P. Charles Lin

Endothelial Cell Adhesion Molecules and Cancer Progression

The role of cell adhesion molecules (CAMs), such as intercellular cell adhesion molecule-1 (ICAM-1), vascular endothelial cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin, has been studied extensively in the process of inflammation. These molecules are responsible for recruiting leukocytes onto the vascular endothelium before extravasation to the injured tissues. Some circulating cancer cells have been shown to extravasate to a secondary site using a process similar to inflammatory cells. The most studied ligands for CAMs expressed on cancer cells, sialyl Lewis (a/x) antigens, are shown to be involved in adhesion to endothelial cells by binding to E-selectin. This process, shared by inflammatory cells and cancer cells, may partially explain the link between inflammation and tumorigenesis. Furthermore, this process may elucidate the therapeutic benefit of anti-inflammatory drugs in cancer treatment. The complexity of the tumor microenvironment has been revealed in the past decade. Currently, intense investigation is aimed at various aspects of the tumor microenvironment in addition to the tumor cells themselves. Here, we review the role of CAMs in extravasation of circulating cancer cells, a key step in metastasis.

Molecular characterization of IL-32 in human endothelial cells

IL-32 is a newly discovered protein found in human and certain primates, but absent in rodent. Various reports suggest its role as a proinflammatory mediator. Since vascular endothelium is critical in inflammation, we investigate IL-32 in endothelial cells. We found that the gene is expressed in human endothelial cells and Akt strongly induces its expression. Sequence analysis indicates IL-32 beta as the major isoform in endothelial cells. Surprisingly, we did not detect any secretion of IL-32 beta in human endothelial cells; instead we observed co-localization of IL-32 beta with endoplasmic reticulum, suggesting IL-32 beta is an intracellular protein in these cells. Promoter analysis identified a minimum required region for IL-32 transcription at -0.1 to +0.5 kb around the initially identified transcription start site. We also defined a transcriptional suppressor-binding site at -2.0 to -1.5 kb. Importantly, RNA ligase mediated rapid amplification of cDNA ends in endothelial cells determined the transcription start site at the 328 bp downstream from the original identified site. Finally, we found a positive correlation of IL-32 levels with human breast cancer and glioblastoma multiforme (GBM). These findings improve our understanding of IL-32 in vascular endothelium. IL-32 expression might be valuable as a biomarker for cancer.

Interleukin-32β Propagates Vascular Inflammation and Exacerbates Sepsis in a Mouse Model

Background Inflammation is associated with most diseases, which makes understanding the mechanisms of inflammation vitally important. Methodology/Principal Findings Here, we demonstrate a critical function of interleukin-32β (IL-32β) in vascular inflammation. IL-32β is present in tissues from humans, but is absent in rodents. We found that the gene is highly expressed in endothelial cells. Three isoforms of IL-32, named IL-32α, β, and ε, were cloned from human endothelial cells, with IL-32β being the major isoform. Pro-inflammatory cytokines (TNFα and IL-1β) induced IL-32β expression through NF-κB. Conversely, IL-32β propagated vascular inflammation via induction of vascular cell adhesion molecules and inflammatory cytokines. Accordingly, IL-32β increased adhesion of inflammatory cells to activated endothelial cells, a paramount process in inflammation. These results illustrate a positive feedback regulation that intensifies and prolongs inflammation. Importantly, endothelial/hematopoietic expression of IL-32β in transgenic mice elevated inflammation and worsened sepsis. This was demonstrated by significant elevation of leukocyte infiltration and serum levels of TNFα and IL-1β, increased vascular permeability and lung damage, and accelerated animal death. Together, our results reveal an important function of IL-32 in vascular inflammation and sepsis development. Conclusions/Significance Our results reveal an important function of IL-32 in vascular inflammation and sepsis development.

Angiogenesis links chronic inflammation with cancer.

Angiogenesis, the formation of new blood vessels from existing vessels, is tightly linked to chronic inflammation and cancer. Angiogenesis is one of the molecular events bridging the gap between inflammation and cancer. One of the events linking inflammation and cancer is an increase in cellular adhesion molecules that are expressed on the luminal surface of endothelium upon inflammation. Cellular adhesion molecules are involved in leukocyte recruitment and subsequently lead to extravasation of leukocytes to the injury site. These adhesion molecules are known to be shared by some cancer cells and have the ability to contribute to metastasis. Thus, an elevation of these molecules in chronic inflammation may be a risk factor for metastasis. In this chapter, we discuss the method used to determine the adhesion molecules expressed on endothelium, and leukocyte adhesion to endothelium.

IκB Kinase-α Regulates Endothelial Cell Motility and Tumor Angiogenesis

The transcription factor nuclear factor-kappaB (NF-kappaB) is constitutively activated in many types of cancers and has been implicated in gene expression important for angiogenesis, tumor growth, progression, and metastasis. Here, we show that the NF-kappaB activator, IkappaB kinase-alpha (IKKalpha), but not IKKbeta, promotes endothelial cell motility and tumor angiogenesis. IKKalpha is elevated in tumor vasculature compared with normal endothelium. Overexpression of IKKalpha in endothelial cells promoted cell motility and vascular tubule formation in a three-dimensional culture assay, and conversely, knockdown of IKKalpha in endothelial cells inhibited cell motility, compared with controls. Interestingly, blocking NF-kappaB activation totally abolished IKKalpha-induced angiogenic function. Furthermore, using a tumor and endothelial cell cotransplantation model, we show that overexpression of IKKalpha in endothelial cells significantly increased tumor vascular formation compared with controls, which contributed to increased tumor growth and tumor cell proliferation, and decreased tumor cell apoptosis. Collectively, these findings have identified a new function for IKKalpha through the canonical NF-kappaB pathway in tumor angiogenesis.

Interleukin-32 positively regulates radiation-induced vascular inflammation.

PURPOSE To study the role of interleukin-32 (IL-32), a novel protein only detected in human tissues, in ionizing radiation (IR)-induced vascular inflammation. METHODS AND MATERIALS Irradiated (0-6 Gy) human umbilical vein endothelial cells treated with or without various agents--a cytosolic phospholipase A2 (cPLA2) inhibitor, a cyclooxygenase-2 (Cox-2) inhibitor, or lysophosphatidylcholines (LPCs)--were used to assess IL-32 expression by Northern blot analysis and quantitative reverse transcriptase-polymerase chain reaction. Expression of cell adhesion molecules and leukocyte adhesion to endothelial cells using human acute monocytic leukemia cell line (THP-1) cells was also analyzed. RESULTS Ionizing radiation dramatically increased IL-32 expression in vascular endothelial cells through multiple pathways. Ionizing radiation induced IL-32 expression through nuclear factor kappaB activation, through induction of cPLA2 and LPC, as well as induction of Cox-2 and subsequent conversion of arachidonic acid to prostacyclin. Conversely, blocking nuclear factor kappaB, cPLA2, and Cox-2 activity impaired IR-induced IL-32 expression. Importantly, IL-32 significantly enhanced IR-induced expression of vascular cell adhesion molecules and leukocyte adhesion on endothelial cells. CONCLUSION This study identifies IL-32 as a positive regulator in IR-induced vascular inflammation, and neutralization of IL-32 may be beneficial in protecting from IR-induced inflammation.

Tie2 Signaling Regulates Osteoclastogenesis and Osteolytic Bone Invasion of Breast Cancer

Breast to bone metastasis is a common occurrence in the majority of patients with advanced breast cancer. The metastases are often incurable and are associated with bone destruction and high rates of morbidity. Understanding the underlying mechanisms of how metastatic tumor cells induce bone destruction is critically important. We previously reported that Tie2, a receptor tyrosine kinase, is significantly increased in human breast cancer tissues compared with normal and benign breast tumors and regulates tumor angiogenesis. In this study, we identify a new function of Tie2 in osteoclastogenesis and osteolytic bone invasion of breast cancer. Tie2 is present in hematopoietic stem/precursor cells. Genetic deletion of Tie2 or neutralization of Tie2 function using soluble Tie2 receptor impaired osteoclastogenesis in an embryonic stem cell differentiation assay. In contrast, deletion of Tie2 has no effect on osteoblastogenesis. As CD11b myeloid cells have the potential to become osteoclasts and Tie2 is present in a certain population of these cells, we isolated Tie2(+) and Tie2(-) myeloid cells. We observed a significant reduction of osteoclastogenesis in Tie2(-) compared with Tie2(+) CD11b cells. Consistently, neutralization of Tie2 activity in vivo significantly inhibited osteolytic bone invasion and tumor growth in a mammary tumor model, which correlated with a significant reduction of osteoclasts and tumor angiogenesis. Collectively, these data reveal a direct and novel role of Tie2 signaling in osteoclast differentiation. These findings identify Tie2 as a therapeutic target for controlling not only tumor angiogenesis but also osteolytic bone metastasis in breast cancer.

Spontaneous immortalization of human dermal microvascular endothelial cells.

AIM To establish and characterize a spontaneously immortalized human dermal microvascular endothelial cell line, iHDME1. METHODS We developed a spontaneous immortalization method. This approach is based on the application of optimized culture media and culture conditions without addition of any exogenous oncogenes or carcinogens. Using this approach, we have successfully established a microvascular endothelial cell line, iHDME1, from primary human dermal microvascular endothelial cells. iHDME1 cells have been maintained in culture dishes for more than 50 passages over a period of 6 mo. Using a GFP expressing retrovirus, we generated a GFP-stable cell line (iHDME1-GFP). RESULTS iHDME1 retain endothelial morphology and uniformly express endothelial markers such as VEGF receptor 2 and VE-cadherin but not α-smooth muscle actin (α-SM-actin) and cytokeratin 18, markers for smooth muscle cells and epithelial cells respectively. These cells retain endothelial properties, migrate in response to VEGF stimulation and form 3-D vascular structures in Matrigel, similar to the parental cells. There is no significant difference in cell cycle profile between the parental cells and iHDME1 cells. Further analysis indicates enhanced stemness in iHDME1 cells compared to parental cells. iHDME1 cells display elevated expression of CD133 and hTERT. CONCLUSION iHDME1 cells will be a valuable source for studying angiogenesis.

Angiopoietin/Tie2 Signaling Regulates Tumor Angiogenesis

Tie2 was identified as a receptor tyrosine kinase (RTK) expressed principally on vascular endothelium. Disrupting Tie2 function in mice resulted in embryonic lethality with defects in embryonic vasculature. Multiple ligands for Tie2, named angiopoietin (Ang), have been identified. Ang1, an agonist, stimulates Tie2 phosphorylation in endothelial cells (EC). Ang2 has been considered to be an antagonist of Tie2 that blocks Tie2 activation induced by Ang1 in EC. Disrupting the function of Ang1 or overproduction of Ang2 yielded a phenotype similar to the Tie2 knockout, confirming the importance of the Ang/Tie2 pathway during embryonic vascular development. The genetic evidence suggests that the VEGF pathway and the Tie2 pathway seem to work in a complementary and coordinated fashion during vascular development. Many clinical and animal studies show critical roles of Ang/Tie2 pathway in tumor angiogenesis. The interaction of the Ang/Tie2 pathway with the VEGF pathway has also been demonstrated. Elevated VEGF and Ang2 expressions are associated with increased tumor angiogenesis. Ang2 corresponds positively with tumor development and progression as well as with metastasis and correlates negatively with patient survival in many studies. Blocking the Ang/Tie2 pathway has been shown to inhibit tumor angiogenesis, growth, and metastasis, demonstrating the potential of future clinical therapies based on this pathway. As anti-angiogenic therapies move from the bench to a clinical setting, an understanding of the role of the Ang/Tie2 pathway is vital.