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2011Vol. 2 No. 2:2doi: 10:3823/424
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cells . EPCs, bone marrow derived CD34-positive stem cellsthat mature into endothelial cells, replenish aging or damagedECs that line blood vessels [17-19]. Decreasing EPC numbers areassociated with coronary diseases (including atherosclerosis,diabetes, and hypertension) and increased cardiovascular risk [20-23]. The eects of toxic metals on the cellular oxidation-reduction balance can damage progenitor cells. For example,lead is not only cytotoxic toward stem cells but also interfereswith their dierentiation [24-25]. This suggests a mechanism inwhich toxic metals can harm the vascular system by inducinggeneration of ROS, which in turn hinder the replenishment of vessel walls by endothelial progenitor cells.Chelation therapy has been proposed for clearing toxicheavy metals from the body [26-29], as it not only removescontaminating metals but also decreases free radicalproduction . The two most commonly used chelationagents to date are ethylenediaminetetraacetic acid (EDTA) anddimercaptosuccinic acid (DMSA). Safety and ecacy for thesetwo agents have been studied [29, 31]. In the present study,we focused on how toxic metal exposure, and subsequentchelation therapy, aects circulating CD34 positive cellnumbers. We also present results concerning the toxicityof lead and mercury to stem cells (mesenchymal stem cells,MSCs), endothelial progenitor cells and dierentiated cells(endothelial cells and broblasts).
Materials and Methods
Data were obtained with full IRB approval and consent fromover four-hundred subjects given chelation therapy for long-term non-occupational toxic metal exposure. The breakdownof specic treatments is as follows: oral DMSA (500
g) for 160subjects; intravenous low dose Na-EDTA (1 g) for 50 subjects;and intravenous high dose EDTA (Ca-EDTA or Na-EDTA) (3 g)for 200 subjects. Prior to treatment, each subject was screenedfor adequate kidney functioning by assaying fasting serumcreatinine (levels below 1.7 mg/dL were considered acceptable).Urine samples were collected pre and post treatment for toxicmetal excretion analysis.For ve subjects, selected for long-term exposure and atleast three amalgam llings, we conducted a more detailedanalysis: these subjects were given a series of ve DMSA dosesand blood samples were collected at the beginning and endof their treatments. In these subjects, we measured completeblood count (CBC), circulating CD34-positive cell levels, andurine metals.
Measurements of samples from subjects
Body burdens and excretion rates of toxic and essential metalswere determined through urine analysis. For patients treatedby EDTA, aluminum, lead, cadmium and manganese levelsin urine were measured by the Perkin-Elmer 4100ZL atomicabsorption spectrometer with graphite furnace by standardprotocols. Calcium, magnesium, iron, copper and zinc were alsoanalyzed on a Perkin-Elmer Inductively Coupled Plasma atomicemission spectrometer (Model 3300 DV). For patients treatedby DMSA, heavy metal analyses of urine were undertaken bythe King James Laboratory (Cleveland, OH). All measurementsof metals were performed using Inductively Coupled Plasma-Atomic Emission spectroscopy (ICP-AES). The procedure of stem/progenitor cells measurements wasbased on gating strategy dened by the ISHAGE guidelines. Cells were selected that expressed bright CD34 antigenand dim CD45 antigen along with low side-angle light scattercharacteristics. In our case, we considered circulating CD34positive cells as precursor of EPCs. Our focus on the CD34marker is work , in which blood–derived cells from whichendothelial cells in culture were developed were describedas cells expressing this antigen. Moreover, recent studiesdemonstrate that CD34+ cells not expressing leukocyteantigen (CD45-) form endothelial colony-forming units andthose expressing CD45 demonstrate hematopoietic properties.PBMCs were separated from whole blood, re-suspended in100
l (0.5M cells per 100
l) of a buer (PBS+0.5% BSA), andstained by 20
l of antibodies (Stem cells kit, Beckman Coulter)during 15 min. Flow cytometry was performed on “Cell LabQuant SC” (Beckman Coulter) equipped with 22 mW argon lasertuned at 488 nm. Quantication of CD34+ cells was performedon separated PBMCs by two–color immunostaining usinganti-CD45-FITC and anti-CD34-PE. The percentage of CD34positive cells was calculated based on the measured number of leukocytes (CD45-positive cells) and the number of circulatingCD34-positive cells per micro liter was recalculated bynormalization on the concentration of leukocytes determinedby the Complete blood count (CBC) test.
In Vitro studies
We conducted in vitro experiments using human endothelialcells (HUVEC, Cascade Biologics), lung broblasts (CCD 18lu,ATCC), mesenchymal stem cells (BioE Co) and EPCs developedin our laboratory by long-term culture from PBMCs. Endothelialcells and EPCs were grown in medium M-200 (CascadeBiologics) supplemented with 2% fetal bovine serum (FBS),hydrocortisone, human epidermal factor, broblast growthfactor, and heparin. CCD 18lu cells and MSCs were grown inDMEM medium (ATCC) and MSCGM (Lonza) with 10% FBS.Chelating agent concentrations for in vitro studies were basedon clinical dosages used for chelation by EDTA-Ca and DMSA.Cellular lead levels were determined as described previously. Cells seeded in the 24–well plates were exposed todierent concentrations of lead and antidotes in the mediumwith a dierent concentration of serum (including serum freemedium) for 24h to 48h. After exposure, cells were harvestedby rinsing with 2 mM EDTA in PBS and then detached fromthe wells by trypsin. Cells were counted with hemacytometer