Enrico Pola

Sonic Hedgehog Regulates Angiogenesis and Myogenesis During Post-Natal Skeletal Muscle Regeneration

Sonic hedgehog (Shh) is a morphogen‐regulating crucial epithelial‐mesenchymal interactions during embryonic development, but its signalling pathway is considered generally silent in post‐natal life. In this study, we demonstrate that Shh is de novo expressed after injury and during regeneration of the adult skeletal muscle. Shh expression is followed by significant up‐regulation of its receptor and target gene Ptc1 in injured and regenerating muscles. The reactivation of the Shh signalling pathway has an important regulatory role on injury‐induced angiogenesis, as inhibition of Shh function results in impaired up‐regulation of prototypical angiogenic agents, such as vascular endothelial growth factor (VEGF) and stromal‐derived factor (SDF)‐1alpha, decreased muscle blood flow and reduced capillary density after injury. In addition, Shh reactivation plays a regulatory role on myogenesis, as its inhibition impairs the activation of the myogenic regulatory factors Myf‐5 and MyoD, decreases the up‐regulation of insulin‐like growth factor (IGF)‐1 and reduces the number of myogenic satellite cells at injured site. Finally, Shh inhibition results in muscle fibrosis, increased inflammatory reaction and compromised motor functional recovery after injury. These data demonstrate that the Shh pathway is functionally important for adult skeletal muscle regeneration and displays pleiotropic angiogenic and myogenic potentials in post‐natal life. These findings might constitute the foundation for new therapeutic approaches for muscular diseases in humans.

Clinical Factors Associated with an Increased Risk of Perioperative Blood Transfusion in Nonanemic Patients Undergoing Total Hip Arthroplasty

BACKGROUND The aim of this study was to identify clinical factors associated with an increased need for perioperative blood transfusion in nonanemic patients undergoing total hip arthroplasty. METHODS We evaluated eighty-five consecutive nonanemic patients who underwent elective, unilateral, cementless, primary total hip arthroplasty and met our inclusion criteria. We attempted to determine whether clinical parameters influencing perioperative blood loss, such as age, gender, hypertension, and body mass index, were also associated with the need for perioperative blood transfusion. RESULTS Perioperative blood transfusion was required in twenty-four (28%) of the eighty-five nonanemic patients. When considered alone, age, gender, hypertension, and body mass index were not significantly associated with an increased risk of perioperative blood transfusion, on the basis of the numbers available. In contrast, there was a significantly increased risk of blood transfusion when two or more of these clinical parameters were present (p = 0.02). CONCLUSIONS Our findings indicate that clinical variables such as age, gender, hypertension, and body mass index may have a synergistic effect on the risk of transfusion in patients undergoing elective total hip arthroplasty. The simultaneous analysis of these parameters might help to stratify patients with different risks for transfusion and may increase the efficiency and reduce the cost of blood-ordering practices associated with total hip arthroplasty. LEVEL OF EVIDENCE Prognostic study, Level II-1 (retrospective study). See Instructions to Authors for a complete description of levels of evidence.

The K469E polymorphism of the ICAM-1 gene is a risk factor for peripheral arterial occlusive disease

Intercellular adhesion molecule-1 (ICAM-1) plays a crucial role in lymphocyte migration and activation, and is considered important in the pathogenesis of atherosclerosis. K469E is a common polymorphism of the ICAM-1 gene with potential functional significance. The aim of the present case–control study was to evaluate the association between this polymorphism and peripheral arterial occlusive disease (PAOD). ICAM-1 gene polymorphism was examined by polymerase chain reaction and restriction enzyme analysis in 75 Italian subjects affected by PAOD and 227 controls. The distribution of ICAM-1 genotypes in patients affected by PAOD was 32.1% EE, 50.6% EK, and 17.3% KK. The distribution of ICAM-1 genotypes in control subjects was 17.2% EE, 55.1% EK, and 27.7% KK. The EE genotype was significantly more common in patients than controls (P = 0.006). Logistic regression analysis indicated that the presence of the EE genotype significantly increases the risk of PAOD (odds ratio, 3.5; 95% confidence interval, 1.5–8.4;P = 0.004). This is the first study documenting a role of the ICAM-1 gene polymorphism in the pathogenesis of a cardiovascular disease, such as PAOD. Our data support the hypothesis that inflammatory mechanisms are important in the pathophysiology of vascular diseases with an atherosclerotic basis.

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.

Ex vivo-transduced autologous skin fibroblasts expressing human Lim mineralization protein-3 efficiently form new bone in animal models.

Local gene transfer of the human Lim mineralization protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation in rodents. To develop a clinically relevant gene therapy approach to facilitate bone healing, we have used primary dermal fibroblasts transduced ex vivo with Ad.LMP-3 and seeded on a hydroxyapatite/collagen matrix prior to autologous implantation. Here, we demonstrate that genetically modified autologous dermal fibroblasts expressing Ad.LMP-3 are able to induce ectopic bone formation following implantation of the matrix into mouse triceps and paravertebral muscles. Moreover, implantation of the Ad.LMP-3-modified dermal fibroblasts into a rat mandibular bone critical size defect model results in efficient healing, as determined by X-rays, histology and three-dimensional microcomputed tomography (3DμCT). These results demonstrate the effectiveness of the non-secreted intracellular osteogenic factor LMP-3 in inducing bone formation in vivo. Moreover, the utilization of autologous dermal fibroblasts implanted on a biomaterial represents a promising approach for possible future clinical applications aimed at inducing new bone formation.

Association between IL-6 and MMP-3 gene polymorphisms and adolescent idiopathic scoliosis : A case-control study

Study Design. Case-control study. Objective. As inflammation plays a key role in the etiology of intervertebral disc degeneration, we suggest a possible contribution of pro-inflammatory gene polymorphisms in the pathogenesis of adolescent idiopathic scoliosis (AIS). Summary of Background Data. The nucleus pulposus of scoliotic discs responds to exogenous stimuli by secreting interleukin-6 (IL-6) and other inflammatory cytokines. The association between matrix metalloproteinases (MMPs) and disc degeneration has been reported by several investigators. A human MMP-3 promoter 5A/6A gene polymorphism regulates MMP-3 genes expression, while the G/C polymorphism of the promoter region of IL-6 gene influences levels and functional activity of the IL-6 protein. Methods. We conducted a case-control study to investigate whether the 5A/6A polymorphism of the MMP-3 gene and the G/C polymorphism of the promoter region of IL-6 gene were associated with susceptibility to AIS. Results. The frequency of the 5A/5A genotype of MMP-3 gene polymorphism in patients with scoliosis was almost 3 times higher than in controls (30.2% vs. 11.2%, p 0.001), and the frequency of the G/G genotype of IL-6 gene polymorphism in patients with scoliosis was almost 2 times higher than in controls (52.8% vs. 26.2%, P < 0.001). 5A/5A genotype of MMP-3 gene polymorphism and G/G genotype of IL-6 gene polymorphism are independently associated with a higher risk of scoliosis (odds ratio, respectively, 3.34 and 10.54). Conclusion. This is the first study that has evaluated the possibility that gene variants of IL-6 and MMPs might be associated with scoliosis and suggests that MMP-3 and IL-6 promoter polymorphisms constitute important factors for the genetic predisposition to scoliosis.

Efficient bone formation by gene transfer of human LIM mineralization protein-3

LIM mineralization protein (LMP) is a novel positive regulator of the osteoblast differentiation program. In humans, three different LMP splice variants have been identified: LMP-1, LMP-2, and LMP-3. Gene transfer of human LMP-1 (hLMP-1) induces expression of genes involved in bone formation, including certain bone morphogenetic proteins (BMPs), promotes bone nodule formation in vitro, ectopic bone formation in vivo, and is therapeutic in animal models of posterior thoracic and lumbar spine fusion. To examine the osteoinductive properties of the LMP-3 in vitro and in vivo, we have generated plasmid and adenoviral vectors expressing codon-optimized hLMP-3. Here we demonstrate that gene transfer of hLMP-3 induces expression of the bone-specific genes osteocalcin, osteopontin, and bone sialoprotein and induced bone mineralization in preosteoblastic and fibroblastic cells. We also demonstrate that hLMP-3 is able to induce bone mineralization and the expression of the bone-specific genes, BMP-2, OSX, RunX2, and alkaline phosphatase in human mesenchymal stem cells in a dose-dependent manner. Finally, we demonstrate that direct gene transfer of hLMP-3 into murine skeletal muscle results in ectopic bone formation more efficiently than BMP-2. These results demonstrate that hLMP-3 gene transfer can be used to promote bone formation in cell culture and in vivo as or more efficiently than BMP-2, thus establishing feasibility and efficacy of direct gene delivery of hLMP-3 to produce bone in vivo. These results suggest that gene transfer of hLMP-3 could be developed as a bone-inductive therapeutic agent for clinical applications.

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.

Local infusion of norepinephrine reduces blood losses and need of transfusion in total knee arthroplasty

Blood loss after total knee arthroplasty (TKA) is often associated with cardiovascular complications and a high transfusion rate of allogenic blood. In our study we focused our attention on developing a new intra-surgical procedure that appears safe, easy to perform and effective in the reduction of bleeding in TKA. We evaluated 84 patients who underwent TKA and met our inclusion criteria; they were assigned to two groups: 55 controls in which a saline solution was used to wash the surgical field before tourniquet release, and a second group of 29 patients, in which a saline solution containing a low dose of norepinephrine was locally applied before tourniquet release. The local administration of a low dose of norepinephrine has induced a significant reduction of perioperative blood loss and blood transfusion requirements; in addition, this method was characterised by the absence of complications or adverse effects. In conclusion, our data suggest that intraoperative local administration of a low dose of norepinephrine could represent an effective and safe method of reducing blood loss and preventing blood transfusions in patients with TKA.RésuméLes pertes sanguines après prothèse totale du genou sont souvent associées avec des complications cardio-vasculaires et nécessitent un pourcentage important de transfusions sanguines. Nous avons développé pour notre étude une technique chirurgicale destinée à réduire les saignements dans les prothèses totales du genou. Nous avons inclus 84 patients qui ont bénéficié d’une prothèse totale du genou. Nous les avons divisés en deux groupes: un groupe contrôle de 55 patients pour lesquels une solution saline a été utilisée pour laver le champ opératoire avant le lever du garrot et un second groupe de 29 patients pour lequel une solution saline contenant une faible dose de norépinéphrine a été localement appliquée avant l’ablation du garrot. L’administration locale d’une petite dose de norépinéphrine a entraîné une reduction significative du saignement péri-opératoire et de la nécessité de transfusion sanguine. Cette méthode n’a entraîné aucune complication. Conclusion, ces données nous incitent à suggérer que l’administration locale de deux petites doses de norépinéphrine représente une méthode de réduction du saignement et de prévention de la transfusion sanguine dans la mise en place d’une prothèse totale du genou.

A meta-analysis of interleukin-6 promoter polymorphisms on risk of hip and knee osteoarthritis

OBJECTIVE Interleukin-6 is a pro-inflammatory cytokine involved in the pathogenesis of osteoarthritis (OA). We investigated the role of two single nucleotide polymorphisms (SNPs) mapping to the promoter of the IL-6 gene on genetic susceptibility to hip and knee OA. METHODS The -174G/C (rs1800795) and -597G/A (rs1800797) SNPs, implicated in the literature in risk of hip and hand OA, were genotyped in 2511 controls, 1101 hip OA cases and 1904 knee OA cases from four cohorts from the UK and Estonia. Data were analysed in conjuntion with published data on rs1800797 from the Genetics of OA and Lifestyle study (UK) on 791 controls, 1034 knee and 997 hip OA cases and rs1800795 data on 75 hip OA cases and 96 controls from Italy. Cases included both radiographic OA only and radiographic and symptomatic OA. Fixed and random-effects meta-analysis models were tested. RESULTS No significant association was found with hip OA or knee OA with either SNP nor with the haplotypes formed by them. For individual SNPs the smallest P-value for hip OA was observed using a random-effects model for rs1800795 OR(Gallele)=1.066 (95% CI 0.89-1.28) P<0.49, and significant heterogeneity between cohorts (I(2)=65%, P<0.034) was detected. For knee OA the smallest P-value was seen for rs1800797 OR(Aallele)=1.055 (95%CI 0.98-1.12) P<0.18, no significant heterogeneity was observed (I(2)=0%, P<0.68). CONCLUSIONS Our data do not support a role for the -174 and -597 IL-6 promoter polymorphisms in genetic susceptibility to knee or hip OA in Caucasian populations.