Background and objectives: The incidence of cutaneous melanoma has been increasing. Melanoma is a... more Background and objectives: The incidence of cutaneous melanoma has been increasing. Melanoma is an aggressive form of skin cancer irresponsive to radiation and chemotherapy, rendering this cancer a disease with poor prognosis: In order to surpass some of the limitations addressed to melanoma treatment, alternatives like vitamins have been investigated. In the present study, we address this relationship and investigate the possible role of vitamin A. Materials and Methods: We perform a co-culture assay using a macrophage cell model and RAW 264.7 from mouse, and also a murine melanoma cell line B16-F10. Macrophages were stimulated with both Escherichia coli lipopolysaccharides (LPS) as control, and also with LPS plus vitamin A. Results: Using B16-F10 and RAW 264.7 cell lines, we were able to demonstrate that low concentrations of vitamin A increase cytotoxic activity of macrophages, whereas higher concentrations have the opposite effect. Conclusion: These findings can constitute a new...
A marked decrease in human cancers, including breast cancer, bone cancer, and cervical cancer, ha... more A marked decrease in human cancers, including breast cancer, bone cancer, and cervical cancer, has been linked to the consumption of vegetable and fruit, and the corresponding chemoprotective effect has been associated with the presence of several active molecules, such as kaempferol. Kaempferol is a major flavonoid aglycone found in many natural products, such as beans, bee pollen, broccoli, cabbage, capers, cauliflower, chia seeds, chives, cumin, moringa leaves, endive, fennel, and garlic. Kaempferol displays several pharmacological properties, among them antimicrobial, anti-inflammatory, antioxidant, antitumor, cardioprotective, neuroprotective, and antidiabetic activities, and is being applied in cancer chemotherapy. Specifically, kaempferol-rich food has been linked to a decrease in the risk of developing some types of cancers, including skin, liver, and colon. The mechanisms of action include apoptosis, cell cycle arrest at the G2/M phase, downregulation of epithelial-mesenchy...
Adrenocortical tumors (ACT) are common adrenal tumors. The majority of ACTs are non-functioning a... more Adrenocortical tumors (ACT) are common adrenal tumors. The majority of ACTs are non-functioning and benign, while adrenocortical carcinomas (ACC) are rare, usually very aggressive and often metastasized when first diagnosed. Our aim was to assess whether blood and lymph vessel density within ACTs correlate with the malignancy character or tumor functionality. For that, the microvascular distribution was evaluated by immunohistochemistry staining with D2-40 antibody, for lymph vessels and CD-31 antibody, for blood vessels, in ACCs (n = 15), adenomas with Cushing syndrome (n = 9) and non-functioning adenomas (n = 10). The percentage of stained area was quantified by computerized morphometric analysis. D2-40 expression was significantly lower in ACC as compared to adenomas with Cushing syndrome (p < 0.01) and correlated positively with the expression of the steroidogenic acute regulatory protein (StAR) (R(2) = 0.553, p < 0.001). CD31 expression was found to be significantly highe...
Adrenocortical carcinomas (ACC) are most frequently highly aggressive tumors. We assessed the tel... more 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 Cushing's 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....
Melanoma is an aggressive form of skin cancer refractory to conventional therapies. Obesity has r... more 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.
Vascularization is a major challenge in tissue engineering. Different strategies exist, but cell-... more Vascularization is a major challenge in tissue engineering. Different strategies exist, but cell- based approaches have emerged as a promising therapy to achieve successful vascularization. The use of en- dothelial cells, namely outgrowth endothelial cells (OECs), has been extensively investigated to engineer vas- cularized tissues. In previous works, it has already been demonstrated that co-cultures of endothelial cells with supporting cells, like osteoblasts, mesenchymal stem cells, fibroblasts, or smooth muscle cells, result in a beneficial effect concerning capillary-like structures formation. Therefore, the main goals of the present work are (a) to determine whether fibroblasts improve neovascularization over time, evaluating their potential to stabilize capillary-like structures formed by OECs, that have been shown to exhibit a higher proliferative ca- pacity and increased regeneration potential – and (b) to characterize the extracellular matrix produced in these co-culture sy...
In vitroresistance to amphotericin B is an extremely rare event among pathogenic yeasts. However,... more In vitroresistance to amphotericin B is an extremely rare event among pathogenic yeasts. However,in vivoresponse 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;...
Diabetic chronic foot ulceration represents a major medical, social, and economic problem. Key fe... more Diabetic chronic foot ulceration represents a major medical, social, and economic problem. Key features of the non-healing ulcer in diabetic patients are persistent inflammation and impaired blood vessel regeneration (angiogenesis). Angiogenesis is a complex physiological process that requires normal functions and properly orchestrated interaction between fibroblasts and endothelial cells (EC). In diabetic chronic ulcer, those cellular activities and functions are impaired. The fibroblasts-endothelial cell interactions in impaired angiogenesis are poorly understood, primarily due to the lack of an in vitro wound-healing angiogenesis model. In order to improve the knowledge of this issue in the present work human outgrowth endothelial cells (OECs) and mature ECs were co-cultured with different types of human fibroblasts. Experimentally, OECs were isolated from human umbilical cord blood samples and characterized by immunofluorescence, western blot and imaging flow cytometry. Two type...
ABSTRACT Introduction: Vascularizationconstitutesachallengeintissueengineer- ing. Cell-based appr... more ABSTRACT Introduction: Vascularizationconstitutesachallengeintissueengineer- ing. Cell-based approaches, namely co-culture systems of endothelial cells (ECs) with supporting cells, like fibroblasts, have emerged as a promising therapy to achieve successful vascularization. In this work, it was hypothesized that mature and outgrowth endothelial cells (OECs) respond differently when co-cultured with different types of human fi- broblasts, in terms of formation of capillary-like structures. Materialsandmethods: OECswereisolatedfromhumanumbilicalcord blood samples and characterized by immunofluorescence and flow cytometry. Two types of human dermal fibroblasts were used (neonatal human foreskin fibroblasts, HFF-1, and juvenile human dermal fibro- blasts, HDF), being characterized in terms of podoplanin (PDPN) and transglutaminase-2 (TG2) expression. Co-culture systems were estab- lished using either human umbilical vein ECs (HUVECs) or OECs with HFF-1 or HDF. The formation of capillary-like structures was assessed by immunocytochemistry against CD31 and vWF proteins, after 14 days in culture. Parameters such as the number, length and thick- ness of structures were evaluated. The presence of extracellular matrix (ECM) components, including laminin, fibronectin, collagen types I and IV, was evaluated by immunofluorescence. Results: OECs expressed CD31, VE-cadherin and vWF, as well as VEG- FR2 and CD34. HFF-1 exhibited a higher expression of TG2 than that observed for HDF, while HDF expressed higher amounts of PDPN. Fi- broblasts influenced the formation of capillary-like structures by ECs, which were only observed in co-cultures with HDF and not with HFF-1. In terms of ECM synthesis, HUVECs were found to secrete collagen type IV, fibronectin and laminin to the extracellular media, whereas in OECs these proteins were only detected intracellularly. HDF were able to secrete all the investigated components, primarily collagen I, which was not secreted by HUVECs, OECs and HFF-1. In co-cultures systems, it was observed that all investigated ECM components were present, with collagen types I and IV being mainly expressed in sites of capil- lary-like structures formation. Discussion and conclusions: HDF seems to be a preferential cell source for enhancing vascularization, both in HUVECs and OECs. Given the already described advantages of OECs, these findings open a new field of research regarding the use of specific fibroblast populations co-cul- tured with OECs, as efficient partners for vascular development with tissue regeneration purposes. Acknowledgments: The authors acknowledge Prof. Carla Ramalho from Serviço de Ginecologia e Obstetr�ıcia at Hospital de São João (Porto, Por- tugal) for providing umbilical cord blood samples and Prof. James Kirk- patrick from REPAIR-lab (University of Mainz, Germany) for providing HUVECs and HDF cells. This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia (PEst-OE/SAU/UI0038/2011). Disclosures: The authors have nothing to disclose.
Background and objectives: The incidence of cutaneous melanoma has been increasing. Melanoma is a... more Background and objectives: The incidence of cutaneous melanoma has been increasing. Melanoma is an aggressive form of skin cancer irresponsive to radiation and chemotherapy, rendering this cancer a disease with poor prognosis: In order to surpass some of the limitations addressed to melanoma treatment, alternatives like vitamins have been investigated. In the present study, we address this relationship and investigate the possible role of vitamin A. Materials and Methods: We perform a co-culture assay using a macrophage cell model and RAW 264.7 from mouse, and also a murine melanoma cell line B16-F10. Macrophages were stimulated with both Escherichia coli lipopolysaccharides (LPS) as control, and also with LPS plus vitamin A. Results: Using B16-F10 and RAW 264.7 cell lines, we were able to demonstrate that low concentrations of vitamin A increase cytotoxic activity of macrophages, whereas higher concentrations have the opposite effect. Conclusion: These findings can constitute a new...
A marked decrease in human cancers, including breast cancer, bone cancer, and cervical cancer, ha... more A marked decrease in human cancers, including breast cancer, bone cancer, and cervical cancer, has been linked to the consumption of vegetable and fruit, and the corresponding chemoprotective effect has been associated with the presence of several active molecules, such as kaempferol. Kaempferol is a major flavonoid aglycone found in many natural products, such as beans, bee pollen, broccoli, cabbage, capers, cauliflower, chia seeds, chives, cumin, moringa leaves, endive, fennel, and garlic. Kaempferol displays several pharmacological properties, among them antimicrobial, anti-inflammatory, antioxidant, antitumor, cardioprotective, neuroprotective, and antidiabetic activities, and is being applied in cancer chemotherapy. Specifically, kaempferol-rich food has been linked to a decrease in the risk of developing some types of cancers, including skin, liver, and colon. The mechanisms of action include apoptosis, cell cycle arrest at the G2/M phase, downregulation of epithelial-mesenchy...
Adrenocortical tumors (ACT) are common adrenal tumors. The majority of ACTs are non-functioning a... more Adrenocortical tumors (ACT) are common adrenal tumors. The majority of ACTs are non-functioning and benign, while adrenocortical carcinomas (ACC) are rare, usually very aggressive and often metastasized when first diagnosed. Our aim was to assess whether blood and lymph vessel density within ACTs correlate with the malignancy character or tumor functionality. For that, the microvascular distribution was evaluated by immunohistochemistry staining with D2-40 antibody, for lymph vessels and CD-31 antibody, for blood vessels, in ACCs (n = 15), adenomas with Cushing syndrome (n = 9) and non-functioning adenomas (n = 10). The percentage of stained area was quantified by computerized morphometric analysis. D2-40 expression was significantly lower in ACC as compared to adenomas with Cushing syndrome (p < 0.01) and correlated positively with the expression of the steroidogenic acute regulatory protein (StAR) (R(2) = 0.553, p < 0.001). CD31 expression was found to be significantly highe...
Adrenocortical carcinomas (ACC) are most frequently highly aggressive tumors. We assessed the tel... more 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 Cushing's 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....
Melanoma is an aggressive form of skin cancer refractory to conventional therapies. Obesity has r... more 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.
Vascularization is a major challenge in tissue engineering. Different strategies exist, but cell-... more Vascularization is a major challenge in tissue engineering. Different strategies exist, but cell- based approaches have emerged as a promising therapy to achieve successful vascularization. The use of en- dothelial cells, namely outgrowth endothelial cells (OECs), has been extensively investigated to engineer vas- cularized tissues. In previous works, it has already been demonstrated that co-cultures of endothelial cells with supporting cells, like osteoblasts, mesenchymal stem cells, fibroblasts, or smooth muscle cells, result in a beneficial effect concerning capillary-like structures formation. Therefore, the main goals of the present work are (a) to determine whether fibroblasts improve neovascularization over time, evaluating their potential to stabilize capillary-like structures formed by OECs, that have been shown to exhibit a higher proliferative ca- pacity and increased regeneration potential – and (b) to characterize the extracellular matrix produced in these co-culture sy...
In vitroresistance to amphotericin B is an extremely rare event among pathogenic yeasts. However,... more In vitroresistance to amphotericin B is an extremely rare event among pathogenic yeasts. However,in vivoresponse 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;...
Diabetic chronic foot ulceration represents a major medical, social, and economic problem. Key fe... more Diabetic chronic foot ulceration represents a major medical, social, and economic problem. Key features of the non-healing ulcer in diabetic patients are persistent inflammation and impaired blood vessel regeneration (angiogenesis). Angiogenesis is a complex physiological process that requires normal functions and properly orchestrated interaction between fibroblasts and endothelial cells (EC). In diabetic chronic ulcer, those cellular activities and functions are impaired. The fibroblasts-endothelial cell interactions in impaired angiogenesis are poorly understood, primarily due to the lack of an in vitro wound-healing angiogenesis model. In order to improve the knowledge of this issue in the present work human outgrowth endothelial cells (OECs) and mature ECs were co-cultured with different types of human fibroblasts. Experimentally, OECs were isolated from human umbilical cord blood samples and characterized by immunofluorescence, western blot and imaging flow cytometry. Two type...
ABSTRACT Introduction: Vascularizationconstitutesachallengeintissueengineer- ing. Cell-based appr... more ABSTRACT Introduction: Vascularizationconstitutesachallengeintissueengineer- ing. Cell-based approaches, namely co-culture systems of endothelial cells (ECs) with supporting cells, like fibroblasts, have emerged as a promising therapy to achieve successful vascularization. In this work, it was hypothesized that mature and outgrowth endothelial cells (OECs) respond differently when co-cultured with different types of human fi- broblasts, in terms of formation of capillary-like structures. Materialsandmethods: OECswereisolatedfromhumanumbilicalcord blood samples and characterized by immunofluorescence and flow cytometry. Two types of human dermal fibroblasts were used (neonatal human foreskin fibroblasts, HFF-1, and juvenile human dermal fibro- blasts, HDF), being characterized in terms of podoplanin (PDPN) and transglutaminase-2 (TG2) expression. Co-culture systems were estab- lished using either human umbilical vein ECs (HUVECs) or OECs with HFF-1 or HDF. The formation of capillary-like structures was assessed by immunocytochemistry against CD31 and vWF proteins, after 14 days in culture. Parameters such as the number, length and thick- ness of structures were evaluated. The presence of extracellular matrix (ECM) components, including laminin, fibronectin, collagen types I and IV, was evaluated by immunofluorescence. Results: OECs expressed CD31, VE-cadherin and vWF, as well as VEG- FR2 and CD34. HFF-1 exhibited a higher expression of TG2 than that observed for HDF, while HDF expressed higher amounts of PDPN. Fi- broblasts influenced the formation of capillary-like structures by ECs, which were only observed in co-cultures with HDF and not with HFF-1. In terms of ECM synthesis, HUVECs were found to secrete collagen type IV, fibronectin and laminin to the extracellular media, whereas in OECs these proteins were only detected intracellularly. HDF were able to secrete all the investigated components, primarily collagen I, which was not secreted by HUVECs, OECs and HFF-1. In co-cultures systems, it was observed that all investigated ECM components were present, with collagen types I and IV being mainly expressed in sites of capil- lary-like structures formation. Discussion and conclusions: HDF seems to be a preferential cell source for enhancing vascularization, both in HUVECs and OECs. Given the already described advantages of OECs, these findings open a new field of research regarding the use of specific fibroblast populations co-cul- tured with OECs, as efficient partners for vascular development with tissue regeneration purposes. Acknowledgments: The authors acknowledge Prof. Carla Ramalho from Serviço de Ginecologia e Obstetr�ıcia at Hospital de São João (Porto, Por- tugal) for providing umbilical cord blood samples and Prof. James Kirk- patrick from REPAIR-lab (University of Mainz, Germany) for providing HUVECs and HDF cells. This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia (PEst-OE/SAU/UI0038/2011). Disclosures: The authors have nothing to disclose.
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