Methods for reducing and/or preventing excessive cellular apoptosis

Abstract

The invention is directed to methods for reducing the number of apoptotic cell deaths in a population of cells undergoing excessive cellular apoptosis. The invention is also directed to methods for preventing apoptotic cell death in a population of cells at risk for developing excessive cellular apoptosis. In particular, the invention is directed to methods for reducing or preventing excessive cellular apoptosis comprising exposing cells exhibiting or at risk for developing excessive cellular apoptosis to a cellular factor-containing composition called Amnion-derived Cellular Cytokine Solution (referred to herein as ACCS), which is obtained from the culturing of Amnion-derived Multipotent Progenitor (AMP) cells, or AMP cells.

Claims

1. A method for reducing the number of apoptosis-induced cell deaths in a population of cells undergoing excessive cellular apoptosis, the method comprising the step of contacting the population of cells undergoing excessive cellular apoptosis with a therapeutically effective dose of a composition selected from the group consisting of Amnion-derived Cellular Cytokine Solution (ACCS) and Amnion-derived Multipotent Progenitor (AMP) cells, such that the number of apoptosis-induced cell deaths is reduced.

2. The method of claim 1 wherein the ACCS comprises physiologic concentrations of VEGF, TGF2, Angiogenin, PDGF, TIMP-1 and TIMP-2, wherein the physiologic concentration is about 5.0-16 ng/mL for VEGF, about 3.5-4.5 ng/mL for Angiogenin, about 100-165 pg/mL for PDGF, about 2.5-2.7 ng/mL for TGF2, about 0.68 g/mL for TIMP-1, and about 1.04 g/mL for TIMP-2.

3. The method of claim 1 wherein the excessive cellular apoptosis occurs in a subject as a result of a condition selected from the group consisting of a radiation-induced injury, and an injury to nervous tissue.

4. The method of claim 1 wherein the ACCS or AMP cells are administered in combination with another agent.

5. The method of claim 4 wherein the other agent is selected from the group consisting of cytokines, chemokines, growth factors, antibodies, inhibitors, antibiotics, anti-fungals, anti-virals, immunosuppressive agents, anti-oxidants, and cells.

6. A method of protecting a population of cells at risk for developing excessive cellular apoptosis, the method comprising the step of contacting the population of cells at risk for developing excessive cellular apoptosis with a therapeutically effective dose of a composition selected from the group consisting of ACCS and AMP cells, such that the cells are protected from developing excessive cellular apoptosis.

7. The method of claim 6 wherein the ACCS comprises physiologic concentrations of VEGF, TGF2, Angiogenin, PDGF, TIMP-1 and TIMP-2, wherein the physiologic concentration is about 5.0-16 ng/mL for VEGF, about 3.5-4.5 ng/mL for Angiogenin, about 100-165 pg/mL for PDGF, about 2.5-2.7 ng/mL for TGF2, about 0.68 g/mL for TIMP-1, and about 1.04 g/mL for TIMP-2.

8. The method of claim 7 wherein the protecting of cells from excessive cellular apoptosis occurs in a subject that is at risk of developing a condition selected from the group consisting of, a radiation-induced injury, and an injury to nervous tissue.

9. The method of claim 6 wherein the protecting of cells from excessive cellular apoptosis occurs in a donated organ or tissue.

10. The method of claim 6 wherein the ACCS or AMP cells are administered in combination with another agent.

11. The method of claim 10 wherein the other agent is selected from the group consisting of cytokines, chemokines, growth factors, antibodies, inhibitors, antibiotics, anti-fungals, anti-virals, immunosuppressive agents, anti-oxidants, and cells.

Description

EXAMPLES

(1) The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the compositions and methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.

(2) Detailed information and methods on the preparation of AMP cell compositions, generation of ACCS, generation of pooled ACCS, detection of cytokines in non-pooled and pooled ACCS using ELISA, generation of PCS compositions, and generation of sustained-release CFS compositions can be found in U.S. Pat. Nos. 8,058,066 and 8,088,732, both of which are incorporated herein by reference.

Example 1

Evaluation of the Effect of ACCS on Apoptosis in Jurkat Cells

(3) Apoptosis is programmed cell death and is a necessary event for proper healing and tissue turnover involving a complex network of biochemical pathways. Dis-regulation of apoptosis is a cause of delayed wound healing as well as many chronic diseases.

(4) Depending on the extent of damage, cells will either apoptose or survive. Applicants have discovered that ACCS contains anti-apoptotic factors that may help some cells survive various insults that would typically lead to apoptosis. From a therapeutic perspective, these anti-apoptotic factors may benefit partially damaged cells such as those found in a wound by helping them survive and repopulate the wound bed quicker.

(5) Initial experiments were performed using a FITC Annexin V staining kit (BD Biosciences) and analyzed by Flow Cytometry.

(6) Brief description of the assay: In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer part of the plasma membrane. Annexin V is a 35-36 kDa Ca.sup.2+-dependent phospholipid-binding protein and has a high affinity for PS, thus it will bind to cells with exposed PS on the outer part of the plasma membrane. Since externalization of PS occurs in the earlier stages of apoptosis, FITC Annexin V staining can identify cells in the early stages of apoptosis. Membranes of dead and damaged cells are permeable to propidium iodide PI. For example, cells that are considered viable are FITC Annexin V and PI negative; cells that are in early apoptosis are FITC Annexin V positive and PI negative; and cells that are in late apoptosis or are already dead are both FITC Annexin V and PI positive.

(7) Initial experiments were done using Jurkat cells cultured in RPMI medium containing 10% FCS and antibiotic. Approximately 210.sup.6 Jurkatcells were treated overnight with 12 M camptothecin, an apoptosis inducer, and 1, 2 or 4ACCS treatment or control media and then stained the next day with Annexin V.

(8) ResultsIn cells treated with camptothecin only, the percentage of apoptotic cells was 48.2%, whereas untreated controls were 6.6% apoptotic. In the presence of both ACCS (1, 2 or 4) and camptothecin, the percentage of apoptotic cells was reduced to approximately 11%. While the percentage of apoptotic cells was reduced in the presence of ACCS, it did not seem to be dose-dependent at the tested doses.

Example 2

Evaluation of the Effect of ACCS on Apoptosis in Human Foreskin Fibroblasts Cells

(9) Additional experiments were performed using an apoptosis assay kit which tests for the activation of both caspase 3 and caspase 7, both of which are key biomarkers of apoptosis.

(10) Brief description of the assay: The Caspase-Glo 3/7 Assay (Promega) provides a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity, luciferase activity and cell lysis. Following caspase cleavage, a substrate for luciferase (aminoluciferin) is released, resulting in the luciferase reaction and the production of light. The higher the luminescence the more caspase activity in the sample.

(11) In this particular experiment, Human Foreskin Fibroblasts (HFF's) (20,000 cells/well) were plated in 96-well plates and then treated for 5 hrs with and without Staurosporine, an apoptosis inducer, in control media, ACCS, irradiated ACCS, or normal growth media. Substrate was added and the luminescence was read on a Biotek plate reader.

(12) ResultsIn the presence of either ACCS or irradiated ACCS, the caspase activation was significantly reduced. These results clearly indicate that there is a reduction in caspase activity with ACCS treatment compared to controls.

Example 3

Evaluation of the Effects of ACCS on Radiation Protection in Jurkat Cells

(13) Based on the above results, it was hypothesized that ACCS may protect cells from exposure to radiation, a known apoptosis inducer. Experiments were performed using Jurkat Cells to evaluate the potential of ACCS as a radioprotection agent. In one such experiment, in a total volume of 1 mL, 210.sup.6 Jurkat cells were irradiated at 1000, 2000 and 4000 RADs and apoptosis was analyzed by flow cytometry with Annexin V and the caspase luminescence assays. Treatments included control medium at 0.75 and ACCS at 0.75. All treatments were added immediately prior to irradiating the cell samples.

(14) ResultsThe results indicate that ACCS protects cells from ionizing radiation as indicated by a reduction (6.6, 19.4 and 18.2% reduction, respectively, for each irradiation dose as compared to control) in the percentage of apoptotic cells and a reduction (48, 42 and 25%, respectively, in caspase activity as compared to media controls) in the ACCS-treated samples.

Example 4

Evaluation of ACCS on Post-Irradiation Apoptosis in Jurkat Cells

(15) An experiment was performed to confirm the prior results as well as to evaluate the effect of ACCS treatment post-irradiation. In this particular experiment, in a total volume of 1 mL, 210.sup.6 Jurkat cells were irradiated at 2000 RADs. Apoptosis was analyzed by the caspase luminescence assay and viability was assessed by flow cytometry.

(16) ResultsIn a previous experiment, protection with ACCS treatment was observed in Jurkat cells at 1000, 2000 and 4000 RAD's. The second experiment confirmed the first result and, compared to controls, protection was also observed 8 hrs post-ACCS treatment and increased viability of the irradiated Jurkat cells was seen in ACCS-treated cells.

(17) In-vitro experiments were preformed to evaluate the ability of ACCS to protect cells when exposed to various inducers of apoptosis. Experiments on Jurkat cells using camptothecin as the induction agent and Annexin-V staining as a read-out demonstrated a protective effect of ACCS when compared to untreated control samples. Additional experiments on the same cell line with gamma irradiation as the inducer of apoptosis also showed protection by ACCS at 1000, 2000 and 4000 RADs. This protective effect was also seen when the ACCS was added several hours post irradiation exposure which suggests that ACCS may be a viable treatment for radiation sickness. ACCS's ability to protect cells from insult was also assessed on various cell lines including fibroblasts and HUVEC cells (data not shown). In all cases, under these test conditions, there was a measurable protective effect of ACCS treatment compared to the untreated controls, The experiments measured the caspase 3/7 activity with two different apoptosis inducers, camptothecin and staurosporine.

Example 5

Evaluation of the Effects of ACCS on Radiation Protection in Hematopoietic Stem and Progenitor Cells

(18) Based on the above results, it is hypothesized that ACCS may protect hematopoietic stem and progenitor cells from exposure to radiation. Experiments are performed using hematopoietic stem and progenitor cells to evaluate the potential of ACCS as a radioprotection agent in these cell types. In a representative experiment, in a total volume of 1 mL, 210.sup.6 hematopoietic stem or progenitor cells are irradiated at 1000, 2000 and 4000 RADs and apoptosis is analyzed by flow cytometry with Annexin V and the caspase luminescence assays. Treatments include control medium at 0.75 and ACCS at 0.75. In certain experiments, the treatments are added immediately prior to irradiating the cell samples. In other experiments, treatments are added after irradiating the cell samples.

Example 6

Evaluation of the Effects of AMP Cells on Radiation Protection in Hematopoietic Stem and Progenitor Cells

(19) Based on the above results, it is hypothesized that AMP cells may protect hematopoietic stem and progenitor cells from exposure to radiation. Experiments are performed using hematopoietic stem and progenitor cells to evaluate the potential of AMP cells as a radioprotection agent in these cell types. Hematopoietic stem or progenitor cells are irradiated and apoptosis is analyzed by flow cytometry with Annexin V and the caspase luminescence assays. Treatments include adding various concentrations of AMP cells at different time points. In certain experimental conditions, treatments are added immediately prior to irradiating the cell samples. In other experiments conditions, treatments are added post-irradiation of the cell samples.

Example 7

Detection of Anti-apoptotic Factors in ACCS

(20) The following anti-apoptotic proteins were detected in ACCS by antibody array analysis: Bcl-2 (B-cell lymphoma 2), Bcl-w, CD40L (CD40 Ligand), cIAP-2, HSP60 (heat shock protein 60), HSP70 (heat shock protein 70), IGF-II (Insulin growth factor II), IGF-1sR (IGF-1 soluble receptor), Livin, P27 (cyclin Kinase inhibitor), Surivin, sTNF-R1, sTNF-R2, TRAILR-3, TRAILR-4.

(21) In addition to the anti-apoptotic proteins listed above, TIMP-1, VEGF, Angiogenin, PDGF-BB, EGF and human serum albumin, detected by ELISA, also exhibit anti-apoptotic activity (Liu, Xu-Wen, et al. Tissue inhibitor of metalloproteinase-1 protects human breast epithelial cells from extrinsic cell death: a potential oncogenic activity of tissue inhibitor of metalloproteinase-1. Cancer research 65.3 (2005): 898-906; Abu-Ghazaleh, Robin, et al. Src mediates stimulation by vascular endothelial growth factor of the phosphorylation of focal adhesion kinase at tyrosine 861, and migration and anti-apoptosis in endothelial cells Biochemical Journal 360.Pt 1 (2001): 255; Li, Shuping, Wenhao Yu, and Guo-Fu Hu. Angiogenin inhibits nuclear translocation of apoptosis inducing factor in a Bcl-2-dependent manner Journal of cellular physiology 227.4 (2012): 1639-1644; Steidinger, Trent U., David G. Standaert, and Talene A. Yacoubian. A neuroprotective role for angiogenin in models of Parkinson's disease Journal of neurochemistry 116.3 (2011): 334-341; Hsieh, Patrick C H, et al. Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers. Journal of Clinical Investigation 116.1 (2006): 237-248; Shao, Hanshuang, Xiao-Ming Yi, and Alan Wells. Epidermal growth factor protects fibroblasts from apoptosis via PI3 kinase and Rac signaling pathways Wound Repair and Regeneration 16.4 (2008): 551-558; Zoellner, Hans, et al. Serum albumin is a specific inhibitor of apoptosis in human endothelial cells Journal of cell science 109.10 (1996): 2571-2580.

(22) The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

(23) Throughout the specification various publications have been referred to. It is intended that each publication be incorporated by reference hi its entirety into this specification.