Isolation methods and culture conditions of human umbilical vein endothelial cells from Malaysian women

Human umbilical vein endothelial cell (HUVEC) isolated from umbilical cord is widely used as endothelial cell model. However, HUVEC has been characteristically hard to maintain and showed molecular heterogeneity depending on the umbilical cord donors. Commercial HUVEC is commonly derived from European and Caucasian population which have different molecular characteristics from Asian women. This study aimed to optimize the isolation and culture condition of HUVEC using combinations of growth factors and extracellular matrix components so that the isolated HUVEC will purely represent the population under study. Umbilical cords were obtained from women post-labour. Different incubation times and digestive enzymes were used during endothelial cells isolation process. The culture conditions were optimized based on the coating materials and the media supplements. The results showed that 0.1% collagenase for 40 min incubation was the optimal isolation condition of HUVEC. HUVEC grown in 0.2% gelatin coated plate with 10% heat-inactivated fetal calf serum showed higher proliferative capacity and reduced cell death compared to other conditions (p<0.05). The results generated from this study provide a basic protocol of HUVEC isolation and culture conditions in order to generate working endothelial cell populations purely represent the Malaysian population

Lymphovascular invasion in melanoma and breast cancer

The theory of metastatic cascade suggests that vasculature plays a central role in the metastatic processes by being the major route of spreading. Two main circulatory systems in the body are responsible for cancer cell dissemination; the blood vascular system and the lymphatic system. However, comparing between these circulatory systems, much less is known about lymphatic vessels, with few studies being conducted about the initial steps of metastasis. In the first part of this project, a series of 202 formalin fixed paraffin embedded (FFPE) cutaneous melanoma sections were stained with D2-40, CD34 and CD68 to identify lymphatics, blood vessels and macrophages respectively, to examine vessel distribution and the involvement of inflammatory infiltrate in mediating vascular invasion (VI). Sections were also stained by conventional haematoxylin and eosin (H&E), to assess VI, and results compared against those obtained by immunohistochemistry (IHC) that allow discrimination of lymphatic and blood vessel invasion. It was found that lymphatics are mainly located at the peritumoural area of the tumour but intratumoural lymphatics are present and appeared to be functional based on the presence of tumour emboli in the vessels. In addition, vascular invasion in melanoma is mainly lymphatic vessel invasion with H&E assessment underestimating its incidence. Lymphatic vessel invasion were significantly associated with markers of aggressive disease which suggest their importance in melanoma. Lymphatic vessel invasion was also associated with a high macrophage count, suggesting a role for macrophage in mediating the process of metastatic via lymphatic vessels. In the second part of this project, the adhesion pattern of melanoma and breast cancer cell lines to blood and lymphatic endothelial cell models; large vessel versus microvessel and primary versus immortalised cells were compared. In addition, the effect of macrophage secreted cytokines; TNF-α and IL-1β, tumour conditioned media and macrophage conditioned media on the adhesive process were also studied. Both melanoma and breast cancer cells exhibited a higher level of adhesion to blood compared to the lymphatic endothelial cells. IL-1β stimulation of endothelial cells, tumour cells or both together showed a significant increased in the percentage of adhered tumour cells to the endothelial cell models with a higher increased to the lymphatic endothelial cells. A significant increased tumour cell adhesion was also observed with macrophage conditioned media and this effect seemed to be associated with the amount of IL-1β present. Interestingly, the increased adhesion effect observed with this supernatant was removed with the use of interleukin-1 converting enzyme (ICE) inhibitor. Expression of adhesion molecules; CLEVER-1, ICAM-1 and VCAM-1 were examined to study which adhesion molecules might regulate the process of tumour-endothelial interactions. Stimulation of endothelial cell models with IL-1β did not show any significant altered CLEVER-1 expression suggesting that although CLEVER-1 is an important lymphatic specific adhesion molecule, it may not be the principle regulator of tumour-endothelial interactions. This process may be regulated by ICAM-1 and VCAM-1 in which the expression increased significantly upon stimulation with IL-1β. In the third part of this study, the effect of TNF-α, IL-1β, tumour conditioned media and macrophage conditioned media stimulation on melanoma and breast cancer cell migration were investigated using wound healing assays. Following exposure to TNF-α and IL-1β, a significant increase in the percentage of wound closure was observed and the increase was higher in IL-1β stimulated cells. Similarly, when tumour cells were exposed to macrophage conditioned media, there was an increase in the percentage of wound closure compared to control cells. The effect of IL-1β and macrophage conditioned media on breast cancer cell migration across blood and lymphatic endothelial cells were also studied using Boyden chamber transmigration assay. Significant increased in tumour cells transmigration was observed with IL-1β stimulation, with similar affinity across both endothelial cell types. However, when cells were stimulated with macrophage supernatant from lipopolysaccharide (LPS) stimulated macrophages, an increase transmigratory effect was notably observed to the lymphatic endothelial cells. Interestingly, the increased adhesion effect was removed with the used of ICE inhibitors. The last part of this study dealt with IL-1β expression in breast tissue samples. 1511 early stage breast cancer tissue microarray samples were stained with commercially available IL-1β antibody to examine the association with lymphatic vessel invasion, clinicopathological variables and clinical outcome. High IL-1β expression in tumour cells was significantly associated with the absence of both intra-tumoural and peri-tumoural lymphatic vessel invasion. A significant association was also observed between low IL-1β expression in tumour cells with breast cancer specific survival and disease free interval. In conclusion, lymphatic vessels have been found to play a significant role in breast cancer and melanoma cells progression by being the major route for vascular dissemination. In the in-vitro settings, this study has shown that IL-1β, with macrophages as the main producer, could regulate tumour cell invasion especially to the lymphatic circulation. This project has yielded some important results towards understanding of the lymphatic vasculature and modulation of lymphatic vessel invasion. However, more studies are needed to enable translation of research into clinical management of cancer

Protective Effects of the Polyphenolic-Rich Fraction of Cornsilk against Oxidative Stress in Streptozotocin-Induced Diabetic Rats

Diabetes Mellitus (DM) has become a significant public health problem worldwide and primarily correlated to hyperglycaemia and abnormal lipid and antioxidant levels. Fruit and vegetable wastes are rich in phenolic compounds thus suitable for antioxidant sources. Cornsilk (CS), a maize cultivar waste, also contains phenolic compounds. The current study investigated the anti-hyperglycemic and antioxidative properties of the Phenolic-Rich Fraction of Cornsilk (PRF-CS) in Streptozotocin (STZ)-induced diabetic rats. Five groups of 30 male Sprague Dawley rats were employed in this study. A sample size of six rats each is placed in five groups: Normal-Control (NC), Diabetic-Control (DC), Diabetic-PRF-CS treated 100 mg/kg (DPRF100) and 200 mg/kg (DPRF200), and Diabetic-Metformin Treated (Dmet) groups. The PRF-CS was administered at 100 and 200 mg/kg doses for 28 consecutive days to the diabetic rats. Treatment with both doses of PRF-CS (DPRF100 and DPRF200) significantly decreased the blood glucose levels of the rats (p&lt;0.05). Additionally, the PRF-treated rats demonstrated significantly decreased (p&lt;0.05) lipid peroxidation (3.60±0.23 and 3.31±0.56 µmol/g, respectively). The hepatic antioxidant enzyme activities of Superoxide Dismutase (SOD) (169.35±4.75 and 175.30±3.69 U/mg, respectively), Catalase (CAT) (1457.51±152.74 and 2011.99±396.96 U/mg), and Glutathione Peroxidase (GSH-Px) (63.43±2.99 and 78.47±4.51 U/mg) were also elevated in contrast to the DC group. Furthermore, the PRF-CS administration improved the histological alterations in the liver tissues of the DPRF100 and DPRF200 rats. In conclusion, PRF-CS treatment exhibited protective effects in the diabetic rat model by decreasing oxidative stress and preserving liver integrity

Macrophage-derived interleukin-1beta promotes human breast cancer cell migration and lymphatic adhesion in vitro

Lymphovascular invasion (LVI), encompassing blood and lymphatic vessel invasion, is an important event in tumourigenesis. Macrophages within the tumour microenvironment are linked to the presence of LVI and angiogenesis. This study investigates the role of macrophage-derived, caspase-1 dependent interleukin-1beta (IL-1β) in an in vitro model of LVI. IL-1β significantly augmented the adhesion and transmigration of breast cancer cell lines MCF7 and MDA-MB-231 across endothelial cell barriers. MDA-MB-231 and MCF7 showed a higher percentage of adhesion to lymphatic endothelial cells than blood endothelial cells following endothelial cell IL-1β stimulation (P<0.001 and P<0.0001 respectively). Supernatants from activated macrophages increased the adhesion of tumour cells to lymphatic and blood endothelium. Secretion of IL-1β was caspase-1 dependent, and treatment with caspase-1 inhibitor reduced IL-1β production by 73% and concomitantly reduced tumour cell adhesion to levels obtained with resting macrophages. Transmigration of MDA-MB-231 cells across blood and lymphatic endothelial monolayers was significantly increased following IL-1β stimulation. Furthermore, supernatants from activated macrophages increased transmigration of MDA-MB-231 cells across endothelial monolayers, which was abolished by caspase-1 inhibition. IL-1β stimulation of tumour cells significantly increased their migratory ability and a significant increase in migration was observed when MDA-MB-231 cells were stimulated with macrophage conditioned media (two of three donors). Results demonstrate that macrophage production of IL-1β plays an important role in the migration of breast cancer cells and their adhesion to, and transmigration across, blood and lymphatic endothelial cells. Results suggest that IL-1β may play a role in the adhesion to lymphatic endothelial cells in particular

A review of the take-home exposure pathway of workplace hazards

Para-occupational exposure has been reported in the literature with increase in the risk of ill health among spouses, children and family members of workers exposed to materials like asbestos, heavy metals and pesticides. The family members are exposed to workplace agents brought home via the pathway of the take-home exposures routes such as on skin, clothes, shoes and cars. Hence the purposes of this review is to: demonstrate the evidence of take-home exposure pathway of chemicals such as pesticides, lead and asbestos; discuss sources and factors of take-home exposure; and consider methods to reduce the risk of take-home exposures

Objective assessment of blood and lymphatic vessel invasion and association with macrophage infiltration in cutaneous melanoma

The aims of this study were to investigate the role of vascular invasion (blood and lymphatic), vessel density and the presence of tumour-associated macrophages as prognostic markers in 202 cutaneous melanoma patients. Sections of primary melanoma were stained with lymphatic-specific antibody D2-40 to assess lymphatic vessel invasion and density in intratumoural and peritumoural areas; an antibody against endothelial marker CD34 was used to determine blood vessel invasion and density, and an antibody against CD68 was used to determine macrophage counts. Immunohistochemically determined vascular invasion (combined blood and lymphatic) was compared with that determined using haematoxylin and eosin (H&E) staining. The use of immunohistochemistry increased detection of vascular invasion from 8–30% of patients, and histological exam of H&E-stained tissue was associated with a false positive rate of 64%. Lymphatic vessel invasion occurred at a much higher frequency than blood vessel invasion (27 and 4% of patients, respectively). Although immunohistochemically detected vessel invasion was significantly associated with histological markers of adverse prognosis, such as increased Breslow thickness, ulceration and mitotic rate (all P<0.001), no associations with relapse-free or overall survival were observed. High macrophage counts were significantly associated with markers of aggressive disease, such as Breslow thickness, ulceration and mitotic rate (P<0.001, P<0.001, P=0.005, respectively), and lymphatic vessel invasion and high microvessel density (P=0.002 and P=0.003, respectively). These results suggest that vascular invasion is more accurately detected using immunohistochemistry and occurs predominantly via lymphatic vessels. The association of vessel characteristics with histological characteristics of the primary melanoma provides evidence for their biological importance in melanoma, but that they were not associated with clinical outcome attests to the value of existing histological prognostic biomarkers. We note that a high macrophage count may be associated with neovascularisation and primary tumour growth, and may also promote invasion through lymphatic vessels

The association of ICAM-1 detected by immunohistochemical staining with triple negative and non-triple negative breast carcinoma

Metastasis of tumour cell greatly contributes to the cause of mortality. Tumour-associated macrophage (TAM) and the intercellular adhesion molecule 1 (ICAM-1) were associated with metastases of breast carcinoma. However, the relationship between lymphatic vessel densities and invasion with TAM and ICAM-1 remained unclear. The aim of this study was to investigate the relationship of lymphovascular densities and invasions with patient’s clinicopathological data. The roles of TAM and ICAM-1 influencing lymphovascular invasions were also investigated. Haematoxylin and eosin (H&E) and immunohistochemical (IHC) staining on a consecutive section of 37 formalin fixed-paraffin embedded (FFPE) breast invasive carcinoma samples were carried out. The D2-40, CD34, CD163, and ICAM-1 antibodies were used to stain lymphatic vessel, blood vessel, TAM, and ICAM-1 receptor, respectively. The total lymphatic vessel density (LVD) was significantly reduced on increased tumor size (p=0.045). The increase of intra-tumoral LVD and lymphatic vessel invasion (LVI) was significantly associated with human epidermal growth factor receptor 2 (HER2) negative status (p=0.022 and p=0.05, respectively). The percentage of LVI was higher than blood vessel invasion (BVI) in 18.5%. Lymphovascular invasions detected in H&E were missed in 49.76% compared with those detected in IHC-stained tissues (206/410). ICAM-1 scores were significantly associated with non-triple negative breast cancer (non-TNBC) (p=0.008). ICAM-1 is significantly overexpressed on non-TNBC sample. Therefore, ICAM-1 might be clinically useful as a targeted molecule for non-TNBC patients. In histological reporting, in addition to H&E staining, IHC staining using D2-40 and CD34 should be considered to increase the accuracy of diagnosis