Research Subject

Research background

1. Blood vessels

Blood vessels are distributed throughout the entire body. They not only deliver blood, oxygen, and nutrients to the body’s tissues but also play diverse roles in the tissue generation process and the development and maintenance of internal organs, as well as in the onset and progression of various diseases. For example, most cases of heart disease and cerebrovascular disorders, in 2012 the second and fourth leading causes of death, respectively, in Japan, are caused by lesions in the blood vessels, and blood vessels also play an important role in the progress of cancer, the leading cause of death in Japan and a condition that will affect one-third of the Japanese public.

The structure of a blood vessel consists of endothelial cells that cover one layer of the lumen and wall cells (pericytes in capillary vessels, and arteriovenous smooth muscle cells) that envelop these from the outside and serve to support, contract, and relax the blood vessel. Angiogenesis is the process by which endothelial cells in existing blood vessels form new lumen in response to stimulus. Endothelial cells sprouting from existing blood vessels are guided to tip cells, which have high migration potency on their tips, and these later connect to the blood vessel through stalk cells and then phalanx cells. In the unstable state in which a blood vessel is formed by endothelial cells alone, a backing forms and develops in the surrounding wall cells of smooth muscle cells and pericytes. These can be categorized by the diameter of the blood vessel.

2. Tumor angiogenesis

Adult angiogenesis is induced by a number of factors (angiogenic factors) that stimulate propagation of the angioendothelium, such as hypoxia in tissues suffering ischemia due to cancer or the healing of a wound and vascular endothelial growth factors (VEGFs).

Tumor angiogenesis is one of the most important types of pathological adult angiogenesis. To get the supply of oxygen and nutrients they need to propagate, cancer cells release angiogenic factors to induce active angiogenesis. In this way, newly formed vascular tumors are extremely important to the progress and metastasis of cancer.

The manifestation of VEGFs also is exacerbated by hypoxia, gene activation, and antioncogene abnormalities. In addition, angiogenic factors aside from VEGFs include basic fibroblast growth factors (bFGFs), angiopoietins, hepatocyte growth factors (HGFs), EGFs, and placental-derived growth factors (PlGFs), among others. In a case of cancer there is an overabundance of such angiogenic factors. In the cancer micro-environment endogenous anti-angiogenic factors, thrombospoidins-1 (TSP-1), and negative control factors such as vasohibin and Down syndrome critical region-1 (DSCR-1) are secreted as well. Furthermore, large quantities of various cells invade the tumor stroma in which vascular tumors are distributed, and these stromal cells too produce angiogenic factors that promote tumor angiogenesis. In this way, cancer angiogenesis occurs through a complex mechanism controlled by the balance between multiple angiogenic factors and anti-angiogenic factors.

3. Anti-angiogenic therapy

Anti-angiogenic therapy is a new therapeutic method for cancer that attempts to starve the cancer by targeting the blood vessels that nourish it. It was first proposed in 1971 by the pediatric surgeon Dr. Judah Folkman. He believed that since all cancer cells were dependent on angiogenesis it might be possible to decrease and eliminate cancer tissue by impeding angiogenesis, and that since the angioendothelium was made of normal cells and was genetically stable, it would be suitable as a target for therapy because it would not develop drug resistance the way cancer cells did. The drug Bevacizumab, the best-known of the angiogenesis inhibitors developed from this concept, is an aneutralizing antibody of the human vascular endothelial cell growth factor (VEGF) that isolates the VEGF signal that induces angiogenesis, secreted from a tumor and from interstitial cells. A variety of tyrosine kinase inhibitors targeting VEGF receptors (VEGFRs), such as Sorafenib and Sunitinib, also have been developed and are seeing clinical application, resulting in improved prognoses for numerous cancer patients. Today, aside from the goal of starving cancer by cutting off its supply of nutrients and oxygen through inhibiting angiogenesis, angiogenesis inhibitors also are used to normalize vascular tumors to make it easier to deliver drugs such as anticancer agents. They do so by improving the structure of vascular tumors, which are weak and liable to leak due to excessive VEGF stimulation.

4. Structural differences between normal blood vessels and vascular tumors

As described above, existing angiogenesis inhibitors play an important role in cancer therapy. However, a number of problems have been identified with these therapeutic methods. One type of such problems is the reported side-effects of high blood pressure, thrombosis, and gastrointestinal perforations. It is thought that these side-effects are caused by damage to normal blood vessels since most of these angiogenesis inhibitors inhibit the VEGF signals needed for normal angioendothelium generation and maintenance.

At the same time, it is known that the vascular tumors that are the target of anti-angiogenic therapy differ from normal blood vessels on many histopathological points. For example, vascular tumors are known to have higher permeability due to lower adhesion between endothelial cells themselves and between wall cells and endothelial cells, and the structures of the blood vessels’ basal membranes are known to differ as well. These abnormal blood-vessel structures are caused by factors such as the disordered, irregular progress of blood through vascular tumors and irregular blood flows. This is known to induce hypoxia, which results in increased malignancy and drug resistance in cancer, due to low blood flow within the cancer tissue even though it contains an abundance of blood vessels.

Subjects of our research

Discovering tumor endothelial cell abnormalities

When research began into tumor angiogenesis, vascular tumors were thought not to differ from normal blood vessels. While the view later arose that blood vessel endothelial cells could differ in light of the above structural differences in blood vessels, the biological properties of tumor endothelial cells remained unknown until the early 21st century. For this reason, normal endothelial cells (NECs) taken from relatively easily accessible sources such as umbilical arteries and skin capillaries were used widely in research on blood vessels.

However, cancer blood vessels exist in an environment that differs considerably from that of the blood vessels in normal tissue. We are the first in the world to have developed and studied an isolation culture for the tumor endothelial cells (TECs) that make up cancer blood vessels. We believe that by identifying the distinctive properties and target molecules of tumor endothelial cells that differ from normal endothelial cells (NECs) it would be possible to develop new angiogenesis inhibitors to attack cancer blood vessels only, leaving normal blood vessels unharmed.

We have learned a lot from our studies of tumor endothelial cell abnormalities. Our current research activities are focused on the following projects:

1. Development of specific inhibitors through elucidation of the distinctive properties of tumor angioendothelium
2. Elucidation of the diversity of tumor angioendothelium
3. The mechanism by which abnormality develops in tumor angioendothelium (from the perspective of the micro-environment of cancer)
4. Analysis of the functions of tumor endothelial cells in cancer stem cells and cancer invasion and metastasis

1. Research intended to develop specific inhibitors through elucidation of the distinctive properties of tumor angioendothelium

We have identified the way tumor endothelial cells differ from normal endothelial cells on a variety of points. For example, we have reported how tumor endothelial cells involve differences in gene expression and are weaker in angiogenic potential (Matsuda, Biochem Biophys Res Commun 2010). Furthermore, we were the first to point out that there were chromosomal abnormalities in tumor endothelial cells in cancer stroma, counter to the commonly accepted view at the time (Hida, Cancer Res 2004, highlighted and selected for the cover). We later showed that such abnormalities were present not only in mice but in human beings as well (Akino, Am J Pathol 2009). Further analysis led to findings such as the fact that it was possible to differentiate tumor endothelial cells because they had higher manifestations of stem cell markers such as Sca-1 and CD90 (Ohga, Am J Pathol 2012). We also identified the fact that tumor angioendothelium has higher manifestations of transporters and develops drug resistance (Akiyama, Am J Pathol 2012). We intend to develop specific drugs for vascular tumors through making clear the differences between them and normal blood vessels (see fig. below). We have used tumor endothelial cell and normal endothelial cell specimens to compare their gene expression, concentrating on those that have significantly higher manifestations in tumor angioendothelium and contribute to the functions of tumor angioendothelium (e.g., high viability and migration potency). These molecules have higher manifestations not only in mouse tumors but in human cancer as well, and we believe that they hold promise as new targets for anti-angiogenic therapy. We have analyzed the following molecules that show higher manifestations in tumor angioendothelium: Biglycan (Yamamoto, Brit J Cancer 2012), Ptgir (Osawa, Cancer Sci 2012), COX-2 (Muraki, Int J Cancer 2012), CXCR7 (Maichi, Pathol Int 2012), Ptgfr (Akiyama , Pathol Int 2013), and Lox (Osawa, Brit J Cancer 2013). Currently we are carrying out research aimed at practical applications using other new molecules. In particular, we are taking part in joint research aimed at practical application of nucleic acid medicine targeting vascular tumors, using the MEND drug-delivery system (DDS) developed by the Laboratory of Innovative Nanomedicine, led by Prof. Hideyoshi Harashima of the Graduate School of Pharmaceutical Sciences (http://www.pharm.hokudai.ac.jp/mirai/) (nine coauthored papers, including Sakurai, J Control Release and Kibria, Biomaterials 2013).

Furthermore, among tumor angioendothelium markers protein secretions also can be used as diagnostic agents, and currently we are analyzing their usefulness using blood from cancer patients.

2. Elucidation of the diversity of tumor angioendothelium

Today, the nature of a cancer is thought to be determined not by abnormalities in the cancer cells alone but also by the effects of the interaction between the micro-environment in which the cancer cells are located and interstitial cells. We are studying the micro-environment of cancer and analyzing the mechanism by which abnormality develops in a tumor endothelial cell. In particular, we have shown that the nature of a tumor endothelial cell (i.e., its angiogenic potential, gene expression, degree of chromosomal abnormality, and drug resistance) varies with differences in the degree of malignancy of a cancer and identified the possibility that differences in the micro-environment of cancer could cause diversity in tumor angioendothelium (Ohga, Am J Pathol 2012; highlighted). These findings concerned the differences in endothelial cells between tumors having high and low levels of metastasis, and today we are studying diversity in tumor endothelial cells through investigating the relationship between the invasiveness of cancer and metastasis, endothelial cells that form the niche of cancer stem cells, and the nature of tumor endothelial stem cells. This will make it possible to understand the angiogenesis that accompanies the progress of cancer as well as the changes in the character of endothelial cells and other factors, and our future goal is to achieve individualized antiangiogenic treatment through ascertaining the degree and status of angiogenesis and selecting drugs and patients accordingly.

3. Analysis of the mechanism by which abnormality develops in tumor angioendothelium (from the perspective of the micro-environment of cancer)

Current thinking holds that the mechanisms of abnormality in tumor endothelial cells include (1) dedifferentiation of the tumor cell to the angioendothelium, change in culture, and cell fusion with the tumor cell, and (2) abnormality developing through factors intrinsic to the micro-environment of cancer (e.g., hypoxia and humoral factors resulting from the tumor cell). While a number of findings have been reported concerning the first of these, many points still remain unclear. We are studying the second mechanism. Already we have reported our research findings that drug resistance in endothelial cells is induced by humoral factors resulting from tumor cells (Akiyama, Am J Pathol 2012) and that incorporation of exosomes from tumor cells by endothelial cells contributes to high levels of angiogenic potential in tumor endothelial cells (Kawamoto, PLoS ONE 2012). Our research also has suggested that conditions of hypoxia also induce ROS in endothelial cells and serve as a cause of chromosomal abnormalities in tumor endothelial cells (Kondoh, PLoS ONE 2013). Furthermore, currently we are carrying out basic research into the contributions to the development of abnormalities in tumor endothelial cells of factors such as miRNA in tumor-derived exosomes. At present it is understood that at least some of the properties of a tumor endothelial cell are maintained during the cell culture process, and we also are studying the possibility that epigenetic structure plays a part in controlling the appearance of tumor endothelial cell-specific molecules.

4. Functional analysis of tumor endothelial cells in cancer stem cells and cancer invasion and metastasis

Vascular tumors are known not just to support the formation of cancer but also to form cancer stem cell niches and to play an important role in cancer metastasis. We also are analyzing the molecular mechanisms by which an abnormal tumor endothelial cell within the micro-environment of cancer contributes to the invasion and metastasis of cancer and to the formation of cancer stem cell niches.

Furthermore, we also are focusing on molecules related to angiogenesis, which are being identified through the process of advancing research on angiogenesis and anti-angiogenesis. We believe that these hold promise for application to revascularization and tissue regeneration for a variety of diseases in the future.