An increase in Wharton's jelly membrane osteocompatibility by a genipin-cross-link.

Wharton's Jelly (WJ) has attracted significant interest in the field of tissue healing thanks to its biological properties, including antibacterial activity and immunomodulation. However, due to the fast degradation and poor mechanical behavior in biological environment, its application in bone regeneration is compromised. Here, we proposed to use genipin as an efficient cross-linking agent to significantly improve the elasticity and the enzymatical stability of the WJ matrix. The degree of cross-linking, linear elastic moduli, and collagenase resistance varied over a wide range depending on genipin concentration. Furthermore, our results highlighted that an increase in genipin concentration led to a decreased surface wettability, therefore impairing cell attachment and proliferation. The genipin cross-linking prevented rapid in vitro and in vivo degradation, but led to an adverse host reaction and calcification. When implanted in the parietal bone defect, a limited parietal bone regeneration to the dura was observed. We conclude that genipin-cross-linked WJ is a versatile medical device however, a careful selection is required with regards to the genipin concentration.

Discoidin Domain Receptor 1 Expression in Colon Cancer: Roles and Prognosis Impact.

Extracellular matrix components such as collagens are deposited within the tumor microenvironment at primary and metastatic sites and are recognized to be critical during tumor progression and metastasis development. This study aimed to evaluate the clinical and prognostic impact of Discoidin Domain Receptor 1 (DDR1) expression in colon cancers and its association with a particular molecular and/or morphological profile and to evaluate its potential role as a prognosis biomarker. Immunohistochemical expression of DDR1 was evaluated on 292 colonic adenocarcinomas. DDR1 was highly expressed in 240 (82.2%) adenocarcinomas. High DDR1 immunostaining score was significantly associated, on univariate analysis, with male sex, left tumor location, BRAF wild type status, KRAS mutated status, and Annexin A10 negativity. High DDR1 immunohistochemical expression was associated with shorter event free survival only. Laser capture microdissection analyses revealed that DDR1 mRNA expression was mainly attributable to adenocarcinoma compared to stromal cells. The impact of DDR1 expression on cell invasion was then evaluated by modified Boyden chamber assay using cell types with distinct mutational profiles. The invasion capacity of colon adenocarcinoma is supported by DDR1 expression. Thus, our results showed that DDR1 was highly expressed in most colon adenocarcinomas and appears as an indicator of worse event free survival.

Apelin expression deficiency in mice contributes to vascular stiffening by extracellular matrix remodeling of the aortic wall.

Numerous recent studies have shown that in the continuum of cardiovascular diseases, the measurement of arterial stiffness has powerful predictive value in cardiovascular risk and mortality and that this value is independent of other conventional risk factors, such as age, cholesterol levels, diabetes, smoking, or average blood pressure. Vascular stiffening is often the main cause of arterial hypertension (AHT), which is common in the presence of obesity. However, the mechanisms leading to vascular stiffening, as well as preventive factors, remain unclear. The aim of the present study was to investigate the consequences of apelin deficiency on the vascular stiffening and wall remodeling of aorta in mice. This factor freed by visceral adipose tissue, is known for its homeostasic role in lipid and vascular metabolisms, or again in inflammation. We compared the level of metabolic markers, inflammation of white adipose tissue (WAT), and aortic wall remodeling from functional and structural approaches in apelin-deficient and wild-type (WT) mice. Apelin-deficient mice were generated by knockout of the apelin gene (APL-KO). From 8 mice by groups, aortic stiffness was analyzed by pulse wave velocity measurements and by characterizations of collagen and elastic fibers. Mann-Whitney statistical test determined the significant data (p < 5%) between groups. The APL-KO mice developed inflammation, which was associated with significant remodeling of visceral WAT, such as neutrophil elastase and cathepsin S expressions. In vitro, cathepsin S activity was detected in conditioned medium prepared from adipose tissue of the APL-KO mice, and cathepsin S activity induced high fragmentations of elastic fiber of wild-type aorta, suggesting that the WAT secretome could play a major role in vascular stiffening. In vivo, remodeling of the extracellular matrix (ECM), such as collagen accumulation and elastolysis, was observed in the aortic walls of the APL-KO mice, with the latter associated with high cathepsin S activity. In addition, pulse wave velocity (PWV) and AHT were increased in the APL-KO mice. The latter could explain aortic wall remodeling in the APL-KO mice. The absence of apelin expression, particularly in WAT, modified the adipocyte secretome and facilitated remodeling of the ECM of the aortic wall. Thus, elastolysis of elastic fibers and collagen accumulation contributed to vascular stiffening and AHT. Therefore, apelin expression could be a major element to preserve vascular homeostasis.

Anti-Tumoral and Anti-Angiogenic Effects of Low-Diluted Phenacetinum on Melanoma.

Melanoma is the most aggressive form of skin cancer and the most rapidly expanding cancer in terms of worldwide incidence. If primary cutaneous melanoma is mostly treated with a curative wide local excision, malignant melanoma has a poor prognosis and needs other therapeutic approaches. Angiogenesis is a normal physiological process essential in growth and development, but it also plays a crucial role in crossing from benign to advanced state in cancer. In melanoma progression, angiogenesis is widely involved during the vertical growth phase. Currently, no anti-angiogenic agents are efficient on their own, and combination of treatments will probably be the key to success. In the past, phenacetin was used as an analgesic to relieve pain, causing side effects at large dose and tumor-inducing in humans and animals. By contrast, Phenacetinum low-dilution is often used in skin febrile exanthema, patches profusely scattered on limbs, headache, or flushed face without side effects. Herein are described the in vitro, in vivo, and ex vivo anti-angiogenic and anti-tumoral potentials of Phenacetinum low-dilution in a B16F1 tumor model and endothelial cells. We demonstrate that low-diluted Phenacetinum inhibits in vivo tumor growth and tumor vascularization and thus increases the survival time of B16F1 melanoma induced-C57BL/6 mice. Moreover, Phenacetinum modulates the lung metastasis in a B16F10 induced model. Ex vivo and in vitro, we evidence that low-diluted Phenacetinum inhibits the migration and the recruitment of endothelial cells and leads to an imbalance in the pro-tumoral macrophages and to a structural malformation of the vascular network. All together these results demonstrate highly hopeful anti-tumoral, anti-metastatic, and anti-angiogenic effects of Phenacetinum low-dilution on melanoma. Continued studies are needed to preclinically validate Phenacetinum low-dilution as a complementary or therapeutic strategy for melanoma treatment.

Expression of the Serrated Markers Annexin A10 or Gremlin1 in Colonic Adenocarcinomas: Morphology and Prognostic Values.

Describe clinical, histological and molecular charatcteristics and prognosis values of the serrated candidate markers AnnexinA10 and Gremlin1 in colon adenocarcinomas. Immunohistochemical expression of AnnexinA10 and Gremlin1 was evaluated on 346 colonic adenocarcinomas. Clinicopathological, molecular features and prognostic characteristics were then evaluated. A total of 40 colonic adenocarcinomas expressed AnnexinA10 (11.6%) and, 115 expressed Gremlin1 (40.4%). AnnexinA10 expression was significantly associated, on univariate analyses, with female gender (p = 0.03), right tumor location (p < 0.001), differentiation grade 3 (p < 0.001), serrated adenocarcinoma subtype (p < 0.001), serrated (p < 0.001), medullary (p = 0.005), and mucinous component (p = 0.004), cytoplasmic eosinophilia (p < 0.001), discernible nuclei (p = 0.001), preserved polarity (p < 0.001), lymphatic invasion (p = 0.01), BRAFV600E mutation (p < 0.001), MSI-H status (p < 0.001) and CIMP-H status (p = 0.019). Multivariate analyses revealed that mucinous component (p = 0.002), lymphatic invasion (p = 0.02) and BRAFV600E mutation (p < 0.001) were independently associated with AnnexinA10 expression. In addition, AnnexinA10 was an indicator of poorer overall survival (OS) in UICC stage IV adenocarcinomas (p = 0.01) only. Gremlin1 expression was neither associated with serrated adenocarcinoma subtype (p = 0.51) nor with AnnexinA10 expression (p = 0,31), but was significantly associated, in univariate analysis with male gender (p = 0.002), younger age (p = 0.002), left tumor location (p = 0.04), and MSS status (p = 0.03). Gremlin1 expression was associated with better OS only in UICC stage III colon adenocarcinomas (p = 0.006). Colon adenocarcinomas expressing AnnexinA10 have distinct clinico-pathological and molecular features. AnnexinA10 expression is an indicator of poorer OS in UICC stage IV patients. Gremlin1 expression is not associated with serrated adenocarcinomas subtype. Its expression was associated with better OS in UICC Stage III patients.

In vivo efficacy of endothelial growth medium stimulated mesenchymal stem cells derived from patients with critical limb ischemia.

BACKGROUND: Cell therapy has been proposed for patients with critical limb ischemia (CLI). Autologous bone marrow derived cells (BMCs) have been mostly used, mesenchymal stem cells (MSCs) being an alternative. The aim of this study was to characterize two types of MSCs and evaluate their efficacy. METHODS: MSCs were obtained from CLI-patients BMCs. Stimulated- (S-) MSCs were cultured in endothelial growth medium. Cells were characterized by the expression of cell surface markers, the relative expression of 6 genes, the secretion of 10 cytokines and the ability to form vessel-like structures. The cell proangiogenic properties was analysed in vivo, in a hindlimb ischemia model. Perfusion of lower limbs and functional tests were assessed for 28 days after cell infusion. Muscle histological analysis (neoangiogenesis, arteriogenesis and muscle repair) was performed. RESULTS: S-MSCs can be obtained from CLI-patients BMCs. They do not express endothelial specific markers but can be distinguished from MSCs by their secretome. S-MSCs have the ability to form tube-like structures and, in vivo, to induce blood flow recovery. No amputation was observed in S-MSCs treated mice. Functional tests showed improvement in treated groups with a superiority of MSCs and S-MSCs. In muscles, CD31+ and αSMA+ labelling were the highest in S-MSCs treated mice. S-MSCs induced the highest muscle repair. CONCLUSIONS: S-MSCs exert angiogenic potential probably mediated by a paracrine mechanism. Their administration is associated with flow recovery, limb salvage and muscle repair. The secretome from S-MSCs or secretome-derived products may have a strong potential in vessel regeneration and muscle repair. Trial registration NCT00533104.

Is the Mirror Image Method Really Useful in Tumor Tissue Bank Quality Control?

Oncology research projects are highly dependent on the quality of tumor samples stored in the biobank. Microscopic control is important to ensure the quality of the frozen sample (Does the sample correspond to tumor tissue? Does the sample contain a sufficient number of tumor cells for molecular analysis?). The aim of this study was to evaluate the value of the mirror image method in quality control of colonic adenocarcinoma samples stored in a tumor bank. Microscopic concordance for the differentiation grade, malignant and normal cell percentages, necrosis, mucinous component, and ulceration was assessed on 82 colon adenocarcinoma banked samples and their paired, formalin-fixed, paraffin-embedded mirror controls. Molecular concordance for KRAS status was evaluated in 76 of these 82 cases. Morphological correspondence between frozen and mirror samples was good for the mucinous component (intraclass correlation coefficient [ICC] = 0.81), moderate for differentiation (Cohen's kappa coefficient [k] = 0.67), fair for malignant cells (ICC = 0.44), and poor for ulceration (k = 0.08), normal tissue (ICC = 0.36), and necrosis (ICC = 0.13) percentages. Molecular correspondence for KRAS status was almost perfect (95% correspondence, k = 0.88) between frozen and mirror samples. In conclusion, the mirror sample method is not a good alternative for microscopic and molecular control of frozen colonic adenocarcinoma samples.

LRP1 expression in colon cancer predicts clinical outcome.

LRP1 (low-density lipoprotein receptor-related protein 1), a multifunctional endocytic receptor, has recently been identified as a hub within a biomarker network for multi-cancer clinical outcome prediction. As its role in colon cancer has not yet been characterized, we here investigate the relationship between LRP1 and outcome. MATERIALS AND METHODS: LRP1 mRNA expression was determined in colon adenocarcinoma and paired colon mucosa samples, as well as in stromal and tumor cells obtained after laser capture microdissection. Clinical potential was further investigated by immunohistochemistry in a population-based colon cancer series (n = 307). LRP1 methylation, mutation and miR-205 expression were evaluated and compared with LRP1 expression levels. RESULTS: LRP1 mRNA levels were significantly lower in colon adenocarcinoma cells compared with colon mucosa and stromal cells obtained after laser capture microdissection. Low LRP1 immunohistochemical expression in adenocarcinomas was associated with higher age, right-sided tumor, loss of CDX2 expression, Annexin A10 expression, CIMP-H, MSI-H and BRAFV600E mutation. Low LRP1 expression correlated with poor clinical outcome, especially in stage IV patients. While LRP1 expression was downregulated by LRP1 mutation, LRP1 promoter was never methylated. CONCLUSIONS: Loss of LRP1 expression is associated with worse colon cancer outcomes. Mechanistically, LRP1 mutation modulates LRP1 expression.

Matricellular TSP-1 as a target of interest for impeding melanoma spreading: towards a therapeutic use for TAX2 peptide.

Thrombospondin-1 (TSP-1) is a matricellular glycoprotein known for being highly expressed within a tumor microenvironment, where it promotes an aggressive phenotype particularly by interacting with the CD47 cell-surface receptor. While it originates from the stromal compartment in many malignancies, melanoma is an exception as invasive and metastatic melanoma cells overexpress TSP-1. We recently demonstrated that a new molecular agent that selectively prevents TSP-1 binding to CD47, called TAX2, exhibits anti-cancer properties when administered systemically by decreasing viable tumor tissue within subcutaneous B16 melanoma allografts. At the same time, emerging evidence was published suggesting a contribution of TSP-1 in melanoma metastatic dissemination and resistance to treatment. Through a comprehensive systems biology approach based on multiple genomics and proteomics databases analyses, we first identified a TSP-1-centered interaction network that is overexpressed in metastatic melanoma. Then, we investigated the effects of disrupting TSP-1:CD47 interaction in A375 human malignant melanoma xenografts. In this model, TAX2 systemic administrations induce tumor necrosis by decreasing intra-tumoral blood flow, while concomitantly making tumors less infiltrative. Besides, TAX2 treatment also drastically inhibits B16F10 murine melanoma cells metastatic dissemination and growth in a syngeneic experimental model of lung metastasis, as demonstrated by histopathological analyses as well as longitudinal and quantitative µCT follow-up of metastatic progression. Altogether, the results obtained by combining bioinformatics and preclinical studies strongly suggest that targeting TSP-1/CD47 axis may represent a valuable therapeutic alternative for hampering melanoma spreading.

Induction and regulation of murine emphysema by elastin peptides.

Emphysema is the major component of chronic obstructive pulmonary disease (COPD). During emphysema, elastin breakdown in the lung tissue originates from the release of large amounts of elastase by inflammatory cells. Elevated levels of elastin-derived peptides (EP) reflect massive pulmonary elastin breakdown in COPD patients. Only the EP containing the GXXPG conformational motif with a type VIII ß-turn are elastin receptor ligands inducing biological activities. In addition, the COOH-terminal glycine residue of the GXXPG motif seems a prerequisite to the biological activity. In this study, we endotracheally instilled C57BL/6J mice with GXXPG EP and/or COOH-terminal glycine deleted-EP whose sequences were designed by molecular dynamics and docking simulations. We investigated their effect on all criteria associated with the progression of murine emphysema. Bronchoalveolar lavages were recovered to analyze cell profiles by flow cytometry and lungs were prepared to allow morphological and histological analysis by immunostaining and confocal microscopy. We observed that exposure of mice to EP elicited hallmark features of emphysema with inflammatory cell accumulation associated with increased matrix metalloproteinases and desmosine expression and of remodeling of parenchymal tissue. We also identified an inactive COOH-terminal glycine deleted-EP that retains its binding-activity to EBP and that is able to inhibit the in vitro and in vivo activities of emphysema-inducing EP. This study demonstrates that EP are key actors in the development of emphysema and that they represent pharmacological targets for an alternative treatment of emphysema based on the identification of EP analogous antagonists by molecular modeling studies.