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Roundabout4 gene

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Related terms
Background
Methods
Research
Implications
Limitations
Safety
Future research
Author information
Bibliography

Related Terms
  • ARMD, angiogenesis, bioinformatic datamining, colorectal cancer, diabetic retinopathy, fibronectin, immunoglobulin, immunohistochemistry, in-situ hybridization, knockout mouse technology, macular degeneration, magic roundabout, magic roundabout4, myogenesis, neurogenesis, non-small cell lung cancer, prostate cancer, Robo genes, Robo1 gene, Robo2 gene, Robo3 gene, ROBO4, Robo4 gene, roundabout genes, Slit/Robo pathway, tumor angiogenesis, tumor specific endothelial marker, vascular endothelial growth factor, VGEF.

Background
  • The roundabout4 (Robo4) gene is one of the genes from the roundabout family of genes, which provide instructions for making the Robo4 protein. This protein basically prevents the extra and uncontrolled growth of blood vessels and stabilizes them in the body. Uncontrolled growth of blood vessels is seen in many medical conditions such as macular degeneration, which is a degenerative disease of the eye causing vision loss; diabetic retinopathy, which is retinal damage in the eye associated with diabetes; and cancer.
  • Genes are considered the building blocks of life because they provide instructions for all of the cells in the body. Genes, located within cells, control an organism's development and functions by instructing cells to make new molecules (usually proteins). Genes are passed down from parents to their children.
  • The growth of new blood vessels from pre-existing blood vessels is called angiogenesis, and the generation of new nerve cells is called neurogenesis. These are finely balanced physiological processes, commonly seen during wound healing. Some molecules that are involved in angiogenesis and neurogenesis have been identified on the surface of the cells that are located on both nerve cells and vascular endothelial cells, which are the innermost layer of cells in a blood vessel and include neuripilin, ephrin, plexin, and roundabout (robo) receptors. The processes of angiogenesis and neurogenesis is mediated by various chemical signaling mechanisms.
  • The roundabout family of genes includes Robo1, Robo2, Robo3, and the recently identified Robo4 gene. These genes provide instructions for making their corresponding class of roundabout protein receptors, which express their features predominantly in nerve cells (for neuronal guidance) and endothelial cells (for their migration to the site of angiogenesis). Receptors are molecules on the surface of cells that helps to bind with other substances, thereby helping in chemical reactions.
  • The Robo4 gene, also called the magic roundabout4 (ROBO4; GenBank acc. no. AF361473), is smaller than other members of the roundabout gene family. The Robo4 protein is structurally different from other Robos. The other Robo family members have five immunoglobulin (Ig) domains and three fibronectin domains, whereas Robo4 has only two Ig domains and two fibronectin in the extracellular (outside the cell) region. Antibodies (immunoglobulins) have domains or folds that help to attach with certain substances. Fibronectin domain is a binding site for fibronectin. Fibronectin is a glycoprotein, which is a combination of protein and carbohydrate.
  • The characteristics of Robo4, also called Robo4 expression, are specific to the endothelial cells. This has been demonstrated both by laboratory experiments (in vitro) and experiments done within living organisms (in vivo), according to recent research reports. According to these research reports, the expression of Robo4 in adults was exclusively restricted to sites of active angiogenesis, especially tumor blood vessels.
  • Robo4 expression is activated by a protein called Slit through the Slit/Robo pathway or signal, which inhibits the vascular endothelial growth factor (VGEF). VGEF is a chemical that signals the creation of new blood vessels. This starts a chain of biochemical events within the endothelium that prevents the uncontrolled growth and destabilization of the blood vessels, thereby allowing growth of the blood vessels only where required.
  • In patients with certain conditions, such as age-related macular degeneration (ARMD) and diabetic retinopathy, certain blood vessels in the eye become weak and leaky, which is followed by the development of abnormal new blood vessels. Activation of Robo4 in such sites stabilizes the leaky blood vessels and curbs the development of new blood vessels.

Methods
  • The roundabout4 (Robo4) gene was discovered with the help of knockout mouse technology and bioinformatic datamining. Its expression and characteristics were determined and experimentally verified with the help of in situ hybridization and immunohistochemistry.
  • Knockout mouse technology: A knockout mouse is a genetically engineered mouse used for research. Genetically engineered mice are created by the manipulation of genetic material in a laboratory to carry out specific experiments. Knockout mice have specific genes that are "knocked out" or deactivated.
  • This type of research allows scientists to learn what specific genes do. When a gene is deactivated, the function of that gene may be revealed by the characteristics of the knockout mouse. This has helped researchers identify and understand the functions of many important genes that are also present in humans. For example, researchers have identified genes that influence aggression, alcoholism, drug addiction, and maternal behavior.
  • Bioinformatics datamining: As the name implies, bioinformatics datamining involves going through large volumes of information within biological databases to uncover and analyze patterns and relationships between biological organisms. This is done with the help of computational programs, statistical analysis, algorithms, and artificial intelligence so as to discover previously unknown relationships within the data.
  • In-situ hybridization:This is a precise method of identifying and detecting specific nucleic acid sequences (DNA/RNA) in its natural or normal place (in situ) such as tissue sections that are well preserved in both structure and form or cell preparations. Here the nucleic acid sequences, which are labeled with fluorescent dye for detection, are allowed to interact and form complexes or hybrids with complementary nucleic acid sequences within the tissues or cells.
  • Immunohistochemistry: This technique involves localization or identification of antigens or proteins within tissue sections. This method uses the principle that specific antibodies bind with specific antigens in biological tissues. Thus, antibodies, which are labeled with markers (e.g., fluorescent dye, enzyme, etc.), bind with specific antigens in the tissue samples and these can be visualized under a microscope

Research
  • The predominant expression of the roundabout4 (Robo4) gene in the vascular endothelium is activated by slit protein, which then inhibits the vascular endothelial growth factor (VGEF). This results in the stabilization of the leaky blood vessels at the sites of angiogenesis in embryonic as well as tumor growths.
  • Macular degeneration: Macular degeneration is a degenerative disease of the retina, a thin layer of nerve cells that lines the back of the eyeball. The condition causes progressive loss of central vision due to the deterioration of vision from the central part of the retina, known as the macula. The risk of developing macular degeneration increases with age, and hence it is called age-related macular degeneration (ARMD). ARMD is the leading cause of vision loss in the United States and worldwide in people over the age of 60 years.
  • In ARMD, certain blood vessels in the eye become weak and leaky, followed by the development of abnormal new blood vessels. Activation of Robo4 in such sites stabilizes the leaky blood vessels and curbs the development of new blood vessels, thereby controlling this degenerative condition to a certain extent.
  • Diabetic retinopathy: Retinopathy occurs when the small blood vessels called capillaries in the retina become damaged. The retina, located at the back of the eye, sends visual images to the brain where they are perceived. Because these capillaries nourish the retina, retinopathy may lead to partial or complete vision loss. The most common cause of retinopathy is high sugar levels in the blood as seen in diabetes. This is because prolonged high blood sugar levels damage the sensitive blood vessels in the eye. Diabetic retinopathy is the leading cause of blindness in Americans between the ages of 20 and 64 years. Robo4 activation can prevent angiogenesis by stabilizing the capillaries, thereby reducing the damage caused by diabetic retinopathy.
  • Severe acute respiratory syndrome (SARS): Many diseases are caused by injury or inflammation, which destabilize the blood vessels, causing them to leak fluid into the adjacent tissues in the lungs. Research is under way in the field of SARS that may contribute to the development of novel vessel-targeting therapies utilizing the concept of Robo4 expression to stabilize the affected blood vessels.
  • Tumor-specific markers: Robo4 is being studied as a tumor-specific endothelial marker and for its role in tumor angiogenesis in various types of cancers, including prostate, colorectal, and non-small cell lung cancer (NSCLC). Because Robo4 is endothelial cell specific and found at the sites of angiogenesis in certain rapidly growing tumors, it may be used for the detection or identification of certain tumors.
  • Myogenesis: Robo4 has also been implicated in functions across multiple cell types such as myogenesis, which is the origin of new muscles from pre-existing muscles, and leukocyte chemotaxis, which is the movement of white blood cells in response to chemical stimulus.

Implications
  • Abnormal blood vessel growth and leakage are two primary factors in age-related macular degeneration (ARMD) and diabetic retinopathy in humans. The same disease conditions were simulated in mice models for research and experimental purposes. Activation of the roundabout4 (Robo4) gene in mice models simulated with these conditions reduced the damage caused by abnormal angiogenesis and destabilization of the blood vessels. This is of significance for the development of novel drugs that activate the Robo4 and may eventually help treat ARMD and diabetic retinopathy.
  • Because Robo4 expression prevents abnormal angiogenesis, it is of significance in developing treatment options that inhibit tumor growth and its spread to the adjacent structures in the body.

Limitations
  • An estimated 15% of gene knockout studies cause the mouse to die before it can develop into an adult mouse. When a mouse dies before adulthood, it makes it difficult to understand a gene's function in relation to human health. This is because some genes function differently during development than they do during adulthood.
  • Sometimes, an inactivated gene in a mouse produces no observable change in the animal's phenotype. Phenotype may be described as the observable physical and biochemical characteristics of a being that is determined by the genetic makeup.

Safety




Future research
  • Studies with magic roundabout4 (Robo4) genes and proteins are ongoing. Researchers are studying the Robo4 as a potential target for anti-cancer and anti-angiogenic therapy in various cancers such as intestinal cancer, prostate cancer, and lung cancer, and conditions such as macular degeneration, diabetic retinopathy, and severe acute respiratory syndrome (SARS).
  • The possible role of the Slit/Robo signaling pathway during luteolysis in women is being evaluated to understand the cellular and molecular mechanisms involved in this natural process. Luteolysis is the structural and functional degeneration of the corpus luteum, which is the remaining part of a follicle on the ovary after the egg has been released, in the absence of pregnancy.

Author information
  • This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

Bibliography
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  2. Dickinson RE, Myers M, Duncan WC. Novel regulated expression of the SLIT/ROBO pathway in the ovary: possible role during luteolysis in women. Endocrinology. 2008 Jun 19.
  3. Englund C, Steneberg P, Falileeva L, et al. Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea. Development. 2002 Nov;129(21):4941-51.
  4. Gorn M, Anige M, Burkholder I, et al. Serum levels of Magic Roundabout protein in patients with advanced non-small cell lung cancer (NSCLC). Lung Cancer. 2005 Jul;49(1):71-6.
  5. Grone J, Doebler O, Loddenkemper C, et al. Robo1/Robo4: differential expression of angiogenic markers in colorectal cancer. Oncol Rep. 2006 Jun;15(6):1437-43.
  6. Huminiecki L, Bicknell R. In silico cloning of novel endothelial-specific genes. Genome Res. 2000 Nov;10(11):1796-806.
  7. Huminiecki L, Gorn M, Suchting S, et al. Magic roundabout is a new member of the roundabout receptor family that is endothelial specific and expressed at sites of active angiogenesis. Genomics. 2002 Apr;79:547-52.
  8. Jones CA, London NR, Chen H, et al. Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability. Nat Med. 2008 Apr;14(4):448-53.
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  14. Yoshikawa M, Mukai Y, Okada Y et al. Ligand-independent assembly of purified soluble magic roundabout (Robo4), a tumor-specific endothelial marker. Protein Expr Purif. 2008 May 21.

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