Diet Quality and Survival in a Population-Based Bladder Cancer Study.

Nutrition may impact bladder cancer survival. We examined the association between diet quality and overall and bladder cancer-specific survival. Bladder cancer cases from a population-based study reported pre-diagnosis diet. Diet quality was assessed using the 2010 Alternate Healthy Eating Index (AHEI-2010). Vital status was ascertained from the National Death Index. Adjusted hazard ratios (HR) and 95% confidence intervals (CI) were estimated using proportional hazards and competing risks regression models. Overall AHEI-2010 adherence was not associated with overall or bladder cancer-specific survival among non-muscle invasive bladder cancer (NMIBC) cases (HR, 1.00; 95% CI, 0.98-1.01; HR, 1.00; 95% CI, 0.97-1.02) or muscle invasive bladder cancer (MIBC) cases (HR, 0.99; 95% CI, 0.96-1.03; HR, 1.01, 95% CI 0.97-1.06). AHEI-2010 sugar-sweetened beverages adherence was associated with poorer overall survival (HR, 1.04; 95% CI, 1.01-1.08) and AHEI-2010 sodium adherence was associated with better overall and bladder cancer-specific survival after NMIBC diagnosis (HR, 0.92, 95% CI, 0.85-1.00; HR, 0.82; 95% CI, 0.68-0.98). AHEI-2010 fruit adherence was associated with poorer overall and bladder cancer-specific survival after MIBC diagnosis (HR, 1.17; 95% CI, 1.02-1.33; HR, 1.26; 95% CI, 1.03-1.55). Consumption of sugar-sweetened beverages, sodium, and fruit, not overall AHEI-2010 adherence, may be associated with bladder cancer survival.

Diet quality, common genetic polymorphisms, and bladder cancer risk in a New England population-based study.

PURPOSE: We examined the interaction between common genetic bladder cancer variants, diet quality, and bladder cancer risk in a population-based case-control study conducted in New England. METHODS: At the time of enrollment, 806 bladder cancer cases and 974 controls provided a DNA sample and completed a diet history questionnaire. Diet quality was assessed using the 2010 Alternate Healthy Eating Index (AHEI-2010) score. Single nucleotide polymorphisms (SNPs) reported in genome-wide association studies to be associated with bladder cancer risk were combined into a polygenic risk score and also examined individually for interaction with the AHEI-2010. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression. RESULTS: A 1-standard deviation increase in polygenic risk score was associated with higher bladder cancer risk (OR, 1.34; 95% CI 1.21-1.49). Adherence to the AHEI-2010 was not associated with bladder cancer risk (OR, 0.99; 95% CI 0.98-1.00) and the polygenic risk score did not appear to modify the association between the AHEI-2010 and bladder cancer risk. In single-SNP analyses, rs8102137 (bladder cancer risk allele, C) modified the association between the AHEI-2010 total score and bladder cancer risk, with the strongest evidence for the AHEI-2010 long chain fat guideline (OR for TT, 0.92; 95% CI 0.87-0.98; OR for CT, 1.02; 95% CI 0.96-1.08; OR for CC, 1.03; 95% CI 0.93-1.14; p for interaction, 0.02). CONCLUSIONS: In conclusion, rs8102137 near the cyclin E1 gene ( CCNE1 ) may be involved in gene-diet interactions for bladder cancer risk.

Small-scale spatial analysis shows the specular distribution of excess mortality between the first and second wave of the COVID-19 pandemic in Italy.

OBJECTIVES: The objective of the study is to compare excess mortality (EM) patterns and spatial correlation between the first and second wave of the pandemic in Lombardy, the Italian region that paid an extremely high COVID-19-related mortality toll in March and April 2020. STUDY DESIGN: We conducted a longitudinal study using municipality-level mortality data. METHODS: We investigated the patterns and spatial correlation of EM of men aged ≥75 years during the first two pandemic waves (March-April 2020 vs November 2020) of COVID-19, using the mortality data released by the Italian National Institute of Statistics. EM was estimated at the municipality level to accurately detect the critical areas within the region. RESULTS: The areas that were mostly hit during the first wave of COVID-19 were generally spared by the second wave: EM of men aged ≥75 years in the municipality of Bergamo plummeted from +472% in March and April to -13% in November, and in Cremona the variation was from +344% to -19%. Conversely, in November 2020 EM was higher in some areas that had been protected in the first wave of the pandemic. Spatial correlation widely corroborates these findings, as large sections of the hot spots of EM detected in the first wave of the pandemic changed into cold spots in the second wave, and vice versa. CONCLUSIONS: Our results reveal the specular distribution of EM between the first and second wave of the pandemic, which may entail the consequences of social distancing measures and individual behaviors, local management strategies, 'harvesting' of the frailer population and, possibly, acquired immune protection. In conclusion, our findings support the need for continuous monitoring and analysis of mortality data using detailed spatial resolution.

TRP channels coordinate ion signalling in astroglia.

Astroglial excitability is based on highly spatio-temporally coordinated fluctuations of intracellular ion concentrations, among which changes in Ca(2+) and Na(+) take the leading role. Intracellular signals mediated by Ca(2+) and Na(+) target numerous molecular cascades that control gene expression, energy production and numerous homeostatic functions of astrocytes. Initiation of Ca(2+) and Na(+) signals relies upon plasmalemmal and intracellular channels that allow fluxes of respective ions down their concentration gradients. Astrocytes express several types of TRP channels of which TRPA1 channels are linked to regulation of functional expression of GABA transporters, whereas TRPV4 channels are activated following osmotic challenges and are up-regulated in ischaemic conditions. Astrocytes also ubiquitously express several isoforms of TRPC channels of which heteromers assembled from TRPC1, 4 and/or 5 subunits that likely act as stretch-activated channels and are linked to store-operated Ca(2+) entry. The TRPC channels mediate large Na(+) fluxes that are associated with the endoplasmic reticulum Ca(2+) signalling machinery and hence coordinate Na(+) and Ca(2+) signalling in astroglia.

Activation of neuronal NMDA receptors induces superoxide-mediated oxidative stress in neighboring neurons and astrocytes.

Excitotoxic neuronal death is mediated in part by NMDA receptor-induced activation of NOX2, an enzyme that produces superoxide and resultant oxidative stress. It is not known, however, whether the superoxide is generated in the intracellular space, producing oxidative stress in the neurons responding to NMDA receptor activation, or in the extracellular space, producing oxidative stress in neighboring cells. We evaluated these alternatives by preparing cortical neuron cultures from p47(phox-/-) mice, which are unable to form a functional NOX2 complex, and transfecting the cultures at low density with GFP-tagged p47(phox) to reconstitute NOX2 activity in widely scattered neurons. NMDA exposure did not induce oxidative stress or cell death in the nontransfected, p47-phox(-/-) cultures, but did produce oxidative stress and neuronal death in neurons surrounding the transfected, NOX2-competent neurons. This cell-to-cell spread of NMDA-induced oxidative injury was blocked by coincubation with either superoxide dismutase or the anion channel blocker 4'-diisothiocyanostilbene-2,2'-disulphonate, confirming superoxide anion as the mediating oxidant. In neurons plated on a preexisting astrocyte layer, NMDA induced oxidative stress in both the neurons and the astrocytes, and this was also prevented by superoxide dismutase. These findings show that activation of NMDA receptors on one neuron can lead to oxidative stress and cell death in neighboring neurons and astrocytes by a process involving the extracellular release of superoxide by NOX2.

Enhanced Ca(2+)-dependent glutamate release from astrocytes of the BACHD Huntington's disease mouse model.

Huntington's disease (HD) causes preferential loss of a subset of neurons in the brain although the huntingtin protein is expressed broadly in various neural cell types, including astrocytes. Glutamate-mediated excitotoxicity is thought to cause selective neuronal injury, and brain astrocytes have a central role in regulating extracellular glutamate. To determine whether full-length mutant huntingtin expression causes a cell-autonomous phenotype and perturbs astrocyte gliotransmitter release, we studied cultured cortical astrocytes from BACHD mice. Here, we report augmented glutamate release through Ca(2+)-dependent exocytosis from BACHD astrocytes. Although such release is usually dependent on cytosolic Ca(2+) levels, surprisingly, we found that BACHD astrocytes displayed Ca(2+) dynamics comparable to those in wild type astrocytes. These results point to a possible involvement of other factors in regulating Ca(2+)-dependent/vesicular release of glutamate from astrocytes. We found a biochemical footprint that would lead to increased availability of cytosolic glutamate in BACHD astrocytes: i) augmented de novo glutamate synthesis due to an increase in the level of the astrocyte specific mitochondrial enzyme pyruvate carboxylase; and ii) unaltered conversion of glutamate to glutamine, as there were no changes in the expression level of the astrocyte specific enzyme glutamine synthetase. This work identifies a new mechanism in astrocytes that could lead to increased levels of extracellular glutamate in HD and thus may contribute to excitotoxicity in this devastating disease.

N-acetylcysteine prevents loss of dopaminergic neurons in the EAAC1-/- mouse.

OBJECTIVE: Dopaminergic neuronal death in Parkinson's disease (PD) is accompanied by oxidative stress and preceded by glutathione depletion. The development of disease-modifying therapies for PD has been hindered by a paucity of animal models that mimic these features and demonstrate an age-related progression. The EAAC1(-/-) mouse may be useful in this regard, because EAAC1(-/-) mouse neurons have impaired neuronal cysteine uptake, resulting in reduced neuronal glutathione content and chronic oxidative stress. Here we aimed to (1) characterize the age-related changes in nigral dopaminergic neurons in the EAAC1(-/-) mouse, and (2) use the EAAC1(-/-) mouse to evaluate N-acetylcysteine, a membrane-permeable cysteine pro-drug, as a potential disease-modifying intervention for PD. METHODS: Wild-type mice, EAAC1(-/-) mice, and EAAC1(-/-) mice chronically treated with N-acetylcysteine were evaluated at serial time points for evidence of oxidative stress, dopaminergic cell death, and motor abnormalities. RESULTS: EAAC1(-/-) mice showed age-dependent loss of dopaminergic neurons in the substantia nigra pars compacta, with more than 40% of these neurons lost by age 12 months. This neuronal loss was accompanied by increased nitrotyrosine formation, nitrosylated α-synuclein, and microglial activation. These changes were substantially reduced in mice that received N-acetylcysteine. INTERPRETATION: These findings suggest that the EAAC1(-/-) mouse may be a useful model of the chronic neuronal oxidative stress that occurs in PD. The salutary effects of N-acetylcysteine in this mouse model provide an impetus for clinical evaluation of glutathione repletion in PD.

Inositol hexakisphosphate kinases differentially regulate trafficking of vesicular glutamate transporters 1 and 2.

Inositol pyrophosphates have been implicated in cellular signaling and membrane trafficking, including synaptic vesicle (SV) recycling. Inositol hexakisphosphate kinases (IP6Ks) and their product, diphosphoinositol pentakisphosphate (PP-IP5 or IP7), directly and indirectly regulate proteins important in vesicle recycling by the activity-dependent bulk endocytosis pathway (ADBE). In the present study, we show that two isoforms, IP6K1 and IP6K3, are expressed in axons. The role of the kinases in SV recycling are investigated using pharmacologic inhibition, shRNA knockdown, and IP6K1 and IP6K3 knockout mice. Live-cell imaging experiments use optical reporters of SV recycling based on vesicular glutamate transporter isoforms, VGLUT1- and VGLUT2-pHluorins (pH), which recycle differently. VGLUT1-pH recycles by classical AP-2 dependent endocytosis under moderate stimulation conditions, while VGLUT2-pH recycles using AP-1 and AP-3 adaptor proteins as well. Using a short stimulus to release the readily releasable pool (RRP), we show that IP6K1 KO increases exocytosis of both VGLUT1-and VGLUT2-pH, while IP6K3 KO decreases the amount of both transporters in the RRP. In electrophysiological experiments we measure glutamate signaling with short stimuli and under the intense stimulation conditions that trigger bulk endocytosis. IP6K1 KO increases synaptic facilitation and IP6K3 KO decreases facilitation compared to wild type in CA1 hippocampal Schaffer collateral synapses. After intense stimulation, the rate of endocytosis of VGLUT2-pH, but not VGLUT1-pH, is increased by knockout, knockdown, and pharmacologic inhibition of IP6Ks. Thus IP6Ks differentially affect the endocytosis of two SV protein cargos that use different endocytic pathways. However, while IP6K1 KO and IP6K3 KO exert similar effects on endocytosis after stimulation, the isoforms exert different effects on exocytosis earlier in the stimulus and on the early phase of glutamate release. Taken together, the data indicate a role for IP6Ks both in exocytosis early in the stimulation period and in endocytosis, particularly under conditions that may utilize AP-1/3 adaptors.

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-1alpha (HIF-1alpha)] 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-1alpha 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.

Mitochondria modulate Ca2+-dependent glutamate release from rat cortical astrocytes.

Vesicular glutamate release from astrocytes depends on mobilization of free Ca(2+) from the endoplasmic reticulum (ER), and extracellular space to elevate cytosolic Ca(2+) (Ca(2+)(cyt)). Although mitochondria in neurons, and other secretory cells, have been shown to sequester free Ca(2+) and have been implicated in the modulation of Ca(2+)-dependent transmitter release, the role of mitochondria in Ca(2+)-dependent glutamate release from astrocytes is not known. A pharmacological approach was taken to manipulate Ca(2+) accumulation in mitochondria and thereby affect Ca(2+)(cyt) of solitary astrocytes in response to mechanical stimuli. Ca(2+)(cyt) responses and levels of glutamate release were measured optically in parallel experiments using a fluorescent Ca(2+) indicator and an enzyme-linked assay, respectively. It was observed that inhibiting mitochondrial Ca(2+) accumulation is correlated to increased Ca(2+)(cyt) and glutamate release, whereas enhancing mitochondrial Ca(2+) accumulation is correlated to decreased Ca(2+)(cyt) and glutamate release. These observations suggest that, in addition to the activity of ER and plasma membrane ion channels, mitochondria modulate Ca(2+)(cyt) dynamics in astrocytes and play a role in Ca(2+)-dependent glutamate release from astrocytes.

Models of astrocytic Ca dynamics and epilepsy.

Neurons have been the focus of neuroscience research. Only recently, however, astrocytes, a subset of glial cells, have been on the neurobiology "radar" owing to their Ca(2+) excitability, which allows them to signal to other astrocytes and neurons. This review summarizes the models for studying astrocytic Ca(2+) dynamics and the consequential Ca(2+)- dependent glutamate release, which plays a role in astrocytic-neuronal signaling and have been implicated in epilepsy.

Skin wound healing in the first generation (F1) offspring of Yorkshire and red Duroc pigs: evidence for genetic inheritance of wound phenotype.

Fibroproliferative scars in humans often demonstrate familial inheritance patterns, and genetics may contribute to healing and scarring. Genetic factors may also influence the scarring phenotype in a porcine model. Healing of full thickness excisional skin wounds in Yorkshire pigs closely resembles normal healing in humans, while identical wounds in red Duroc pigs form hypercontracted, hyperpigmented scars. The present study has evaluated the healing process in the first generation cross (F1) of red Duroc and Yorkshire pigs. Gross and histologic analysis revealed that the F1 animals exhibit an intermediate healing phenotype, with some features of each parent breed. F1 full thickness wounds were significantly hypercontracted and fibrotic, but apigmented. Analysis of mRNA expression patterns for a panel of relevant molecules (N=32) in the F1 animals revealed some similarities to each parent breed, as well as unique patterns for other molecules. Furthermore, a depth dependency to the healing response was observed at the gross, histologic, and molecular levels, with deep dermal wounds healing similar to Yorkshire wounds. These findings suggest that the genetic contribution to scar phenotype in this animal model is complex. However, the results indicate that further understanding in this model may provide insights into risk factors for hypertrophic scarring in human burn patients.

Neuronal Glutathione Content and Antioxidant Capacity can be Normalized In Situ by N-acetyl Cysteine Concentrations Attained in Human Cerebrospinal Fluid.

N-acetyl cysteine (NAC) supports the synthesis of glutathione (GSH), an essential substrate for fast, enzymatically catalyzed oxidant scavenging and protein repair processes. NAC is entering clinical trials for adrenoleukodystrophy, Parkinson's disease, schizophrenia, and other disorders in which oxidative stress may contribute to disease progression. However, these trials are hampered by uncertainty about the dose of NAC required to achieve biological effects in human brain. Here we describe an approach to this issue in which mice are used to establish the levels of NAC in cerebrospinal fluid (CSF) required to affect brain neurons. NAC dosing in humans can then be calibrated to achieve these NAC levels in human CSF. The mice were treated with NAC over a range of doses, followed by assessments of neuronal GSH levels and neuronal antioxidant capacity in ex vivo brain slices. Neuronal GSH levels and antioxidant capacity were augmented at NAC doses that produced peak CSF NAC concentrations of ≥50 nM. Oral NAC administration to humans produced CSF concentrations of up to 10 µM, thus demonstrating that oral NAC administration can surpass the levels required for biological activity in brain. Variations of this approach may similarly facilitate and rationalize drug dosing for other agents targeting central nervous system disorders.

Pregnancy alters the in vitro responsiveness of the rabbit medial collateral ligament to neuropeptides: effect on mRNA levels for growth factors, cytokines, iNOS, COX-2, metalloproteinases and TIMPs.

Explants of tissue derived from the medial collateral ligament (MCL) of normal and pregnant NZW rabbits cultured in the presence of substance P (SP), calcitonin gene-related peptide (CGRP), or both neuropeptides were found to have altered mRNA levels for a number of relevant molecules. Using a very efficient RNA isolation method, semi-quantitative RT-PCR and rabbit-specific primers, mRNA for growth factors (TGFbeta, bFGF, IGF-2, ET-1), cytokines (IL-1, TNF), enzymes (COX-2, iNOS), metalloproteinases (collagenase, stromelysin) and metalloproteinase inhibitors (TIMP-1, TIMP-2) were assessed after culture with or without neuropeptide. The results indicate that SP was effective in lowering mRNA levels for all of the molecules assessed in RNA from normal ligaments except IL-1beta, IGF-2 and TIMP-1, for which there was no significant effect. Similarly, CGRP was effective in lowering mRNA levels for all molecules except TNF, ET-1 and the TIMPs. The extent of the lowering of mRNA levels was both molecule-specific and neuropeptide-specific. When the experiments were repeated with ligament tissue from pregnant animals, a very different pattern of responsiveness to the neuropeptides was observed. While mRNA levels for 9/12 genes assessed were significantly affected by SP when normal MCL tissue was investigated, pregnancy abolished all significant responsiveness to this neuropeptide except for iNOS mRNA levels. In the case of iNOS mRNA, SP induced an increase in the steady-state levels, the opposite to what was observed with tissue from non-pregnant animals. For CGRP and SP+CGRP, tissue from pregnant animals was still responsive, but the pattern of responsiveness was changed from strictly a lowering of steady-state mRNA levels to elevations in mRNA levels for a number of genes. These findings indicate that mRNA levels for a number of genes can be influenced by neuropeptides known to be in ligaments. Thus, neuropeptides likely are important regulators of ligament cell metabolism. As the responsiveness to SP was nearly completely abolished during pregnancy, neuroregulatory influences mediated by this peptide are altered in the pregnant female. This loss of responsiveness to SP may also be one aspect of the analgesia associated with pregnancy.

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.

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.

Surgical menopause initiates molecular changes that do not result in mechanical changes in normal and healing ligaments.

OBJECTIVES: Ligaments which heal spontaneously have a healing process that is similar to skin wound healing. Menopause impairs skin wound healing and may likewise impair ligament healing. Our purpose in this study was to investigate the effect of surgical menopause on ligament healing in a rabbit medial collateral ligament model. METHODS: Surgical menopause was induced with ovariohysterectomy surgery in adult female rabbits. Ligament injury was created by making a surgical gap in the midsubstance of the medial collateral ligament. Ligaments were allowed to heal for six or 14 weeks in the presence or absence of oestrogen before being compared with uninjured ligaments. Molecular assessment examined the messenger ribonucleic acid levels for collagens, proteoglycans, proteinases, hormone receptors, growth factors and inflammatory mediators. Mechanical assessments examined ligament laxity, total creep strain and failure stress. RESULTS: Surgical menopause in normal medial collateral ligaments initiated molecular changes in all the categories evaluated. In early healing medial collateral ligaments, surgical menopause resulted in downregulation of specific collagens, proteinases and inflammatory mediators at 6 weeks of healing, and proteoglycans, growth factors and hormone receptors at 14 weeks of healing. Surgical menopause did not produce mechanical changes in normal or early healing medial collateral ligaments. With or without surgical menopause, healing ligaments exhibited increased total creep strain and decreased failure stress compared with uninjured ligaments. CONCLUSIONS: Surgical menopause did not affect the mechanical properties of normal or early healing medial collateral ligaments in a rabbit model. The results in this preclinical model suggest that menopause may result in no further impairment to the ligament healing process. Cite this article: Bone Joint Res 2015;4:38-44.

Pregnancy induces complex changes in the 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.

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.