endothelial-cell-tube-formation-assay-for-vitro-study

Endothelial Cell Tube Formation Assay for the <em>In Vitro</em> Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

American University

Skin cancer is the most common form of cancer in the USA, with an estimated two million cases diagnosed annually. Tumor progression locus 2 (Tpl2), also known as MAP3K8, is a serine/threonine protein kinase in the mitogen-activated protein kinase signal transduction cascade. Tpl2 was identified by our laboratory as having a tumor suppressor function in skin carcinogenesis, with the absence of this gene contributing to heightened inflammation and increased skin carcinogenesis. In this study, we used gene expression profiling to compare expression levels between Tpl2 (+/+) and Tpl2 (-) (/-) keratinocytes. We identified over 2000 genes as being differentially expressed between genotypes. Functional annotation analysis identified cancer, cell growth/proliferation, cell death, cell development, cell movement and cell signaling as the top biological processes to be differentially regulated between genotypes. Further microarray analysis identified several candidate genes, including Mmp1b, Mmp2, Mmp9 and Mmp13, involved in migration and invasion to be upregulated in Tpl2 (-) (/-) keratinocytes. Moreover, Tpl2 (-/-) keratinocytes had a significant downregulation in the matrix metalloproteinase (MMP) inhibitor Timp3. Real-time PCR validated the upregulation of the MMPs in Tpl2 (-/-) keratinocytes and zymography confirmed that MMP2 and MMP9 activity was higher in conditioned media from Tpl2 (-/-) keratinocytes. Immunohistochemistry confirmed higher MMP9 staining in 12-O-tetradecanoylphorbol-13-acetate-treated skin from Tpl2 (-/-) mice and grafted tumors formed from v-ras(Ha) retrovirus-infected Tpl2 (-/-) keratinocytes. Additionally, Tpl2 (-/-) keratinocytes had significantly higher invasion, malignant conversion rates and increased endothelial cell tube formation when compared with Tpl2 (+/+) keratinocytes. In summary, our studies reveal that keratinocytes from Tpl2 (-/-) mice demonstrate a higher potential to be invasive and metastatic.

Tpl2 knockout keratinocytes have increased biomarkers for invasion and metastasis.

γ-Amino butyric acid type C (GABA(C)) receptors inhibit neuronal firing primarily in retina. Maintenance of GABA(C) receptor protein homeostasis in cells is essential for its function. However, a systematic study of GABA(C) receptor protein homeostasis (proteostasis) network components is absent. Here coimmunoprecipitation of human GABA(C)-ρ1-receptor complexes was performed in HEK293 cells overexpressing ρ1 receptors. To enhance the coverage and reliability of identified proteins, immunoisolated ρ1-receptor complexes were subjected to three tandem mass spectrometry (MS)-based proteomic analyses, namely, gel-based tandem MS (GeLC-MS/MS), solution-based tandem MS (SoLC-MS/MS), and multidimensional protein identification technology (MudPIT). From the 107 identified proteins, we assembled GABA(C)-ρ1-receptor proteostasis network components, including proteins with protein folding, degradation, and trafficking functions. We studied representative individual ρ1-receptor-interacting proteins, including calnexin, a lectin chaperone that facilitates glycoprotein folding, and LMAN1, a glycoprotein trafficking receptor, and global effectors that regulate protein folding in cells based on bioinformatics analysis, including HSF1, a master regulator of the heat shock response, and XBP1, a key transcription factor of the unfolded protein response. Manipulating selected GABA(C) receptor proteostasis network components is a promising strategy to regulate GABA(C) receptor folding, trafficking, degradation and thus function to ameliorate related retinal diseases.

Tracy Tabib

Department of Biology

American University

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Tracy Tabib

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

American University

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Department of Biology