Susana Guerreiro

Pectin-Based Injectable Biomaterials for Bone Tissue Engineering

A variety of natural polymers and proteins are considered to be 3D cell culture structures able to mimic the extracellular matrix (ECM) to promote bone tissue regeneration. Pectin, a natural polysaccharide extracted from the plant cell walls and having a chemical structure similar to alginate, provides interesting properties as artificial ECM. In this work, for the first time, pectin, modified with an RGD-containing oligopeptide or not, is used as an ECM alternative to immobilize cells for bone tissue regeneration. The viability, metabolic activity, morphology, and osteogenic differentiation of immobilized MC3T3-E1 preosteoblats demonstrate the potential of this polysaccharide to keep immobilized cells viable and differentiating. Preosteoblasts immobilized in both types of pectin microspheres maintained a constant viability up to 29 days and were able to differentiate. The grafting of the RGD peptide on pectin backbone induced improved cell adhesion and proliferation within the microspheres. Furthermore, not only did cells grow inside but also they were able to spread out from the microspheres and to organize themselves in 3D structures producing a mineralized extracellular matrix. These promising results suggest that pectin can be proposed as an injectable cell vehicle for bone tissue regeneration.

Xanthohumol inhibits inflammatory factor production and angiogenesis in breast cancer xenografts

Xanthohumol (XN), a natural polyphenol present in beer, is known to exert anti‐cancer effects. However, its precise mechanisms are not yet clearly defined. The aim of this study was to investigate the effect of oral administration of XN in breast cancer xenografts in nude mice. Proliferation and apoptosis were first examined in MCF7 cell cultures after incubation with XN by trypan blue exclusion assay, [3H]‐thymidine incorporation, KI67 immunostaining and TUNEL. Morphological and histological characteristics of tumours from XN‐treated or control (vehicle‐treated) mice were compared. Immunohistochemistry for proliferative, inflammatory and endothelial cell markers was performed and activation of nuclear factor kappa B (NFκB) pathway was assessed by ELISA. In vitro MCF7 cell proliferation decreased in a dose‐dependent manner. Oral administration of XN to nude mice inoculated with MCF7 cells resulted in central necrosis within tumours, reduced inflammatory cell number, focal proliferation areas, increased percentage of apoptotic cells and decreased microvessel density. Anti‐angiogenic effects of XN were further confirmed by immunoblotting for factor VIII expression in XN‐treated tumours as compared to controls. Decreased immunostaining for NFκB, phosphorylated‐inhibitor of kappa B and interleukin‐1β were also observed as well as a significant decrease in NFκB activity to 60% of control values. These novel findings indicate that XN is able to target both breast cancer and host cells, namely inflammatory and endothelial cells, suggesting its potential use as a double‐edge anti‐cancer agent. J. Cell. Biochem. 104: 1699–1707, 2008.

Red wine increases adipose tissue aromatase expression and regulates body weight and adipocyte size

OBJECTIVE Obesity is an important component of the metabolic syndrome in constituting a risk factor for cardiovascular disease, diabetes, and cancer. Estrogens influence lipid accumulation in adipocytes, acting indirectly or directly on adipose tissue. In this study we aimed to investigate the influence of red wine ingestion on the expression of aromatase (estrogen synthase) in adipose tissue. METHODS Red wine or ethanol solution, in the concentration found in red wine, was provided to Wistar rats as the sole drinking fluid for 8 wk. Food and drink intakes and body weight were monitored throughout treatment and adipocyte size and aromatase expression in the adipose tissue were determined at the end of the experimental period. RESULTS Red wine and ethanol increased aromatase expression in the adipose tissue and red wine decreased adipocyte size (P < 0.05). In addition, animals treated with red wine or ethanol had significantly lower weight gain than controls, despite a similar energy intake. CONCLUSION Thus, the ingestion of red wine may alter the production of estrogens by adipose tissue, body weight gain, and adipocyte size. Some of these red wine effects are attributable to ethanol. This relation among estrogen availability, adipocyte biology, and weight gain is most interesting and deserves further study because it may lead to new strategies to reduce metabolic syndrome incidence.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 +/- 3.43 nmol p-nitrophenol.mg protein-1.min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). beta-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Elucidating progesterone effects in breast cancer: Cross talk with PDGF signaling pathway in smooth muscle cell

Several studies indicate that progesterone exerts relevant effects in breast tissue. However, the exact role of this steroid in breast cancer development and progression has not been elucidated. Here, we show that platelet‐derived growth factor (PDGF)‐A is one of the progesterone target genes on breast cancer MCF7 and T47D cells. A paracrine role for PDGF‐A was investigated, since its receptor expression was down‐regulated from breast cancer cells. Progesterone increased PDGF‐A protein release as evaluated by Western blotting and ELISA. Medium from Progesterone‐treated MCF7 cells resulted in phosphorylation of smooth muscle cells PDGF receptor α. This effect was not observed after treatment with PDGF inhibitor. MCF7 cells‐secreted PDGF‐A was able to increase smooth muscle cell viability and proliferation and decrease apoptosis, effects that were prevented by the use of a PDGF‐A neutralizing antibody. Notably, cell invasion was not influenced by PDGF‐A secreted by MCF7 cells. Our results elucidated for the first time the cross talk between progesterone and PDGF signaling pathway. The fact that MCF7‐secreted PDGF elicited crucial roles in vascular wall smooth muscle cells, suggested a paracrine pathway for progesterone. Targeting these progesterone‐induced processes may provide novel therapeutic strategies for hormone‐dependent human breast cancer. J. Cell. Biochem. 100: 174–183, 2007.

Implanted neonatal human dermal fibroblasts influence the recruitment of endothelial cells in mice.

The vascularization of new tissue within a reasonable time is a crucial prerequisite for the success of different cell- and material-based strategies. Considering that angiogenesis is a multi-step process involving humoral and cellular regulatory components, only in vivo assays provide the adequate information about vessel formation and the recruitment of endothelial cells. The present study aimed to investigate if neonatal human dermal fibroblasts could influence in vivo neovascularization. Results obtained showed that fibroblasts were able to recruit endothelial cells to vascularize the implanted matrix, which was further colonized by murine functional blood vessels after one week. The vessels exhibited higher levels of hemoglobin, compared with the control matrix, implanted without fibroblasts, in which no vessel formation could be observed. No significant differences were detected in systemic inflammation. The presence of vessels originated from the host vasculature suggested that host vascular response was involved, which constitutes a fundamental aspect in the process of neovascularization. Fibroblasts implanted within matrigel increased the presence of endothelial cells with positive staining for CD31 and for CD34 and the production of collagen influencing the angiogenic process and promoting the formation of microvessels. New strategies in tissue engineering could be delineated with improved angiogenesis using neonatal fibroblasts.

New insights regarding yeast survival following exposure to liposomal amphotericin B

ABSTRACT In vitro resistance to amphotericin B is an extremely rare event among pathogenic yeasts. However, in vivo response is sometimes reduced, resulting in an unfavorable outcome. Such adverse outcomes might be related to subfungicidal plasma concentrations. We aimed to clarify the mechanisms of liposomal amphotericin B (AMB-L; AmBisome)-induced lesions and the mechanisms responsible for yeast cell recovery following exposure at plasma concentrations. The physiological statuses developing following exposure to AMB-L at simulated plasma concentrations (20 to 0.1 mg/liter) and at a constant concentration (3 mg/liter) were assessed in a 24-h time course assay. Time-kill experiments also were carried out under the same AMB-L treatment conditions. Our results suggest that yeast cells develop compensatory responses related to membrane polarization, metabolic activity, and reactive oxygen species (ROS) production after exposure to high plasma concentrations (20 to 5 mg/liter) during the first 6 h; in the remaining 18 h, when exposed to lower concentrations, cells reveal almost full recovery with no evidence of fungicidal activity. In contrast, whenever cells are exposed to a constant concentration above the MIC, despite initially exhibiting compensatory stress responses, soon afterwards they exhibit membrane depolarization, a decrease of metabolic activity, increasing ROS production, and lastly, programmed cell death and necrosis, resulting in succumbing to AMB-L fungicidal effects. This study may represent a step forward in the support of AMB-L use for clinical treatment of invasive fungal infections, since it demonstrates the importance of maintaining levels of AMB-L above the MIC in plasma and tissues to ensure it produces its fungicidal effects.

Telomerase and N-Cadherin Differential Importance in Adrenocortical Cancers and Adenomas†

Adrenocortical carcinomas (ACC) are most frequently highly aggressive tumors. We assessed the telomerase reverse transcriptase (TERT) and N‐cadherin role in the biology of ACC and their potential utility as molecular biomarkers, in different types of tumoral adrenocortical tissue. A total of 48 adrenal cortex samples (39 tumoral and 9 normal adrenal glands) were studied. TERT promoter mutations were searched by PCR and Sanger sequencing in two hotspots positions (−124 and −146). Also, telomerase and N‐cadherin expression were evaluated by immunohistochemistry. TERT promoter mutations were not detected in any of the samples either malignant or benign. Telomerase nuclear expression was present in 26.6% of ACC and in 45.5% of non‐functioning adenomas. It was absent in benign Cushings lesions and in normal adrenal glands. Contrarily, N‐cadherin was always expressed in the cellular membranes of benign adenomas or normal adrenals but no expression was detected in the majority of ACC. Nuclear telomerase and membrane N‐cadherin expression were positively correlated in ACCs. We conclude that in ACC, the loss of N‐cadherin is a frequent phenomenon while the existence of TERT promoter mutations is not and nuclear telomerase expression is present in only a minority of cases. Since the loss of N‐cadherin expression was identified in both high and low proliferative ACC, this marker should be considered important for diagnostic application. Our study also suggests the existence of a TERT non‐canonical function in cell adhesion. J. Cell. Biochem. 118: 2064–2071, 2017.

Xanthohumol and 8-prenylnaringenin reduce type 2 diabetes–associated oxidative stress by downregulating galectin-3

Background: Galectin-3 (Gal3) expression is associated with accumulation of Advanced Glycation End products (AGE), a common feature in diabetes mellitus (DM). The role of Gal3 in oxidative stress is, however, controversial, being considered in the literature to play either a protective role or exacerbating disease. Methods: Herein, we examined the interplay between Gal3 and oxidative stress in a high-fat diet -induced type 2 DMC57Bl/6 mice model. Because natural polyphenols are known to play antioxidant and anti-inflammatory roles and to modulate metabolic activity, we further evaluated the effect of xanthohumol and 8-prenylnaringenin polyphenols in this crosstalk. Results: Gal3 expression was accompanied by 3-nitrotyrosine and AGE production in liver and kidney of diabetic mice compared to healthy animals (fed with standard diet). Oral supplementation with polyphenols decreased the levels of these oxidative biomarkers as evaluated by immunohistochemistry and western blotting. Interestingly, blocking Gal3 by incubating human microvascular endothelial cells with modified citrus pectin increased 3-nitrotyrosine protein expression. Conclusions: These findings imply that Gal3 overexpression is probably controlling oxidative stress in endothelial cells. In conclusion, our results indicate that supplementation with 8-prenylnaringenin or xanthohumol reverses diabetes-associated oxidation in liver and kidney, and consequently decreases this diabetic biomarker that predispose to cardiovascular complications.

Melanoma and obesity: Should antioxidant vitamins be addressed?

Melanoma is an aggressive form of skin cancer refractory to conventional therapies. Obesity has reached epidemic dimensions acting as a risk factor for several cancer types, such as melanoma. Several reactive species of oxygen are also involved in melanoma initiation and progression. Low levels of antioxidant content and/or activity in lightly pigmented cells could expose them to an extremely oxidative environment and rise the susceptibility to oxidative damage and consequently loss of cell homeostasis. Despite the knowledge about melanoma biology, pathogenesis and developed therapies, is extremely important to understand the antioxidant modulation of melanoma under an environment of obesity, especially the effect of some natural compounds of the diet, such as antioxidant vitamins A, C and E and selenium in order to establish alternatives to conventional therapies, which are known to be ineffective against melanoma.