Degraded RNA from Human Anterior Cruciate Ligaments Yields Valid Gene Expression Profiles.

Correlating gene expression patterns with biomechanical properties of connective tissues provides insights into the molecular processes underlying the tissue growth and repair. Cadaveric specimens such as human knees are widely considered suitable for biomechanical studies, but their usefulness for gene expression experiments is potentially limited by the unavoidable, nuclease-mediated degradation of RNA. Here, we tested whether valid gene expression profiles can be obtained using degraded RNA from human anterior cruciate ligaments (ACLs). Human ACL RNA (N = 6) degraded in vitro by limited ribonuclease digestion resemble highly degraded RNA isolated from cadaveric tissue. PCR threshold cycle (Ct) values for 90 transcripts (84 extracellular matrix, 6 housekeeping) in degraded RNAs variably ranged higher than values obtained from their corresponding non-degraded RNAs, reflecting both the expected loss of target templates in the degraded preparations as well as differences in the extent of degradation. Relative Ct values obtained for mRNAs in degraded preparations strongly correlated with the corresponding levels in non-degraded RNA, both for each ACL as well as for the pooled results from all six ACLs. Nuclease-mediated degradation produced similar, strongly correlated losses of housekeeping and non-housekeeping gene mRNAs. RNA degraded in situ yielded comparable results, confirming that in vitro digestion effectively modeled degradation by endogenous ribonucleases in frozen and thawed ACL. We conclude that, contrary to conventional wisdom, PCR-based expression analyses can yield valid mRNA profiles even from RNA preparations that are more than 90% degraded, such as those obtained from connective tissues subjected to biomechanical studies. Furthermore, legitimate quantitative comparisons between variably degraded tissues can be made by normalizing data to appropriate housekeeping transcripts.

The Codon 72 TP53 Polymorphism Contributes to TSC Tumorigenesis through the Notch-Nodal Axis.

We discovered that 90.3% of patients with angiomyolipomas, lymphangioleiomyomatosis (LAM), and tuberous sclerosis complex (TSC) carry the arginine variant of codon 72 (R72) of TP53 and that R72 increases the risk for angiomyolipoma. R72 transactivates NOTCH1 and NODAL better than the proline variant of codon 72 (P72); therefore, the expression of NOTCH1 and NODAL is increased in angiomyolipoma cells that carry R72. The loss of Tp53 and Tsc1 within nestin-expressing cells in mice resulted in the development of renal cell carcinomas (RCC) with high Notch1 and Nodal expression, suggesting that similar downstream mechanisms contribute to tumorigenesis as a result of p53 loss in mice and p53 polymorphism in humans. The loss of murine Tp53 or expression of human R72 contributes to tumorigenesis via enhancing epithelial-to-mesenchymal transition and motility of tumor cells through the Notch and Nodal pathways. IMPLICATIONS: This work revealed unexpected contributions of the p53 polymorphism to the pathogenesis of TSC and established signaling alterations caused by this polymorphism as a target for therapy. We found that the codon 72 TP53 polymorphism contributes to TSC-associated tumorigenesis via Notch and Nodal signaling.

Liposome-induced immunosuppression and tumor growth is mediated by macrophages and mitigated by liposome-encapsulated alendronate.

Liposomal nanoparticles are the most commonly used drug nano-delivery platforms. However, recent reports show that certain pegylated liposomal nanoparticles (PLNs) and polymeric nanoparticles have the potential to enhance tumor growth and inhibit antitumor immunity in murine cancer models. We sought herein to identify the mechanisms and determine whether PLN-associated immunosuppression and tumor growth can be reversed using alendronate, an immune modulatory drug. By conducting in vivo and ex vivo experiments with the immunocompetent TC-1 murine tumor model, we found that macrophages were the primary cells that internalized PLN in the tumor microenvironment and that PLN-induced tumor growth was dependent on macrophages. Treatment with PLN increased immunosuppression as evidenced by increased expression of arginase-1 in CD11b+Gr1+ cells, diminished M1 functionality in macrophages, and globally suppressed T-cell cytokine production. Encapsulating alendronate in PLN reversed these effects on myeloid cells and shifted the profile of multi-cytokine producing T-cells towards an IFNγ+ perforin+ response, suggesting increased cytotoxic functionality. Importantly, we also found that PLN-encapsulated alendronate (PLN-alen), but not free alendronate, abrogated PLN-induced tumor growth and increased progression-free survival. In summary, we have identified a novel mechanism of PLN-induced tumor growth through macrophage polarization and immunosuppression that can be targeted and inactivated to improve the anticancer efficacy of PLN-delivered drugs. Importantly, we also determined that PLN-alen not only reversed protumoral effects of the PLN carrier, but also had moderate antitumor activity. Our findings strongly support the inclusion of immune-responsive tumor models and in-depth immune functional studies in the preclinical drug development paradigm for cancer nanomedicines, and the further development of chemo-immunotherapy strategies to co-deliver alendronate and chemotherapy for the treatment of cancer.

Notch transactivates Rheb to maintain the multipotency of TSC-null cells.

Differentiation abnormalities are a hallmark of tuberous sclerosis complex (TSC) manifestations; however, the genesis of these abnormalities remains unclear. Here we report on mechanisms controlling the multi-lineage, early neuronal progenitor and neural stem-like cell characteristics of lymphangioleiomyomatosis (LAM) and angiomyolipoma cells. These mechanisms include the activation of a previously unreported Rheb-Notch-Rheb regulatory loop, in which the cyclic binding of Notch1 to the Notch-responsive elements (NREs) on the Rheb promoter is a key event. This binding induces the transactivation of Rheb. The identified NRE2 and NRE3 on the Rheb promoter are important to Notch-dependent promoter activity. Notch cooperates with Rheb to block cell differentiation via similar mechanisms in mouse models of TSC. Cell-specific loss of Tsc1 within nestin-expressing cells in adult mice leads to the formation of kidney cysts, renal intraepithelial neoplasia, and invasive papillary renal carcinoma.

Age, sex, body anthropometry, and ACL size predict the structural properties of the human anterior cruciate ligament.

Anterior cruciate ligament (ACL) injury continues to be at the forefront of sports injury concerns because of its impact on quality of life and joint health prognosis. One strategy is to reduce the occurrence of this injury by identifying at-risk subjects based on key putative risk factors. The purpose of our study was to develop models that predict the structural properties of a subject's ACL based on the combination of known risk factors. We hypothesized that the structural properties of the ACL can be predicted using a multi-linear regression model based on significant covariates that are associated with increased risk of injury, including age, sex, body size, and ACL size. We also hypothesized that ACL size is a significant contributor to the model. The developed models had predictive capabilities for the structural properties of the ACL: load at failure (R2 = 0.914), elongation at failure (R2 = 0.872), energy at failure (R2 = 0.913), and linear stiffness (R2 = 0.756). Furthermore, sex, age, body mass, BMI, and height were contributors (p < 0.05) to all predicted structural properties. ACL minimal area was a contributor to elongation, energy at failure, and linear stiffness (p < 0.05), but not to load at failure. ACL volume was also a contributor to elongation and energy at failure (p < 0.05), but not to linear stiffness and load at failure models. ACL length was not a significant contributor to any structural property. The clinical significance of this research is its potential, after continued development and refinement of the model, for application to prognostic studies that are designed to identify individuals at increased risk for injury to the ligament.

Shallow medial tibial plateau and steep medial and lateral tibial slopes: new risk factors for anterior cruciate ligament injuries.

BACKGROUND: The geometry of the tibial plateau has been largely ignored as a source of possible risk factors for anterior cruciate ligament injury. Discovering the anterior cruciate ligament injury risk factors associated with the tibial plateau may lead to delineation of the existing sex-based disparity in anterior cruciate ligament injuries and help develop strategies for the prevention of anterior cruciate ligament injuries regardless of gender. HYPOTHESIS: Individuals with a shallower medial tibial depth of concavity, while having increased posteriorly directed slope of their tibial plateau, are at increased risk of suffering an anterior cruciate ligament injury compared with those with decreased posterior slope and increased medial tibial depth. Furthermore, these relationships are different between men and women. STUDY DESIGN: Case-control study (prevalence); Level of evidence, 3. METHODS: The medial, lateral, and coronal tibial plateau slopes as well as the medial tibial depth of concavity in 55 uninjured controls (33 women and 22 men) and 49 anterior cruciate ligament-injured cases (27 women and 22 men) were measured using magnetic resonance images. First, a preliminary t test was performed to establish any existing differences between groups. Next, a logistic regression model was developed to determine the probability of anterior cruciate ligament injury in an individual based on the measured covariates. RESULTS: The female anterior cruciate ligament-injured cases had increased lateral tibial slope (P = .03) and shallower medial tibial depth (P = .0003) compared with the uninjured controls, while male cases had increased lateral and medial tibial slope (P = .02) and shallower medial tibial depth (P = .0004) compared with controls. The logistic regression analysis and odds ratio estimates showed that medial tibial depth is an important risk factor (odds ratio = 3.03 per 1 mm decrease in its value), followed by lateral tibial slope (odds ratio = 1.17 per 1 degrees increase in its value) in all participants. The medial tibial slope (odds ratio = 1.18 per 1 degrees increase in its value) was a risk factor only in men. CONCLUSION: A combination of increased posterior-directed tibial plateau slope and shallow medial tibial plateau depth could be a major risk factor in anterior cruciate ligament injury susceptibility regardless of gender. Different injury risk models may be needed for men and women as other key risk factors are identified.

The geometry of the tibial plateau and its influence on the biomechanics of the tibiofemoral joint.

BACKGROUND: The geometry of the tibial plateau is complex and asymmetric. Previous research has characterized subject-to-subject differences in the tibial plateau geometry in the sagittal plane on the basis of a single parameter, the posterior slope. We hypothesized that (1) there are large subject-to-subject variations in terms of slopes, the depth of concavity of the medial plateau, and the extent of convexity of the lateral plateau; (2) medial tibial slope and lateral tibial slope are different within subjects; (3) there are sex-based differences in the slopes as well as concavities and convexities of the tibial plateau; and (4) age is not associated with any of the measured parameters. METHODS: The medial, lateral, and coronal slopes and the depth of the osseous portion of the tibial plateau were measured with use of sagittal and coronal magnetic resonance images that were made for thirty-three female and twenty-two male subjects, and differences between the sexes with respect to these four parameters were assessed. Within-subject differences between the medial and lateral tibial slopes also were assessed. Correlation tests were performed to examine the existence of a linear relationship between various slopes as well as between slopes and subject age. RESULTS: The range of subject-to-subject variations in the tibial slopes was substantive for males and females. However, the mean medial and lateral tibial slopes in female subjects were greater than those in male subjects (p < 0.05). In contrast, the mean coronal tibial slope in female subjects was less than that in male subjects (p < 0.05). The correlation between medial and lateral tibial slopes was poor. The within-subject difference between medial and lateral tibial slopes was significant (p < 0.05). No difference in medial tibial plateau depth was found between the sexes. The subchondral bone on the lateral part of the tibia, within the articulation region, was mostly flat. Age was not associated with the observed results. CONCLUSIONS: The geometry of the osseous portion of the tibial plateau is more robustly explained by three slopes and the depth of the medial tibial condyle.

The human anterior cruciate ligament: sex differences in ultrastructure and correlation with biomechanical properties.

The purpose of this study was to investigate the existence of sex-based differences in the ultrastructural characteristics of the human anterior cruciate ligament (ACL) as the underlying cause of differences in the structural and mechanical properties between sexes. The ACL of six male and six female cadaveric donors were randomly chosen from a pool of 10 male and 10 female ACLs that had previously been tested for their structural and mechanical properties. Eighteen tissue samples from the distal, proximal, and middle sections of the anteromedial and posterolateral bundles were analyzed by transmission electron microscopy. Female ACLs exhibited both lower fibril concentration and lower percent area occupied by collagen fibrils (p < 0.05) compared to males. There was also a difference in the fibril diameters (p < 0.05); donor age, height, body mass, and body mass index contributed significantly to this difference. In females, ACL stiffness and modulus of elasticity were highly correlated to fibril concentration (r = 0.96 and 0.97, respectively); in males ACL failure load and strength were highly correlated to percent area occupied by collagen (r = 0.96 and 0.96, respectively). These differences in ultrastructure may underlie differences in ACL properties between sexes.

Sex-based differences in the tensile properties of the human anterior cruciate ligament.

After immense amounts of research, the root cause for the significantly higher rates of anterior cruciate ligament (ACL) failure incidents in females as compared to males still remains unknown and the existing sex-based disparity has not diminished. To date, the possibility that the female ACL is mechanically weaker than the male ACL has not been directly investigated. Although it has been established in the literature that the female ACL is smaller in size, the differences in the structural and material properties of the ACL between sexes have not been studied. The aim of this cadaveric study was to determine if any sex-based differences in the tensile properties of the human ACL exist when considering age as well as ACL and body anthropometric measurements as covariates. Ten male and 10 female unpaired cadaveric knees (mean age 36.75 years) were used for this study. The geometry of the ACL (including length, cross-sectional area, and volume) was analyzed using a 3-D scanning system. The femur-ACL tibia complex was tested to failure along the longitudinal axis of the ligament in a tensile testing machine. The structural properties of the ACL as well as its mechanical properties were determined. Analysis of covariance was performed to assess the effect of sex on tensile properties. The female ACL was found to have a lower mechanical properties (8.3% lower strain at failure; 14.3% lower stress at failure, 9.43% lower strain energy density at failure, and most importantly, 22.49% lower modulus of elasticity) when considering age, ACL, and body anthropometric measurements as covariates.