KR101594248B1 - An Enhancing Angiogenes of IL-8-treated Stem Cells and the Use thereof - Google Patents

Abstract

The present invention relates to the remarkable angiogenic function of IL-8-treated stem cells, and more particularly to a method for stimulating the secretion of VEGF through a mechanism of IL-8-mediated mesenchymal stem cell phosphorylation of AKt and ERK And inducing angiogenesis to effectively treat ischemic diseases such as stroke.

Description

An Enhancing Angiogenes of IL-8-treated Stem Cells and the Use thereof < RTI ID = 0.0 >

The present invention relates to the remarkable angiogenesis function of IL-8-treated stem cells, and more specifically, to a method of stimulating the production of VEGF and phosphorylating AKt and ERK in mesenchymal stem cells treated with IL-8 To induce angiogenesis to effectively treat ischemic diseases such as stroke, myocardial ischemia, and spinal cord injury.

Stroke is the leading cause of death worldwide. 85% of strokes come from cerebral infarction due to occlusion of the blood vessels. The main cause of occlusion is thrombus formation, embolization due to the heart or large blood vessels. When the blood vessel is clogged, an ischemic core is formed, which is the center of irreversible damage, and an ischemic penumbra around the ischemic core affected by various factors. Thereafter, necrosis and apoptosis are caused by various mechanisms in the tissues.

In summary, the disruption of the nerve cell ion channel results in the release of the excitatory neurotransmitter, glutamate, which causes the N-methyl-D-aspartate (NMDA) receptor and the voltage-dependent calcium channel , VDCC). ≪ / RTI > In addition, in response to DNA damage, nuclear protein poly (ADP-ribose) polymerase is activated, resulting in decreased intracellular concentration of NAD + and decreased energy production.

Therefore, reperfusion, the rapid recovery of blood flow, is the best way to minimize damage to brain tissue. The peripheral portion of the cerebral infarction is the main site where new neurons and blood vessels are generated. This inherent neurogenesis is also associated with vascular remodeling, in which newborn cells migrate to the site of cerebral infarction and differentiate into the required form. In this process, neurogenesis and angiogenesis are closely linked through neovascular stromal-derived factor 1 (SDF1) and angiopoietin 1 (Ang1).

To date, thrombolytic therapy, the only treatment approved for acute phase in stroke patients, is only applicable to 3% of stroke patients due to temporal problems and accessibility problems. The initial stroke mortality rate varies from country to country, ranging from 8 to 50%, and surviving patients will achieve functional recovery based on rehabilitation therapy. However, about 50% of patients suffer from persistent functional disabilities, 30% of whom are admitted to a nursing home or live in a similar environment. About 20% or more of the patients require help from caregivers or caregivers in their daily activities.

Therefore, new therapies with restorative and regenerative abilities capable of minimizing and restoring neurological disorders after stroke are indispensable.

Recently, there have been many reports on the safety and efficacy of stem cell therapy to increase neurological recovery from stroke. Stroke has several advantages over stem cell therapy. Unlike other neurological diseases, there is one local damage site, which is a direct target for cell injection and can be expected to restore function rather than alleviate the stabilization or worsening of the disease. Stem cell research has shown that stem cell therapies are capable of regenerating cell transplantation, inducing differentiation into brain tissue, and improving function.

However, the treatment of stroke patients using stem cells is still at an early stage. There are still many challenges to be overcome if stem cells are considered as a candidate therapy to overcome the functional impairment caused by stroke. In addition, there is a need for additional studies on the timing, method, cell type, and cell number of stem cells after stroke.

Accordingly, the present inventors have completed the present invention by confirming the mechanism by which IL-8-treated mesenchymal stem cells promote the production of VEGF and induce angiogenesis through a mechanism of phosphorylating AKt and ERK.

1. KR 20100056318 A 2. KR 20100023715 A 3. US 20070059288 A1

1.Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, Bartkowski HM. (1986) Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17: 1304-1308. (2003) Intravenous Administration of Human Bone Marrow Stromal Cells Induces Angiogenesis in the Ischemic Boundary Zone After Stroke in Chungbuk National University, Cheongju, Republic of Korea); Chen JL; Zhang ZG; Li Y; Wang L; Xu YX; Gautam SC; Lu M; Zhu ZP; Rats. Circulation Research 92: 692-699. 3. Deng YB, Ye WB, Hu ZZ, Yan Y, Wang Y, Takon BF, Zhou GQ, Zhou YF. (2010) Intravenously administered BMSCs reduce neuronal apoptosis and promote neuronal proliferation through the release of VEGF after stroke in rats. Neurological Research 32: 148-156. 4. Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, Gonzalez-Baron M. (2004) PI3K / Akt signaling pathway and cancer. Cancer Treatment Reviews 30: 193-204. 5. Glynn PC, Henney E, Hall IP. (2002) The selective CXCR2 antagonist SB272844 blocks interleukin-8 and growth-related oncogene-alpha-mediated inhibition of spontaneous neutrophil apoptosis. Pulmonary Pharmacology and Therapeutics 15: 103-110. 6. Monocyte function and plasma levels of interleukin-8 in acute ischemic stroke. (2006) Grau AJ, Reise, Buggle F, Al-Khalaf A, Werle E. Valois N. Bertram M. Becher H. Grond-Ginsbach C. Journal of the Neurological Sciences 192: 41-47. 7. Herrmann JL, Weil BR, Abarbanell AM, Wang Y, Poynter JA, Manukyan MC, Meldrum DR. (2011) IL-6 and TGF-α costimulate mesenchymal stem cell vascular endothelial growth factor production by ERK-, JNK-, and PI3K-mediated mechanisms. Shock 35: 512-516 8. Jain KK. (2009) Cell therapy for CNS trauma. Molecular Biotechnology 42: 367-376.

The present invention relates to an exceptionally prominent VEGF-producing ability and related mechanism of IL-8-treated stem cells, and a main object of the present invention is to provide a method for treating ischemic diseases containing IL-8-treated stem cells And a method for treating ischemic diseases using the same.

In order to solve the above problems, the present invention provides a method for inducing angiogenesis by promoting the production of VEGF and further promoting phosphorylation of AKt and ERK in stem cells treated with IL-8, preferably mesenchymal stem cells Provides the use of one mechanism.

At this time, the IL-8 treatment induces angiogenesis by promoting the production of VEGF and phosphorylation of AKT and ERK without affecting the survival and differentiation ability of stem cells.

As a specific example, the present invention provides a cell therapy agent for treatment and prevention of ischemic brain diseases, which comprises the stem cell treated with IL-8 of the present invention. The ischemic brain disease is most preferably a stroke.

At this time, the stem cells can be selected from stem cells derived from bone marrow, umbilical cord blood, fat, blood, liver, skin, gastrointestinal tract, placenta, nerve, adrenal, epithelium and uterus And more preferably selected from these mesenchymal stem cells. In one embodiment of the present invention, bone marrow-derived mesenchymal stem cells were used.

In addition, IL-8 is preferably treated with about 150 to 250 ng / ml of the stem cells for about 1 to 3 days.

In addition, the present invention may also provide other uses that utilize the exceptionally prominent angiogenic potential of IL-8 treated stem cells.

The present invention utilizes a mechanism related to the remarkable production ability of VEGF in IL-8-treated stem cells. In the stem cell treated with IL-8, AKT and ERK are involved in the phosphorylation promoting signal pathway, Promotes new life. Therefore, the present invention can be effectively used for the treatment of ischemic brain diseases such as stroke by inducing angiogenesis very effectively by a simple method of treating a stem cell with a specific cytokine IL-8.

Figure 1 shows the results of Western blot (A) and ELISA (B) for VEGF expression and production in IL-8 treated hBM-MSCs.
FIG. 2 shows the results of comparing the viability and the differentiation potential of hBM-MSCs according to IL-8 treatment as a result of confirming the effect of IL-8 on hBM-MSCs.
Figure 3 shows the effect (A) of each inhibitor to identify PI3K / Akt and MAPK / ERK signaling pathways involved in IL-8-induced VEGF production in hBM-MSCs, Akt, ERK, p38, and ERK, p38, and JNK knockdown in western blot analysis of JNK knockdown (B) and IL-8 treated hBM-MSCs.
FIG. 4 shows the results of confirming the ability of IL-8 to stimulate Akt and ERK phosphorylation in hBM-MSCs in the presence or absence of PI3K and ERK inhibitors.
FIG. 5 is a result of observing the tube-forming ability of IL-8-induced VEGF in vitro and confirming the angiogenic ability of MBECs.
FIG. 6 shows the results of confirming the ability of IL-8-induced angiogenesis control by PI3K / Akt and MAPK / ERK signal transduction pathways [A: tube forming ability, B: statistical analysis graph]
Figure 7 shows the results of statistical analysis of the ischemic lesion volume measured by TTC staining for the effect of IL-8-treated hBM-MSCs on lesion size in MCAO rats.
Figure 8 shows the results of transplanting PKH26-labeled IL-8-treated hBMMSCs into the ischemic brain.
FIG. 9 shows results of endothelial cell proliferation observed by double labeling of brain tissue frozen with endothelial marker BrdU and CD31 in order to examine the effect of IL-8-treated hBM-MSCs on angiogenesis after stroke.

Representative terms used in the present invention are as follows.

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, rat, rabbit, chicken, It is preferably a human. In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included.

"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

The term "polynucleotide" or "nucleic acid" refers to nucleotide polymers of any length, including ribonucleotides as well as deoxyribonucleotides. This term refers only to the primary structure of the molecule, and thus to DNA or RNA of a double or single chain.

"Transfection or transfection" refers to the process by which extracellular DNA enters host cells in the absence and presence of entities. "Transfected cell" refers to a cell in which extracellular DNA is introduced into a cell and contains extracellular DNA. DNA can be introduced into the cell and the nucleic acid can be inserted into the chromosome or replicated as an extrachromosomal material.

"Stem cell" means a cell capable of developing into any tissue. There are two basic features: self-renewal, which creates itself by repeated division, and the ability to differentiate into cells with specific functions depending on the environment.

"Mesenchymal stem cells" are a kind of undifferentiated adult stem cells isolated from human or mammalian tissues and can be derived from various tissues. In adult stem cells, hematopoietic stem cells are mainly non-adherent, whereas mesenchymal stem cells are mainly adherent cells. Particularly, it is possible to provide a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, Placenta-derived mesenchymal stem cells. Techniques for separating stem cells from each tissue are well known in the art.

The term "differentiation" refers to a phenomenon in which cells and tissues specialize in structure or function, while cells grow and proliferate, that is, the form or function changes to carry out the task given to each of them . In general, a relatively simple system is separated into two or more qualitatively different systems.

"Undifferentiated" means a property that indicates undifferentiated state which can be confirmed by expression of at least one undifferentiated marker or various antigen molecules in stem cells. The undifferentiated state of stem cells refers to a state in which stem cells are capable of proliferating almost permanently or prolonged cells, exhibiting a normal nucleus (chromosomal) shape, and capable of differentiating into cells of all lineages of trimesters at appropriate conditions.

By "inhibitor" is meant a substance that inhibits, blocks or reduces the expression or activity of a particular gene. The mechanism of activation of the inhibitor is not particularly limited. Examples include organic or inorganic compounds, proteins, carbohydrates, polymeric compounds such as lipids, and composites of various compounds.

"Treatment" means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, "treatment" may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. &Quot; Palliating " a disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression, It means to lose.

A "cell therapeutic agent" is a cell therapeutic agent that can be used to multiply, select, or alter the biological properties of a cell in vitro, such as autologous, allogenic, or xenogenic cells to restore cell and tissue function It is used for the purpose of treatment, diagnosis and prevention through a series of actions. It can be divided into somatic cell treatment agent and stem cell treatment agent depending on the type of cell and degree of differentiation. The present invention relates to a stem cell therapeutic agent.

An "effective amount" is an appropriate amount that affects a beneficial or desired clinical or biochemical outcome. An effective amount may be administered one or more times. For purposes of the present invention, an effective amount of an inhibitor compound is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, slow down, or delay the progression of a disease state. If the recipient animal is capable of enduring the administration of the composition, or the administration of the composition to the animal is suitable, the composition will be "pharmaceutically or physiologically acceptable ". If the dose administered is physiologically significant, it can be said that the formulation is administered in a "therapeutically effective amount ". The formulation is physiologically relevant if the presence of the formulation results in a physiologically detectable change in the recipient.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprises "and" comprising ", when not explicitly required in the context, include a stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to the treatment and prevention of ischemic diseases.

In the present invention, the ischemic disease refers to ischemic diseases such as cerebral ischemia, cardiac ischemia, diabetic cardiomyopathy, heart failure, myocardial hypertrophy, retinal ischemia, ischemic colitis, ischemic acute renal failure, stroke, brain trauma, Alzheimer's disease, , Neonatal hypoxia, glaucoma, diabetic neuropathy, and the like. Among these, preferred is the treatment and prevention of ischemic brain diseases, for example, most preferably stroke.

Stroke is divided into two types: ischemic stroke which occurs as ischemic state of brain tissue due to decrease or interruption of blood supply to brain tissue, and hemorrhagic stroke which causes bleeding in brain tissue due to blood vessel burst Cerebral ischemia is a serious disease that accounts for about 80% of all stroke patients

The present invention is characterized by using stem cells for the treatment and prevention of ischemic brain diseases. In particular, the stem cells are characterized by treatment with IL-8.

A stem cell is a cell capable of self-replicating and capable of differentiating into two or more cells. In the present invention, preferably a stem cell such as a fat, a uterus, a bone marrow, a muscle, a placenta, Tissue-derived stem cells such as cord blood or skin (epithelium) can be used. In particular, mesenchymal stem cells (MSCs) are preferred. Techniques for separating stem cells from each tissue are well known in the art.

The mesenchymal stem cells are generally stroma for helping hematopoiesis in bone marrow, and have the ability to differentiate into various mesodermal cells including bone, cartilage, fat and muscle cells, and the undifferentiated state And it is easy to obtain and expand in vivo, and there is almost no ethical problem associated with these uses.

In one embodiment of the present invention, bone marrow-derived MSCs were used. These mesenchymal stem cells can be obtained from the patient himself or using a database of blood banks to separate the human leukocyte antigen (HLA) type from the matched bone marrow, thereby minimizing the immune rejection among individuals.

The stem cells according to the present invention can be proliferated and cultured according to methods known in the art.

Suitable media are those which have been developed for the cultivation of animal cells and in particular mammalian cells, or for any use which may be produced in the laboratory together with suitable components necessary for animal cell growth, such as assimilable carbon, nitrogen and / or micronutrients Possible media can be used. The medium may be any basic medium suitable for animal cell growth, such as, but not limited to, Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's modified Eagles medium (DMEM) Alpha MEM (Gibco, Gibco), RPMI 1640, or any other suitable commercial media. To the base medium may be added an assimilable source of carbon, nitrogen and micronutrients, such as, but not limited to, serum sources, growth factors, amino acids, antibiotics, vitamins, reducing agents, and / or sugar sources.

The "IL-8 treated stem cells" of the present invention can be prepared by treating the cultured stem cells with IL-8 at a concentration of 100 to 300 ng / ml, preferably 150 to 250 ng / ml have. IL-8 is preferably treated for 1 to 3 days.

Interleukin-8 (IL-8), one of the CXC chemokines, was originally reported to be a chemotaxis factor of neutrophils and secreted not only in leukocytes but also in vascular endothelial cells and fibroblasts in response to various stimulus sources. In addition, IL-8 acts on receptors present in vascular endothelial cells, resulting in abnormalities of vascular endothelial cells and promotes vascular inflammation and angiogenesis. Recently, IL-8 has been reported to act as a permeability factor of vascular endothelial cells in the early stage of angiogenesis (Biffl et al., 1995; Petreaca et al., 2007).

In the present invention, the IL-8 protein can be used by expressing it using any nucleic acid having a nucleotide sequence encoding IL-8 that is commercially available or known in the art. At this time, the nucleic acid may have a nucleotide sequence encoding a functional equivalent of IL-8. The 'functional equivalents' are those having at least 70%, preferably 80%, more preferably 90% or more sequence homology with IL-8 as a result of amino acid addition, substitution or deletion, 8 < / RTI >

<Mechanism>

The present inventors have identified the molecular mechanisms involved in promoting angiogenesis by enhancing the following vascular endothelial growth factor (VEGF) production capacity by IL-8-treated stem cells.

Therefore, the present invention relates to a mechanism that induces the stimulation of angiogenesis by promoting the production of VEGF and further promoting the phosphorylation of AKt and ERK, and a mechanism of using the same to induce angiogenesis in IL-8-treated stem cells, preferably mesenchymal stem cells .

(1) IL-8-treated stem cells promote the production of VEGF.

In order to maintain normal brain function, sufficient blood supply by angiogenesis is essential. Angiogenesis requires a variety of complex processes such as simple vascular endothelial cell growth as well as endothelial basement membrane penetration, migration and differentiation, and capillary formation.

The most important angiogenesis promoting factor is vascular endothelial growth factor (VEGF), which acts on the receptor on the surface of vascular endothelial cells, KDR / Flk-1. VEGF is a 32-44 kDa secreted in almost all cells and is a vascular permeablity factor with a strong blood-leaking effect of over 50,000 histamine (Senger et al., 1983). This characteristic of blood flow helps the formation of new blood vessels by moving proteins in the blood out of the blood vessels. VEGF receptors are commonly found in vascular endothelial cells, but are expressed in neurons, Kaposi's sarcoma, hematopoietic stem cells, etc. and VEGFR-1 (flt-1), VEGFR-2 (Kdr / flk- 4) were reported. When VEGF is bound, signal transduction is mediated by receptor dimerization along with phosphorylation of the receptor tyrosine residues. VEGFR-1 indicates endothelial cell migration, and VEGFR-2 indicates endothelial cell growth and blood flow efficacy.

Particularly, in the present invention, neurogenesis and angiogenesis due to the increase of VEGF production appear in the cerebral infarction area due to stroke, which is explained by the mechanism and regeneration ability inherent in IL-8-treated stem cells.

In order for the stem cells to function properly, the stem cells must arrive at the correct position, and several chemokines can gather the stem cells into the periphery of the cerebral cortex and make it possible to improve the reproduction environment of the stem cells. Also, local inflammation caused by cerebral infarction plays a role in attracting stem cells. In particular, various factors necessary for angiogenesis are involved in areas where neurogenesis and angiogenesis are active.

The treatment of IL-8 with stem cells of the present invention has no negative influence on the viability and the ability to differentiate into stem cells. This was also confirmed in one embodiment of the present invention.

That is, the treatment of IL-8 of the present invention induces an increase in the production of VEGF without being affected by the ability of the stem cells to regenerate the regeneration environment.

(2) IL-8-treated stem cells induce VEGF production through PI3K / Akt and MAPK / ERK signal transduction pathways.

Two pathways are known for the signal transduction involved in angiogenesis of various signaling materials.

The first is the signal transduction pathway that activates HIF-a through phosphoinositol (PI) 3-kinase / Akt (Akagi et al., 1998), second is the activation of Erk-1/2 or P38 MAPK (mitogen-activated protien kinase) (Jung et al., 1999;

The "IL-8 treated stem cells" of the present invention activate such angiogenesis-related signal transduction by inducing such Akt and ERK phosphorylation, thereby stimulating VEGF production and consequently causing angiogenesis ).

Therefore, the treatment of IL-8 of the present invention activates the PI3K / Akt and MAPK / ERK signaling pathways following Akt and ERK phosphorylation without affecting the regenerating ability of the stem cells themselves, and induces angiogenesis ) Induces an increase in the secretion of VEGF, thereby promoting the angiogenic potency of stem cells themselves.

<Treatment>

Based on the discovery of the mechanisms described above, the present invention covers all uses that utilize the exceptionally prominent angiogenic potential of IL-8 treated stem cells.

As a specific application, the present invention provides an effective cell therapy agent and its uses for ischemic diseases, preferably ischemic brain diseases, most preferably stroke, containing stem cells treated with L-8.

The therapeutic agent of the present invention may further comprise a pharmaceutically acceptable excipient, carrier and diluent. "Pharmaceutically acceptable" refers to a composition that is physiologically tolerated and, when administered to a human, does not normally cause an allergic reaction such as a gastrointestinal disorder, dizziness, or the like

In addition, the therapeutic agent according to the present invention can be formulated into a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. In particular, the therapeutic agent is preferably formulated in the form of an injection suitable for injecting into tissues or organs, and may be formulated into an isotonic sterile solution or a dry preparation which may be an injectable solution by adding sterilized water or physiological saline In particular freeze-dried preparations)

Examples of carriers, excipients and diluents that can be contained in the therapeutic agent of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The amount of the therapeutic agent to be used according to the present invention may vary depending on the age, sex and body weight of the patient, and may vary depending on the condition of the subject to be treated, the specific category or type of cancer to be treated, Dependent stroke susceptibility

The therapeutic agents of the present invention are administered in a manner such that they are transplanted or transferred directly to the desired tissue site to reconstitute or regenerate the functionally deficient site. Preferably as a pharmaceutical composition for the treatment of ischemic brain diseases such as stroke.

For example, Bjorklund and Stenevi, Brain Res. 177, 555-560 (1979) or Lindvall et al., Arch Neurol., 46, 615-31 (1989)).

The dosage can be increased or decreased in consideration of various related factors such as the disease to be treated, the severity of the disease, route of administration, body weight of the patient, age and sex.

The therapeutic agent of the present invention has no immune rejection reaction after transplantation when the active ingredient mesenchymal stem cells are harvested from the patient's own bone marrow, and even when obtained from the bone marrow in which the HLA type of the blood bank is matched, Because of this, it is possible to continue administration until the treatment is completed. In addition, mesenchymal stem cells can be mass cultured without the use of oncogenes and have an advantage that they can be obtained in a sufficient amount for patient transplantation.

In addition, the present invention may provide other uses that exploit the exceptionally prominent angiogenic potential of IL-8 treated stem cells.

As described above, the present invention provides a method for stimulating VEGF production using stem cells treated with IL-8, preferably IL-8-mediated mesenchymal stem cells, and inducing angiogenesis through the mechanism of phosphorylating AKt and ERK And may be used variously for the treatment of ischemic diseases.

<Examples>

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Materials and methods

1. Cell culture and reagents

hBM-MSCs were purchased from Lonza (Walkersville, IN, USA) and cells were melted and cultured. Mouse brain endothelial cells (MBECs) were purchased from ATCC (Manassas, VA, USA) and cultured in endothelial growth medium. All cells were incubated at 37 ° C in a humidified atmosphere containing 5% CO ₂ .

Recombinant human IL-8 was purchased from Sigma (St. Louis, MO, USA) and Neutralizing VEGF antibody was purchased from Abcam (Cambridge, MA, USA). Growth Factor Reduced Basement Membrane Matrix was purchased from BD Biosciences (San Jose, Calif., USA), and the PI3K inhibitor LY294002, the MAPK / ERK inhibitor PD098059 and the JNK (c-jun N-terminal kinase) SP600125 was purchased from Cell Signaling Technology (Beverly, MA, USA) and p38 / MAPK inhibitor SB203580 was purchased from Calbiochem (La Jolla, CA, USA)

2. Survival rate and characteristics

hBM-MSCs were seeded in 24-well plates (8 x 10 ³ / well) and IL-8 hBMMSC-specific cytotoxicity was measured by MTT assay (3- (4,5-dimethylthiazol- ) -2,5-diphenyltetrazolium) (Sigma).

It has been previously reported that the differentiation of hBM-MSCs into the fat or osteogenic lineage is induced by the presence or absence of IL-8 (Pittenger et al, 1999). After 3-4 weeks incubation in the absence or presence of IL-8 (200 ng / ml), the differentiated cells were fixed with 10% formaldehyde. Fat cells were detected by staining lipid droplets in cells for 10 minutes using 0.3% Oil Red O staining method. Bone cells were detected by calcium phosphate deposition using 0.2% Alizarin Red S staining for 20 minutes.

3. ELISA  analysis( Enzyme - linked immunosorbent assay )

VEGF production was confirmed by ELISA using a commercial kit from R & D Systems (Madison, WI, USA), and the conjugated antibody was added to the plate after 3 washes and incubated at room temperature for 2 hours. Subsequently, addition of the substrate solution and incubation at room temperature for 30 minutes were carried out. The reaction was finally stopped with a stop solution and the absorbance of each well was measured at 450 nm with a microplate reader (Molecular Devices, Sunnyvale, Calif., USA).

4. Inhibitor Experiment

After reaching confluence 70%, hBM-MSCs were seeded into 24-well plates (2 x 10 ⁴ cells / well) and cells were treated with the following reagents for 48 h: (a) -MSCs growth medium; (b) promoter IL-8 alone; (c) Accelerator + Inhibitor: PD098059 (50 [mu] M); LY294002 (10 [mu] M); SB203580 (20 [mu] M); SP600125 (10 [mu] M); (d) Inhibitor alone. The capacity of the inhibitor is based on data from known literature (Wang et al., 2008; Herrmann et al., 2011). After 48 hours of treatment, the supernatant was recovered

5. RNA  Interference experiment

SiRNA for human ERK, p38, Akt, JNK and control siRNA were purchased from Cell Signaling Technology. hBM-MSCs were cultured to 70% confluence and transfected with siRNA duplex using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA). After 18 hours of transfection, the cells were treated with IL-8 for 48 hours, and the supernatant and cells were harvested to confirm the VEGF concentration and transfection efficiency, respectively.

6. Western Blot  analysis

Cell protein was degraded by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and β-actin (Sigma), VEGF, ERK, phospho-ERK, Akt, And phospho-Akt (Cell Signaling) with the respective primary antibodies. The membranes were then incubated with horseradish peroxidase (HRP) -conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and Amersham ECL detection reagents (GE Healthcare Life Sciences, Pittsburgh, Pa. To detect bands.

7. Matrix gel-phase angiogenesis assay Angiogenesis assay )

The gel was polymerized in a 96-well plate at 37 DEG C for 30 minutes. MBECs were seeded at 2 x 10 ⁴ / well and grown in different media as follows: (a) endothelial cell basal medium; (b) hBMMSCs growth medium; (c) hBM-MSCs supernatant; (d) IL-8-treated hBM-MSCs supernatant; And (e) IL-8-treated hBM-MSCs supernatant + VEGF neutralizing antibody.

After 18 hours, tube formation was observed under an optical microscope and four pictures were captured from different regions of each sample for analysis by a computer system under a × 100 objective. All images were verified on a computer using MetaMorph software version 7.5 (Molecular Devices).

8. Ischemic animal models and Experimental group

All animal protocols have been approved by the Catholic Medical Laboratory Experimental Animal Care Committee. Transient MCAO (middle cerebral artery occlusion) was induced by a slight modification of the known literature (Longa et al., 1989). Briefly, the right common carotid artery (CCA), the external carotid artery (ECA), and the internal carotid artery (ICA) are exposed through the abdominal midline incision and a rounded tip The 4-0 nylon monofilament suture was introduced into the CCA lumen and gently moved to the ICA until the bifurcating origin of the MCA (middle cerebral artery) was blocked. The tips were removed after 2 hours of onset.

Twenty-four hours after surgery, 18 rats were randomly divided into three groups: PBS (20 μl, intrathecal injection with LP (lumbar puncture), n = 6), hBM-MSCs X 10 ⁶ cells, LP-injected spinal n = 6), and IL-8-treated hBM-MSCs (1.0 x 10 ⁶ cells in 20 μL PBS per rat, LP injected, n = 6). BrdU (Bromodeoxyuridine) (Sigma) was intraperitoneally administered after cell transplantation.

9. Dyeing and quantitative analysis of infarct volume

14 days after cell administration, the rats were sacrificed, fresh brain was removed and sectioned into four equal sized (2 mm) corona blocks using a rodent brain matrix. Each section was stained with 2% 2,3,5-triphenylterazolium (TTC) for 30 minutes at 37 ° C. Unstained areas were considered as infarcted areas (Bederson et al., 1986). The total infarct volume for each slice was calculated by the infarct sum of all brain slices using MetaMorph software, version 7.5 (Molecular Devices).

10. Double-labeled immunofluorescence analysis ( Double - Labeling immunofluorescence )

After 14 days of cell transplantation, frozen brain slices from the 3 groups were collected and incubated overnight at 4 ° C with the following antibodies: monoclonal mouse anti-BrdU (Dako, Glostrup, Denmark), polyclon rabbit anti-CD31 Chemicon International, Temecula, CA, USA). Primary antibodies were visualized with anti-mouse Cy3-conjugated anti-mouse antibody (Jackson ImmunoResearch, West Grove, Pa., USA), Alexa 488-conjugated anti-rabbit antibody (Invitrogen).

The specificity of the immunoreactivity was confirmed by the absence of immunohistochemistry in the sections omitting the primary or secondary antibodies. In all sections, control sections of cell nuclei were stained by incubating sections with 4-6-diamidino-2-phenylindole (DAPI; Roche, Penzberg, Germany) for 10 minutes. All images were obtained using an LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany). .

11. Statistical analysis

Values were expressed as mean ± SE. Statistical analysis of VEGF production in inhibitor and siRNA experiments was performed by two-way ANOVA according to the Tukeys post hoc test. Phosphorylation and tube formation of the transduction pathway were compared by the one-way ANOVA according to the Bonferroni post hoc test for the control and various treatment groups. The total tube length was expressed as the sum of the single edges under the object magnification ratio of the photograph. In vivo data, all statistical comparisons between groups were performed using one-way ANOVA with post-hoc Bonferroni corrections. p <0.05 is considered statistically significant.

Example  One: hBM - MSCs in IL -8 VEGF  Identify generation stimuli

VEGF expression and production in IL-8 (0-200 ng / ml) treated hBM-MSCs were detected by Western blot and ELISA, respectively.

IL-8 dose-dependently increased VEGF expression. VEGF production was slightly increased when stimulated with low concentrations of IL-8 (25 or 50 ng / ml) and significantly increased in cells exposed to high concentrations (100 ng / ml). And when stimulated with 200 ng / ml (p <0.05, Fig. 1A). IL-8 also increased VEGF expression and production in a time-dependent manner (p < 0.05, Fig. 1B) in hBM-MSCs.

IL-8 did not affect hBM-MSC viability until cells were exposed to 200 ng / ml capacity at 72 h (Fig. 2A). Based on these results, we selected an IL-8 concentration of 200 ng / ml and a treatment time of 48 hours for subsequent experiments. This selective IL-8 concentration did not affect the ability of hBM-MSCs to differentiate into fat or osteogenic lineage (Fig. 2B).

Example  2: hBM - MSCs in IL -8-Induced VEGF  Involved in the creation of PI3K / Akt  And MAPK / ERK  Signal path

To investigate IL-8-induced VEGF production mechanisms in hBM-MSCs, we performed experiments involving blocking of various signal pathways.

IL-8-induced VEGF production was attenuated by inhibition of PI3K (LY294002), ERK (PD098059), p38 (SB203580), and JNK (SP600125). In contrast, inhibition of ERK, p38, and JNK significantly inhibited basal VEGF production (p < 0.01, Figure 3A). Two-way ANOVA by Tukeys post hoc test showed that PI3K and ERK inhibition markedly attenuated IL-8-induced VEGF production (p <0.01), whereas p38 and JNK inhibition inhibited IL- Did not reduce 8-induced VEGF production.

These data suggest that PI3K and ERK signal transduction pathways are involved in IL-8-induced VEGF production in hBM-MSCs; And PI3K, ERK, and P38 signaling pathways are involved in autocrine generation in hBM-MSCs.

In order to identify signal transduction pathways involved in the effect of IL-8 on VEGF production in hBM-MSCs, we further transfected hBM-MSCs with siRNA for Akt, ERK, p38, and JNK.

As a result, the siRNA downregulated Akt, ERK, p38, and JNK proteins (Fig. 3B) and showed similar VEGF production patterns such as inhibitors (Fig. 3C).

These results suggest that IL-8 induces VEGF production through the PI3K / Akt and MAPK / ERK signal transduction pathways in hBM-MSCs.

Example  3: hBM - MSCs in IL -8 Akt  And ERK  Phosphorylation phosphorylation ) Induction ability  Confirm

IL-8 was treated with hBM-MSCs for 2 hours after PI3K and ERK inhibitors were present or absent, to confirm the effect of IL-8 on PI3K / Akt and MAPK / ERK signaling activity.

As a result, IL-8 stimulation increased Akt and ERK phosphorylation, as shown in Fig. Treatment of the PI3K inhibitor (LY294002) or the ERK inhibitor (PD098059) prevents this phosphorylation (p < 0.05, Figure 4).

These results further demonstrate that IL-8 activates the PI3K / Akt and MAPK / ERK signal transduction pathways to stimulate VEGF production in hBM-MSCs.

Example  4: Matrigel ( Matrigel ) Prize IL -8-Induced VEGF of MBECs of Vascularity  Confirm increase

The formation of capillary-like tubes by matrigel endothelial cells is an important method for screening a variety of factors that promote or inhibit angiogenesis (Ponce, 2009; Muramatsu et al., 2013). Thus, the inventors of the present invention also performed tube formation analysis to visualize IL-8 effects on the secretion production of VEGF in hBM-MSCs.

As a result, the MBECs cultured on the matrigel did not form tubes in the serum-free basal medium (data not shown).

However, by contrast, MBECs formed tubes in hBM-MSC growth medium (Fig. 5A, a). The tubes were significantly longer in hBM-MSC supernatants as compared to growth medium (p < 0.05, FIG. 5A, b and FIG. 5B). In addition, the tube length was significantly increased in IL-8-treated hBM-MSC supernatants as compared to hBM-MSC supernatant (p < 0.05, Fig. And, this induction effect of IL-8 was greatly reduced by VEGF-neutralizing antibody (p <0.05, FIGS. 5A and 5B).

These results suggest that IL-8-treated hBM-MSCs increase angiogenesis and promote functional recovery in damaged tissues. Also. PI3K / Akt and MAPK / ERK signaling pathways are involved in IL-8-induced angiogenesis (Figure 6).

Example  5: IL -8-treated hBM - MSCs By administration of Stroke  Reduction of post-infarction area

To examine the effect of IL-8-treated hBM-MSCs on lesion size in MCAO rats, the size of the ischemic lesion was measured by TTC staining and shown in Figure 7A.

As shown in Figure 7, hBM-MSC treatment significantly reduced the infarct volume compared to the PBS treated group (p < 0.05). In addition, administration of IL-8-treated hBM-MSCs significantly reduced the infarct volume compared to the hBM-MSC treated group (p < 0.05, Figure 7B).

On the other hand, since intrathecal transplantation of MSCs by LP is a useful and feasible form for the treatment of brain injury (Lim et al., 2011), we also found that PKH26-labeled IL-8- The results of transplanting hBMMSCs into the ischemic brain are shown in Fig. Likewise, it was confirmed that transplantation of IL-8-treated hBMMSCs into the ischemic brain reduced the infarct volume.

Example  6: IL -8- Treatment hBM - MSCs of Stroke  Posterior vessel formation Possible  Confirm

To examine the effect of IL-8-treated hBM-MSCs on post-stroke angiogenesis, frozen brain tissue was double-labeled with endothelial markers BrdU and CD31 and endothelial cell proliferation was detected.

As a result, the number of double-labeled cells in the ischemic area was significantly higher in the group treated with IL-8-treated hBM-MSCs than in the hBM-MSCs alone group and the PBS-treated group (p <0.05, 9 B and C).

These results suggest that IL-8-treated hBM-MSCs may increase angiogenesis in damaged tissue.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (7)

A cell therapy agent for treating and preventing ischemic brain diseases, comprising IL-8 treated mesenchymal stem cell. The method according to claim 1,
Wherein the treatment of IL-8 induces angiogenesis by promoting the production of VEGF and phosphorylation of AKt and ERK without affecting the survival and differentiation ability of stem cells. . delete delete The method according to claim 1,
Wherein said IL-8 is treated to a stem cell at a concentration of 150 to 250 ng / ml. The method according to claim 1,
Wherein said IL-8 is treated for 1 to 3 days. The method according to claim 1,
Wherein the ischemic brain disease is a stroke.

Images