Experimental diabetes induces structural, inflammatory and vascular changes of Achilles tendons.

This study aims to demonstrate how the state of chronic hyperglycemia from experimental Diabetes Mellitus can influence the homeostatic imbalance of tendons and, consequently, lead to the characteristics of tendinopathy. Twenty animals were randomly divided into two experimental groups: control group, consisting of healthy rats and diabetic group constituted by rats induced to Diabetes Mellitus I. After twenty-four days of the induction of Diabetes type I, the Achilles tendon were removed for morphological evaluation, cellularity, number and cross-sectional area of blood vessel, immunohistochemistry for Collagen type I, VEGF and NF-κB nuclear localization sequence (NLS) and nitrate and nitrite level. The Achilles tendon thickness (µm/100g) of diabetic animals was significantly increased and, similarly, an increase was observed in the density of fibrocytes and mast cells in the tendons of the diabetic group. The average number of blood vessels per field, in peritendinous tissue, was statistically higher in the diabetic group 3.39 (2.98) vessels/field when compared to the control group 0.89 (1.68) vessels/field p = 0.001 and in the intratendinous region, it was observed that blood vessels were extremely rare in the control group 0.035 (0.18) vessels/field and were often present in the tendons of the diabetic group 0.89 (0.99) vessels/field. The immunohistochemistry analysis identified higher density of type 1 collagen and increased expression of VEGF as well as increased immunostaining for NFκB p50 NLS in the nucleus in Achilles tendon of the diabetic group when compared to the control group. Higher levels of nitrite/nitrate were observed in the experimental group induced to diabetes. We conclude that experimental DM induces notable structural, inflammatory and vascular changes in the Achilles tendon which are compatible with the process of chronic tendinopathy

Glycemic Expression ml/dL at three evaluations.

<p>CG – Control group and DG – Diabetic group. *- <i>p</i> = 0.001. <i>Student t</i> test to determine statistical differences of CG and DG.</p

Level of nitric oxide product – nitrite/nitrate (NOx) in the Achilles tendon in the Control Group – CG and the Diabetic Group – DG.

<p>* – p<0.05 Student-t Test for independent samples between the groups studied.</p

A greater degree of Immunomarking of NF-κB nuclear expression was observed in the diabetic group.

<p><b>B;</b> when compared to the control group – <b>A</b>. Highlights of immunomarking of NF-κB nuclear expression in the diabetic group – <b>C</b>. *-<i>p</i> = 0,001 Student-t Test for independent samples between the groups studied.</p

Immunomarking the density of Type I Collagen and VEGF. Density of type I Collagen in the Achilles tendon in the Control Group.

<p><b>A;</b> Increased of the density of type I Collagen in the Achilles tendon in the Diabetic Group and disorganization in the Extracellular Matrix –<b>B</b>; The absence of VEGF expression in the control group –<b>D</b>; Expression of VEGF in the diabetic group –<b>E</b>; Negative Controls –<b>C</b> and <b>F</b>. 400X. Relative stained area was quantified using National Institutes of Health ImageJ software. The bar graph summarizes average values of each group for type 1 collagen –<b>G</b> and VEGF –<b>H</b>. The values are expressed as means and Standard Deviation of the Mean (SEM). * –p<0.05 Student-T Test for independent samples showing statistical differences between the groups studied.</p

Thickness of the Achilles tendons in the Control Group – CG and the Diabetic Group – DG with results are exhibited in terms of thickness value/100 g of the animal's weight.

<p><b>A</b>. Quantification of cell density – Fibrocytes, fibroblast and Total Cells in the Control Group – CG and the Diabetic Group – <b>B</b>. H.E sections (10 most proximal X400 viewing fields).The values are expressed as means and standard errors. * – p<0.05 Student-t Test for independent samples showing statistical differences between the groups studied.</p

Density of mast cells in the Achilles tendon in the Control Group – CG and the Diabetic Group – DG.

<p><b>Normal tendon. A;</b> Tendon of the diabetic group with arrows pointing to mast cells –<b>B</b>. * –p<0.05 Student-t Test for independent samples between the groups studied –400X – Toluidine blue.</p

Signaling pathways in diabetic tendinopathy.

<p>Initially, the state of chronic hyperglycemia results in the activation of NF-κB, which leads to the increased expression of target genes such as VEGF and NOSi. These, in turn, result in increased NO and vascularization. This increased vascularization associated with cell proliferation and possible migration causes hypercellularity and the rise of disorganized deposition of type 1 collagen. Mast cells increased as a result of an inflammatory process of the tendon denoted by the increase of nitrite and nitrate indicative of increased NO also contribute to the increase of VEGF and therefore to increased vascularity.</p

Lycopene and beta-carotene induce growth inhibition and proapoptotic effects on ACTH-secreting pituitary adenoma cells.

Pituitary adenomas comprise approximately 10-15% of intracranial tumors and result in morbidity associated with altered hormonal patterns, therapy and compression of adjacent sella turcica structures. The use of functional foods containing carotenoids contributes to reduce the risk of chronic diseases such as cancer and vascular disorders. In this study, we evaluated the influence of different concentrations of beta-carotene and lycopene on cell viability, colony formation, cell cycle, apoptosis, hormone secretion, intercellular communication and expression of connexin 43, Skp2 and p27(kip1) in ACTH-secreting pituitary adenoma cells, the AtT20 cells, incubated for 48 and 96 h with these carotenoids. We observed a decrease in cell viability caused by the lycopene and beta-carotene treatments; in these conditions, the clonogenic ability of the cells was also significantly decreased. Cell cycle analysis revealed that beta-carotene induced an increase of the cells in S and G2/M phases; furthermore, lycopene increased the proportion of these cells in G0/G1 while decreasing the S and G2/M phases. Also, carotenoids induced apoptosis after 96 h. Lycopene and beta-carotene decreased the secretion of ACTH in AtT20 cells in a dose-dependent manner. Carotenoids blocked the gap junction intercellular communication. In addition, the treatments increased the expression of phosphorylated connexin43. Finally, we also demonstrate decreased expression of S-phase kinase-associated protein 2 (Skp2) and increased expression of p27(kip1) in carotenoid-treated cells. These results show that lycopene and beta-carotene were able to negatively modulate events related to the malignant phenotype of AtT-20 cells, through a mechanism that could involve changes in the expression of connexin 43, Skp2 and p27(kip1); and suggest that these compounds might provide a novel pharmacological approach to the treatment of Cushing's disease

Aberrant levels of Wnt/β-catenin pathway components in a rat model of endometriosis

Endometriosis is a benign gynecological disease affecting approximately 10-15% of women of reproductive age and 25-50% of all infertile women. It is characterized by the presence of glands and/or endometrial stroma outside the uterine cavity. Angiogenesis is a crucial process for the development and maintenance of endometriotic lesions. The Wnt/βcatenin pathway is a major promoter of angiogenesis in both physiological and pathological conditions. In the present study, we evaluated the expression of molecules related to the Wnt/β-catenin pathway in a rat model of peritoneal endometriosis. mRNA analyses showed significantly increased expression of Wnt4 and Wnt7b and decreased expression of Gsk3beta and E-cadherin in endometriotic lesions. However, there were no differences in β-catenin and Fzd2 mRNA expression. In addition, we observed a significant increase of nuclear β-catenin in endometriotic lesions, a hallmark of Wnt/ β -catenin pathway activation. Stromal β-catenin staining was found in 45.4% of endometrial tissues and 77.8% of endometriotic lesions. β-catenin nuclear localization was found in 18.2% of the endometrial tissues and 33.3% of endometriotic lesions. Finally, the expression of galectin-3, a regulator of this pathway, was increased in endometriosis. In summary, this pattern of Wnt/βcatenin components expression suggests an increased activity of this pathway in endometriosis