Cell Signaling & Tumor Angiogenesis

Cell Signaling, Retinal & Tumor
Angiogenesis

Overview

Sudhakar Akul Yakkanti is interested in understanding the molecular basis of extra cellular matrix derived molecules in cell adhesion and the signaling mechanisms in human vascular diseases such as tumor angiogenesis and macular degeneration.

Background

Most cells in human body adhere to the neighboring cells and to the extra-cellular matrix (ECM, a fibrillar meshwork like structure) in blood vessels. Cellular adhesion to ECM through integrins receptors, play important roles in the normal functions such as cellular organization, proliferation, migration, survival and metabolism including gene expression. During the embryonic development, cell adhesion is important for the correct movement of cells modeling the embryo. In the adult development, appropriate cell adhesion is necessary for numerous physiological processes that can be deranged in diseased conditions including tumor angiogenesis, macular degeneration, arthritis, cancer etc.

Our laboratory seeks to understand the ECM containing type IV collagen derived anti-angiogenic proteins that are present in normal circulation, which are involved in cell signaling and the way these proteins control adhesion and migration of cells in pathological processes. Type IV collagen derived anti-angiogenic proteins, by binding to cell surface integrins, transduce the signaling mechanisms in regulating anti-angiogenic activity. Thus, integrins serve as transmembrane linkers between the ECM and cytoskeleton for outside-in signaling. Both type IV collagen and several integrin receptors are essential for angiogenesis (a process in which new blood vessel formation). Presently, we are analyzing type IV collagen derived non-collagenous domains (NC1) binding to certain integrins and regulate anti-angiogenic and anti-tumorogenic actions specifically targeting tumor microvascular endothelial cells. Our recent work has led to the new interpretations on the efficacy of certain type IV collagen NC1 domains working as antiangiogenic and anti-tumorigenic drugs (Science 2002, 295, 140-143; PNAS 2003, 100, 4766-4771; JCI 2005, 115, 2801-2810; Blood 2007, 110; 1168-1177 etc).

Cell Signaling, Retinal and Tumor Angiogenesis Laboratory Facilities

The laboratory consists of 1,200 square feet of newly renovated space with a separate tissue culture room (150 sq. ft.) that contains the most modern 2 cell culture incubators. All of the equipments required to conduct the experiments for this proposal are available within the laboratory. This space is adjacent to all utilities; deionizer water runs from the special tap in the lab. There is also a separate cold cabinet for chromatography equipment and protein purification in the lab and a 37oC bacterial incubator in the lab. Autoclaving and dishwashing are done in the building. Major equipment within the laboratory includes a class II safety hood for bacterial transfers, medium and low rate centrifuges, 2 microfuges, 2 refrigerated centrifuges, 3 vertical and 1 horizontal electrophoresis equipment, and 4 western blotting apparatus. Equipment for handling and staining of slides, 1 bacterial and 2 cell culture incubators, two refrigerators, two -20oC and one -80oC freezers, one liquid nitrogen storage tank (can store more than 2500 tubes), 2 PCR thermocycler (MJ Research Inc; Labnet), 1 Bio-rad BioLogic LP chromatography system and BioFrac Fraction collector for protein purification, 1 ELISA plate reader, one 37oC incubator, 4 magnetic stirrers, 2 Accumet pH meters (AB 15), 1 QuicGene automated DNA extraction system,1 inverted microscope, 4 sets of electroporation equipment, tissue culture room with laminar hood and 2 CO2 incubators, one 27oC (for Sf9 cells) and one 37oC, 2 microbalances, 2 shakers, 3 water baths, 1 speed-vac, 1 chromatographic refrigerator, and other basic molecular biology equipment. All of the necessary minor equipment required to carry out cell signaling work is also available.

A separate cell culture suite (approximately 120 sq. ft.), which is dedicated only to Cell Signaling, Retinal and Tumor Angiogenesis laboratory, includes a HEPA-filtered positive-pressure room with wrapped ceiling tiles and germicidal lamps. Equipment includes a doublewide laminar flow hood, two stacked double-chamber CO2 incubators, a low speed centrifuge, two Olympus CK-2 inverted microscope, 37oC water bath, Hypoxia chamber with flow meter, Sf-9 cell culture spinner flasks, separate magnetic stirrers in the CO2 incubators and two refrigerator freezers. In addition, the cell sorting (primary mouse lung endothelial cells preparation) equipment includes surgical/dissecting tools, 4 sets of stainless steel cannula (14g)-VWR BD1789, 4”, sieve-bellco glass Co.1-800-257-7043 and 1985-18500 Cellector pan 1985-00100 140µm screen. In addition recently our laboratory acquired JULI a microscope to capture live videos and images of cells which are undergoing apoptosis. The JULI a microscope is connected to a data acquisition and transfer PC.

The Cell Signaling Laboratory has 7 PCs and 4 Mac with all the required software. The computers provide access to the World Wide Web services. One exclusively dedicated color laser printer is located in the Cell Signaling Lab on the first floor. Individual LASER printer (HP LASER Jet 4000 TN) is located in the office of Dr. Sudhakar. In addition, Dr. Sudhakar has exclusive use of portable MacPro, MacAir NoteBooks and iPAD-2 for data analysis, manuscript preparation and presentations. MacPro Note Book and desk top Mac computers also have the Mac Vector 5.0 to perform primary and secondary structure analysis on DNA and protein sequences and other research laboratory software includes Endnote X1, Adobe Photoshop CS2, Adobe Illustration CS3, CanvasTM 8; NIH Image 1.62 and CA-Cricket Graph III, etc.

Staff

Sudhakar Akul Yakkanti, Ph.D., Director of Cell Signaling, Retinal & Tumor Angiogenesis Laboratory

Chandra S. Boosani, Ph.D., (Research Associate-1): Dr. Boosani is an expert in molecular cloning, transgenic mice and adenoviruses development. His work in the laboratory includes cloning, expression, purification and in-vitro and in-vivo functional characterization of anti-angiogenic and anti-tumorigenic proteins which were developed in the laboratory.

Venugopal Gunda, Ph.D., (Post doctoral Fellow-1): Dr. Gunda is an expert in recombinant DNA technology including molecular cloning, transgenic mice and knockout mice generation. His work includes functional characterization of transgenic and knockout mice, expression, purification and functional characterization of various endogenous angioinhibitor molecules.

RaJ K. Verma, Ph.D., (Post doctoral Fellow-2): Dr. Verma is an expert in recombinant protein expression, purification and functional characterization of proteins including apoptosis and cell signaling studies.

Smita Pawar, Ph.D., (visiting Professor from Osmania University, India): Dr. Pawar DST-BOYSCAST FELLOWS studies partly at Cell Signaling Laboratory related to the molecular mechanism(s) underlying the regulation of tumor angiogenesis and tumor growth and also partly at University of Nebraska Medical Center, about protein-protein interactions in regulation of angiogenesis.

Representative Publications (citations Dec 2011):

  1. Sudhakar, A., Krishnamoorthy, T., Jain, A., Chatterjee, U., Hasnain, S.E., Kaufman, R.J. & Ramaiah, K.V.A. (1999). Serine 48 in initiation factor 2 alpha (eIF2 alpha) is required for high-affinity interaction between eIF2 alpha(P) and eIF2B. Biochem. 38, 15398-15405. Citation 23.
  2. Sudhakar, A., Ramachandran, A., Ghosh, S., Hasnain, S.E., Kaufman, R.J. & Ramaiah, K.V.A. (2000). Phosphorylation of serine 51 in initiation factor 2 alpha (eIF2 alpha promotes complex formation between eIF2 alpha(P) and eIF2B and causes inhibition in the guanine nucleotide exchange activity of eIF2B. Biochem. 39, 12929-12938. Citation 63.
  3. Hamano, Y., Grunkemeyer, J.A., Sudhakar, A., Zeisberg, M., Cosgrove, D., Morello, R., Lee, B., Sugimoto, H. & Kalluri, R. (2002). Determinants of vascular permeability in the kidney glomerulus. J. Biol. chem. 277(34), 31154-31162.
  4. Sudhakar, Ax, Maeshima, Yx, Lively, J.C., Ueki, K., Kharbanda, S., Kahn, C.R., Sonenberg, N., Hynes, R.O. & Kalluri R. (2002). Tumstatin, an endothelial cell-specific inhibitor of protein synthesis. Science 295(5552), 140-143 (xequal contributing authors). Citation 359.
  5. Sudhakar, A.x, Tsukaguchi, Hx, , Le, T.C., Trang, N., Yao, J., Schwimmer, J.A., Schachter, A.D., Poch, E., Abreu, P.F., Appel, G.B., Pereira, A.B., Kalluri, R. & Pollak, M.R. (2002). NPHS2 mutations in late-onset focal segmental glomerulosclerosis: R229Q is a common disease-associated allele. J. Clin. Invest. 110(11), 1659-1666. (xequal contributing authors]. Citation 143.
  6. Hamano, Y., Zeisberg, M., Sugimoto, H., Lively, J., Maeshima, Y., Yang, C., Hynes, R.O., Werb, Z., Sudhakar, A. & Kalluri, R. (2003). Physiological levels of tumstatin, a fragment of collagen IV α3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via αvβ3 integrin. Cancer Cell 3, 589-601. Citation 304.
  7. Sudhakar, A., Sugimoto, H., Yang, C.Q., Lively, J., Zeisberg, M. & Kalluri R. (2003). Human tumstatin and human endostatin exhibits distinct antiangiogenic activities mediated by &alphaVβ3 and α5β1 integrins. Proc. Nat. Acad. Sci. 100, 4766-4771. Citation 319.
  8. Sugimoto, H., Hamano, Y., Charytan, D., Cosgrove, D., Kieran, M., Sudhakar, A. & Kalluri R. (2003). Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria. J. Biol. Chem. 278(15), 12605-12608. Citation 294.
  9. Yang, C., Zeisberg, M., Mosterman, B., Sudhakar, A., Yerramalla, U.L., Holthaus, K.A., Xu, L., Eng, F., Afdhal, N. & Kalluri, R. (2003). Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors. Gastroenterol. 124, 147-159.
  10. Hamano, Y., Sugimoto, H., Soubasakos, M.A., Kiemar, M., Olsen, B.R., Lawler, J., Sudhakar, A. & Kalluri, R. (2004). Thrombospondin-1 associated with tumor microenvironment contributes to low-dose cyclophosphamide-mediated endothelial cell apoptosis and tumor growth suppression. Cancer Res. 64, 1570-1574. Citation 119.
  11. Krishna, T.H., Mahipal, S., Sudhakar, A., Sugimoto, H., Kalluri, R. & Rao, K.S. (2005). Reduced DNA gap repair in aging rat neuronal extracts and its restoration by DNA polymerase beta and DNA-ligase. J. Neurochem. 92(4), 818-823.
  12. Sudhakar, A., Nyberg, P., Keshamouni, V.G., Mannam, A.P., Li, J., Sugimoto, H., Cosgrove, D.E. & Kalluri, R. (2005). Human α1 type IV collagen NC1 domain exhibits distinct antiangiogenic activity mediated by α1β1 integrin. J. Clin. Invest. 115(10), 2801-2810. Citation 76.
  13. Sund, M., Hamano, Y., Sugimoto, H., Sudhakar, A., Soubasakos, M., Yerramalla, U., Benjamin, L.E., Lawler, J., Kieran, M., Shah, A. & Kalluri, R. (2005). Function of endogenous inhibitors of angiogenesis as endothelium-specific tumor suppressors. Proc. Natl. Acad. Sci. 102, 2934-2939. Citation 141.
  14. Boosani, C.S. & Sudhakar, A. (2006). Cloning, purification, and characterization of a non-collagenous anti-angiogenic protein domain from human α1 type IV collagen expressed in Sf9 cells. Protein Expr. Purif. 49(2), 211-218. Citation 15.
  15. Keshamouni, V.G., Michailidis, G., Grasso, C.S., Anthwal, S., Strahler, J.R., Walker, A., Arenberg, D.A., Reddy, R.C., Sudhakar, A., Thannickal, V.J., Standiford, T.J., Andrews, P.C. & Omenn, G.S. (2006). Differential protein expression profile by iTRAQ-2DLC-MS/MS reveals a metastatic phenoltype in TGF-b-induced epithelial-mesenchymal transition in human lung cancer cells. J. Proteome Res. 5(5), 1143-1154.
  16. Boosani, C.S., Mannam, A.P., Cosgrove. D., Silva, R., Hodivala-Dilke, K.M., Keshamouni, G.V. & Sudhakar, A. (2007). Regulation of COX-2 mediated signaling by α3 type IV non-collagenous domain in tumor angiogenesis. Blood 110(4), 1168-1177. Citation 25.
  17. Boosani, C.S. & Sudhakar, A. (2008). Molecular cloning and functional characterization of mouse 3(IV)NC1. Clin. Med.: Oncol. 2, 73-81. Citation 10.
  18. Nyberg, P., Xie, L., Sugimoto, H., Colorado, P., Sund, M., Holthaus, K., Sudhakar, A., Salo, T. & Kalluri, R. (2008). Characterization of the anti-angiogenic properties of arresten, an α1β1 integrin-dependent collagen-derived tumor suppressor. Exp. Cell Res. 314, 3292-3305. Citation 16.
  19. Sudhakar, A. & Boosani, C.S. (2008). Inhibition of tumor angiogenesis by tumstatin: Insights into signaling mechanisms and implications in cancer regression. Pharm. Res. 25, 2731-2739. Citation 24.
  20. Boosani, C.S., Nalabothula., N, Munugalvadla, V., Cosgrove, D., Keshamouni, V.G., Sheibani, N. & Sudhakar, A. (2009). FAK and p38-MAP kinase-dependent activation of apoptosis and caspase-3 in retinal endothelial cells by α1(IV)NC1. Invest. Ophthalmol. Vis. Sci. 50, 4567-4575. Citation 6.
  21. Boosani, C. S., Varma, A. K & Sudhakar, A. (2009). Identification of an expression system for determining the purity and in-vitro and iv-vivo activity of tumstatin. J Canc Sci Ther. 1(1): 008-018.
  22. Boosani, C. S., Nalabothula, N., Sheibani, N & Sudhakar, A. (2010). Inhibitory effects of α1(IV)NC1 on bFGF induced proliferation, migration and matrix metalloproteinase-2 activation of mouse retinal endothelial cells. Curr Eye Res. 35(1):45-55. Citation 7.
  23. Bhanu, R. K., Ramaiah, K. V. A., Hinnebusch, A. G., Bhuyan, A. K., Sivasai K. S. R., Sudhakar, A and Burela, L. (2010). Trends in wheat germ cell free protein expression system with an emphasis on up scaling and industrial application. Ind J of Sci & Tech. 3 (3): 349-354).
  24. Palil, R., Das, S., Stanley, A., Yadav, L., Sudhakar A and Varma, A. K (2010). Optimized hydrophobic interactions and hydrogen bonding at the target-ligand interface lead the pathways of drug-designing. PLoS One. 2010 Aug 16;5(8). pii: e12029.
  25. Gunda, V., Wang, S., Sheibani, N. & Sudhakar, A. (2011). Inhibitory effect of tumstatin on corneal neovascularization both in-vitro and in-vivo. J. Clinic Experiment Ophthalmol. (Accepted for publication, publication ahead of print).
  26. Boosani, C.S., Gunda, V., Varma, A.K & Sudhakar, A.Y. (2011). Functional characterization of a fusion angioinhibitor molecule combostatin. J. Canc. Sci. Ther (Accepted for publication, publication ahead of print).
  27. Boosani, C.S., Gunda, V., Varma, A.K., Overall C. M., Wang, S., Sheibani, N. & Sudhakar, A.Y. (2011). Tumstatin inhibits choroidal neovascularization by inhibiting MMP-2 activation in-vitro and in-vivo. Mol. Vision (Accepted for Publication).
  28. Boosani, C.S., Gunda V., Munugalavadla, V., Kalluri, R & Sudhakar, A.Y. (2011). Tumstatin inhibits tumor angiogenesis by inducing caspase-3 mediated endothelial cell apoptosis both in-vitro and in-vivo. Clinical Cancer Res. (under review, MS # CCR-09-2910).
  29. Gunda, V., Boosani, C.S., Verma, R. K., Guda, C and Sudhakar, A. Y (2012). Purification of soluble, anti-angiogenic non-collagenous protein domain of human α6 type IV collagen using L-arginine mediated renaturation and size exclusion chromatography. Protein Expr. Purif. (Under review, Ms. No.: PREP-11-448).

b. Review articles:

  1. Sudhakar, A. (2007). Signaling mechanisms of collagen derived endogenous angiogenesis inhibitors. Sterling Life Sciences Journal, August 2007, pages 1-9.
  2. Sudhakar, A. & Boosani, C.S. (2007). Signaling mechanisms of endogenous angiogenesis inhibitors derived from type IV collagen. Gene Regulation and System Biology 1, 217-226. Citation 10.
  3. Sudhakar, A. & Boosani, C.S. (2008). Inhibition of tumor angiogenesis by tumstatin: Insights into signaling mechanisms and implications in cancer regression. Pharm. Res. 25, 2731-2739. Citation 24.
  4. Hong, H., Perry, G., Sörgel, F., Gedela, S., Sudhakar, A. (2009). What is the Journal of Bioequivalence & Bioavailability. JBB 1(1), 001-002.
  5. Sudhakar, A. (2009). The matrix reloaded: new insights from type IV collagen derived endogenous angiogenesis inhibitors and their mechanism of action. JBB 1(2), 052-062. Citation 5.
  6. Sudhakar, A. (2010). History of Cancer, Ancient and Modern Treatment Methods. J Canc Sci Ther. 1(2): i-iv.
  7. Singh, R. K., Sudhakar, A and Lokeshwar B. L. (2010). Role of Chemokines and Chemokine Receptors in Prostate Cancer Development and Progression. J. Canc. Sci. Ther. 2(4): 089-094.
  8. Singh, R. K., Sudhakar, A and Lokeshwar B. L. (2011). From Normal Cells to Malignancy: Distinct Role of Pro-inflammatory Factors and Cellular Redox Mechanism in Human Carcinogenesis. J Canc Sci Ther. 3(4): 070-075.
  9. Gunda V and Sudhakar, A.Y (2011). Extra Cellular Matrix Derived Endogenous Angiogenesis Inhibitors and Their Applications. Nova Science Publishers invited review article (Invited review article).
  10. Boosani, C. S., and Sudhakar, A.Y (2011). Proteolytically derived endogenous angioinhibitors originating from the Extracellular Matrix. Pharmaceuticals Special Issue "Angiogenesis Inhibitors (accepted for publication).
  11. Gunda V and Sudhakar, A.Y (2011). Regulation of Tumor Angiogenesis and Choroidal Neovascularization by Endogenous Angioinhibitors. J. Clinic Experiment Ophthalmol (Acceptedfor publication) (Invited review article).

c. Book chapters:

  1. Sudhakar, A. & Kalluri, R. (2010). Molecular mechanism of angiostasis. In M.R. Dana (Ed.), Encyclopedia of the Eye. 3: 852-859.
  2. Sudhakar, A. & Kalluri, R. (2010). Molecular mechanism of angiostasis. In Darlene A. Dartt, Reza Dana, Patricia D”Amore, Jerry Y. Niederkorn (Ed.). Immunology, Inflammation and Diseases of the Eye. 270-277.
  3. Gunda V and Sudhakar, A.Y (2011). Regulation of angiogenesis in Choroidal Neovascularization of Age Related Macular Degeneration by Endogenous Angioinhibitors. InTech Publishers (Ophthalmology Book Chapter) (Invited expert review article).

Underlined: Corresponding author

Summary of Research Program

For Physicians and Scientists

The central focus of Cell Signaling, Retinal and Tumor Angiogenesis laboratory is the study of cellular microenvironment as determined by extra cellular matrix (ECM) and basement membranes (BM) in the regulation of the cellular behavior during health and disease. This fundamental interest is being translated into two major areas in the laboratory.

Tumor Angiogenesis Area

The Cell Signaling Angiogenesis laboratory was established in early 2004, and is screening important angioinhibitory/anti-cancer molecules from vascular basement membrane (VBM). This approach aims to understand the process of angiogenesis in the laboratory that led to the identification of new angioinhibitory protein fragments from basement membranes. Discoveries of such matrix derived endogenous angioinhibitors are providing new insights into the progression of cancer. This new area of research developed in our laboratory includes the development of multi-domain fusion protein, named as Combostatin, and other non-collagenous domain from Type IV collagen.

Retinal Angiogenesis & Macular Degeneration Area:

The extracellular matrix (ECM), cell signaling and the emerging role of integrin signaling processes are important factors modulating macular degeneration. The new areas of research being developed in my laboratory from last 6 years include studies on the role of extracellular matrix derived angioinhibitors and their molecular signaling in regulation of Macular degeneration. Further research recently published/accepted five research articles aimed at understanding the signaling mechanism mediated by type IV collagen derived angioinhibitors role in choroidal neovascularisation in age related macular degeneration (CNV of AMD).

For Families

Cancer is currently one of the most prevalent causes of death in the United States of America. Current therapeutic options aim only to slow the progression of cancer disease. Therefore, a renewed effort must be made to identify relevant endogenous cancer inhibitor molecules that could be exploited as therapeutic drugs. Cell Signaling and Tumor Angiogenesis laboratory is screening important anti-cancer molecules, which are released into circulating blood of cancer patients. Several of these endogenous circulating molecules were cloned in the laboratory and identified them as inhibitors of solid tumor growth (cancer). It should be noted that this method of treatment is not likely to have side effects, since these proteins are normally found in the body. Such a treatment option would be far superior to surgery, radiation, and chemotherapy that have quite severe side effects.

Age related macular degeneration (AMD) is also one of the major causes of blindness in the elderly people in the world. Researchers long ago noticed that progression of AMD related to abnormal growth of blood vessels and leakiness in the macula region or retina in the eye leads to this devastating eye disease. We have tested several of the endogenous circulating anti-angiogenesis molecules that were inhibiting proliferation of different endothelial cells and may be used to treat AMD in near future. We therefore expect that our laboratory studies on endogenous angiogenesis inhibitor molecules will advance cure for cancer and AMD.
 

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