Probes for LONG

Determination of possible sex differences in mechanisms promoting migraine progression and the contribution of prolactin and the prolactin long (PRLR-L) and short (PRLR-S) receptor isoforms.The majority of patients with chronic migraine and medication overuse headache are female. Prolactin is present at higher levels in women and increases migraine. Prolactin signaling at the PRLR-S selectively sensitizes nociceptors in female rodents, while expression of the PRLR-L is protective.Medication overuse headache was modeled by repeated sumatriptan administration in male and female mice. Periorbital and hindpaw cutaneous allodynia served as a surrogate of migraine-like pain. PRLR-L and PRLR-S isoforms were measured in the trigeminal ganglion with western blotting. Possible co-localization of PRLR with serotonin 5HT1B and 5HT1D receptors was determined with RNAscope. Cabergoline, a dopamine receptor agonist that inhibits circulating prolactin, was co-administered with sumatriptan. Nasal administration of CRISPR/Cas9 plasmid was used to edit expression of both PRLR isoforms.PRLR was co-localized with 5HT1B or 5HT1D receptors in the ophthalmic region of female trigeminal ganglion. A single injection of sumatriptan increased serum PRL levels in female mice. Repeated sumatriptan promoted cutaneous allodynia in both sexes but down-regulated trigeminal ganglion PRLR-L, without altering PRLR-S, only in females. Co-administration of sumatriptan with cabergoline prevented allodynia and down-regulation of PRLR-L only in females. CRISPR/Cas9 editing of both PRLR isoforms in the trigeminal ganglion prevented sumatriptan-induced periorbital allodynia in females.We identified a sexually dimorphic mechanism of migraine chronification that involves down-regulation of PRLR-L and increased signaling of circulating prolactin at PRLR-S. These studies reveal a previously unrecognized neuroendocrine mechanism linking the hypothalamus to nociceptor sensitization that increases the risk of migraine pain in females and suggest opportunities for novel sex-specific therapies including gene editing through nasal delivery of CRISPR/Cas9 constructs.

Although pheochromocytomas and paragangliomas (PPGLs) are usual low-grade neoplasms, the metastatic forms of these lesions are associated with high morbidity and mortality. Recent studies have discovered multiple aberrantly expressed long non-coding RNAs (lncRNAs) in cancers that may have regulatory roles in tumor pathogenesis and metastasis; however, the roles of some lncRNAs in PPGLs are still unknown. The expression levels of lncRNAs including metastasis-associated lung adenocarcinoma transcript (MALAT1), prostate cancer antigen 3 (PCA3), and HOX transcript antisense intergenic RNA (HOTAIR) in PPGLs were analyzed by in situ hybridization, using two tissue microarrays (TMAs). The pheochromocytoma (PCC) TMA consisted of normal adrenal medulla (N = 25), non-metastatic PCCs (N = 76) and metastatic PCCs (N = 5) while the paraganglioma (PGL) TMA had 73 non-metastatic PGLs and 5 metastatic PGLs. Immunohistochemical staining was performed on all samples with an anti-SDHB antibody. The correlations between lncRNA expression, loss of SDHB expression and clinical characteristics including tumor progression and disease prognosis were investigated. The expression levels of MALAT1 and PCA3 were significantly elevated (2.5-3.9 folds) in both non-metastatic and metastatic PCCs compared to normal adrenal medulla, although there were no significant differences between the non-metastatic and metastatic neoplasms. In contrast to non-metastatic PGLs, metastatic PGLs had significantly upregulated expression of MALAT1, PCA3, and HOTAIR. SDHB loss was more frequently observed in PGLs (25 of 78), especially in metastatic PGLs (5 of 5), compared to PCCs (2 of 81) and in 0 of 5 metastatic PCCs. Patients with SDHB loss, in contrast to SDHB retained, were younger at diagnosis, had higher rates of tumor recurrence, metastatic disease, and mortality. In addition, PGLs with SDHB loss had significantly increased expression of PCA3 compared to tumors with intact SDHB expression. Our findings suggest that specific lncRNAs may be involved in the SDHx signaling pathways in the tumorigenesis and in the development of PPGL.

With variable potencies atrial-, brain-type and c-type natriuretic peptides (NP)s, best documented for ANP and its analogues, promote sodium and water excretion, renal blood flow, lipolysis, lower blood pressure, and suppress renin and aldosterone secretion through interaction predominantly with cGMP-coupled NPR-A receptor. Infusion of especially ANP and its analogues up to 50 ng/kg/min in patients with high risk of acute kidney injury (cardiac vascular bypass surgery, intraabdominal surgery, direct kidney surgery) protects kidney function (GFR, plasma flow, medullary flow, albuminuria, renal replacement therapy, tissue injury) at short term and also long term and likely additively with the diuretic furosemide. This documents a pharmacologic potential for the pathway. Neprilysin (NEP, neutral endopeptidase) degrades NPs, in particular ANP, and angiotensin II. The drug LCZ696, a mixture of the neprilysin inhibitor sacubitril and the ANGII-AT1 receptor blocker valsartan, was FDA approved in 2015 and marketed as Entresto . In preclinical studies of kidney injury, LCZ696 and NPs lowered plasma creatinine, countered hypoxia and oxidative stress, suppressed proinflammatory cytokines, and inhibited fibrosis. Few randomized clinical studies exist and were designed with primary cardiac outcomes. The studies showed that LCZ696/entresto stabilized and improved glomerular filtration rate in patients with chronic kidney disease. LCZ696 is safe to use concerning kidney function and stabilizes or increases GFR. In perspective, combined AT1 and neprilysin inhibition is a promising approach for long-term renal protection in addition to AT1 receptor blockers in acute kidney injury and chronic kidney disease.

Intestinal homeostasis disorder is critical for developing Crohn's disease (CD). Maintaining mucosal barrier integrity is essential for intestinal homeostasis, preventing intestinal injury and complications. Among the remarkably altered long non-coding RNAs (lncRNAs) in CD, we aimed to investigate whether metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) modulated CD and consequent disruption of intestinal homeostasis. Microarray analyses on intestinal mucosa of CD patients and controls were performed to identify dysregulated lncRNAs. MALAT1 expression was investigated via qRT-PCR and its distribution in intestinal tissues was detected using BaseScope. Intestines from MALAT1 knockout mice with colitis were investigated using histological, molecular and biochemical approaches. Effects of intestinal epithelial cells transfected with MALAT1 lentiviruses and Smart Silencer, on monolayer permeability and apical junction complex (AJC) proteins were analysed. MiR-146b-5p were confirmed as a critical MALAT1 mediator in cells transfected with miR-146b-5p mimic/inhibitor and in colitis mice administered with agomir-146b-5p/antagomir-146b-5p. Interaction between MALAT1 and miR-146b-5p was predicted via bioinformatics and validated using Dual-luciferase reporter assay and Ago2-RIP. MALAT1 was aberrantly downregulated in the intestine mucosa of CD patients and mice with experimental colitis. MALAT1 knockout mice were hypersensitive to DSS-induced experimental colitis. MALAT1 regulated intestinal mucosal barrier and regained intestinal homeostasis by sequestering miR-146b-5p and maintaining the expression of the AJC proteins NUMB and CLDN11. Downregulation of MALAT1 contributed to the pathogenesis of CD by disrupting AJC. Thus, a specific MALAT1-miR-146b-5p-NUMB/CLDN11 pathway that plays a vital role in maintaining intestinal mucosal homeostasis may serve as a novel target for CD treatment.

Sarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve, which governs lower limb muscles, in aging mice.Sciatic nerve and gastrocnemius muscle were obtained from female C57BL/6JN mice aged 5, 18, 21 and 24 months old (n = 6 per age group). Sciatic nerve RNA was extracted and underwent RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) were validated using quantitative reverse transcription PCR (qRT-PCR). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups (adjusted P-value < 0.05, likelihood ratio test [LRT]) was performed. Pathological skeletal muscle aging was confirmed between 21 and 24 months by a combination of molecular and pathological biomarkers. Myofiber denervation was confirmed with qRT-PCR of Chrnd, Chrng, Myog, Runx1 and Gadd45ɑ in gastrocnemius muscle. Changes in muscle mass, cross-sectional myofiber size and percentage of fibres with centralized nuclei were analysed in a separate cohort of mice from the same colony (n = 4-6 per age group).We detected 51 significant DEGs in sciatic nerve of 18-month-old mice compared with 5-month-old mice (absolute value of fold change > 2; false discovery rate [FDR] < 0.05). Up-regulated DEGs included Dbp (log2 fold change [LFC] = 2.63, FDR < 0.001) and Lmod2 (LFC = 7.52, FDR = 0.001). Down-regulated DEGs included Cdh6 (LFC = -21.38, FDR < 0.001) and Gbp1 (LFC = -21.78, FDR < 0.001). We validated RNA-seq findings with qRT-PCR of various up- and down-regulated genes including Dbp and Cdh6. Up-regulated genes (FDR < 0.1) were associated with the AMP-activated protein kinase signalling pathway (FDR = 0.02) and circadian rhythm (FDR = 0.02), whereas down-regulated DEGs were associated with biosynthesis and metabolic pathways (FDR < 0.05). We identified seven significant clusters of genes (FDR < 0.05, LRT) with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in age-related changes in skeletal muscles and/or sarcopenia initiation including extracellular matrix organization and an immune response (FDR < 0.05).Gene expression changes in mouse peripheral nerve were detected prior to disturbances in myofiber innervation and sarcopenia onset. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies are warranted to confirm the disease modifying and/or biomarker potential of the key changes we report here.

Transthyretin (TTR) distributes thyroxine in the cerebrospinal fluid of mammals. Choroid plexus epithelial cells produce and secrete TTR, and were long recognized as the only CNS source of TTR. However, research over the last years has reported neuronal-specific expression as well, but without a clear function. Recently, we found Ttr transcripts in cells of the adult mouse subventricular zone (SVZ), the largest neural stem cell (NSC) region, but the protein was undetectable. We therefore investigated in more detail what role TTR might play in the SVZ, and when. We mapped temporal-spatial Ttr expression by re-analysing publicly available single-cell RNA-Seq data obtained from dissected mouse SVZs at E14-E17-P2-P7-P20-P61. We observed a peak in Ttr expression in NSCs, neural progenitors and differentiating cells at postnatal day 7 (P7). That is one week prior to when thyroxine serum levels peak and T3 activates SVZ-NSCs that start generating neurons and glia at a constant rate. RNAscope on P7 brain sections confirmed that few Ttr transcripts are present in a many SVZ-progenitors, oligodendrocyte precursors and neuroblasts. Unexpectedly though, no protein was detectable using commercially available antibodies, signal amplification and appropriate controls. This might suggest TTR is rapidly secreted to affect nearby cells. To test this hypothesis, we prepared neurospheres from dissected SVZ-progenitors at P7. After 7 days of proliferation, cells were dissociated, and allowed to differentiate for 1 or 5 days. In parallel with controls, we treated them once at day 0 of differentiation with a low (2.5 µg/ml) or a high dose (25 µg/ml) of human recombinant TTR, or with 5 nM T3. Low TTR doses reduced cell mitosis at day 1, as did T3. After 5 days, we counted a 30% lower proportion of differentiated neuroblasts with the highest TTR dose. That proportion had dropped 3-fold in the presence of T3. Proportions of oligodendroglia after 5 days of differentiation were only significantly higher in T3 conditions. As a result, the neuron/glia balance shifted in favour of oligodendrogenesis under T3, and borderline-significantly following high TTR doses. Altogether, the murine SVZ represents a novel region containing cells that express Ttr, with a peak at P7, despite seeming absence of the protein itself, precluding deducing its exact role. Single-cell RNA-Seq on treated neurospheres could reveal how exogenous TTR affects intracellular pathways, and whether its action is TH-dependent or not. This can help unravelling the pathophysiology of familial amyloid polyneuropathy, in which misfolded TTR proteins cause neurodegeneration.

Following tissue injury, latent sensitization (LS) of nociceptive signaling can persist indefinitely, kept in remission by compensatory µ-opioid receptor constitutive activity (MORCA) in the dorsal horn of the spinal cord. To demonstrate LS, we conducted plantar incision in mice and then waited 3-4 weeks for hypersensitivity to resolve. At this time (remission), systemic administration of the opioid receptor antagonist/inverse agonist naltrexone reinstated mechanical and heat hypersensitivity. We first tested the hypothesis that LS extends to serotonergic neurons in the rostral ventral medulla (RVM) that convey pronociceptive input to the spinal cord. We report that in male and female mice, hypersensitivity was accompanied by increased Fos expression in serotonergic neurons of the RVM, abolished upon chemogenetic inhibition of RVM 5-HT neurons, and blocked by intrathecal injection of the 5-HT3R antagonist ondansetron; the 5-HT2AR antagonist MDL-11,939 had no effect. Second, to test for MORCA, we microinjected the MOR inverse agonist CTAP and/or neutral opioid receptor antagonist 6β-naltrexol. Intra-RVM CTAP produced mechanical hypersensitivity at both hindpaws. 6β-naltrexol had no effect by itself, but blocked CTAP-induced hypersensitivity. This indicates that MORCA, rather than an opioid ligand-dependent mechanism, maintains LS in remission. We conclude that incision establishes LS in descending RVM 5-HT neurons that drives pronociceptive 5-HT3R signaling in the dorsal horn, and this LS is tonically opposed by MORCA in the RVM. The 5-HT3 receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic post-surgical pain.Significance statementSurgery leads to latent pain sensitization and a compensatory state of endogenous pain control that is maintained long after tissue healing. Here we show that either chemogenetic inhibition of serotonergic neuron activity in the rostral ventromedial medulla (RVM), or pharmacological inhibition of 5-HT3 receptor signaling at the spinal cord blocks behavioral signs of post-surgical latent sensitization. We conclude that µ-opioid receptor constitutive activity (MORCA) in the RVM opposes descending serotonergic facilitation of LS, and that the 5-HT3 receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic post-surgical pain.

Background Pain is the most common cause of disability in IBD. What causes inter-individual variability in chronic pain after successful treatment of inflammation remains elusive. We have shown that activation of TRPV1+ colonic nociceptors is essential for the establishment of persistent pain in DSS colitis. Nociceptor development coincides with microbial colonization, while early life dysbiosis can lead to visceral hypersensitivity in adulthood. Whether the microbiota dictates nociceptor development and pain susceptibility remains unknown. Here we test the hypothesis that the microbiota programs nociceptor specification during early development, rendering them more susceptible to sensitization later in life. We have identified the aryl hydrocarbon receptor (AHR) that senses bacterial-derived metabolites as a candidate target that orchestrates transcriptional regulation in nociceptors. Aims We investigated the developmental regulation of nociceptors by the microbiome and how it influences pain sensitivity. We will determine the effects of AHR activation on nociceptor lineage and function as well as the long term impact of AHR signaling on pain sensitivity. Methods We have developed a germ-free (GF) TRPV1-GFP reporter mouse that was used to phenotype and visualise TRPV1+ nociceptors in the absence of a microbiota. We will isolate TRPV1+ neurons by FACS to identify genes that are under the control of the microbiota and to characterise the phosphoproteome of TRPV1+ nociceptors in GF conditions. Finally, we will investigate the role of AHR signaling in nociceptors both acutely and during development. Results We showed a reduction in thermal pain threshold and a reduction in capsaicin test responses in GF mice. The number and size of DRG neurons was unchanged in GF mice. Examination of molecular markers for peptidergic (CGRP) and non-peptidergic (IB4) neurons did not show a difference. Finally, there was no difference in the expression of TRPV1, suggesting post-translational modification of the channel. In cultured DRG neurons, we found a decrease in capsaicin induced action potentials and a decrease in the amplitude of the capsaicin response in GF mice. Using RNAscope, we showed that TRPV1+ neurons express AHR. Conclusions Our results highlight the importance of bacterial composition in regulating the development of nociceptors and pain sensitivity in adulthood. Furthermore, we are the first to demonstrate the expression of AHR in sensory neurons. These findings point to a role of the microbiota in programming nociceptors during development. My work will advance our understanding of the role of commensal bacteria in regulating pain and could lead to recommendations for the treatment of neonates in early life to reduce their risk of developing chronic pain later in life. Funding Agencies CAG, CIHR

Introduction: Conventional 2D mono-culture in vitro models using immortalized cell lines are still widely used in experimental nephrology, albeit their limited translatability and predictivity for the in vivo situation. The feasibility of more sophisticated assays is often reduced by complex protocols and long lasting procedures. We aimed to establish and validate an easy-to-use but yet (patho-) physiologically relevant 3D cell culture assay that mimics key aspects of the in vivo situation of renal tubules, including a leak-thight epithelium with a luminal and baso-lateral side, interstitial matrix, a peri-tubular capillary and circulating blood cells inside its lumen. Methods: We utilized the 3-lane OrganoPlate system (Mimetas, Leiden, Netherlands) as a scaffold. After infusing a collagen I matrix in the middle channel (C2), primary human renal progenitor cells are seeded into the upper channel (C1), adhering to the C2-matrix. The plate is put on a perfusion rocker, that facilitates continuous gravity-triggered bidirectional perfusion in all channels. Thereafter the cells form a leaktight tubular structure with a continuous lumen. Next, human endothelial cells are seeded into the bottom channel (C3), which adhered to the opposite site of C2 and formed a vessel-like structure with a continuous lumen, as well. Finally, primary human white blood cells (WBCs) were isolated and seeded into C3 (figure A). Results: The leak-tightness of the 3D-tubule increased significantly over time, as measured by tracing the diffusion of a 150 kDa FITClabeled dextran from C1 to C2 (time-to-leakage day 1: 3.3 2.6 min; day 9: 36.2 10.7 min), indicating the stability of the co-culture system as well as a cellular maturation resulting in significant barrier functionality as seen in vivo (figure B). In accordance with this and other studies, the primary human tubular cells expressed higher levels of functionally relevant proteins in 3D than under 2D, no-flow conditions, as indicated by cell-number normalized mean fluorescence intensity measured by immunofluorescence, e..g ZO-1 (2.1 0.4 vs. 82.2 20.8) and Na-KATPase (2.3 0.3 vs. 52.8 5.4). Additionally, the growth conditions of the OrganoPlate rendered the cells more resilient to stimuli of acute tubular necrosis, e.g. extracellular histones, as compared to standard cell culture, indicated by cell number normalized lactate dehydrogenase release.The primary WBCs seeded inside the endothelial lumen (C3) did not leave the compartment under normal culture conditions, but displayed extravasation and directed migration from C3 through C2 towards C1 when attracted by chemokines released from tubular cells in C1. This effect was inhibitable by pre-emptive treatment of the endothelium with an selective monoclonal anti-P-selectin antibody (percent migrated cells, medium: 0 0, chemokines: 4.59 0.6, chemokines + Pselectin AB: 1.0 0.5, figure C). This serves as a proof of principle, that the system is applicable to study complex cell-cell and cell-substrate interactions, such as chemokine-mediate immune cell homing. Conclusions: The results of this study suggest, that sophisticated 3D co-culture models of a renal tubule including an interstitial compartment, a peri-tubluar capillary and circulating immune cells are feasible and potentially suited for in depth mechanistic studies in vitro.

Purpose: As skeletal maturity is approached, long bone elongation draws to a close and the cartilaginous growth plate (GP) ossifies and fuses as bone bridges form. This is likely a pivotal moment for the appendicular skeleton, but our mechanistic appreciation of how this process is orchestrated is limited. We have been studying how chondrocytes integrate biological cues, such as growth factor signalling, and mechanical forces, and have investigated the mechanosensitivity of epiphyseal fusion and roles for putative mechanotransduction machinery, including the primary cilium, in these contexts. Here we asked whether primary cilia have a mechanotransduction role in the juvenile GP and adolescent epiphyseal fusion. Methods: We used an inducible aggrecan (ACAN) Cre mouse model, enabling temporal deletion of the core ciliary protein IFT88 in cartilage to investigate GP narrowing dynamics and closure from 4 - 10 weeks of age. Both control (Ift88fl/fl) and cKO (Ift88fl/fl;ACANCreERT2) were injected with tamoxifen (I.P.). Cre activity was validated using a ROSA26TdTomato reporter line. Animals were exposed to (i) sciatic and femoral double neurectomy (DN) to off-load the right hind limb (immobilised DN) whilst the left bears full weight (contralateral DN) at 8 weeks of age, or (ii) voluntary wheel exercise between 8 and 10 weeks of age. Joints were scanned by μCT before histomorphometric analyses of tibial GP using Safranin-O/fast green, TUNEL, Collagen type X (ColX) immunohistochemistry, Von Kossa and TRAP. Cryosections of mouse GPs were analysed by confocal microscopy to investigate primary cilia prevalence and RNA scope was used to identify molecular mechanisms in situ. Medians +/- 95% confidence intervals quoted throughout below, Two-way ANOVA statistical comparisons. Results: We have, for the first time, investigated the role of cilia beyond 4 weeks of age. μCT analysis showed GP length in wild-type mice reduces from ∼260 μm to 130 μm between 4 and 10 weeks of age. Deletion of IFT88 in juvenile mice at 4 or 6 weeks of age resulted in longer GPs in cKO mice at every timepoint compared with control mice (Fig. 1A, 1st and 2nd panel and 1B). Thus, two weeks after tamoxifen, cKO GP lengths were not statistically significantly different to controls at time of treatment, indicating inhibition of GP closure. Deleting IFT88 at 8 weeks of age also resulted in longer GPs (p< 0.0001, n=12 controls, n=23 cKO). Interestingly, some cKO mice exhibited extremely elongated GPs at the edges of the tibia, which appeared as large holes by μCT (Fig. 1A), whilst the centre of the GP appeared less affected. Histology confirmed longer GPs were predominantly characterised by increases in hypertrophic chondrocyte populations. The large, often bi-lateral “holes”, observed by μCT were largely filled with disorganised hypertrophic chondrocytes, as indicated by IHC labelling for ColX. Interestingly limb immobilisation, (DN), at 8 weeks of age, rescued the GP phenotype observed in IFT88 cKO mice (Fig. 1A, 2nd and 3rd panels, and 1C), whilst the contralateral, unoperated (increased load-bearing) limb exhibited bi-lateral failure of ossification, similar to that observed in IFT88 cKO mice. Compared with naïve controls, wheel-exercised mice also displayed elongated GP (p< 0.0001, n=12 controls, n=10 wheel exercised) (Fig. 1A, 4th panel, and 1C) at 10 weeks of age. These expanded GP were, again, most pronounced at the edges of the tibia, whilst the centre of the GP appeared less affected and again was largely filled with disorganised, differentiated, ColX positive hypertrophic chondrocytes. In both wheel exercised and IFT88 cKO mice, regions of failed ossification, but not middle regions, were associated with loss of osteoclast activity. Confocal imaging and analysis revealed a statistically significant (p< 0.001) decrease in cilia positive cells in wheel exercised mice (32.9%, n=5) compared with control (40.7%, n=4) and IFT88cKO mice (p< 0.0001, (23.4%, n=4) compared with controls (40.7% n=4) at 10 weeks of age. Ongoing experiments are investigating 3D spatial analysis of fusion mechanisms, and the status of ciliary Hh signalling (Gli1, by RNAscope) within GP from control, DN, exercised, and cKO mice to dissect the apparently negative, regulatory role IFT88 is plays in the mechanical regulation of epiphyseal fusion. Conclusions: We conclude that IFT88 unequivocally plays a role in GP closure, its removal resulting in failed ossification of the GP, without disruption to chondrocytic lineage differentiation. This phenomenon, observed in cKO animals, is mechanosensitive with limb immobilisation rescuing the phenotype, suggesting, paradoxically, that IFT88 is dampening a mechanically-induced signal in the GP. Wheel exercise also resulted in impaired ossification thus these data collectively unveil both the acute response of the adolescent mouse GP to exercise and, through Ift88 deletion (cKO), a novel mechanoregulatory mechanism orchestrated by ciliary IFT. The effects of altered mechanics and mechanotransduction are most pronounced in the hypertrophic zone where cells are apparently trapped short of transdifferentiation. Osteoclast recruitment and/or activity is impaired, and bone formation inhibited. These Results may have implications for our understanding of hypertrophic chondrocyte biology in articular cartilage in OA. Moreover, it has been proposed that changes to mechanical inputs during adolescence and associated cam morphology contribute to hip OA development. In adolescent patient cohorts, high levels of exercise lead to cartilaginous hypertrophy, epiphyseal extension, cam development, and reduced rates of GP closure. Femoral and tibial epiphyseal extension has also been observed in adolescent athletes that sustain repetitive trauma through high intensity exercise. This research is crucial to a holistic understanding of skeletal mechano-biological health, and the effects of exercise, on the maturing appendicular skeleton