Luigi Aurelio Nasto

NF-κB inhibition delays DNA damage–induced senescence and aging in mice

The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB-activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.

Mitochondrial-derived reactive oxygen species (ROS) play a causal role in aging-related intervertebral disc degeneration

Oxidative damage is a well‐established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O2) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O2). Human disc cells grown at 20% O2 showed increased levels of mitochondrial‐derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria‐targeted reactive oxygen species (ROS) scavenger XJB‐5‐131 blunted the adverse effects caused by 20% O2. Importantly, we demonstrated that treatment of accelerated aging Ercc1−/Δ mice, previously established to be a useful in vivo model to study age‐related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial‐derived ROS contributes to age‐associated IDD in Ercc1−/Δ mice. Collectively, these data provide strong experimental evidence that mitochondrial‐derived ROS play a causal role in driving changes linked to aging‐related IDD and a potentially important role for radical scavengers in preventing IDD.

Spine degeneration in a murine model of chronic human tobacco smokers

OBJECTIVE To investigate the mechanisms by which chronic tobacco smoking promotes intervertebral disc degeneration (IDD) and vertebral degeneration in mice. METHODS Three month old C57BL/6 mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 6 months) to model long-term smoking in humans. Total disc proteoglycan (PG) content [1,9-dimethylmethylene blue (DMMB) assay], aggrecan proteolysis (immunobloting analysis), and cellular senescence (p16INK4a immunohistochemistry) were analyzed. PG and collagen syntheses ((35)S-sulfate and (3)H-proline incorporation, respectively) were measured using disc organotypic culture. Vertebral osteoporosity was measured by micro-computed tomography. RESULTS Disc PG content of smoke-exposed mice was 63% of unexposed control, while new PG and collagen syntheses were 59% and 41% of those of untreated mice, respectively. Exposure to tobacco smoke dramatically increased metalloproteinase-mediated proteolysis of disc aggrecan within its interglobular domain (IGD). Cellular senescence was elevated two-fold in discs of smoke-exposed mice. Smoke exposure increased vertebral endplate porosity, which closely correlates with IDD in humans. CONCLUSIONS These findings further support tobacco smoke as a contributor to spinal degeneration. Furthermore, the data provide a novel mechanistic insight, indicating that smoking-induced IDD is a result of both reduced PG synthesis and increased degradation of a key disc extracellular matrix protein, aggrecan. Cleavage of aggrecan IGD is extremely detrimental as this results in the loss of the entire glycosaminoglycan-attachment region of aggrecan, which is vital for attracting water necessary to counteract compressive forces. Our results suggest identification and inhibition of specific metalloproteinases responsible for smoke-induced aggrecanolysis as a potential therapeutic strategy to treat IDD.

An overview of underlying causes and animal models for the study of age-related degenerative disorders of the spine and synovial joints

As human lifespan increases so does the incidence of age‐associated degenerative joint diseases, resulting in significant negative socioeconomic consequences. Osteoarthritis (OA) and intervertebral disc degeneration (IDD) are the most common underlying causes of joint‐related chronic disability and debilitating pain in the elderly. Current treatment methods are generally not effective and involve either symptomatic relief with non‐steroidal anti‐inflammatory drugs and physical therapy or surgery when conservative treatments fail. The limitation in treatment options is due to our incomplete knowledge of the molecular mechanism of degeneration of articular cartilage and disc tissue. Basic understanding of the age‐related changes in joint tissue is thus needed to combat the adverse effects of aging on joint health. Aging is caused at least in part by time‐dependent accumulation of damaged organelles and macromolecules, leading to cell death and senescence and the eventual loss of multipotent stem cells and tissue regenerative capacity. Studies over the past decades have uncovered a number of important molecular and cellular changes in joint tissues with age. However, the precise causes of damage, cellular targets of damage, and cellular responses to damage remain poorly understood. The objectives of this review are to provide an overview of the current knowledge about the sources of endogenous and exogenous damaging agents and how they contribute to age‐dependent degenerative joint disease, and highlight animal models of accelerated aging that could potentially be useful for identifying causes of and therapies for degenerative joint diseases.

ISSLS prize winner: inhibition of NF-κB activity ameliorates age-associated disc degeneration in a mouse model of accelerated aging.

Study Design. NF-&kgr;B activity was pharmacologically and genetically blocked in an accelerated aging mouse model to mitigate age-related disc degenerative changes. Objective. To study the mediatory role of NF-&kgr;B-signaling pathway in age-dependent intervertebral disc degeneration. Summary of Background Data. Aging is a major contributor to intervertebral disc degeneration (IDD), but the molecular mechanism behind this process is poorly understood. NF-&kgr;B is a family of transcription factors that play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic NF-&kgr;B activation as a culprit in many aging-related diseases, but its role in aging-related IDD has not been adequately explored. We studied the effects of NF-&kgr;B inhibition on IDD, using a DNA repair-deficient mouse model of accelerated aging (Ercc1−/&Dgr; mice) previously been reported to exhibit age-related IDD. Methods. Systemic inhibition of NF-&kgr;B activation was achieved either genetically by deletion of 1 allele of the NF-&kgr;B subunit p65 (Ercc1−/&Dgr;p65+− mice) or pharmacologically by chronic intraperitoneal administration of the Nemo Binding Domain (8K-NBD) peptide to block the formation of the upstream activator of NF-&kgr;B, I&kgr;B Inducible Kinase (IKK), in Ercc1−/&Dgr; mice. Disc cellularity, total proteoglycan content and proteoglycan synthesis of treated mice, and untreated controls were assessed. Results. Decreased disc matrix proteoglycan content, a hallmark feature of IDD, and elevated disc NF-&kgr;B activity were observed in discs of progeroid Ercc1−/&Dgr; mice and naturally aged wild-type mice compared with young wild-type mice. Systemic inhibition of NF-&kgr;B by the 8K-NBD peptide in Ercc1−/&Dgr; mice increased disc proteoglycan synthesis and ameriolated loss of disc cellularity and matrix proteoglycan. These results were confirmed genetically by using the p65 haploinsufficient Ercc1−/&Dgr;p65+/− mice. Conclusion. These findings demonstrate that the IKK/NF-&kgr;B signaling pathway is a key mediator of age-dependent IDD and represents a therapeutic target for mitigating disc degenerative diseases associated with aging.

Investigating the role of DNA damage in tobacco smoking-induced spine degeneration

BACKGROUND CONTEXT Tobacco smoking is a key risk factor for spine degeneration. However, the underlying mechanism by which smoking induces degeneration is not known. Recent studies implicate DNA damage as a cause of spine and intervertebral disc degeneration. Because tobacco smoke contains many genotoxins, we hypothesized that tobacco smoking promotes spine degeneration by inducing cellular DNA damage. PURPOSE To determine if DNA damage plays a causal role in smoking-induced spine degeneration. STUDY DESIGN To compare the effect of chronic tobacco smoke inhalation on intervertebral disc and vertebral bone in normal and DNA repair-deficient mice to determine the contribution of DNA damage to degenerative changes. METHODS Two-month-old wild-type (C57BL/6) and DNA repair-deficient Ercc1(-/Δ) mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 7 weeks) to model first-hand smoking in humans. Total disc proteoglycan (PG) content (1,9-dimethylmethylene blue assay), PG synthesis ((35)S-sulfate incorporation assay), aggrecan proteolysis (immunoblotting analysis), and vertebral bone morphology (microcomputed tomography) were measured. RESULTS Exposure of wild-type mice to tobacco smoke led to a 19% increase in vertebral porosity and a 61% decrease in trabecular bone volume. Intervertebral discs of smoke-exposed animals also showed a 2.6-fold decrease in GAG content and an 8.1-fold decrease in new PG synthesis. These smoking-induced degenerative changes were similar but not worse in Ercc1(-/Δ) mice. CONCLUSIONS Short-term exposure to high levels of primary tobacco smoke inhalation promotes degeneration of vertebral bone and discs. Disc degeneration is primarily driven by reduced synthesis of proteoglycans needed for vertebral cushioning. Degeneration was not exacerbated in congenic DNA repair-deficient mice, indicating that DNA damage per se does not have a significant causal role in driving smoke-induced spine degeneration.

GH receptor isoforms and skeletal fragility in acromegaly

OBJECTIVE Acromegaly is associated with an increased prevalence of vertebral fractures (VFs) in close relationship with GH hypersecretion. Two isoforms of the GH receptor (GHR) have been identified; the two isoforms differ or not by the expression of the protein fragment encoded by exon 3 of the GHR gene. Deletion of the exon 3 may influence the functional properties of the GHR and affect fracture risk in acromegalic patients. DESIGN A cross-sectional study was designed to investigate the association between the d3-GHR isoform and the prevalence of VFs in patients with acromegaly. METHODS In this study, 109 acromegalic patients were included (M/F, 48/61): 73 with controlled/cured acromegaly and 36 with active disease. GHR genotype was assessed in each patient. All patients were evaluated for VFs and bone mineral density at lumbar spine and hip. Serum IGF1 levels and bone metabolism markers were measured. A multivariate analysis was performed to establish risk factors for VFs in our population. RESULTS d3-GHR carriers showed an increased prevalence of VFs when compared with patients expressing full-length GHR (35/55 vs 12/54; P<0.001). The association between GHR deletion and VFs was demonstrated both in patients with active disease and in those with controlled/cured disease. Out of 35 patients who were prospectively evaluated, 13 (37.1%) developed incident VFs. The incidence of VFs was significantly higher in patients for whom the GHR gene has been deleted when compared with those harboring the fl gene (P=0.04). In multivariate analysis, male sex (odds ratio (OR), 3.250; P=0.041), IGF1 levels (OR, 1.183; P=0.031), length of active diseases (OR, 1.038; P=0.001), and d3-GHR genotype (OR, 3.060; P=0.015) were all confirmed as risk factors of VFs in our population. CONCLUSIONS This study suggests for the first time that exon 3 deletion of GHR may predispose patients with active and controlled acromegaly to a higher risk of VFs.

Genotoxic stress accelerates age-associated degenerative changes in intervertebral discs.

Intervertebral disc degeneration (IDD) is the leading cause of debilitating spinal disorders such as chronic lower back pain. Aging is the greatest risk factor for IDD. Previously, we demonstrated IDD in a murine model of a progeroid syndrome caused by reduced expression of a key DNA repair enzyme. This led us to hypothesize that DNA damage promotes IDD. To test our hypothesis, we chronically exposed adult wild-type (Wt) and DNA repair-deficient Ercc1(-/Δ) mice to the cancer therapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage and measured the impact on disc structure. Proteoglycan, a major structural matrix constituent of the disc, was reduced 3-5× in the discs of MEC- and IR-exposed animals compared to untreated controls. Expression of the protease ADAMTS4 and aggrecan proteolytic fragments was significantly increased. Additionally, new PG synthesis was reduced 2-3× in MEC- and IR-treated discs compared to untreated controls. Both cellular senescence and apoptosis were increased in discs of treated animals. The effects were more severe in the DNA repair-deficient Ercc1(-/Δ) mice than in Wt littermates. Local irradiation of the vertebra in Wt mice elicited a similar reduction in PG. These data demonstrate that genotoxic stress drives degenerative changes associated with IDD.

Growth hormone receptor isoforms and fracture risk in adult-onset growth hormone-deficient patients.

Growth hormone deficiency is considered the most important factor determining skeletal fragility in hypopituitary patients. Osteoblasts and chondrocytes express growth hormone (GH) receptor. Two GH receptor isoforms (GHRi) have been identified: they differ for the presence/absence of a protein fragment encoded by exon 3 of GHR gene. Consequently, three genotypes were identified: carriers of both the full‐length proteins (flfl‐GHR), carriers of one full‐length protein and one deleted protein (fld3‐GHR) and carriers of both deleted proteins (d3d3‐GHR). This polymorphism confers a higher sensitivity to endogenous GH and to recombinant human GH (rhGH); its effect on bone metabolism and skeletal fragility is unknown. The aim of this article was to investigate the role of GHRi in predicting skeletal fragility in adult‐onset GHD (AO‐GHD) patients.

Onset of a Charcot spinal arthropathy at a level lacking surgical arthrodesis in a paraplegic patient with traumatic cord injury

The study design included a case report of Charcot spinal arthropathy treated with posterior and anterior spinal instrumentation. The objective of the study was to report an unusual case of Charcot spinal arthropathy as a late complication of traumatic spinal cord injury in a patient previously treated with a long posterior thoraco-lumbar instrumentation and postero-lateral fusion. A 33-year-old man with T10–T11 complete paraplegia presented with focal low back pain, kyphotic deformity of the lumbar region with L2–L3 fracture–dislocation and hardware failure. Our treatment consisted of a circumferential arthrodesis performed with a combined anterior and posterior approach. Spinal stabilization was achieved and the patient was pain free and able to resume a sitting posture. This report suggests that the development of a Charcot spine arthropathy must always be considered as a late complication of a spinal cord injury. Moreover, we would emphasize the fundamental role of a strict clinical and radiological follow-up in order to detect an early Charcot spine complication.