Local Renin-Angiotensin System in Normal Hematopoietic and Multiple Myeloma-Related Progenitor Cells

Objective: The prominent functions of the local renin-angiotensin system (RAS) in primitive hematopoiesis further support the hypothesis that local autocrine bone marrow RAS could also be active in neoplastic hematopoiesis. The aim of this study is to examine critical RAS elements in normal CD34+ hematopoietic stem cells and multiple myeloma (MM)-related progenitor cells. Materials and Methods: The study group comprised the total bone marrow cells (CBM) of 10 hematologically normal people, the CD34+ stem cell samples (CD34+CBM) of 9 healthy donors for allogeneic peripheral stem cell transplantation, and the CD34+ stem cell samples (CD34+MM) of 9 MM patients undergoing autologous peripheral stem cell transplantation. We searched for the gene expression of the major RAS components in healthy hematopoietic cells and myeloma cells by quantitative real-time polymerase chain reaction analysis. Results: RENIN, angiotensinogen (ANGTS), and angiotensin converting enzyme-I (ACE I) mRNA expression levels of CBM were significantly higher than those in myeloma patients (p=0.03, p=0.002, and p=0.0008, respectively). Moreover, RENIN and ANGTS mRNA expression levels were significantly higher in CD34+ stem cell samples of healthy allogeneic donors compared to those in myeloma patients (p=0.001 and p=0.01). However, ACE I expression levels were similar in CD34+CBM and CD34+MM hematopoietic cells (p=0.89). Conclusion: Although found to be lower than in the CBM and CD34+CBM hematopoietic cells, the local RAS components were also expressed in CD34+MM hematopoietic cells. This point should be kept in mind while focusing on the immunobiology of MM and the processing of autologous cells during the formation of transplantation treatment protocols.


Introduction
The local hematopoietic renin-angiotensin system (RAS) affects the essential steps of hematopoiesis in the bone marrow (BM) microenvironment [1,2,3,4]. Myelopoiesis, erythropoiesis, thrombopoiesis, and other cellular lineages are influenced by the actions of the local BM RAS [3]. Besides those cellular effects, the local RAS [4,5] is also active in the BM stromal niche for the crucial governing of hematopoietic functions [6,7]. The local BM RAS affects hematopoiesis via both altering the internal signals of transcription factors regulating gene expressions and mediating the external signals from the growth factors secreted from the BM microenvironmental hematopoietic and stromal cells [8,9,10,11].
The RAS affects numerous biological events that are important for the formation and function of blood cells. Apoptosis, cellular proliferation, intracellular signaling, mobilization, angiogenesis, fibrosis within the cytokine network, and many other essential pathobiological events are affected by the critical RAS molecules [3,4,5,12,13]. Malignant blood cells, including multiple myeloma (MM) cells, are derived from the clonal neoplastic stem cells within a complex series of pathological proliferative steps. The local BM RAS could affect neoplastic tumoral blood cell production [14,15,16,17,18,19,20,21,22]. The local RAS is effective even in embryonic hematopoiesis [23,24,25,26]. The prominent functions of the local RAS in primitive hematopoiesis further support the hypothesis that the local autocrine BM RAS could also be active in neoplastic hematopoiesis [3].
The aim of this study is to examine critical RAS elements in normal CD34+ hematopoietic stem cells and MM-related progenitor cells. For this purpose, CD34+ hematopoietic cells obtained from healthy peripheral allogeneic hematopoietic stem cell transplantation donors and from MM patients undergoing peripheral autologous stem cell transplantation were analyzed via quantitative real-time polymerase chain reaction analysis (qRT-PCR). Normal BM cells obtained from hematologically normal people were also studied to detect the impact of precursor cell compartments on RAS expressions. Elucidation of the status of the local RAS molecules in early and neoplastic hematopoiesis represents a clinically relevant basic research area for better understanding of the biology of the diseases [4,5].

Study Population
The study group comprised the total bone marrow cells (CBM) of 10 normal people, the CD34+ stem cell samples (CD34+CBM) of 9 healthy donors for allogeneic peripheral stem cell transplantation, and the CD34+ stem cell samples (CD34+MM) of 9 MM patients undergoing autologous peripheral stem cell transplantation. The diagnoses of MM were reached based on the criteria of the International Myeloma Working Group [27]. At the time of the sample collection, all of the patients were in good health and well hydrated. Written informed consent was obtained from all participants and the study protocol was approved by the local ethics committee of Hacettepe University.

Quantitative Real-Time Polymerase Chain Reaction Analysis
We searched for the gene expression of the major RAS components including RENIN, angiotensinogen (ANGTS), angiotensin converting enzyme-I (ACE I), ACE II, angiotensin receptor-I (AGTR I), and AGTR II in healthy hematopoietic cells and myeloma cells by qRT-PCR. Previously designed primer-probes were used [28]. mRNA levels were normalized to CYPA and B-ACTIN genes.
The qRT-PCR analyses were performed using a LightCycler 480 instrument (Roche Diagnostics). Real-time amplification was performed with a final reaction mixture of 20 µL containing 5 µM of each primer, 0.5 µM of each probe, LightCycler 480 Probe Master Mix, and 100 ng/µL of cDNA. Each sample was studied in duplicate and all runs were repeated twice. The PCR protocol was as follows: initial denaturation at 95 °C for 7 min, and amplification segment at 5 s at 95 °C, 10 s at 60 °C, and 10 s at 72 °C for 45 cycles. The 2-Ct method was used to calculate relative expression levels determined from the qRT-PCR experiments [29] and results were given as percentages.
Statistical SPSS 15.0 (SPSS Inc., USA) was used for all statistical analyses. The results were given as mean ± standard error for the data with normal distribution, as median (min-max) for the data without a normal distribution, and as ratio for the nominal data. Data distribution was tested with the Kolmogorov-Smirnov test. Homogeneities of variances were evaluated with Levene's test. Nonparametric tests were used since the sample sizes of the groups were small. Differences between 2 groups were assessed by the Mann-Whitney U test. A p-value of ≤0.05 was considered statistically significant.

Patient Characteristics
Five female and 4 male myeloma patients with a median age of 52 (40-62) years were recruited. Based on the International Staging System, 4 had stage I, 2 had stage II, and 2 had stage III disease. One patient could not be evaluated due to the lack of initial data.

Relative mRNA Expressions of the RAS Components in the Studied Cellular Samples
RENIN, ANGTS, and ACE I mRNA expression levels of CBM were significantly higher than those in myeloma patients (p=0.03, p=0.002, and p=0.0008, respectively; Figures 1, 2, 3). Moreover, RENIN and ANGTS mRNA expression levels were significantly higher in CD34+ stem cell samples of healthy allogeneic donors compared to those in myeloma patients (p=0.001 and p=0.01; Figures 1  and 2). However, ACE I expression levels were similar in the CD34+CBM and CD34+MM groups (p=0.89; Figure  3). Relative expression levels of RENIN, ANGTS, and ACE I genes in CD34+MM cells compared to CD34+CBM cells are given in Table 1. Other RAS pathway members' (ACE II, AGTR I, and AGTR II) expressions were also examined and were not found to be at detectable levels, and no significant differences were determined between any groups.

Discussion
In the present study, RENIN and ANGTS mRNA expressions were significantly higher in CD34+ hematopoietic stem cells of healthy allogeneic donors in comparison to myeloma-related progenitor cells. Likewise, RENIN, ANGTS, and ACE I mRNA expression levels of CBM were significantly higher than those in the myeloma patients. However, ACE I expression levels were similar in CD34+CBM and CD34+MM hematopoietic cells. These findings support our original hypothesis that there is a biologically active local RAS in the hematopoietic system in normal and pathological states [    and AGTR I, are detectable in the normal rat BM cellular compartment and rat microenvironmental stroma at the molecular and protein levels [6]. In the present study, we have found RENIN, ANGTS, and ACE I mRNA expressions in CD34+ stem cell samples of normal human subjects. ACE/CD143 was implicated in enhancing the recruitment of primitive stem cells into the S-phase by degrading AcSDKP [9,30,31,32,33]. ACE, converting Ang-I into Ang-II, is an important peptide for almost all aspects of hematopoiesis [23,31,34,35,36]. Myelopoietic effects of ACE and Ang-II are evident at the hematopoietic stem cell level, extending to the committed myeloid and erythroid lineages [35]. Hence, the local hematopoietic RAS seems to be effective in all species for cellular development. Hematopoiesis [37], myelopoiesis [35], erythropoiesis [38], thrombopoiesis [9], and other cellular lineages [37,39,40,41] are regulated by the actions of the peptides of the local BM RAS. The local BM RAS mediates those complicated networks of BM hematopoiesis in an autocrine/paracrine/intracrine fashion. The growth, production, proliferation and differentiation of the blood cells are affected by the hematopoietic RAS [1,4,5].
The local RAS is also expressed in the cellular compartment of the immunohematological system. The RAS is active in the production and function of distinct blood cell lineages such as dendritic cells, mast cells, T lymphocytes, monocytes, macrophages, and antigen-presenting cells [4,5]. For instance, ACE degrades substance P present in the BM microenvironment, lymphoblasts, and lymphocytes by cleaving a C-terminal dipeptide or tripeptide [41]. Stegbauer et al. reported the up-regulation of renin, ACE, and AGTR I in the immune system, including antigen-presenting cells in myelin-oligodendrocyte glycoproteininduced experimental autoimmune diseases [42]. In this study, CD34+MM hematopoietic cells also locally expressed RENIN, ANGTS, and ACE I mRNA, indicating the activity of RAS in myeloma-related progenitor cells. ACE activity was also linked to MM [43,44]. BM AGTR I expression levels of myeloma patients showed a positive correlation with their BM infiltration pattern and tumor load, indicated by serum β2 microglobulin levels [44]. Our results about myelomarelated progenitors in this study provide an additional clue for the local RAS effects in the pathobiology of MM.
Recent studies [23,24,25,26] focused on the status of ACE within the context of the local RAS, the earliest human embryonic hematopoietic stem cells [45], and the developmental sequence underlying the ontogeny of human blood cells. The local RAS regulates the genesis and function of the hematopoietic system starting from embryonic life [25]. Human embryonic stem cell-derived ACE+CD45-CD34+/-cells are the common yolk sac-like progenitors for not only the endothelium, but also for both primitive and definitive human lymphohematopoietic stem cells [26]. Human angiohematopoiesis initiates from an ACE-hemangioblastic progenitor of primitive and definitive hematopoiesis under the functional activities of the local RAS [26]. Sinka et al. [24] searched for the presence of ACE in the earliest pre-aorta-gonad-mesonephros stages of human intraembryonic angiohematopoiesis. At the earliest stages of human development, hematopoietic potential in the splanchnopleura is restricted to emerging CD34-ACE+ precursors. ACE expression has functions in the maintenance of embryonic hematopoiesis [24]. The results of the present study show that the main RAS components are present in early hematopoietic stem cells and progenitors as well as BM stem, progenitor, and precursor cells. Therefore, manipulation of RAS action could be an important strategy for the expansion of multipotent hematopoietic progenitors during hematopoietic stem cell-related management procedures [46].
The development of MM depends upon deregulation in a complex series of neoplastic pathobiological events. The local tissue RAS influences tumor growth and metastases in an autocrine and paracrine fashion, via the modulation of numerous carcinogenic events such as angiogenesis, apoptosis, cellular proliferation, immune responses, cell signaling, and extracellular matrix formation. Potential manipulation of the local RAS with many enzymes, peptides, and feedback mechanisms can even represent a therapeutic target for the clinical management of cancer [34,47,48]. Our findings about the CD34+MM hematopoietic cells, which locally express critical RAS components in the myeloma-related progenitor cells, could be a starting point for future studies functions on the immunobiology of MM and processing of autologous cells during the formation of transplantation treatment protocols.
Future experimental and clinical studies are needed to elucidate the puzzling functions of local tissue RASs, including the local BM RAS. These efforts should focus on dissecting local RAS interactions with the complicated pathobiological characteristics of neoplastic disorders and on manipulating autocrine-paracrine-intracrine systems for better clinical management of patients with hematological neoplastic disorders.