Shared and Compartment-Specific Processes in Nucleus Pulposus and Annulus Fibrosus During Intervertebral Disc Degeneration.

Elucidating how cell populations promote onset and progression of intervertebral disc degeneration (IDD) has the potential to enable more precise therapeutic targeting of cells and mechanisms. Single-cell RNA-sequencing (scRNA-seq) is performed on surgically separated annulus fibrosus (AF) (19,978; 26,983 cells) and nucleus pulposus (NP) (20,884; 24,489 cells) from healthy and diseased human intervertebral discs (IVD). In both tissue types, depletion of cell subsets involved in maintenance of healthy IVD is observed, specifically the immature cell subsets - fibroblast progenitors and stem cells - indicative of an impairment of normal tissue self-renewal. Tissue-specific changes are also identified. In NP, several fibrotic populations are increased in degenerated IVD, indicating tissue-remodeling. In degenerated AF, a novel disease-associated subset is identified, which expresses disease-promoting genes. It is associated with pathogenic biological processes and the main gene regulatory networks include thrombospondin signaling and FOXO1 transcription factor. In NP and AF cells thrombospondin protein promoted expression of genes associated with TGFß/fibrosis signaling, angiogenesis, and nervous system development. The data reveal new insights of both shared and tissue-specific changes in specific cell populations in AF and NP during IVD degeneration. These identified mechanisms and molecules are novel and more precise targets for IDD prevention and treatment.

Mohawk is a transcription factor that promotes meniscus cell phenotype and tissue repair and reduces osteoarthritis severity.

Meniscus tears are common knee injuries and a major osteoarthritis (OA) risk factor. Knowledge gaps that limit the development of therapies for meniscus injury and degeneration concern transcription factors that control the meniscus cell phenotype. Analysis of RNA sequencing data from 37 human tissues in the Genotype-Tissue Expression database and RNA sequencing data from meniscus and articular cartilage showed that transcription factor Mohawk (MKX) is highly enriched in meniscus. In human meniscus cells, MKX regulates the expression of meniscus marker genes, OA-related genes, and other transcription factors, including Scleraxis (SCX), SRY Box 5 (SOX5), and Runt domain-related transcription factor 2 (RUNX2). In mesenchymal stem cells (MSCs), the combination of adenoviral MKX (Ad-MKX) and transforming growth factor-ß3 (TGF-ß3) induced a meniscus cell phenotype. When Ad-MKX-transduced MSCs were seeded on TGF-ß3-conjugated decellularized meniscus scaffold (DMS) and inserted into experimental tears in meniscus explants, they increased glycosaminoglycan content, extracellular matrix interconnectivity, cell infiltration into the DMS, and improved biomechanical properties. Ad-MKX injection into mouse knee joints with experimental OA induced by surgical destabilization of the meniscus suppressed meniscus and cartilage damage, reducing OA severity. Ad-MKX injection into human OA meniscus tissue explants corrected pathogenic gene expression. These results identify MKX as a previously unidentified key transcription factor that regulates the meniscus cell phenotype. The combination of Ad-MKX with TGF-ß3 is effective for differentiation of MSCs to a meniscus cell phenotype and useful for meniscus repair. MKX is a promising therapeutic target for meniscus tissue engineering, repair, and prevention of OA.

FOXO1 and FOXO3 transcription factors have unique functions in meniscus development and homeostasis during aging and osteoarthritis.

The objective of this study was to examine FoxO expression and FoxO function in meniscus. In menisci from human knee joints with osteoarthritis (OA), FoxO1 and 3 expression were significantly reduced compared with normal menisci from young and old normal donors. The expression of FoxO1 and 3 was also significantly reduced in mouse menisci during aging and OA induced by surgical meniscus destabilization or mechanical overuse. Deletion of FoxO1 and combined FoxO1, 3, and 4 deletions induced abnormal postnatal meniscus development in mice and these mutant mice spontaneously displayed meniscus pathology at 6 mo. Mice with Col2Cre-mediated deletion of FoxO3 or FoxO4 had normal meniscus development but had more severe aging-related damage. In mature AcanCreERT2 mice, the deletion of FoxO1, 3, and 4 aggravated meniscus lesions in all experimental OA models. FoxO deletion suppressed autophagy and antioxidant defense genes and altered several meniscus-specific genes. Expression of these genes was modulated by adenoviral FoxO1 in cultured human meniscus cells. These results suggest that FoxO1 plays a key role in meniscus development and maturation, and both FoxO1 and 3 support homeostasis and protect against meniscus damage in response to mechanical overuse and during aging and OA.

Role of heparan sulfate 6-0 endosulfatases in intervertebral disc homeostasis.

The expression of heparan sulfate endosulfatases (Sulfs) was investigated in the intervertebral disc (IVD) to clarify their role in IVD homeostasis. Sulf-1 and -2 expression were elucidated in normal and degenerated human IVD. Age-related effects on Sulf expression, type II collagen levels, and structural changes were analyzed in IVDs of wild-type (WT) and Sulf-1 knockout (Sulf-1⁻/⁻) mice. The effect of recombinant Sulf-1 (100 ng/ml) and Sulf-1 knockdown on heparan sulfate proteoglycan and collagen expression in ATDC5 cells were examined. Finally, the effect of Sulf-1 on transforming growth factor (TGF) ß1-induced signaling was evaluated. Results show that Sulf-1 and -2 levels were higher in degenerated human IVDs. In WT mice, Sulf-1 and -2 expression generally declined as the animals aged. In particular, Sulf-1 in the nucleus pulposus was higher compared with Sulf-2 at the age of 1 and 6 months and significantly declined with aging. Sulf-1⁻/⁻ mice showed more severe IVD pathology than WT mice, with lower type II collagen levels in nucleus pulposus, and degeneration with type I collagen in annulus fibrosus. In vitro, Sulf-1 induced type II collagen expression and significantly increased TGF-ß1-induced Smad2/3 phosphorylation in ATDC5 cells. In conclusion, Sulf-1 might play a critical role from development to maintenance of IVD homeostasis by regulating collagen expression.

FOXO are required for intervertebral disk homeostasis during aging and their deficiency promotes disk degeneration.

Intervertebral disk (IVD) degeneration is a prevalent age-associated musculoskeletal disorder and a major cause of chronic low back pain. Aging is the main risk factor for the disease, but the molecular mechanisms regulating IVD homeostasis during aging are unknown. The aim of this study was to investigate the function of FOXO, a family of transcription factors linked to aging and longevity, in IVD aging and age-related degeneration. Conditional deletion of all FOXO isoforms (FOXO1, 3, and 4) in IVD using the Col2a1Cre and AcanCreER mouse resulted in spontaneous development of IVD degeneration that was driven by severe cell loss in the nucleus pulposus (NP) and cartilaginous endplates (EP). Conditional deletion of individual FOXO in mature mice showed that FOXO1 and FOXO3 are the dominant isoforms and have redundant functions in promoting IVD homeostasis. Gene expression analyses indicated impaired autophagy and reduced antioxidant defenses in the NP of FOXO-deficient IVD. In primary human NP cells, FOXO directly regulated autophagy and adaptation to hypoxia and promoted resistance to oxidative and inflammatory stress. Our findings demonstrate that FOXO are critical regulators of IVD homeostasis during aging and suggest that maintaining or restoring FOXO expression can be a therapeutic strategy to promote healthy IVD aging and delay the onset of IVD degeneration.

FoxO transcription factors modulate autophagy and proteoglycan 4 in cartilage homeostasis and osteoarthritis.

Aging is a main risk factor for osteoarthritis (OA). FoxO transcription factors protect against cellular and organismal aging, and FoxO expression in cartilage is reduced with aging and in OA. To investigate the role of FoxO in cartilage, Col2Cre-FoxO1, 3, and 4 single knockout (KO) and triple KO mice (Col2Cre-TKO) were analyzed. Articular cartilage in Col2Cre-TKO and Col2Cre-FoxO1 KO mice was thicker than in control mice at 1 or 2 months of age. This was associated with increased proliferation of chondrocytes of Col2Cre-TKO mice in vivo and in vitro. OA-like changes developed in cartilage, synovium, and subchondral bone between 4 and 6 months of age in Col2Cre-TKO and Col2Cre-FoxO1 KO mice. Col2Cre-FoxO3 and FoxO4 KO mice showed no cartilage abnormalities until 18 months of age when Col2Cre-FoxO3 KO mice had more severe OA than control mice. Autophagy and antioxidant defense genes were reduced in Col2Cre-TKO mice. Deletion of FoxO1/3/4 in mature mice using Aggrecan(Acan)-CreERT2 (AcanCreERT-TKO) also led to spontaneous cartilage degradation and increased OA severity in a surgical model or treadmill running. The superficial zone of knee articular cartilage of Col2Cre-TKO and AcanCreERT-TKO mice exhibited reduced cell density and markedly decreased Prg4 In vitro, ectopic FoxO1 expression increased Prg4 and synergized with transforming growth factor-ß stimulation. In OA chondrocytes, overexpression of FoxO1 reduced inflammatory mediators and cartilage-degrading enzymes, increased protective genes, and antagonized interleukin-1ß effects. Our observations suggest that FoxO play a key role in postnatal cartilage development, maturation, and homeostasis and protect against OA-associated cartilage damage.

Transthyretin deposition promotes progression of osteoarthritis.

Deposition of amyloid is a common aging-associated phenomenon in several aging-related diseases. Osteoarthritis (OA) is the most prevalent joint disease, and aging is its major risk factor. Transthyretin (TTR) is an amyloidogenic protein that is deposited in aging and OA-affected human cartilage and promotes inflammatory and catabolic responses in cultured chondrocytes. Here, we investigated the role of TTR in vivo using transgenic mice overexpressing wild-type human TTR (hTTR-TG). Although TTR protein was detected in cartilage in hTTR-TG mice, the TTR transgene was highly overexpressed in liver, but not in chondrocytes. OA was surgically induced by destabilizing the medial meniscus (DMM) in hTTR-TG mice, wild-type mice of the same strain (WT), and mice lacking endogenous Ttr genes. In the DMM model, both cartilage and synovitis histological scores were significantly increased in hTTR-TG mice. Further, spontaneous degradation and OA-like changes in cartilage and synovium developed in 18-month-old hTTR mice. Expression of cartilage catabolic (Adamts4, Mmp13) and inflammatory genes (Nos2, Il6) was significantly elevated in cartilage from 6-month-old hTTR-TG mice compared with WT mice as was the level of phospho-NF-κB p65. Intra-articular injection of aggregated TTR in WT mice increased synovitis and significantly increased expression of inflammatory genes in synovium. These findings are the first to show that TTR deposition increases disease severity in the murine DMM and aging model of OA.

Age-related reduction in the expression of FOXO transcription factors and correlations with intervertebral disc degeneration.

Aging is a main risk factor for intervertebral disc (IVD) degeneration, the main cause of low back pain. FOXO transcription factors are important regulators of tissue homeostasis and longevity. Here, we determined the expression pattern of FOXO in healthy and degenerated human IVD and the associations with IVD degeneration during mouse aging. FOXO expression was assessed by immunohistochemistry in normal and degenerated human IVD samples and in cervical and lumbar IVD from 6-, 12-, 24-, and 36-month-old C57BL/6J mice. Mouse spines were graded for key histological features of disc degeneration in all the time points and expression of two key FOXO downstream targets, sestrin 3 (SESN3) and superoxide dismutase (SOD2), was assessed by immunohistochemistry. Histological analysis revealed that FOXO proteins are expressed in all compartments of human and mouse IVD. Expression of FOXO1 and FOXO3, but not FOXO4, was significantly deceased in human degenerated discs. In mice, degenerative changes in the lumbar spine were seen at 24 and 36 months of age whereas cervical IVD showed increased histopathological scores at 36 months. FOXO expression was significantly reduced in lumbar IVD at 12-, 24-, and 36-month-old mice and in cervical IVD at 36-month-old mice when compared with the 6-month-old group. The reduction of FOXO expression in lumbar IVD was concomitant with a decrease in the expression of SESN3 and SOD2. These findings suggest that reduced FOXO expression occurs in lumbar IVD during aging and precedes the major histopathological changes associated with lumbar IVD degeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2682-2691, 2017.

Regulated in Development and DNA Damage Response 1 Deficiency Impairs Autophagy and Mitochondrial Biogenesis in Articular Cartilage and Increases the Severity of Experimental Osteoarthritis.

OBJECTIVE: Regulated in development and DNA damage response 1 (REDD1) is an endogenous inhibitor of mechanistic target of rapamycin (mTOR) that regulates cellular stress responses. REDD1 expression is decreased in aged and osteoarthritic (OA) cartilage, and it regulates mTOR signaling and autophagy in articular chondrocytes in vitro. This study was undertaken to investigate the effects of REDD1 deletion in vivo using a mouse model of experimental OA. METHODS: OA severity was histologically assessed in 4-month-old wild-type and REDD1-/- mice subjected to surgical destabilization of the medial meniscus (DMM). Chondrocyte autophagy, apoptosis, mitochondrial content, and expression of mitochondrial biogenesis markers were determined in cartilage and cultured chondrocytes from wild-type and REDD1-/- mice. RESULTS: REDD1 deficiency increased the severity of changes in cartilage, menisci, subchondral bone, and synovium in the DMM model of OA. Chondrocyte death was increased in the cartilage of REDD1-/- mice and in cultured REDD1-/- mouse chondrocytes under oxidative stress conditions. Expression of key autophagy markers (microtubule-associated protein 1A/1B light chain 3 and autophagy protein 5) was markedly reduced in cartilage from REDD1-/- mice and in cultured human and mouse chondrocytes with REDD1 depletion. Mitochondrial content, ATP levels, and expression of the mitochondrial biogenesis markers peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and transcription factor A, mitochondrial (TFAM) were also decreased in REDD1-deficient chondrocytes. REDD1 was required for AMP-activated protein kinase-induced PGC-1α in chondrocytes. CONCLUSION: Our findings suggest that REDD1 is a key mediator of cartilage homeostasis through regulation of autophagy and mitochondrial biogenesis and that REDD1 deficiency exacerbates the severity of injury-induced OA.

Increased DNA Methylation and Reduced Expression of Transcription Factors in Human Osteoarthritis Cartilage.

OBJECTIVE: To analyze the methylome of normal and osteoarthritic (OA) knee articular cartilage and to determine the role of DNA methylation in the regulation of gene expression in vitro. METHODS: DNA was isolated from human normal (n = 11) and OA (n = 12) knee articular cartilage and analyzed using the Infinium HumanMethylation450 BeadChip array. To integrate methylation and transcription, RNA sequencing was performed on normal and OA cartilage and validated by quantitative polymerase chain reaction. Functional validation was performed in the human TC28 cell line and primary chondrocytes that were treated with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). RESULTS: DNA methylation profiling revealed 929 differentially methylated sites between normal and OA cartilage, comprising a total of 500 individual genes. Among these, 45 transcription factors that harbored differentially methylated sites were identified. Integrative analysis and subsequent validation showed a subset of 6 transcription factors that were significantly hypermethylated and down-regulated in OA cartilage (ATOH8, MAFF, NCOR2, TBX4, ZBTB16, and ZHX2). Upon 5-aza-dC treatment, TC28 cells showed a significant increase in gene expression for all 6 transcription factors. In primary chondrocytes, ATOH8 and TBX4 were increased after 5-aza-dC treatment. CONCLUSION: Our findings reveal that normal and OA knee articular cartilage have significantly different methylomes. The identification of a subset of epigenetically regulated transcription factors with reduced expression in OA may represent an important mechanism to explain changes in the chondrocyte transcriptome and function during OA pathogenesis.

Metabolic Benefit of Chronic Caloric Restriction and Activation of Hypothalamic AGRP/NPY Neurons in Male Mice Is Independent of Ghrelin.

Aging is associated with attenuated ghrelin signaling. During aging, chronic caloric restriction (CR) produces health benefits accompanied by enhanced ghrelin production. Ghrelin receptor (GH secretagogue receptor 1a) agonists administered to aging rodents and humans restore the young adult phenotype; therefore, we tested the hypothesis that the metabolic benefits of CR are mediated by endogenous ghrelin. Three month-old male mice lacking ghrelin (Ghrelin-/-) or ghrelin receptor (Ghsr-/-), and their wild-type (WT) littermates were randomly assigned to 2 groups: ad libitum (AL) fed and CR, where 40% food restriction was introduced gradually to allow Ghrelin-/- and Ghsr-/- mice to metabolically adapt and avoid severe hypoglycemia. Twelve months later, plasma ghrelin, metabolic parameters, ambulatory activity, hypothalamic and liver gene expression, as well as body composition were measured. CR increased plasma ghrelin and des-acyl ghrelin concentrations in WT and Ghsr-/- mice. CR of WT, Ghsr-/-, and Ghrelin-/- mice markedly improved metabolic flexibility, enhanced ambulatory activity, and reduced adiposity. Inactivation of Ghrelin or Ghsr had no effect on AL food intake or food anticipatory behavior. In contrast to the widely held belief that endogenous ghrelin regulates food intake, CR increased expression of hypothalamic Agrp and Npy, with reduced expression of Pomc across genotypes. In the AL context, ablation of ghrelin signaling markedly inhibited liver steatosis, which correlated with reduced Pparγ expression and enhanced Irs2 expression. Although CR and administration of GH secretagogue receptor 1a agonists both benefit the aging phenotype, we conclude the benefits of chronic CR are a consequence of enhanced metabolic flexibility independent of endogenous ghrelin or des-acyl ghrelin signaling.

Antisense RNA controls LRP1 Sense transcript expression through interaction with a chromatin-associated protein, HMGB2.

Long non-coding RNAs (lncRNAs), including natural antisense transcripts (NATs), are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1), referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2) and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimer's disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion.

Transthyretin deposition in articular cartilage: a novel mechanism in the pathogenesis of osteoarthritis.

OBJECTIVE: Amyloid deposits are prevalent in osteoarthritic (OA) joints. We undertook this study to define the dominant precursor and to determine whether the deposits affect chondrocyte functions. METHODS: Amyloid deposition in human normal and OA knee cartilage was determined by Congo red staining. Transthyretin (TTR) in cartilage and synovial fluid was analyzed by immunohistochemistry and Western blotting. The effects of recombinant amyloidogenic and nonamyloidogenic TTR variants were tested in human chondrocyte cultures. RESULTS: Normal cartilage from young donors did not contain detectable amyloid deposits, but 7 of 12 aged normal cartilage samples (58%) and 12 of 12 OA cartilage samples (100%) had Congo red staining with green birefringence under polarized light. TTR, which is located predominantly at the cartilage surfaces, was detected in all OA cartilage samples and in a majority of aged normal cartilage samples, but not in normal cartilage samples from young donors. Chondrocytes and synoviocytes did not contain significant amounts of TTR messenger RNA. Synovial fluid TTR levels were similar in normal and OA knees. In cultured chondrocytes, only an amyloidogenic TTR variant induced cell death as well as the expression of proinflammatory cytokines and extracellular matrix-degrading enzymes. The effects of amyloidogenic TTR on gene expression were mediated in part by Toll-like receptor 4, receptor for advanced glycation end products, and p38 MAPK. TTR-induced cytotoxicity was inhibited by resveratrol, a plant polyphenol that stabilizes the native tetrameric structure of TTR. CONCLUSION: These findings are the first to suggest that TTR amyloid deposition contributes to cell and extracellular matrix damage in articular cartilage in human OA and that therapies designed to reduce TTR amyloid formation might be useful.

FoxO transcription factors support oxidative stress resistance in human chondrocytes.

OBJECTIVE: A major signaling pathway that regulates cellular aging is the insulin/insulin-like growth factor 1 (IGF-1)/phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO transcription factor axis. We previously observed that FoxO transcription factors are dysregulated in aged and OA cartilage. The objective of this study was to investigate the impact of down-regulated FoxO transcription factors on chondrocytes. METHODS: Small interfering RNAs (siRNAs) targeting FOXO1 (siFOXO1) and FOXO3 (siFOXO3) were transfected into human articular chondrocytes. Cell viability following treatment with the oxidant tert-butyl-hydroperoxide (tBHP) was measured by MTT assay. Caspase 3/7 activation and apoptotic cells were examined. Gene and protein expression of antioxidant proteins and autophagy-related proteins and changes in inflammatory mediators following treatment with interleukin-1ß were assessed. Cells transfected with FOXO plasmids were also analyzed. RESULTS: Cell viability was significantly reduced by siFOXO after treatment with tBHP. Apoptosis accompanied by caspase activation was significantly increased in siFOXO-transfected chondrocytes. Knockdown of FOXO1 and FOXO1+3 resulted in significant reductions in levels of glutathione peroxidase 1 (GPX-1), catalase, light chain 3 (LC3), Beclin1, and sirtuin 1 (SIRT-1) proteins following treatment with tBHP. In contrast, the constitutive active form of FOXO3 increased cell viability while inducing GPX-1, Beclin1, and LC3 in response to tBHP. Expression and production of ADAMTS-4 and chemerin were significantly increased in siFOXO-transfected chondrocytes. CONCLUSION: Reduced expression of FoxO transcription factors in chondrocytes increased susceptibility to cell death induced by oxidative stress. This was associated with reduced levels of antioxidant proteins and autophagy-related proteins. Our data provide evidence for a key role of FoxO transcription factors as regulators of chondrocyte oxidative stress resistance and tissue homeostasis.

Palmitate has proapoptotic and proinflammatory effects on articular cartilage and synergizes with interleukin-1.

OBJECTIVE: Obesity is a major risk factor for the development of osteoarthritis (OA) that is associated with a state of low-grade inflammation and increased circulating levels of adipokines and free fatty acids (FFAs). The aim of this study was to analyze the effects of saturated (palmitate) and monounsaturated (oleate) FFAs on articular chondrocytes, synoviocytes, and cartilage. METHODS: Human articular chondrocytes and fibroblast-like synoviocytes obtained from young healthy donors and OA chondrocytes from patients undergoing total knee replacement surgery were treated with palmitate or oleate alone or in combination with interleukin-1ß (IL-1ß). Cell viability, caspase activation, and gene expression of proinflammatory factors, extracellular matrix (ECM) proteins, and proteases were studied. In addition, chondrocyte viability, IL-6 production, and matrix damage were assessed in bovine and human articular cartilage explants cultured with FFAs in the presence or absence of IL-1ß. RESULTS: Palmitate, but not oleate, induced caspase activation and cell death in IL-1ß-stimulated normal chondrocytes, and up-regulated the expression of IL-6 and cyclooxygenase 2 in chondrocytes and fibroblast-like synoviocytes through Toll-like receptor 4 (TLR-4) signaling. In cartilage explants, palmitate induced chondrocyte death, IL-6 release, and ECM degradation. Palmitate synergized with IL-1ß in stimulating proapoptotic and proinflammatory cellular responses. Pharmacologic inhibition of caspases or TLR-4 signaling reduced palmitate and IL-1ß induced cartilage damage. CONCLUSION: Palmitate acts as a proinflammatory and catabolic factor that, in synergy with IL-1ß, induces chondrocyte apoptosis and articular cartilage breakdown. Collectively, our data suggest that elevated levels of saturated FFAs that are often found in patients who are obese may contribute to the pathogenesis of OA.

Growth hormone improves growth retardation induced by rapamycin without blocking its antiproliferative and antiangiogenic effects on rat growth plate.

Rapamycin, an immunosuppressant agent used in renal transplantation with antitumoral properties, has been reported to impair longitudinal growth in young individuals. As growth hormone (GH) can be used to treat growth retardation in transplanted children, we aimed this study to find out the effect of GH therapy in a model of young rat with growth retardation induced by rapamycin administration. Three groups of 4-week-old rats treated with vehicle (C), daily injections of rapamycin alone (RAPA) or in combination with GH (RGH) at pharmacological doses for 1 week were compared. GH treatment caused a 20% increase in both growth velocity and body length in RGH animals when compared with RAPA group. GH treatment did not increase circulating levels of insulin-like growth factor I, a systemic mediator of GH actions. Instead, GH promoted the maturation and hypertrophy of growth plate chondrocytes, an effect likely related to AKT and ERK1/2 mediated inactivation of GSK3ß, increase of glycogen deposits and stabilization of ß-catenin. Interestingly, GH did not interfere with the antiproliferative and antiangiogenic activities of rapamycin in the growth plate and did not cause changes in chondrocyte autophagy markers. In summary, these findings indicate that GH administration improves longitudinal growth in rapamycin-treated rats by specifically acting on the process of growth plate chondrocyte hypertrophy but not by counteracting the effects of rapamycin on proliferation and angiogenesis.

Rapamycin induces growth retardation by disrupting angiogenesis in the growth plate.

Rapamycin, a potent immunosuppressant used in renal transplantation, has been reported to impair longitudinal growth in experimental studies. Rapamycin is both antiproliferative and antiangiogenic; therefore, it has the potential to disrupt vascular endothelial growth factor (VEGF) action in the growth plate and to interfere with insulin-like growth factor I (IGF-I) signaling. To further investigate the mechanisms of rapamycin action on longitudinal growth, we gave the 4-week-old rats rapamycin daily for two weeks. Compared with a vehicle-treated group, rapamycin-treated animals were severely growth retarded and had marked alterations in the growth plate. Vascular invasion was disturbed in the rapamycin group, there was a significant reduction in osteoclast cells near the chondro-osseus junction, and there was lower VEGF protein and mRNA expression in the terminal chondrocytes of the growth cartilage. Compared with the control group, the rapamycin group had higher levels of circulating IGF-I as well as the mRNAs for IGF-I and of the receptors of IGF-I and growth hormone in the liver but not in the growth cartilage. Thus our findings explain the adverse effect of rapamycin on growth plate dynamics. This should be taken into account when the drug is administered to children.

Longitudinal growth in chronic hypokalemic disorders.

Growth retardation remains a major complication in children with primary tubular disorders, despite adequate supplemental treatment with electrolytes, water and bicarbonate. Chronic hypokalemia, characteristic of some tubulopathies, impairs growth by mechanisms that are not well known. Association with growth hormone deficiency has been reported in patients with Bartter's or Gitelman's syndrome. Tissue-specific alterations of growth hormone and insulin-like growth factor I axis have been described in experimental models of potassium depletion. Hypokalemic rats gain less body length and weight than pair-fed normokalemic animals and, by contrast, develop renal hypertrophy. These rats have low circulating concentrations of insulin-like growth factor I, depressed messenger ribonucleic acid (mRNA) levels of this peptide in the tibial growth plate, and they are resistant to the longitudinal growth-promoting effects of exogenous growth hormone. The reason for this resistance remains to be defined. No alterations in the intracellular signaling for growth hormone have been found in the liver of hypokalemic rats. However, treatment with high doses of growth hormone is unable to normalize hypertrophy of the epiphyseal cartilage chondrocytes, which are severely disturbed in potassium depletion and likely play an important role in the pathogenia of growth impairment in this condition.