Gene expression profiling after angiogenesis inhibitor treatment


The angiogenic process can be summarized as cell activation by a lack of oxygen releases angiogenic molecules that attract inflammatory and endothelial cells and promote their proliferation. Several protein fragments produced by the digestion of the blood-vessel walls intensify the proliferative activity of endothelial cells. Acetyl salicylic acid is often used as an analgesic drug to relieve minor aches and pains, a drug with antitumour activity and an anti-inflammatory medication. In our experiments we propose using the angiogenesis model and photodynamic therapy (PDT) for observing changes in angiogenesis after treatment, and also for increasing the effect of PDT by addition of angiogenesis inhibitors.

Keywords:
angiogenesis, photodynamic therapy, acetyl salicylic acid


Authors: Adéla Hanáková 1,2;  Klara Pizova 1,2;  Outi Huttala 3;  Jertta-Riina Sarkanen 3;  Tuula Heinonen 3;  Dagmar Jirova 4;  Kristina Kejlova 4;  Hana Kolarova 1,2
Authors place of work: Department of Medical Biophysics, Faculty of Medicine and Dentistry Palacky University, Olomouc, Czech Republic 1;  Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry Palacky University, Olomouc, Czech Republic 2;  Finnish Center for Alternative Methods, Medical School, University of Tampere, Tampere, Finland 4National Institute of Public Health, Prague, Czech Republic 3
Published in the journal: Lékař a technika - Clinician and Technology No. 1, 2014, 44, 33-38
Category: Původní práce

Summary

The angiogenic process can be summarized as cell activation by a lack of oxygen releases angiogenic molecules that attract inflammatory and endothelial cells and promote their proliferation. Several protein fragments produced by the digestion of the blood-vessel walls intensify the proliferative activity of endothelial cells. Acetyl salicylic acid is often used as an analgesic drug to relieve minor aches and pains, a drug with antitumour activity and an anti-inflammatory medication. In our experiments we propose using the angiogenesis model and photodynamic therapy (PDT) for observing changes in angiogenesis after treatment, and also for increasing the effect of PDT by addition of angiogenesis inhibitors.

Keywords:
angiogenesis, photodynamic therapy, acetyl salicylic acid

Introduction

The vascular network regulates the body homeostasis by transporting oxygen, liquids, nutrients, cells and signaling molecules to all part of the body and disposing of waste from the tissues [1].

Normal blood vessel formation takes place in two main ways by: vasculogenesis and angiogenesis [2, 3].

Vasculogenesis is the differentiation de novo of vascular endothelial cells from precursor cells known as angioblasts during embryonic development [4].The endothelial cell lattice created by vasculogenesis then serves as a scaffold for abiogenesis [3].

Angiogenesis is the formation of new capillary sprouts from pre-existing blood vessels [5]. After the primary capillary plexus is formed, it is remodeled by the sprouting and branching of new vessels from preexisting ones. The normal mechanism of angiogenesis depends on the coordination of several independent processes, for example removal of pericytes from the endothelium and destabilization of the vessel by angiopoietin-2 (Ang2) and shift of endothelial cells from a stable, growth-arrested state to a plastic, proliferative phenotype [3].

VEGF is other candidate as a potential regulator of angiogenesis. This emanates from the fact that VEGF is a secreted protein, its binding sites are selectively expressed in endothelial cells in vitro and in vivo and the expression of its mRNA correlates with blood vessel growth. Vascular endothelial growth factor (VEGF, VEGF-A) is a major regulator of physiological and pathological angiogenesis. Several VEGF inhibitors have been approved by the FDA for the treatment of advanced cancer and neovascular age-related macular degeneration [6]. Further, vascular endothelial growth factor (VEGF)-induced hyperper-meability allows for local extravasation of proteases and matrix components from the bloodstream [3].

Angiogenesis is also stimulated by fibroblast growth factor (FGF) [4]. FGF is synthesized and secreted by human adipocytes and the concentration of bFGF correlates with the BMI in blood samples [7]. Additionally, bFGF is a critical component of human embryonic stem cell culture medium; the growth factor is necessary for the cells to remain in an undifferentiated state, although the mechanisms by which it does this are poorly defined [8]. 

It must be pointed out, that the remodelling and growth of the vasculature is a normal bodily process, particularly in wound healing, in pathological disease processes such as cancer and in the female menstrual cycle and hence in certain cases it may be important that the effects of gene therapy, once delivered, are strictly controlled [3, 4, 9].

One potentially useful toxic gene product is the tumour necrosis factor-α (TNF-α). TNF-α is toxic for proliferating, but not quiescent, microvascular endothelium. TNF-α is by itself active and therefore needs to be regulated at a transcriptional level. However, its wide-ranging toxicity against tumours make it well worth further investigation [9].

Another interesting gene platelet-derived growth factor (PDGF) is released in high concentrations during tissue repair and inflammatory processes, first from platelet alpha granules and subsequently by activated macrophages. Other peptide growth factors that have been shown to be actively released from platelets and activated macrophages, together with PDGF, include epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, TGF-beta, and basic fibroblast growth factor (bFGF) [10].

Experiments

The study aimed to investigate the use of acetyl salicylic acid (ASA) for inhibiting angiogenesis (angiogenesis model in vitro was designed by a scientific team from FICAM). The experiments were carried out with co-culture of two types of cells: hASC (human adipose stem cells, 22 000 cells/well) and HUVEC (human umbilical vein endothelial cells, 4 000 cells/well). Cells were treated with 1mM ASA in concentrations 2, 1, 0.5, 0.25 and 0.125 mmol/l.

Second, the isolation of RNA was performed using RNeasy mini kit (Qiagen). Consequently RNA was transcribed to cDNA (RT2 Prolifer PCR Array). Finally, real-time PCR was carried out using 96-well array: 84 target genes, 5 reference genes and 7 control genes. The expression of genes was assessed using the program BIO-RAD CFX Manager.

Graphs 1–7 show the first results of real-time PCR for selected interesting genes only. All results represent samples treated by ASA. The experiments were performed using 96-well arrays.

Graphs 1, 2 and 3 show the the curves of three selected reference genes Beta-2-microglobulin, very often used Glyceraldehyde-3-phosphate dehydrogenace and Hypoxanthine phosphoribosyltransferase. 

Fig. 1: Curve of Beta-2-microglobulin.
Fig. 1: Curve of Beta-2-microglobulin.

Fig. 2: The curve of Glyceraldehyde-3-phosphate dehydrogenace.
Fig. 2: The curve of Glyceraldehyde-3-phosphate dehydrogenace.

Fig. 3: The curve of Hypoxanthine phosphoribosyltransferase.
Fig. 3: The curve of Hypoxanthine phosphoribosyltransferase.

The amplification curve (graph 4) is for Fibroblast growth factor 2 (FGF2) which mediates the formation of new blood vessels (angiogenesis) during wound healing of normal tissues and tumour development. It is an important mediator responsible for induction of endothelial precursor cells from the mesoderm [11]. It is apparent that the FGF2 amplification curve began to increase several cycles after reference curves but it is still very early to draw any conclusions.

Fig. 4: The curve of Fibroblast growth factor 2 (basic).
Fig. 4: The curve of Fibroblast growth factor 2 (basic).

Angiopoietin 1 (graph 5) is an important protein because of its role in vascular development and angiogenesis. Its amplification curve predicates very high and early expression. It plays a critical role in mediating reciprocal interactions between the endothelium and surrounding matrix and mesenchyme [12, 13].

Fig. 5: Curve of Angiopoetin 1.
Fig. 5: Curve of Angiopoetin 1.

Platelet-derived growth factor alpha (graph 6) polypeptide is the factor for cells of mesenchymal origin.

Fig. 6: The curve of Platelet-derived growth factor alpha polypeptide.
Fig. 6: The curve of Platelet-derived growth factor alpha polypeptide.

Transforming growth factor alpha (TGFα) (graph 7) is a ligand for the epidermal growth factor receptor and activates a signaling pathway for cell proliferation, differentiation and development [14].  

Fig. 7: The curve of Transforming growth factor alpha (TGFα)
Fig. 7: The curve of Transforming growth factor alpha (TGFα)

Conclusion

Our research laboratory is focused on photodynamic therapy (PDT) in vitro. This combinates visible light of an appropriately wavelength and non-toxic chemical agent called photosensitiser. This combination is used to destroy cancer cells or to eradicate bacterial cells [15-25].   

Currently new strategies and alternatives are needed to increase the options for treatment of cancer.Photodynamic therapy (PDT) has become progressively established as a mode of treatment of malignant as well as non-malignant diseases which are characterized by the occurrence of unwanted or harmful cells [26].   

Combining angiogenic inhibitors with chemotherapy and radiotherapy has demonstrated the potential to be an attractive and effective approach to cancer treatment. Ferrario and colleagues first demonstrated that PDT efficacy could be enhanced by combining angiogenic inhibitors with the treatment regimen [27].

Acetylsalicylic acid (ASA, aspirin) appears to be a significant inhibitor of angiogenesis. It has remained the most commonly-used drug for relieving pain, inflammatory symptoms, and fever. Recently, ASA has been shown to reduce the risk for colorectal cancer by as much as 40%, a property that is shared with other nonsteroidal anti-inflammatory drugs (NSAIDS) [28].

Acknowledgements

Work was supported by grants CZ.1.05/2.1.00/01.0030 and CZ.1.07/2.4.00/17.0015, and NT 14375-3/2013 from the Ministry of Health.

Mgr. Adéla Hanáková, Ph.D.

Department of Medical Biophysics

Faculty of Medicine and Dentistry

Palacky University in Olomouc

Hněvotínská 3

775 15 Olomouc

Czech Republic

E-mail: a.hanakova@upol.cz

tel.: +420 585 632 200


Zdroje

[1] Rivron, N. C., Liu, J., Rouwkema, J., Boer de, J., Blitterswijk van, C. A. Engineering vascularised tissues in vitro. European Cells & Materials, 2008, vol. 15. p. 27-40.

[2] Moon, J. J, West, J. L. Vascularization of engineered tisssues: approaches to promote angiogenesis in biomaterials. Current Topics in Medicinal Chemistry, 2008, vol. 8, no. 4, p. 300-310 (11).

[3] Papetti, M., Herman, I. M. Mechanisms of normal and tumor-derived angiogenesis. Am J Physiol Cell Physiol., 2002, vol. 282, no. 5, p. C947-70.

[4] Klagsbrun, M. and Moses, M. A. Molecular angiogenesis. Chem Biol., 1999, vol. 6, no. 8, p. R217-24.

[5] Carmeliet, P., Jain, R. K. Molecular mechanisms and clinical applications of angiogenesis. Nature, 2011, vol. 473, no. 7347, p. 298-307.

[6] Ferrara, N. Vascular Endothelial Growth Factor. Arterioscler Thromb Vasc Biol., 2009, vol. 29, p. 789-791.

[7] Kühn, M. C, Willenberg, H. S, Schott, M., Papewalis, C., Stumpf, U., Flohé, S., Scherbaum, W. A., Schinner, S. Adipocyte-secreted factors increase osteoblast proliferation and the OPG/RANKL ratio to influence osteoclast formation. Mol Cell Endocrinol., 2012, vol. 349, no. 2, p. 180–188.

[8] Pereira, R. C., Economides, A. N., Canalis, E. Bone morphogenetic proteins induce gremlin, a protein that limits their activity in osteoblasts. Endocrinology, 2000, vol. 141, no. 12, p. 4558–63.

[9] Fan, T. P., Jaggar, R., Bicknell, R. Controlling the vasculature: Angiogenesis, anti-angiogenesis and vascular targeting of gene therapy. Trends in pharmacological science, 1995, vol. 16, no. 2, p. 57–66.

[10] Pinzani, M., Gesualdo, L., Sabbah, G. M., Abboud, H. E. Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells. J Clin Invest., 1989, vol. 84, no. 6, p. 1786–1793.

[11] Mehta, D., Malik, A. B. Signaling Mechanisms Regulating Endothelial Permeability. Physiol Rev., 2006, vol. 86, p. 279-367.

[12] Davis, S., Aldrich, T. H., Jones, P.F., Acheson, A., Compton, D. L., Jain, V., Ryan, T. E., Bruno, J., Radziejewski, C., Maisonpierre, P. C., Yancopoulos, G. D. Isolation of Angiopoietin-1, a Ligand for the TIE2 Receptor, by Secretion-Trap Expression Cloning. Cell, 1996, vol. 87, no. 7, p. 1161–1169.

[13] Gale, N. W., Yancopoulos, G. D. Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, Angiopoietins, and ephrins in vascular development. Genes Dev., 1999, vol. 13, no. 9, p. 1055-1066.

[14] Kumar, V., Bustin, S.A., McKay, I. A. Transforming growth factor alpha. Cell Biology International, 1995, vol. 19, no. 5, p. 373–388.

[15] Kolarova, H., Nevrelova, P., Bajgar, R., Jirova, D., Kejlova, K., Strnad, M. In vitro photodynamic therapy on melanoma cell lines with phthalocyanine. Toxicol In Vitro, 2007,vol. 21, no. 2, p. 249-253.

[16] Kolarova, H., Bajgar, R., Tomankova, K., Nevrelova, P., Mosinger, J. Comparison of sensitizers by detecting reactive oxygen species after photodynamic reaction in vitro. Toxicol In Vitro, 2007, vol. 21, no. 7, p.1287-1291.

[17] Kolarova, H., Lenobel, R., Kolar, P., Strnad, M. Sensitivity of differentcell lines to phototoxiceffect of disulfonated chloroaluminium phthalocyanine. Toxicol In Vitro, 2007, vol. 21, no. 7, p. 1304-1306.

[18] Kolarova, H., Bajgar, R., Tomankova, K., Krestyn, E., Dolezal, L., Halek, J. In vitro study of reactive oxygen species production during photodynamic therapy in ultrasound-pretreated cancer cells. Physiol Res, 2007, vol. 56 Suppl 1), p. S27-32.

[19] Kolarova, H., Nevrelova, P., Tomankova, K., Kolar, P., Bajgar, R., Mosinger, J. Production of reactive oxygen species after photodynamic therapy by porphyrin sensitizers. Gen Physiol Biophys, 2008, vol. 27, no. 2, p. 101-105.

[20] Tomankova, K., Kolarova, H., Bajgar, R. Study of photodynamic and sonodynamic effect on A549 cell line by AFM and measurement of ROS production. Phys Stat Sol (a), 2008, vol. 205, no. 6, p. 1472–1477.

[21] Kolarova, H., Tomankova, K., Bajgar, R., Kolar, P., Kubinek, R. Photodynamic and sonodynamic treatment by phthalocyanine on cancer cell lines. Ultrasound Med Biol, 2009, vol. 35, no. 8, p. 1397-1404.

[22] Krestyn, E., Kolarova, H., Bajgar, R., Tomankova, K. Photodynamic properties of ZnTPPS4, ClAlPcS2 and ALA in human melanoma G361 cells. Toxicol In Vitro, 2010, vol. 24, no. 1, p. 286-291.

[23] Binder, S., Kolarova, H., Tomankova, K., Bajgar, R., Daskova, A., Mosinger, J. Phototoxic effect of TPPS4 and MgTPPS4 on DNA fragmentation of HeLa cells. Toxicol In Vitro, 2011, vol. 25, no. 6, p. 1169-1172.

[24] Hanakova, A., Bogdanova, K., Tomankova, K., Binder, S., Bajgar, R., Langova, K., Kolar, M., Mosinger, J., Kolarova, H. Study of photodynamic effects on NIH 3T3 cellline and bacteria. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 2012, vol. 156, doi: 10.5507/bp.2012.057.

[25] Hanakova, A., Bogdanova, K., Tomankova, K., Pizova, K., Malohlava, J., Binder, S., Bajgar, R., Langova, K., Kolar, M., Mosinger, J., Kolarova, H. The application of antimicrobial photodynamic therapy on S. aureus and E. coli using porphyrin photosensitizers bound to cyclodextrin. Microbiol Res, 2014, vol. 169, no. 2-3, p. 163-170.

[26] Plaetzer, K., Kiesslich, T., Verwanger, T., Krammer, B. The Modes of Cell Death Induced by PDT: An Overview. Med. Laser, 2003, vol. 18, p. 7–19.

[27] Bhuvaneswari, R., Gan, Y. Y., Soo, K. C., Olivo, M. The effect of photodynamic therapy on tumor angiogenesis. Cell. Mol. Life Sci., 2009, vol. 66, no. 14, p. 2275–2283.

[28] Yu, H. G., Huang, J. A., Yang, Y. N., Huang, H., Luo, H. S., Yu, J. P., Meier, J. J., Schrader, H., Bastian, A., Schmidt, W. E., Schmitz, F. The effects of acetylsalicylic acid on proliferation, apoptosis, and invasion of cyclooxygenase-2 negative colon cancer cells. European Journal of Clinical Investigation, 2002, vol. 32, no. 11, p. 838–846.

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Článek vyšel v časopise

Lékař a technika

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2014 Číslo 1

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