|PARACRINE ACTION OF MESENCHYMAL STEM CELLS
|Year : 2016 | Volume
| Issue : 1 | Page : 3-9
Vascular endothelial growth factor and insulin growth factor as an underlying paracrine action of mesenchymal stem cells transfused for the regeneration of stage II and III chronic kidney disease
Gamal Saadi1, Mervat El Ansary2, May A Hassaballa1, Mona Roshdy1, Eman A El-Aziz2, Irene Bishai2, Samah Mohamed2, Mahmoud El Gaafary1, Mahmoud Zidan1
1 Department of Internal Medicine and Nephrology, Cairo University, Cairo, Egypt
2 Department of Clinical Pathology, Cairo University, Cairo, Egypt
|Date of Submission||13-Oct-2015|
|Date of Acceptance||13-Dec-2015|
|Date of Web Publication||22-Mar-2016|
Department of Internal Medicine and Nephrology, Cairo University, Cairo
Source of Support: None, Conflict of Interest: None
Mesenchymal stem cells (MSCs) are a group of multipotent cells found in cord blood, adipose tissue, bone marrow, and the stroma of various organs with a great potential for mesoderm-like cell differentiation. The aim of the present work was to study the paracrine effect of MSC transfusion in stage II and III chronic kidney disease, which is measured through the level of insulin growth factor-1 and vascular endothelial growth factor. Human bone marrow MSCs were isolated, expanded, and harvested after an average of 21-30 days not only morphologically, when the cells presented as a uniform spindle fibroblast and reached 70-80% confluence with a good cellular yield, but also through their immunophenotypic analysis, which showed positivity for CD29 and negativity for CD34. They were reinjected intravenously in 10 renal patients. To study the effect of such manipulation on the kidney, creatinine and creatinine clearance were measured at the day of injection (baseline), and the first and third month following injection. In addition, other modulators were measured during the first week of injection (day 0, 2, and 7) using enzyme-linked immunosorbent assay. To illustrate, for the first 3 months the creatinine and creatinine clearance reflected a significant renal improvement with an overall decrease of 14% and an increase of 23%, respectively. Although the third month's results may appear worse off than the first month's, they still were better than the baseline before transfusion. Therefore, such an improvement may be attributed to the growth factors released by the MSCs. In other words, both the vascular endothelial growth factor and insulin growth factor-1 showed an overall rise of 3 and 53%, respectively, in their level during the first week after transfusion. Therefore, MSCs transfused to the patients lead to the rise in such modulators, which in turn caused a significant improvement in renal functions. In conclusion, these findings may provide a novel therapy of regenerative medicine especially for chronic kidney disease where dialysis and renal transplantation are inevitable.
Keywords: Chronic kidney disease, end stage renal disease, mesenchymal stem cells, insulin growth factor-1, vascular endothelial growth factor, bone marrow mesenchymal stem cells
|How to cite this article:|
Saadi G, El Ansary M, Hassaballa MA, Roshdy M, El-Aziz EA, Bishai I, Mohamed S, El Gaafary M, Zidan M. Vascular endothelial growth factor and insulin growth factor as an underlying paracrine action of mesenchymal stem cells transfused for the regeneration of stage II and III chronic kidney disease. J Egypt Soc Nephrol Transplant 2016;16:3-9
|How to cite this URL:|
Saadi G, El Ansary M, Hassaballa MA, Roshdy M, El-Aziz EA, Bishai I, Mohamed S, El Gaafary M, Zidan M. Vascular endothelial growth factor and insulin growth factor as an underlying paracrine action of mesenchymal stem cells transfused for the regeneration of stage II and III chronic kidney disease. J Egypt Soc Nephrol Transplant [serial online] 2016 [cited 2017 Dec 17];16:3-9. Available from: http://www.jesnt.eg.net/text.asp?2016/16/1/3/179198
| Introduction|| |
Chronic kidney disease (CKD) is a significant and growing global public health problem. It is defined as pathogenic abnormality with markers of damage that classify it to five-stage schema based on its severity . In developing countries, the national economy plays a great role in the prevalence of renal diseases ,. To illustrate, infections and intoxication constitute two of the main causes of tropical CKD . In Egypt, the principle causes of end-stage renal disease (ESRD) are hypertension (35%), unknown etiologies (20%), diabetes (15%), interstitial nephritis (15%), glomerulonephritis (10%), polycystic kidney, and obstructed uropathy (5%) .
Current therapeutic approaches to ESRD are nonspecific. They do not fully replace either the loss of endocrine and metabolic functions or the filtrative function of the kidney. Renal dialysis and transplantation may be the only treatment for late stages of CKD. The call for better management in ESRD has drawn the attention toward cellular-based therapies, particularly targeting the use of stem cells.
Mesenchymal stem cells (MSCs) could be considered as the most promising alternative therapy. They, however, offer a new hope for such patients as they have endocrine and paracrine effects as well as differentiative capability. In other words, they may produce growth factors that not only inhibit inflammation but also induce survival and differentiation into tubular epithermal cells. In addition, MSCs secrete factors that aid in glomerular regeneration (Giuseppe et al., 2010).
This current study focuses on the use of bone marrow (BM)-MSCs in stage II and III CKD. The study was carried out by isolating and expanding a sufficient number of multipotent MSCs and injecting them intravenously in the renal patients and then assessing the renal performance.
Not only renal functions (creatinine and creatinine clearance) but also insulin growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) were considered the main parameters that depict the effect of the transfused BM-MSCs. In other words, the secretion and increased levels of growth factors reflect the paracrine role of MSCs and hence the enhancement of kidney functions.
| Materials and methods|| |
Sampling and specimen collections
The present work was conducted on a total number of 10 patients with CKD grade II-III. All patients were chosen from Kasr El Aini Hospital. There were six male (60%) and four female patients (40%), ranging from 16 to 53 years of age.
Blood samples were collected under aseptic condition by means of clean venipucture using a vacuum collection tube; about 6 ml of venous blood was withdrawn from all patients into a sterile serum tube for creatinine, creatinine clearance, and enzyme-linked immunosorbent assay. Kidney functions were assessed at day 0 (before MSCs injection), first month, and third month, and growth factors (VEGF and IGF-1) were measured on days 0, 2, and 7 after the manipulation.
Twelve stage II and III CKD patients of the same age range were selected randomly as controls, and their creatinine and creatinine clearance were measured.
Bone marrow-mesenchymal stem cells collection, processing, and harvesting
Under complete aseptic conditions, using a preservative-free heparin in a sterile syringe, 90 ml of BM blood was aspirated from three donors with matching blood group (nonautologous transfer). The aspirate was then diluted at ratio 6 : 1 with PBS. Thereafter, the diluted cell suspension (35 ml) was carefully layered over 15 ml of ficoll hypage in 50 ml conical tubes. Mononuclear cells were separated and transferred to a new 15 ml conical tube. The cells were then washed and the mononuclear cells were suspended in a 5 ml complete culture medium containing fetal calf serum (FCS) (1 ml), α-MEM (4 ml), gentamycin (100 μl), fungi zone (100 μl), and fibroblast growth factor (2 μl). The mixture was mixed well and divided into tissue culture flasks. The flasks were incubated in 5% CO 2 incubator at 37°C for 48 h. The medium was changed after 24 h by throwing the contents (the media and nonadherent cells); another complete medium was prepared as mentioned earlier and the flasks returned to the incubator. These cells were left for another 3-4 weeks in the incubator and the medium was changed every week using the same procedure as denoted earlier. After the third week, the cells were immunophenotypically tested for cell surface markers (CD29 and CD34) using flowcytometry. The cells were also examined with an inverted microscope for confluence and morphology; besides, they were counted using a hemocytometer. In case of adequate number of cells (90% confluence), the contents were thrown and the cells were harvested.
On reaching such confluence, the media was discarded and each flask was rinsed with PBS to remove any FCS. A volume of3 ml of prewarmed trypsin-EDTA solution (0.05%/0.53 μm EDTA) was added to each flask, and then incubated at 37°C for 10 min. After trypsinization, the cells were dissociated from the flask wall using a scrapper in a zigzag manner, followed by gentle tapping to detach the MSCs. Later, the MSCs were resuspended in 5 ml of complete media. Finally, 15 million MSCs in 5 ml saline were injected into the patients intravenously in one session.
Immunophenotypic characterization of bone marrow-mesenchymal cells
After harvesting and counting MSCs using hemocytometer, the cells were tested for mesenchymal markers CD29 and hematopoietic stem cell marker CD34. Approximately 100 000-200 000 MSCs in DPBS were stained for 20 min at room temperature with 10 μl of antibody, as determined from the manufacturer's recommendation (mesenchymal markers CD29 PE and exclusion marker CD34 FITC (hematopoietic stem cell marker). A volume of 2 ml of PBS was added to the MSCs and then the tubes were centrifuged at 200g for 5 min at room temperature. The supernatant was discarded and the labeled MSCs were finally resuspended in 0.5 ml flow buffer (FACS wash) (5% FBS+95% PBS). The cells were analyzed on a flowcytometer Coulter (Beckman coulter Elite) collecting 10,000 events. As a control, unstained cells were applied first to exclude the effect of autoflourescence of the cultured cells.
Measuring vascular endothelial growth factor and insulin growth factor-1 level using enzyme linked immunosorbent assay
Antibody-coated plates for VEGF and IGF-1 plates were prepared. Reagents and samples were allowed to equilibrate at room temperature before use. A volume of 50 μl of standards and test samples was added to test well and then incubated for 1 h. A volume of 50 μl of biotinylated VEGF/IGF-1 antibody was added to the test well and incubated for another hour at room temperature and then washed (5×). Subsequently, 50 μl of HRP-streptavidin was added to the test well for 30 min at room temperature and then washed (5×). Following wash, 50 μl of TMB substrate was added to each well and incubated for 20 min at room temperature, and finally 25 μl of stop solution was added to each well, and readings were recorded at 450 nm against 630 nm immediately.
| Results|| |
Isolation, expansion, and injection of bone marrow-mesenchymal stem cells in stage II-III chronic kidney disease patients
Spindle-like cells were expanded from the 10 samples. Most of them were double-protruding long spindle-shaped, short rod-like or flat-shaped fibroblast-like cells. After an average of 21-30 days, the cells presented in a uniform spindle morphology shape, and reached 70-80% confluence. The mean cellular yield of the samples was 1.1-5.0 × 10 7 cells at 21-30 days after processing. Thereafter, an average of 15 million MSCs were transfused to each patient, with a mean age of 27.2 ± 10.5 SD. As shown in [Table 1], the study correlates the different demographic, clinical, and laboratory variables (creatinine, creatinine clearance, etc.) with the VEGF and IGF-1 level at 0, 2, and 7 days after transfusion, as these growth factors reflect the MSCs' paracrine effect.
The demographic and numeric variables among the study group are shown in [Table 1].
The patients' ages ranged from 16 to 53 years.
A control group comprising 12 patients showed a continuous rise in creatinine and fall in creatinine clearance, as shown in [Table 2] and [Table 3].
|Table 3: Comparison between the serum creatinine levels at 0, 1, and 3 months after mesenchymal stem cell infusion|
Click here to view
The creatinine level decreased by 22% after the first month; however, it started to increase at a slow rate. Despite this slight increase, an overall creatinine decrease of 14% during the 3-month period was noticed, which reflects a renal improvement, as shown in [Figure 1].
|Figure 1: Comparison between the serum creatinine levels at 0, 1 month, and 3 months after mesenchymal stem cell (MSC) infusion.|
Click here to view
[Table 4] also shows a statistically significant difference in creatinine clearance results. For example, a remarkable elevation of 32% was noticed in the first month, followed by a decline. Despite the 7% decrease by the end of the 3-month study, the renal performance in the third month remains better than the patients' pretransfusion state, with an overall improvement of 23% [Figure 2]
|Figure 2: Comparison between serum creatinine clearance at day 0, 1 month, and 3 months after mesenchymal stem cell (MSC) infusion.|
Click here to view
|Table 4: Comparison between the creatinine clearance levels at 0, 1, and 3 months after mesenchymal stem cell infusion|
Click here to view
In summary, there was a statistically significant improvement in serum creatinine and creatinine clearance level. Despite such a progress decline in the third month, an overall improvement of 14 and 23% in creatinine and creatinine clearance, respectively, was noticed. This, in turn, could demonstrate the necessity of a second booster dose of MSCs to maintain the good renal functions. This search was extended to study one of the reasons behind such improvement in renal parameters through the measurement of MSC paracrine effect. Such an effect could be reflected through two main modulators VEGF and IGF-1, which are measured during the first week following transfusion.
[Table 5] shows a statistically significant difference between day 0 (before transfusion) and the second and even the seventh day after MSCs injection [Figure 3]
|Figure 3: Comparison between serum vascular endothelial growth factor (VEGF) levels at day 0, 2, and 7 after mesenchymal stem cell (MSC) infusion.|
Click here to view
|Table 5: Comparison between serum vascular endothelial growth factor at day 0, 2, and 7 after mesenchymal stem cell infusion|
Click here to view
A significant increase of 12% in the serum VEGF level was noticed on the second day. Despite a 7.7% decrease at the seventh day compared with the second one, the VEGF level showed an overall rise of 3%. This in turn goes in parallel with the previous result pattern of the serum creatinine and creatinine clearance demonstrated earlier in [Figure 1], [Figure 2].
[Table 6] shows a significant increase in the IGF-1 level (another mediator of MSC paracrine action) during the first week after MSC injection of VEGF.
As demonstrated in [Figure 4], there was a continuous increase in IGF-1 level during the first week of measurement of 41% followed by 9% on day 7. In other words, there was an overall increase of 53% during the first week.
|Figure 4: Comparison between serum insulin growth factor-1 (IGF-1) levels at day 0, 2, and 7 after mesenchymal stem cell (MSC) infusion.|
Click here to view
|Table 6: Comparison between serum insulin growth factor-1 at day 0 and at day 2 and day 7 after mesenchymal stem cells transfusion|
Click here to view
Studying those growth factors as parameters for testing the paracrine effect of MSCs, it was important to demonstrate a comparison between their results, as can be shown in [Figure 5]. It was noticed that the median % change of IGF-1 was higher (45.71%) than that of VEGF (10.8%).
|Figure 5: Comparison between % change of insulin growth factor (IGF) and vascular endothelial growth factor (VEGF) of the studied group.|
Click here to view
| Discussion|| |
MSCs are fibroblast-like cells that are found in the BM and have many significant properties that set them as a perfect choice for therapy of different disorders and malfunctions. They have a very high potential to expand and differentiate into different cell types, as well as multiple immunomodulatory properties, besides its ability to secrete cytokines and growth factors . The secretion of these paracrine factors not only has an anti-inflammatory effect but also they can aid in the repair and remodeling of damaged tissues . In practice, they have been tested successfully on a number of animal models with different diseases, which, in turn, encouraged the application on a number of clinical disorders such as myocardial infarction, graft versus host disease, Cohn's disease, and others (Giordano et al., 2007). Complete repair is rather a very complicated process as it requires full engraftment and stability among the regenerating cells as well as their signaling pathway. Homing of the intravenously infused MSCs has showed great success in myocardial repair (Nagaya et al., 2004) diabetes type I , multiple sclerosis , and skin graft survival . Despite these successful trials, there are some limitations that need thorough research. In other words, the dose to be administered, the number of doses and their frequency are not yet confirmed and need to be addressed for general applications. According to Bonfield , the dose required for each disease may be highly dependent on its potency and efficacy. In the context of a specific disease entity, dosing may be defined by the effectiveness and success of the MSC expansion and harvesting on one hand and the status and phenotype of the disease on the other hand. For example, Jane  recommended two doses for MSCs transfusion, high dose (eight million cells/kg of body weight) and low dose (two million cells/kg body weight), which were evaluated in cases of graft versus host disease. In this study, about 0.7-1.0 million MSCs/kg body weight has been achieved; despite this relatively small dose, improvement in the kidney functions was attained.
At the start of this century, the number of patients suffering from ESRD was expected to increase by almost 85%, eventually more financial, human, and social problems were to follow. On the other hand, the current management of such patients is never satisfactory in terms of replacement of the endocrine, metabolic, and filtrative functions lost by the kidney . As a consequence of such shortage in therapeutic management, MSCs offer a very promising alternative through its immunomodulatory functions and paracrine effects .
In the current search, the paracrine effects of MSCs were analyzed by measuring IGF-1 and VEGF level using the enzyme-linked immunosorbent assay technique at 0, 2, and 7 days after transfusion and were correlated with different clinical and laboratory variables including creatinine and creatinine clearance at day 0, and after 1 and 3 months. An overall improvement in renal functions was noticed during the course of the study, which reflected in a 14% decrease in creatinine and 23% increase in creatinine clearance. These results go hand in hand with another study conducted by Saadi et al. (2012), who demonstrated correction of serum creatinine level in 30 patients with impaired kidney functions after MSC injection. A highly statistically significant difference was noticed before and after MSC injection at 1, 3, and 6 months in the serum creatinine and creatinine clearance levels. These results would reflect the role of MSCs in transdifferentiation into mature cells as well as both its immunomodulatory action and paracrine effect .
The paracrine effect of MSCs could be demonstrated through the measurement of growth factors such as IGF-1 and VEGF during the first week of starting MSC infusion. For example, when comparing serum IGF-1 level at day 0, 2, and after 7 days, a continuous increase was noticed, which is a little fast by the second day and then slowed down by the end of the first week, having an average increase of 53%. On the other hand, VEGF showed an accelerating rate of increase of 8% on the second day, followed by a decrease in level on the seventh day, yet with an overall 3% increase compared with the baseline value at day 0. Such growth factors have improved renal functions through its IGF/IGF-IR pathway as well as its very unique phenomenon of self-renewal, propagation, and transdifferentiation besides its regulatory role in cell cycle progression, apoptosis, and the translation of proteins .
Another study conducted by Hladunewich et al.  showed an increase in renal blood flow, recovery of kidney functions, and repair of injured nephrons after systemic administration of IGF-1 directly. It also provides a suitable environment promoting the tubular cell growth and repair . Therefore, the rise of the IGF-1 after MSC infusion reflects its role in the improvement and recovery of renal functions. VEGF also plays an important role in glomerulogenesis as it aids in the endothelial and vascular system regeneration. To illustrate, its expression in kidney plays a role in the permeability and integrity of renal vessels ,.
Comparing the level of these two mediators, it was found that the median % change of IGF was higher (45.71%) than that of VEGF (10.8%). This, however, does not mean a greater effect of the former, as the importance of each factor could exceed its absolute change or may be a part of a greater cytokine-induced changes. Therefore, a thorough search, however, is needed to study the reason for such pattern.
Thus, it can be concluded that MSC infusion provides a novel therapy of regenerative medicine for renal diseases. As the survival and recovery of kidney functions are usually not good, such significant and very promising findings of MSCs are highly valuable and they show an improvement over dialysis and renal transplantation. Although the latter is always costly and lacks the proper donor, BM-MSCs are feasible and a readily accessible source of stem cells for renal-based cellular therapy .
Finally, given these research results, this work should be tested on a large number of humans for a longer period of time to better evaluate the profile of changes induced by MSC transfusion. In addition, further search should be extended to study and compare other cytokine mediators affecting the inflammation, cellular proliferation, integrity, and apoptosis; this should be correlated with an initial and follow-up pathology to correlate the functional changes. Moreover, a more thorough research should be conducted to find out the right timing, dose, frequency, and route of administration of the transdifferentiated or transfected MSCs. In a nut shell, this offers a breakthrough in renal diseases not only in degenerative but also in regenerative medicine.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
National Kidney Foundation. K/DOQI Clinical Practice Guidelines for Chronic Kidney Disease: evaluation, classification and stratification. Am J Kidney Dis 2002; 39
Barsoum RS. Overview: end-stage renal disease in the developing world. Engl J Med 1996; 26
Barsoum RS. Chronic kidney disease in the developing world. Engl J Med 2006; 10
Barsoum RS, Sitprija V. Tropical nephrology. In: Schrier RW (ed) Schrier. Diseases of the kidney and urinary tract
. 8th ed. 2013: 2055. 2006.
Barsoum RS. JISN. Kidney Int Suppl 2013; 164-166.
Porada CD, Zanjani ED, Almeida-Porada G, et al
. Adult mesenchymal stem cells: a pluripotent population with multiple applications. Curr Stem Cell Res Ther 2006; 1
Bartholomew A, Sturgeon C, Siatskas M, et al
. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002; 30
Lee RH, Hsu SC, Munoz J, Jung JS, Lee NR, Pochampally R, Prockop DJ. A subset of human rapidly-self renewing marrow stromal cells (MSCs) preferentially engraft in mice. Blood 2006; 107
Zappia E, Casazza S, Pedemonte E, et al
. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 2005; 106
Bonfield T. Adult MSCs an innovative therapeutic for lung disease. Discov Med 2010; April
Jane E. 2008. Parenchymal adult human mesenchymal stem cells for treatment of moderate-to-severe Crohn′s disease. Available at: http:\\clinical.trials.gov\ct2\show\NCT00294112
Uccelli A, Pistoia V, Moretta L. Mesenchymal stem cells: a new strategy for immunosuppression. Epub 2007; 5
Leri A. Human cardiac stem cells: the heart of a truth. Circulation. 2009; 120
Bi B, Schmitt R, Israilova M, et al
. Stromal cells protect against acute tubular injury via an endocrine effect. J Am Soc Nephrol 2007; 18
Hladunewich MA, Corrigan G, Derby GC, Ramaswamy D, Kambham N, Scandling JD, Myers BD. A randomized, placebo-controlled trial of IGF-1 for delayed graft function: A human model to study post ischemic ARF. Kidney Int 2003; 64
Imberti B, Morigi M, Tomasoni S, Rota C, Corna D, Longaretti L, et al
. Insulin-like growth factor-1 sustains stem cell mediated renal repair. Am Soc Nephrol 2007; 18
Villanueva S, Ce´spedes C, Vio CP. Ischemic acute renal failure induces the expression of a wide range of nephrogenic proteins. Am J Physiol Regul Integr Comp Physiol 2006; 290
Kelly DJ, Hepper C, Wu LL, Cox AJ, Gilbert S, Eichler H, et al
. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2003; 24
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]