Kweon Haeng Lee

Cellular Toxicity of Various Inhalable Metal Nanoparticles on Human Alveolar Epithelial Cells

Nanoparticles (NPs) have a greater potential to travel through an organism via inhalation than any other larger particles, and could be more toxic due to their larger surface area and specific structural/chemical properties. The aim of this study was to evaluate in vitro biological effects of various inhalable metallic NPs (TiO2, Ag, Al, Zn, Ni). Human alveolar epithelial cells (A549) were exposed to various concentrations of NPs for 24 h. The extent of morphological damage was in the order of m-TiO2 > n-TiO2 > m-silica ≫ n-Ni ≈ n-Zn ≈ n-Ag ≈ n-Al and was affected in a dose-dependent manner. The extent of apoptotic damage measured with two-color flow cytometry was in the order of n-Zn > n- Ni > m-silica ≫ n- TiO2 > m- TiO2 > n-Al > n-Ag. The extent of apoptotic damage measured with DNA fragmentation was in the order of n-Zn ≈ m-silica > n- Ni ≫ m- TiO2 ≈ n- TiO2 ≈ n-Al > n-Ag, indicating no significant difference in the damages by both m-TiO2 and n-TiO2. The extents of apoptotic damages were also affected in a dose-dependent manner. Uptake of no other NPs but n-TiO2 and m-TiO2 into the cells was observed after 24 h exposure. The intracellular generation of ROS was significant with n-Zn but not with the other particles. These results demonstrated that various inhalable metallic NPs (TiO2, Ag, Al, Zn, Ni) could cause cell damages directly or indirectly. More detailed studies on the influence of size, structure, and composition of the NPs are needed to better understand their toxic mechanisms.

HOX gene analysis of endothelial cell differentiation in human bone marrow-derived mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have been shown to possess multilineage differentiation potential. HOX genes function in transcriptional regulators, and are involved in stem cell differentiation. The aim of the present study was to demonstrate HOX genes that are related to angiogenesis. To identify the expression patterns of 37 HOX genes in the endothelial cell differentiation of hMSCs, we analyzed HOX genes through profiling with multiplex RT-PCR. The results showed that the expression patterns of four HOX genes, HOXA7, HOXB3, HOXA3, and HOXB13, significantly changed during angiogenesis. The expression levels of HOXA7 and HOXB3 were dramatically increased, whereas those of HOXA3 and HOXB13 were decreased during endothelial cell differentiation. When further analysis of the expressions of these HOX genes was performed with real-time PCR and an immunoblot assay, the expression patterns were also found to be well-matched with the results of multiplex RT-PCR. Here, we report that HOXA7, HOXB3, HOXA3, and HOXB13 might be involved in the angiogenesis of hMSCs.

Cytotoxicity of yellow sand in lung epithelial cells

The present study was carried out to observe the cytotoxicity of yellow sand in comparison with silica and titanium dioxide in a rat alveolar type II cell line (RLE-6TN). Yellow sand (China Loess) was obtained from the loess layer in the Gunsu Province of China. The mean particle diameter of yellow sand was about 0003 ± 0.001 mm. Major elements of yellow sand were Si(27.7 ± 0.6%), Al(6.01± 0.17%), and Ca(5.83 ± 0.23%) in that order. Silica and yellow sand significantly decreased cell viability and increased [Ca2+]i. All three particles increased the generation of H2O2. TiO2 did not change Fenton activity, while silica induced a slight increase of Fenton activity. In contrast, yellow sand induced a significant increase of Fenton activity. Silica, yellow sand and TiO2 induced significant nitrite formations in RLE-6TN cells. Silica showed the highest increase in nitrite formation, while yellow sand induced the least formation of nitrite. Silica and yellow sand increased the release of TNF-α. Based on these results, we suggest that yellow sand can induce cytotoxicity in RLE-6TN cells and reactive oxygen species, Fenton activity and reactive nitrogen species might be involved in this toxicity.

Diesel exhaust particles induce apoptosis via p53 and Mdm2 in J774A.1 macrophage cell line.

Diesel exhaust particles (DEP) are known to cause cardiopulmonary diseases due to their proinflammatory and cytotoxic effects. Continuous exposure to DEP potentiates chronic inflammatory processes and acute symptomatic responses in the respiratory tract. Recent studies have emphasized that alveolar cell apoptosis is a crucial step in chronic inflammation and lung injury. The phenomenon of apoptosis is a key event that successfully clears damaged cells, and its failure leads to the development of more serious diseases, such as lung cancer. The mechanism and molecular target of DEP-induced apoptosis in the respiratory tract remain unclear. In this study, J774A.1 macrophage cells were used to investigate the p53-mediated apoptotic pathway induced by DEP exposure. The results showed that murine double minute 2 (Mdm2), a negative regulator of p53, was downregulated at the protein level by DEP exposure. In contrast, the pro-apoptotic protein Bcl-2-associated X protein (Bax), an endogenous target of p53-dependent transcriptional activation, was continuously upregulated at the mRNA and protein levels by DEP exposure. Furthermore, pifithrin-alpha (p53 inhibitor) blocked DEP-induced apoptosis as well as p53 activation. Taken together, the findings of the present study suggest that DEP trigger apoptosis in J774A.1 macrophage cells via the activation of p53, followed by Bax.

Bone morphogenic protein-2 (BMP-2) immobilized biodegradable scaffolds for bone tissue engineering

Recombinant human bone morphogenic protein-2 (rhBMP-2), which is known as one of the major local stimuli for osteogenic differentiation, was immobilized on the surface of hyaluronic acid (HA)-modified poly(ε-caprolactone) (PCL) (HA-PCL) scaffolds to improve the attachment, proliferation, and differentiation of human bone marrow stem cells (hBMSCs) for bone tissue engineering. The rhBMP-2 proteins were directly immobilized onto the HA-modified PCL scaffolds by the chemical grafting the amine groups of proteins to carboxylic acid groups of HA. The amount of covalently bounded rhBMP-2 was measured to 1.6 pg/mg (rhBMP/HA-PCL scaffold) by using a sandwich enzyme-linked immunosorbant assay. The rhBMP-2 immobilized HA-modified-PCL scaffold exhibited the good colonization, by the newly differentiated osteoblasts, with a statistically significant increase of the rhBMP-2 release and alkaline phosphatase activity as compared with the control groups both PCL and HA-PCL scaffolds. We also found enhanced mineralization and elevated osteocalcin detection for the rhBMP-2 immobilized HA-PCL scaffolds, in vitro.

Role of Tumor Necrosis Factor-α, Interleukin-8, and Dexamethasone in the Focal Adhesion Kinase Expression by Human Nucleus Pulposus Cells

Study Design. Human nucleus pulposus cells from intervertebral disc specimens were cultured to study the effects of tumor necrosis factor (TNF)-&agr; and interleukin (IL)-8 on the focal adhesion kinase (FAK) expression by these cells. The effect of co-stimulation with dexamethasone on the FAK expression by nucleus pulposus cells was also studied. Objectives. To evaluate the possible role of activated FAK expressed by the human nucleus pulposus cells and its correlation with inflammatory cytokines (TNF-&agr;, IL-8) and dexamethasone. Summary of Background Data. There have been no reported studies showing the correlation between the activated FAK expression by human nucleus pulposus cells with inflammatory cytokines and dexamethasone. Methods. The FAK expression in cultured human nucleus pulposus cells was studied, and Western blot and immunofluorescence analysis were performed to assess its relation to TNF-&agr;, IL-8, and dexamethasone. Results. Treatments of TNF-&agr; and IL-8 up-regulated the activated FAK expression. Dexamethasone attenuated the cytokine-induced FAK expression. The effects of inflammatory cytokines on the FAK expression were found to be concentration dependent, with greater correlation shown by IL-8 than TNF-&agr;. Conclusion. TNF-&agr; and IL-8 stimulation up-regulated the FAK expression of human nucleus pulposus cells, and the coadministration of dexamethasone attenuated it.

Use of soybean protein hydrolysates for promoting proliferation of human keratinocytes in serum-free medium

Human keratinocytes are generally cultured in media containing bovine pituitary extract (BPE), an animal product that can be a source of infectious contaminants. We investigated whether a safer plant product could replace BPE in the culture medium. Medium containing both BPE and soy protein hydrolysates (Bacto Soytone and Soy Hydrolysate) produced the largest number of viable cells, followed in descending order by medium supplemented only with BPE, only with the hydrolysates, and without supplementation (basal medium only). Soybean protein is thus an excellent source of nutrients for the growth of adherent keratinocytes, although they do not fully substitute for BPE.