Polymorphism of Genes Encoding Inflammatory Interleukins and the Risk of Anterior Cruciate Ligament Injury: A Systematic Review and Meta-Analysis.

Sport injuries, including the anterior crucial ligament rupture (ACLR) seem to be related to complex genetic backgrounds, including the genes responsible for inflammatory response. This review and meta-analysis investigated the contribution of the polymorphisms of genes encoding inflammatory cytokines and their receptors to the risk of ACLR. The scientific databases Science Direct, EBSCO host, Scopus, PubMed, and Google Scholar were screened (completed on 14 June 2023) according to the established inclusion/exclusion criteria (only fully accessible, original, human case-control studies written in English concerning the effect of interleukin genes' polymorphisms on the occurrence of ACL injury were included) and statistical meta-analysis using R version 4.0.3 was performed. The PRISMA methodology was used to review articles. The review protocol was registered under the number CRD42024514316 in the Prospero database. Eighty-nine studies were identified and narrowed down to three original case-control studies used for the meta-analysis. The studies analyzed Polish, South African, and Swedish cohorts, altogether 1282 participants. The candidate polymorphisms indicated in the studies involved IL6 rs1800795, IL6R rs2228145 and IL1B rs16944. The systematic review showed the relationships between IL6 rs1800795 polymorphism and ACLR in the Polish subpopulation, and IL6R rs2228145 and IL1B rs16944 in the South African subpopulations. The meta-analysis revealed that the IL6 rs1800795 CG genotype was over-represented (OR = 1.30, 95% CI 1.02-1.66), while the CC genotype was under-represented (OR = 0.75, 95% CI 0.54-1.03) in ACLR subjects, but no significant impact of IL6R rs2228145 was shown. Additionally, a tendency of the IL1B rs16944 CT genotype to be protective (OR 0.89, 95% CI 0.70-1.14), while the TT to be a risk genotype (OR 1.19, 95% CI 0.84-1.68) was observed. Thus, the relationship between the interleukin receptor IL6R rs2228145 and ACLR risk was not confirmed. However, the impact of genes coding pleiotropic IL6 rs1800795 on the incidences of ACLR was clear and the effect of pro-inflammatory IL1B rs16944 was possible.

Correlation between vascular endothelial growth factor A gene polymorphisms and tendon and ligament injury risk: a systematic review and meta-analysis.

BACKGROUND: Relevant evidence suggests that angiogenic factors contribute significantly to fibril matrix reconstruction following physical injuries to tendon ligaments. Vascular endothelial growth factor A (VEGFA), with its potent angiogenic effect, has been studied extensively, and its functional polymorphisms, including rs699947, rs1570360, and rs2010963, have been the focus of numerous investigations. Some scholars have explored the association between gene polymorphisms in the VEGFA and the risk of tendon ligament injury, but the findings are not entirely consistent. OBJECTIVES: The purpose of this study was to investigate the association between rs699947, rs1570360, and rs2010963 gene polymorphisms in VEGFA and the risk of tendon and ligament injuries. METHODS: After including articles about the association of VEGFA rs699947, rs1570360, and rs2010963 polymorphisms with tendon and ligament injuries according to the search strategy, we assessed their quality and conducted meta-analyses to examine the link between these polymorphisms and the risk of tendon and ligament injuries using odds ratios and 95% confidence intervals. RESULTS: Of 86 related articles, six were included in the meta-analysis. Some of these suggest an association between VEGFA rs2010963 and the risk of tendon and ligament injury in the population, with the specific C allele being one of the adverse factors for knee injury. Some studies suggest that VEGFA rs699947 and VEGFA rs1570360 single-nucleotide polymorphisms are associated with anterior cruciate ligament rupture. The risk of non-contact anterior cruciate ligament rupture is nearly doubled in individuals with the rs699947 CC genotype compared to the control group. Our analysis did not find any significant relationship between VEGFA gene polymorphisms (rs699947, rs1570360, and rs2010963) and the chance of tendon and ligament injury without consideration of race. However, the European population reveals that the CC genotype of VEGFA rs699947 can result in a greater risk of tendon and ligament injury, whereas the AG genotype for rs1570360 provides some protection. Additionally, rs2010963 was significantly associated with tendon and ligament injury; individuals with the C allele and the CC genotype had higher risk. False-positive report probability confirmed the high credibility of our results. CONCLUSION: Overall, this study found no significant association between VEGFA rs699947, rs1570360, and rs2010963 polymorphisms and the risk of tendon ligament injury. However, in subgroup analysis, some genotypes of VEGFA rs699947, rs1570360, and rs2010963 were found to increase the risk of tendon ligament injury in European populations.

Overexpression of manganese superoxide dismutase mitigates ACL injury-induced muscle atrophy, weakness and oxidative damage.

Oxidative stress has been implicated in the etiology of skeletal muscle weakness following joint injury. We investigated longitudinal patient muscle samples following knee injury (anterior cruciate ligament tear). Following injury, transcriptomic analysis revealed downregulation of mitochondrial metabolism-related gene networks, which were supported by reduced mitochondrial respiratory flux rates. Additionally, enrichment of reactive oxygen species (ROS)-related pathways were upregulated in muscle following knee injury, and further investigation unveiled marked oxidative damage in a progressive manner following injury and surgical reconstruction. We then investigated whether antioxidant protection is effective in preventing muscle atrophy and weakness after knee injury in mice that overexpress Mn-superoxide dismutase (MnSOD+/-). MnSOD+/- mice showed attenuated oxidative damage, atrophy, and muscle weakness compared to wild type littermate controls following ACL transection surgery. Taken together, our results indicate that ROS-related damage is a causative mechanism of muscle dysfunction after knee injury, and that mitochondrial antioxidant protection may hold promise as a therapeutic target to prevent weakness and development of disability.

IL-6 induces periostin production in human ACL remnants: a possible mechanism causing post-traumatic osteoarthritis.

OBJECTIVE: Perostin (POSTN) and IL-6 consistently elevated after ACL injury, and ACL has been proposed as the major source of POSTN. However, there is a lack of evidence whether IL-6 induces ACL remnants to produce POSTN. This study aimed to investigate the effect of IL-6 on POSTN production in ACL fibroblasts, which may help us understand more about the mechanism of PTOA after ACL injury and ACL reconstruction. METHODS: ACL remnants were harvested from 27 patients undergoing ACL reconstruction. Quantitative real-time polymerase chain reaction (PCR) was performed to examine the POSTN gene expression of ACL fibroblasts after treatment of different concentrations of IL-6. The POSTN protein production of ACL fibroblasts was determined using western blot analysis. The blockers of possible signaling pathways, including PI3K/Akt, Ras/MAPK, and JAK/STAT pathways, were added to test whether the effect of IL-6 on ACL fibroblast could be attenuated. ACL fibroblast and chondrocyte co-culture was carried out to determine the influence of ACL and IL-6 on chondrocytes. RESULTS: Quantitative real-time PCR showed that IL-6 time-dependently and dose-dependently increased POSTN gene expression of ACL fibroblast. Western blot analysis also revealed that IL-6 dose-dependently induced POSTN protein production. Regarding the chronicity of ACL injury, the POSTN protein production was comparable between ACL remnants which were derived within 3 months of injury and at least 6 months after injury. PI3K/Akt blockers could attenuate the effect of IL-6 on ACL remnants, whereas Ras/MAPK and JAK/STAT did not decrease POSTN production. The coexistence of ACL and IL-6 induced more MMP-13 and ADAMTS-4 by chondrocytes. CONCLUSIONS: IL-6 induced ACL remnants to produce POSTN. This effect could be attenuated by the PI3K/Akt blocker. Coexistence of IL-6 and ACL remnants may accelerate post-traumatic arthritis.

Anterior cruciate ligament injury and its postoperative outcomes are not associated with polymorphism in COL1A1 rs1107946 (G/T): a case-control study in the Middle East elite athletes.

BACKGROUND: It is unclear what role COL1A1 polymorphisms play in anterior cruciate ligament (ACL) injury pathophysiology. The present study investigated the relationship between COL1A1-1997 guanine (G)/thymine (T) (rs1107946) polymorphism and ACL injury. Moreover, the possible effect of this polymorphism on the postoperative outcomes of ACL reconstruction surgery was evaluated. METHODS: This prospective case-control study was performed on 200 young professional men with an ACL tear who underwent arthroscopic ACL reconstruction surgery. Moreover, 200 healthy athletes without a history of tendon or ligament injury who were matched with the case group were selected as the control group. DNA was extracted from the leukocytes of participants, and the desired allele was genotyped. Clinical outcomes were collected for the case group before and one year after surgery. RESULTS: The genotype distribution was in accordance with the Hardy-Weinberg principle. In the ACL injury group, the G allele frequency was non-significantly higher than the healthy controls, with an odds ratio [95% CI] of 1.08 [0.79-1.47] (P = 64). We did not find a significant difference between the genotype of individuals-GG, GT, and TT-in the case and control groups (P > 0.05). Clinical outcomes of the ACL tear group were significantly improved in terms of preoperative values. However, none of them were significantly different between the three genotypes (GG, GT, and TT). CONCLUSION: According to the findings of the present investigation, single-nucleotide polymorphism (SNP) at COL1A1 rs1107946 (G/T) was not a predisposing genetic factor for ACL injury in a young professional male athlete population in the Middle East. Furthermore, patients' responses to treatment were not different between distinct genotypes.

Influence of type I collagen polymorphisms and risk of anterior cruciate ligament rupture in athletes: a case-control study.

BACKGROUND: Anterior cruciate ligament (ACL) rupture is a common and severe knee injury in sports and occurs mostly due to noncontact injuries. There is an increasing amount of evidence associating ACL rupture to single nucleotide polymorphisms (SNPs), and SNPs in the collagen type I genes can change its expression and tissue mechanical features. This study aimed to investigate the association between SNPs in COL1A1 and COL1A2 with sports-related ACL tears. METHODS: A total of 338 athletes from multiple sports modalities were analyzed: 146 were diagnosed with ACL rupture or underwent an ACL reconstruction surgery and 192 have no musculoskeletal injuries. SNPs were genotyped using validated TaqMan assays. The association of the polymorphisms with ACL rupture was evaluated by a multivariable logistic regression model, using odds ratios (OR) and 95% confidence intervals (CI). RESULTS: The age, sport modality, and training location were associated with an increased risk of a non-contact ACL tear. COL1A2 SNPs (rs42524 CC and rs2621215 GG) were associated with an increased risk of non-contact ACL injury (6 and 4-fold, respectively). However, no significant differences were detected in the distribution of COL1A1 rs1107946 and COL1A2 rs412777 SNPs between cases and controls. There was a protective association with ACL rupture (OR = 0.25; 95% CI = 0.07-0.96) between COL1A1 rs1107946 (GT or TT) and the wildtype genotypes of the three COL1A2 (rs412777, rs42524, rs2621215). COL1A2 rs42524 and rs2621215 SNPs were associated with non-contact ACL risk. CONCLUSION: The combined analysis of COL1A1-COL1A2 genotypes suggests a gene-gene interaction in ACL rupture susceptibility.

Risk modelling further implicates the angiogenesis pathway in anterior cruciate ligament ruptures.

The aim of this study was to explore the interactions between the interleukins and the angiogenesis signalling pathway, following a pathway-based approach. Statistical modelling tools were used to develop a preliminary polygenic risk assessment model for anterior cruciate ligament (ACL) ruptures, incorporating the angiogenesis signalling genes (VEGFA and KDR) and interleukins (IL1B, IL6, IL6R) which also function to regulate angiogenesis. Multivariate logistic regression analysis was used to identify the most informative contributors to ACL rupture risk from a range of eleven potential intrinsic risk factors: age, sex, BMI and eight genetic polymorphisms within five genes, namely, IL1B rs16944 C/T, IL6 rs1800795 G/C, IL6R rs2228145 C/A, VEGFA rs699947 C/A, VEGFA rs1570360 G/A, VEGFA rs2010963 C/G, KDR rs2071559 A/G and KDR rs1870377 T/A. A total of 232 asymptomatic controls (CON) and 234 participants with surgically diagnosed ACL ruptures, of which 135 participants reported a non-contact mechanism of injury (NON subgroup), were previously genotyped for the selected polymorphisms. The polygenic risk model identified the VEGFA rs699947 CC genotype (p = 0.024, odds ratio (OR): 3.35, 95% confidence interval (CI): 1.17-9.62), VEGFA rs2010963 GC genotype (p = 0.049, OR: 2.43, 95% CI: 1.00-5.87), age (p = 0.011, OR: 0.97, 95% CI: 0.95-0.99) and BMI (p = 0.009, OR:1.09, 95% CI: 0.57-2.11) as the most significant predictors of ACL rupture risk from the data included. The results of this study highlight VEGFA, age and BMI as biologically significant components of this network requiring further investigation in the context of musculoskeletal soft tissue injury risk.HighlightsThe findings of this study highlight the VEGFA gene, age and BMI as biologically significant contributors to ACL rupture susceptibility.Upon further validation of these risk factors, they may be included in genetic risk assessment tools to design pre-habilitation strategies, prescribe appropriate treatment strategies after injury or to assess how an individual is likely to respond to load.Polygenic risk models aid in highlighting the components of the complex ECM remodelling pathway requiring further investigation, using a multidisciplinary approach.VEGFA is a key angiogenic protein contributing to ECM homeostasis and may therefore have potential therapeutic implications that need to be explored.

Investigation of multiple populations highlight VEGFA polymorphisms to modulate anterior cruciate ligament injury.

Polymorphisms in VEGFA and KDR encoding proteins have been associated with anterior cruciate ligament (ACL) injury risk. We leveraged a collective sample from Sweden, Poland, and Australia to investigate the association of functional polymorphisms in VEGFA and KDR with susceptibility to ACL injury risk. Using a case-control genetic association approach, polymorphisms in VEGFA and KDR were genotyped and haplotypes inferred from 765 controls, and 912 cases clinically diagnosed with ACL rupture. For VEGFA, there was a significant overrepresentation of the rs2010963 CC genotype (p = 0.0001, false discovery rate [FDR]: p = 0.001, odds ratio [OR]: 2.16, 95% confidence interval [CI]: 1.47-3.19) in the combined ACL group (18%) compared to the combined control group (11%). The VEGFA (rs699947 C/A, rs1570360 G/A, rs2010963 G/C) A-A-G haplotype was significantly (p = 0.010, OR: 0.85, 95% CI: 0.69-1.05) underrepresented in the combined ACL group (23%) compared to the combined control group (28%). In addition, the A-G-G construct was significantly (p = 0.036, OR: 0.81, 95% CI: 0.64-1.02) underrepresented in the combined ACL group (12%) compared to the combined CON group (16%). Our findings support the association of the VEGFA rs2010963 CC genotype with increased risk and (ii) the VEGFA A-A-G haplotype with a reduced risk, and are in alignment with the a priori hypothesis. Collectively identifying a genetic interval within VEGFA to be implicated in ACL risk modulation and highlight further the importance of vascular regulation in ligament biology.

Inhibition of microRNA-27b-3p relieves osteoarthritis pain via regulation of KDM4B-dependent DLX5.

Osteoarthritis (OA) represents a progressive degenerative disorder that predominantly affects the synovial membranes of joints. Recent studies have highlighted the significant role played by microRNAs (miRNAs) in OA development. The current study aimed to elucidate the underlying modulatory role of miR-27b-3p in the development of OA. The expression of miR-27b-3p in the OA patients and rat models post anterior cruciate ligament transection operation was measured using reverse transcription quantitative polymerase chain reaction, through which overexpressed miR-27b-3p was found in both of the samples. To further explore the miR-27b-3p functions in OA, western blot analysis, enzyme-linked immunosorbent assay, and ß-galactosidase activity assay were conducted with the results showing that knockdown of miR-27b-3p promoted expression of the osteogenic differentiation markers while inhibiting expression of the adipogenic differentiation markers, inflammatory factors, and cellular senescence of bone marrow mesenchymal stem cells (BMSCs). After that, the interactions between miR-27b-3p, lysine Demethylase 4B (KDM4B), and Distal-Less Homeobox 5 (DLX5) identified using dual-luciferase reporter gene assay and ChIP assay revealed that miR-27b-3p inhibited KDM4B and further reduced expression of DLX5. Finally, the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were assessed in rat models, and increased PWT and PWL were detected after miR-27b-3p silencing. In conclusion, suppression of miR-27b-3p could enhance KDM4B and DLX5 to alleviate OA pain, shedding light on a new potential therapeutic target for OA.

Contralateral and siblings' knees are at higher risk of ACL tear for patients with a positive history of ACL tear.

PURPOSE: Recent studies have shown that several genetic factors can cause susceptibility to anterior cruciate ligament (ACL) rupture. The aim of the present study was to evaluate certain underlying factors that increase the risk of ACL rupture. METHODS: Eight hundred thirty-six patients with ACL rupture who underwent ACL reconstructive surgery from 2010 to 2013 at an academic center completed a minimum of 5 years post-operation follow-up. The collected variables included sex, age, height, weight, exercise level, time interval between ACL rupture in the first knee and contralateral ACL rupture, dominant leg, side of the involved knee and sibling history of ACL rupture. RESULTS: The median follow-up duration was 6.5 (range: 5-8) years. Eighty-three patients (9.9%) had a contralateral ACL rupture, and 155 patients (18.5%) had siblings with a history of ACL rupture. The rate of contralateral ACL rupture was three times higher in women than in men and in patients with siblings with a history of ACL rupture than in those without such history. In addition, the risk of contralateral ACL rupture was higher in those younger than 30 years of age, those with a BMI of 20-25 kg/m2 and those who participated in regular sports activity. However, whether the involved knee was on the dominant or nondominant side had no effect on the incidence of contralateral ACL rupture. The results of the study showed that 69 (83.1%) of the contralateral ACL ruptures occurred within the first 2 years after the primary operation. CONCLUSION: In a 5- to 8-year follow-up, one out of every ten patients had a contralateral ACL rupture, and two out of every ten patients had siblings with a history of ACL rupture. The findings suggest that having a sibling with a history of ACL rupture and being female are important risk factors for ACL rupture of the contralateral knee. LEVEL OF EVIDENCE: III.

Functional polymorphisms within the inflammatory pathway regulate expression of extracellular matrix components in a genetic risk dependent model for anterior cruciate ligament injuries.

OBJECTIVES: To investigate the functional effect of genetic polymorphisms of the inflammatory pathway on structural extracellular matrix components (ECM) and the susceptibility to an anterior cruciate ligament (ACL) injury. DESIGN: Laboratory study, case-control study. METHODS: Eight healthy participants were genotyped for interleukin (IL)1B rs16944 C>T and IL6 rs1800795 G>C and classified into genetic risk profile groups. Differences in type I collagen (COL1A1), type V collagen (COL5A1), biglycan (BGN) and decorin (DCN) gene expression were measured in fibroblasts either unstimulated or following IL-1ß, IL-6 or tumor necrosis factor (TNF)-α treatment. Moreover, a genetic association study was conducted in: (i) a Swedish cohort comprised of 116 asymptomatic controls (CON) and 79 ACL ruptures and (ii) a South African cohort of 100 CONs and 98 ACLs. Participants were genotyped for COL5A1 rs12722 C>T, IL1B rs16944 C>T, IL6 rs1800795 G>C and IL6R rs2228145 G>C. RESULTS: IL1B high-risk fibroblasts had decreased BGN (p=0.020) and COL5A1 (p=0.012) levels after IL-1ß stimulation and expressed less COL5A1 (p=0.042) following TNF-α treatment. Similarly, unstimulated IL6 high-risk fibroblasts had lower COL5A1 (p=0.012) levels than IL6 low-risk fibroblasts. In the genetic association study, the COL5A1-IL1B-IL6 T-C-G (p=0.034, Haplo-score 2.1) and the COL5A1-IL1B-IL6R T-C-A (p=0.044, Haplo-score: 2.0) combinations were associated with an increased susceptibility to ACL injury in the Swedish cohort when only male participants were evaluated. CONCLUSIONS: This study shows that polymorphisms within genes of the inflammatory pathway modulate the expression of structural and fibril-associated ECM components in a genetic risk depended manner, contributing to an increased susceptibility to ACL injuries.

Are IL1B, IL6 and IL6R Gene Variants Associated with Anterior Cruciate Ligament Rupture Susceptibility?

Cytokines, such as interleukins, are crucial in regulating critical cell signaling pathways as well as being major contributors to inflammatory response and are upregulated during ligament and tendon injuries. The genes encoding key interleukins, such as IL1B and IL6 as well as interleukin receptor IL6R, were chosen as candidate genes for association with soft tissue injuries. The aim of the case-control study was to verify the hypothesis that sequence variants rs1143627, rs16944, rs1800795, rs2228145 in the IL1B, IL6 and IL6R genes are associated with ACL rupture susceptibility in a Polish population. Among four analyzed SNPs, the rs1800795 IL6 gene polymorphism was found to be the only one significantly associated with ACL rupture (p = 0.010, p = 0.022, p = 0.004 for codominant, recessive and overdominant models, respectively; odds ratio = 1.74, 95% CI 1.08-2.81, sex adjusted p = 0.032 for recessive model). With reference to the other analyzed polymorphisms, we failed to show significant differences in the genotype and allele frequencies for IL6R rs2228145as well as IL1B rs16944 and rs1143627 (analyzed alone or in haplotype combination) between the ACL rupture group and the healthy control group among Polish participants. Due to the nature of case-control studies, the results of this study need to be confirmed in independent studies with larger sample sizes.

The potential European genetic predisposition for non-contact anterior cruciate ligament injury.

PURPOSE: Previous research has provided evidence of a hereditary predisposition for anterior cruciate ligament (ACL) injury. The purpose of this study was to evaluate the association between ancestral population genetics and risk of non-contact ACL injuries. METHODS: Blood samples were collected from 177 individuals with a history of non-contact ACL injury and 556 non-injured control individuals for analysis of the genetic material through the use of a panel of 48 INDELs ancestry genetic markers from three ancestral origins. RESULTS: Among patients with non-contact ACL injury, 82% were male and 18% were female. In the control group, 78% were male, and 22% were female. The mean age of the non-contact ACL injury group was 31.7 years (± 10.2), and the control group was 33.8 years (± 13.2). The individual genetic contribution from INDELs of each ancestral origin varied considerably: ranging between 1.5-94.8% contribution for INDELs of African origin (mean of 21.4% of INDELs); between 2 and 96.1% contribution for INDELs of European origin (mean of 66.7% of INDELs); and between 1.3-96.4% contribution for INDELs of Amerindian origin (mean of 11.7% of INDELs). When comparing paired subjects from the non-contact ACL and control groups, the genetic analysis showed that the European ancestry score was higher in the non-contact ACL group than control group (0.70 ± 0.21 vs 0.63 ± 0.22 respectively, p < 0.001), whereas African ancestry scores (ACL group 0.18 ± 0.18 vs control group 0.24 ± 0.21, p < 0.001) and Amerindian ancestry scores (ACL group 0.11 ± 0.09 vs control group 0.12 ± 0.10, n.s.) were lower among the non-contact ACL group than in controls. CONCLUSION: European INDELs markers were found to represent a potential genetic predisposition for non-contact ACL injuries when compared to African and Amerindian INDELs. This study has the potential to correlate a measurable and distinct genetic marker with risk of a non-contact ACL injury. Thus, it increases knowledge base and volume of molecular and genetical factors associated with this pathology. Furthermore, this study provides guidance and evidence for the development of genetic risk-screening panels for non-contact ACL injury. LEVEL OF EVIDENCE: Level III Diagnostic Study.

Higher Gene Expression of Healing Factors in Anterior Cruciate Ligament Remnant in Acute Anterior Cruciate Ligament Tear.

BACKGROUND: Anterior cruciate ligament (ACL) reconstruction with remnant preservation has been described and related to potential advantages. Literature is lacking regarding gene expression of potential factors related to ligament healing in the ACL remnant and its relation to time from injury. HYPOTHESIS: The mRNA expression of ligament healing factors in the ACL remnant would be higher in acute tears (<3 months from injury) than in intermediate (3-12 months) and chronic (>12 months) injuries. STUDY DESIGN: Controlled laboratory study. METHODS: Gene expression of 21 genes related to ligament healing factors was analyzed in 46 ACL remnants biopsied during surgical reconstruction with quantitative real-time polymerase chain reaction technique. Specimens were divided into 3 groups according to time from injury: acute (<3 months from injury; n = 19), intermediate (3-12 months; n = 12), and chronic (>12 months; n = 15). Histological and immunohistochemical evaluation was performed by analysis of hematoxylin and eosin, CD-34, and S-100 staining. RESULTS: Expression of COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL12A1, LOX, PLOD1, and TNC genes in ACL remnant was greater in acute compared with chronic injuries. COL1A1, COL5A1, COL12A1, and TNC genes were also expressed more in the acute group compared with the intermediate group. Furthermore, expression of the genes COL1A1 and COL5A2 was significantly higher in female than in male patients. No difference in the number of blood vessels and mechanoreceptors among groups was observed in the microscopic evaluation. CONCLUSION: The present study demonstrates that expression of COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL12A1, LOX, PLOD1, and TNC genes in ACL remnant is greater in acute (<3 months from injury) compared with chronic (>12 months) injuries. Furthermore, COL1A1, COL5A1, COL12A1, and TNC genes were expressed more in the acute group compared with the intermediate group (3-12 months from injury). CLINICAL RELEVANCE: ACL reconstructions with remnant preservation should be performed in patients with acute injuries, as in these cases the ACL remnant may present the greatest healing potential.

Investigation of angiogenesis genes with anterior cruciate ligament rupture risk in a South African population.

The angiogenesis-signalling pathway is a physiological response after mechanical loading to promote matrix remodelling and thereby maintain tissue homeostasis. Studies have shown increased expression of angiogenic molecules in response to loading and in ruptured ligaments. Recently, polymorphisms within the vascular endothelial growth factor A (VEGFA) and kinase insert-domain receptor (KDR) genes were associated with risk of anterior cruciate ligament (ACL) ruptures and Achilles tendinopathy in Caucasian study groups. A case-control genetic association study was conducted on 100 controls and 98 participants with surgically-diagnosed ACL ruptures; of which 51 participants reported non-contact mechanism of injury (NON). All participants were genotyped for five functional polymorphisms: VEGFA (rs699947, rs1570360, rs2010963) and KDR (rs2071559, rs1870377). Haplotypes were inferred. In the male participants, the KDR rs2071559 AG genotype was significantly over-represented (P = 0.048, OR: 1.90, 95% CI: 1.00-3.59) in the controls. Furthermore, the GG genotype was significantly under-represented in the male controls compared to the male ACL group (P = 0.018, OR: 2.77, 95% CI: 1.17-6.55) and the male NON subgroup (P = 0.013, OR: 3.26, 95% CI: 1.24-8.58). Haplotype analysis implicated the KDR gene in all participants and in male participants separately. Collectively, these results implicate the angiogenesis-signalling pathway as a potentially key biological pathway contributing to ACL injury susceptibility.

Effects of GGCX overexpression on anterior cruciate ligament transection-induced osteoarthritis in rabbits.

Effective therapeutic methods for osteoarthritis (OA) are lacking. γ­glutamyl carboxylase (GGCX) is a key enzyme that regulates carboxylation of cartilage matrix Gla protein (MGP). Whether GGCX overexpression protects against OA remains unknown. The aim of the present study was to explore the effects of GGCX overexpression on anterior cruciate ligament transection (ACLT)­induced OA and its mechanisms in Japanese white rabbits. ACLT surgery was used to establish an OA model in rabbits. A total of 48 rabbits were randomly divided into 4 groups: Sham, OA model + GGCX overexpression plasmid, OA model + saline and OA model + empty vector. The expression of uncarboxylated MGP (ucMGP), carboxylated MGP (cMGP), matrix metalloproteinase (MMP)­13, collagen type X, collagen type II, tumor necrosis factor (TNF)­α and interleukin (IL)­1ß were detected by ELISA, immunohistochemistry, reverse transcription­quantitative polymerase chain reaction and western blotting. Morphological changes to tibial cartilage were assessed by Giemsa and safranin O­fast green staining, respectively. Compared with the Sham control, GGCX expression was significantly decreased in the OA Model group. GGCX expression was increased by injection of a lentivirus­carried overexpression plasmid that encoded GGCX. GGCX overexpression ameliorated ATLC­induced damage in articular cartilage. OA Model rabbits exhibited significantly decreased expression levels of cMGP and collagen type II, and increased expression of ucMGP, collagen type X, MMP­13, IL­1ß and TNF­α. Notably, these expression levels were reversed by GGCX overexpression in OA Model rabbits. Results from the present study indicated that GGCX expression was decreased in OA Model rabbits, whereas overexpression of GGCX was able to promote carboxylation of MGP, reduce inflammation, decrease MMP­13 expression and regulate collagen expression. The results also indicated that GGCX may serve as a therapeutic target for OA.

Human Genetic Variation, Sport and Exercise Medicine, and Achilles Tendinopathy: Role for Angiogenesis-Associated Genes.

Sport and Exercise Medicine is one of the important subspecialties of 21st century healthcare contributing to improving the physical function, health, and vitality of populations while reducing the prevalence of lifestyle-related diseases. Moreover, sport and exercise are associated with injuries such as Achilles tendinopathy, which is a common tendon injury. The angiogenesis-associated signaling pathway plays a key role in extracellular matrix remodeling, with increased levels of angiogenic cytokines reported after cyclic stretching of tendon fibroblasts. We investigated the variants in angiogenesis genes in relation to the risk of Achilles tendinopathy in two population samples drawn independently from South Africa (SA) and the United Kingdom (UK). The study sample comprised 120 SA and 130 UK healthy controls, and 108 SA and 87 UK participants with Achilles tendinopathy. All participants were genotyped for five functional polymorphisms in the vascular endothelial growth factor, A isoform (VEGFA) (rs699947, rs1570360, rs2010963) and kinase insert-domain receptor (KDR) genes (rs1870377, rs2071559). The VEGFA A-G-G inferred haplotype was associated with an increased risk of Achilles tendinopathy in the SA group (15% in controls vs. 20% in cases, p = 0.048) and the combined SA+UK group (14% in controls vs. 20% in cases, p = 0.009). These new findings implicate the VEGFA gene with Achilles tendinopathy risk, while highlighting the potential biological significance of the angiogenesis signaling pathway in the etiology of Achilles tendinopathy. The evidence suggesting a genetic contribution to the susceptibility of sustaining a tendon injury is growing. We anticipate that high-throughput and multi-omics approaches, building on genomics, proteomics, and metabolomics, may soon uncover the pathophysiology of many diseases in the field of Sports and Exercise Medicine, as a new frontier of global precision medicine.

Runx2-Modified Adipose-Derived Stem Cells Promote Tendon Graft Integration in Anterior Cruciate Ligament Reconstruction.

Runx2 is a powerful osteo-inductive factor and adipose-derived stem cells (ADSCs) are multipotent. However, it is unknown whether Runx2-overexpressing ADSCs (Runx2-ADSCs) could promote anterior cruciate ligament (ACL) reconstruction. We evaluated the effect of Runx2-ADSCs on ACL reconstruction in vitro and in vivo. mRNA expressions of osteocalcin (OCN), bone sialoprotein (BSP) and collagen I (COLI) increased over time in Runx2-ADSCs. Runx2 overexpression inhibited LPL and PPARγ mRNA expressions. Runx2 induced alkaline phosphatase activity markedly. In nude mice injected with Runx2-ADSCs, promoted bone formation was detected by X-rays 8 weeks after injection. The healing of tendon-to-bone in a rabbit model of ACL reconstruction treated with Runx2-ADSCs, fibrin glue only and an RNAi targeting Runx2, was evaluated with CT 3D reconstruction, histological analysis and biomechanical methods. CT showed a greater degree of new bone formation around the bone tunnel in the group treated with Runx2-ADSCs compared with the fibrin glue group and RNAi Runx2 group. Histology showed that treatment with Runx2-ADSCs led to a rapid and significant increase at the tendon-to-bone compared with the control groups. Biomechanical tests demonstrated higher tendon pullout strength in the Runx2-ADSCs group at early time points. The healing of the attachment in ACL reconstruction was enhanced by Runx2-ADSCs.