SAMEER KALGHATGI

           

This page gives a brief overview of Angiogenesis and describes my research on Non-Thermal Plasma treatment of endothelial cells for promotion or inhibition of angiogenesis by enhancing or regressing proliferation of endothelial cells via release of fibroblast growth factor 2 (FGF-2)

Angiogenesis

Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels. Angiogenesis is a normal process in growth and development, as well as in wound healing. It also plays an important role during the adult life span, as a "repair mechanism" for damaged tissues. However, it is also a fundamental step in the transition of tumors from a dormant state to a malignant one (1, 2).

The endothelial cells that line the blood vessels play a pivotal regulatory role in the execution of angiogenesis. The sequence of events in endothelial cells that follow the initiation of angiogenesis consists of of three major steps as shown in Figure 1.

1. Proteolysis: Growth factors promote synthesis of proteases that degrade the extracellular matrix of an existing blood vessel in response to either a tumor derived or a natural angiogenic stimulus.

2. Migration/Proliferation: Growth Factors like VEGF/FGF promote migration of endothelial cells to the site of tumor growth or a wound followed by proliferation to increase the number of endothelial cells.

3. Maturation and Differentiation: In the final step the endothelial cells mature and differentiate in to a tube like structure to form a functional vessel

                                                                                                                                                       Figure 1.

Endothelial cells, which line all blood contacting surfaces in the body, control many aspects of the vasculature ranging from vascular tone to coagulation to inflammation. Endothelial cells also play a guiding role in angiogenesis, the growth of new blood vessels from existing vessels. Endothelial cells produce and secrete angiogenic growth factors such as fibroblast growth factor-2 (FGF2), which in conjunction with many other signals induces endothelial cells to invade the surrounding tissue, proliferate, and develop into new blood vessels. Angiogenesis can be both helpful and harmful. In wound healing, angiogenesis is required at the wound site for rapid healing, whereas in cancer, angiogenesis blockade can starve a tumor and prevent its growth (2 - 4).

Non-thermal dielectric barrier discharge plasma is currently being developed for a wide range of medical applications, including skin sterilization, blood coagulation, malignant cell apoptosis, and wound healing. However, the effect of non-thermal plasma on the vasculature is unclear. Blood vessels are affected during plasma treatment of many tissues and may themselves be an important potential target for clinical plasma therapy. Interaction of non-thermal plasma with the vasculature is important to understand prior to treating any vascularized tissue. We further hypothesize that plasma dose can be varied to grow or regress blood vessels.  We studied the effect of non-thermal plasma treatment on endothelial cells, which line the inner surface of blood vessels.

                 Figure 2. Non-Thermal Plasma Treatment Setup                    Figure 3. Non-Thermal Plasma Treatment of Cells

Using an in vitro model, we investigated the effects of non-thermal plasma on endothelial cells. Endothelial cell proliferation and death following non-thermal plasma treatment were measured. FGF2 release from endothelial cells, as well as its effect on cell proliferation, was quantified.

   

Figure 6Endothelial cells release FGF-2 post non-thermal plasma treatment at the dose where we observed enhanced cell proliferation. The level of FGF-2 increases up to 3 hours post plasma treatment. * p < 0.01 as compared to untreated cells.

Figure 7FGF2 release increases up to 3 hours post plasma treatment and then decreases up to 24 hours post plasma treatment. FGF2 was measured in conditioned media samples by ELISA. * p < 0.01 as compared to control (0 h). 

Figure 5Short plasma exposures induce low levels of apoptosis in endothelial cells whereas longer treatments (> 60 s) induce significant apoptosis in endothelial cells. # p < 0.05 as compared to untreated cells, * p < 0.01 as compared to untreated cells.

Figure 4Endothelial cell fold growth is enhanced in non-thermal plasma treated cells 5 days after treatment. Plasma treated cells were counted using a Coulter counter 1 and 5 days after treatment. * p < 0.01 as compared to control.

         

                                                                                                                 

 

 

Non-thermal plasma treatment at short exposures (up to 30 s; 4 J/cm2) was relatively non-toxic to endothelial cells (Figure 3) while treatment at longer exposures (60 seconds and higher; 8 J/cm2) led to apoptosis (Figure 4). Endothelial cells treated with plasma for 30 seconds demonstrated twice as much proliferation as untreated cells five days after plasma treatment. We conclude that high dose non-thermal plasma treatment induces endothelial cell death via apoptosis, lower doses induce endothelial cell proliferation.

             

 

 

 

 

Finally, mechanisms of non-thermal plasma effects were explored. One potential mechanism for this proliferative effect is non-thermal plasma induced release of fibroblast growth factor-2 (FGF-2), a potent angiogenic factor, which is mediated by neutral reactive oxygen species generated by non-thermal plasma (Figure 6 and Figure 7). These data suggest that low power non-thermal plasma treatment is a potential novel therapy for promotion or inhibition of endothelial cell mediated angiogenesis. Future work will investigate the role of FGF-2 in non-thermal plasma enhanced proliferation of endothelial cells and elucidate physical mechanisms of non-thermal plasma induced proliferation of endothelial cells.

References:
  1. Griffioen A, Molema G (2000) Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases and chronic inflammation. Pharmacol Rev 52: 237-268.
  2. Folkman J (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1: 27- 31.

  3. Judah Folkman. 2007. Angiogenesis: An Organizing Principle for Drug Discovery? Nature Reviews: Drug Discovery, 6:273-286.
  4. Judah Folkman. 2006. Angiogenesis, Annu. Rev. Med., 57:1-18.
Relevant Publications, Talks and Posters:
  1. S. Kalghatgi, Gary Friedman, Alexander Fridman, Alisa Morss Clyne. Endothelial Cell Proliferation is Enhanced by Low Dose Non-Thermal Plasma Through Release of Fibroblast Growth Factor - 2, Annals of Biomedical Engineering, Mar 2010, 38 (3): p 748 -757. FULL TEXT (PDF)

  2. S. Kalghatgi, Fridman A, Friedman G, Morss-Clyne A (2009) Proliferation Following Non-Thermal Plasma Is Related to Reactive Oxygen Species Induced Fibroblast Growth Factor-2 Release. Proceedings of the 31st International Conference of the IEEE Engineering in Medicine and Biology (EMBC), Sep 2nd-6th 2009, Minneapolis, Minnesota.

  3. S. Kalghatgi, Alexander Fridman, Alisa Morss-Clyne, Gary Friedman, Non-Thermal Plasma Enhances Endothelial Cell Proliferation Through Fibroblast Growth Factor-2 Release, ASME 2009 Summer Bioengineering Conference, Jun 17 - 21st 20009, Resort at Squaw Creek, Lake Tahoe, CA (poster presentation with Honorable mention at the PhD level student poster competition). Download Abstract (PDF). Download Poster (PDF)

  4. S. Kalghatgi, Alexander Fridman, Alisa Morss-Clyne, Gary Friedman, Non-Thermal Plasma Enhances Endothelial Cell Proliferation Through Fibroblast Growth Factor-2 Release, 36th IEEE International Conference on Plasma Science (ICOPS), May 29th - Jun 4th 2009, San Diego, California, USA. (invited talk). Download Talk (PDF)

  5. S. Kalghatgi, Fridman G, Fridman A, Friedman G, Morss-Clyne A (2008) Non-Thermal Dielectric Barrier Discharge Plasma Treatment of Endothelial Cells. Proceedings of the 30th International Conference of the IEEE Engineering in Medicine and Biology (EMBC), Aug 20th-Aug 24th 2008, Vancouver, Canada. FULL TXT (PDF).

  6. S. Kalghatgi, Alexander Fridman, Alisa Morss-Clyne, Gary Friedman, Non-Thermal Plasma Enhances Proliferation of Endothelial Cells, 2nd International Conference on Plasma Medicine (ICPM-2), Mar 16th – Mar 20th 2009, San Antonio, Texas, USA. (talk). Download Talk (PDF)

  7.  S. Kalghatgi, G Fridman, A Fridman, A Morrs-Clyne , G Friedman,Toxicity of Non-Thermal Plasma Treatment of Endothelial Cells (Contributed Talk), 35th IEEE International Conference on Plasma Science (ICOPS), June 15-19 2008, Karlsruhe, Germany. (talk and poster winning the Best Student Paper Award). Download Talk(PDF)

 

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