones of PCR products was used to verify the identity of the cDNA. Semi-quantitative RT-PCR was used to study mRNA levels for these matrix molecules in normal and healing rabbit ligament at three, six, and fourteen weeks post-injury. The yield of RNA from the ligament scar was increased at three and six weeks post-injury, but it had returned to near normal levels by fourteen weeks. On a microgram RNA basis, it was demonstrated that biglycan, collagen I, collagen III and lumican mRNA levels are significantly elevated, versican mRNA levels significantly depressed, and decorin and fibromodulin mRNA levels showed no significant change in response to tissue injury in the ligament during the course of healing. These findings suggest that differential regulation of mRNA levels for these extracellular matrix molecules occurs during ligament healing.

Gender and Neurogenic Variables in Tendon Biology and Repetitive Motion Disorders

The incidence of repetitive motion disorders is increasing. Numerous studies have indicated that the incidence in females exceeds that in males. Some of the evidence regarding gender related factors in tendon biology is discussed and new data related to the regulation of gene expression in an animal model of tendon overuse, the determination of sex hormone receptors in tendons, and the influence of pregnancy associated factors on gene expression in four different tendons is provided. Furthermore, because neurogenic mechanisms may contribute to inflammatory conditions, new evidence is provided that supports the concept that neurotransmitters can influence expression of genes that could participate in such inflammation. By increasing our understanding of the regulation of tendon cellular and molecular biology, new approaches to preventing disease development and treatment of existing disease may evolve.

Matrix metalloproteinase-13 expression in rabbit knee joint connective tissues: influence of maturation and response to injury.

The hypothesis of the present work was that expression of matrix metalloproteinase-13 (MMP-13, collagenase-3) would be induced during conditions involving important matrix remodeling such as ligament maturation, scar healing and joint instability. Therefore, MMP-13 expression in the medial collateral ligament (MCL) during the variable situations of tissue maturation and healing was assessed. MMP-13 expression in three intra-articular connective tissues of the knee (i.e. articular cartilage, menisci and synovium) following the transection of the anterior cruciate ligament of the knee was evaluated at 3 and 8 weeks post-injury. MMP-13 mRNA (semi-quantitative RT-PCR) and protein (immunohistochemistry and Western blotting) were detected in all of the tissues studied. Significantly higher MCL mRNA levels for MMP-13 were detected during the early phases of tissue maturation (i.e. 29 days in utero and 2-month-old rabbits) compared to later phases (5- and 12-month-old rabbits). This pattern of expression was recapitulated following MCL injury, with very high levels of expression in scar tissue at 3 weeks post-injury and then a decline to levels not significantly different from control values by 14 weeks. Elevated mRNA levels correlated with increased protein levels for MMP-13 in both menisci and synovium following the transection of the anterior cruciate ligament and during medial collateral ligament healing. These results indicate that MMP-13 expression is regulated by a number of variables and that high levels of expression occur in situations when connective tissue remodeling is very active.

Joint immobilization reduces the expression of sensory neuropeptide receptors and impairs healing after tendon rupture in a rat model.

Healing after mobilization versus immobilization was assessed in a model of rat Achilles tendon rupture, by RT‐PCR at 8 and 17 days and by histological analyses at 14 and 28 days postrupture. The expression of mRNA for extracellular matrix (ECM) molecules (collagen type I and type III, versican, decorin, and biglycan), and the subjective histological maturation of the healing area were analyzed. Effects of immobilization on healing were related to changes in the peripheral expression of substance P (NK1)‐ and calcitonin gene‐related peptide (CRLR and RAMP‐1)‐ receptors. At 8 days postinjury, mRNA levels for ECM molecules were equal in both groups. However, by day 17, the ECM mRNA expression in the mobilized group had increased up to ∼14× that of the immobilized group, which were comparable to intact tendon values. Histological analysis confirmed a higher regenerating activity in the mobilized group, with an increased amount of blood vessels, fibroblasts, and new collagen. The expression of sensory neuropeptide receptors in the mobilized group exhibited a significant increase from 8 to 17 days postinjury similar to the increased ECM mRNA expression, whereas the immobilized group at 17 days exhibited levels comparable to the intact tendon values. Therefore, immobilization postrupture appears to hamper tendon healing, a process which may prove to be directly linked to a downregulated peripheral sensitivity to sensory neuropeptide stimulation.

Prolonged immobilization compromises up-regulation of repair genes after tendon rupture in a rat model.

It was hypothesized that mobilization vs immobilization after injury would promote tissue healing by regulating gene expression for molecules associated with repair. Cast immobilization vs free mobilization was studied after rat Achilles tendon rupture. Reverse transcriptase‐polymerase chain reaction was performed at 8 and 17 days post‐rupture to assess different growth factors [brain‐derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and insulin‐like growth factor‐1 (IGF‐1)] and inflammatory mediators [cyclooxygenase 1 and 2 (COX 1 and COX 2), inducible nitric oxide synthase and hypoxia‐inducible factor‐1α (HIF‐1α)] in the healing region. At 8 days post‐injury, tendon mRNA levels were comparable in both groups. However, by day 17, the mRNA levels for BDNF, bFGF, COX 1 and HIF‐1α in the mobilized group had increased significantly. Corresponding mRNA levels in the immobilized group decreased during the same period. There were no significant differences in the expression of NGF, IGF‐1 or COX 2 between the different groups, indicating that injury‐associated expression of these molecules is not overtly influenced by loading. This study supports the notion that prolonged immobilization post‐rupture hampers the healing process by compromising the up‐regulation of repair gene expression in the healing tendon. It might be speculated that a shorter period of immobilization, i.e. 1 week, would not impair the healing process significantly. The findings support the current development of earlier and more active rehabilitation programs after tendon injuries.

PREGNANCY INDUCES COMPLEX CHANGES IN THE PATTERN OF MRNA EXPRESSION IN KNEE LIGAMENTS OF THE ADOLESCENT RABBIT

Knee laxity has been shown to increase during human pregnancy, and the laxity of the rabbit medial collateral ligament also increases during pregnancy. To determine whether the changes in tissue function could be related to alterations in the regulation of gene expression for a subset of relevant molecules in ligaments, RNA was isolated from the medial collateral(MCL) and anterior cruciate(ACL) ligaments of first time pregnant adolescent rabbits. Levels of mRNA for matrix molecules (collagen types I and III and the proteoglycans biglycan, decorin, versican and lumican), proteinases and inhibitors (collagenase, urokinase, PAI-1 and TIMP-1, -2 and -3), growth factors (bFGF, IGF-I, TGF-beta1 and ET-1), cytokines (IL-1beta and TNF) and enzymes responsible for important tissue mediators (COX-2 and iNOS) were assessed by semi-quantitative RT-PCR. In the MCL, levels of transcripts for all of the matrix molecules, growth factors and TIMPs 1 and 2 were significantly depressed at 29 days of pregnancy compared to age-matched non-pregnant controls. In contrast, transcripts for PAI-1 were elevated during pregnancy, while those for collagenase (MMP-1), urokinase, TIMP-3, IL-1beta, TNF, COX-2 and iNOS were not statistically altered. mRNA transcript levels rebounded by 7 days post-partum for most genes studied, indicating that the changes were rapidly reversible. For some molecules, transcript levels were again depressed at 18 days post-partum, indicating that regulatory mechanisms were still not stabilized. Analysis of mRNA from the ACL also revealed changes in the pattern of gene expression, with some similarities and differences from the MCL noted. These results indicate that pregnancy induces reversible changes in mRNA for matrix molecules in ligaments, but differences in responsiveness exist between different ligaments. The complexity of the changes observed indicates that there is probably no simple cause and effect relationship between laxity changes and the molecular alterations during pregnancy.

Gender and Pregnancy Affect Neuropeptide Responses of the Rabbit Achilles Tendon

Tendons such as the Achilles tendon are complex structures that are hypocellular, hypovascular, and hyponeural. The development of pathophysiologic function in this tendon because of overuse is relatively common; however, the mechanisms responsible for the development of paratenonitis and tendinosis remain primarily undefined. To understand better a possible regulatory role for neuropeptides (substance P and calcitonin gene related peptide) known to be present in this tissue, the influence of substance P and calcitonin gene related peptide on messenger ribonucleic acid levels for numerous inflammatory molecules, growth factors, and proteinases and inhibitors have been assessed using a semiquantitative reverse transcription-polymerase chain reaction method and explants of paratenon and Achilles tendon midsubstance tissue from adolescent male and female rabbits and tissue from primigravida females. Most of the significant (p < 0.01) changes observed were at the level of the growth factor transcripts and transcripts for proteinases and inhibitors. Twenty-one significant differences in the responsiveness between tissues from male and female rabbits were observed, and 12 significant differences in responsiveness between virgin females and primigravida rabbits were seen. Differences between paratenon and midsubstance responsiveness to the neuropeptides also were observed within each group of animals. The midsubstance tissue from pregnant animals was hyporesponsive to both neuropeptides. These results indicate that neurotransmitter responsiveness of Achilles tendon tissue differs in a gender specific manner and is influenced by pregnancy associated factors.

Neuropeptides regulate expression of matrix molecule, growth factor and inflammatory mediator mRNA in explants of normal and healing medial collateral ligament

Denervation degrades normal ligament properties and impairs ligament healing. This suggests that secreted neuromediators, such as neuropeptides, could be modulating cell metabolism in ligament and scar tissue. To test this hypothesis we investigated the effect of exogenous substance P (SP), neuropeptide Y (NPY) or calcitonin gene-related peptide (CGRP) on the mRNA levels for proteins associated with inflammation, angiogenesis, and matrix production in tissue-cultured specimens of normal and injured medial collateral ligament. SP and NPY induced increased mRNA levels for several inflammatory mediators in the 2-week post-injury specimens. All three neuropeptides induced decreases in mRNA levels for healing-associated growth factors and matrix molecules, including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and collagen types I and III. The results indicate that neuropeptides strongly influence the metabolic activity of cells in healing ligament, particularly at early time points after injury.

Complex alterations in gene expression occur in the knee ligaments of the skeletally mature multiparous rabbit during pregnancy

Pregnancy is known to influence the function of ligaments of the knee in both humans and rabbits. During pregnancy, ligament laxity increases. The mechanism by which these changes in ligament function occur is unknown. The present study was undertaken to assess changes in the pattern of gene expression for a number of molecules which could be involved in the alterations in tissue function. Using RNA isolated from pregnant and age-matched nonpregnant rabbits, levels of mRNA for matrix molecules, proteinases and inhibitors, cytokines and growth factors and inducible nitric oxide synthase were assessed by semi-quantitative RT-PCR. The results indicate that the pregnancy-induced changes in the pattern of gene expression is very complex and differences in the changes observed for the medial collateral ligament and the anterior cruciate ligament may also indicate that ligament-specific effects of pregnancy occur. mRNA levels of some molecules were significantly elevated or suppressed, while others were unchanged, indicating that the changes observed were specific rather than general. While the extent of the molecules assessed was designed to be representative rather than exhaustive, the results indicate that there is likely no simple cause and effect relationship between the observed mechanical alterations during pregnancy and alterations in gene expression in the affected ligaments.