DIFFERENTIATION OF AORTIC ADVENTITIA CELLS Bradley Ellis, Benjamin Green, MS, Thomas G. Gleason, MD, Julie A. Phillippi, PhD Department of Bioengineering University of Pittsburgh Department of Cardiothoracic Surgery UPMC INTRODUCTION pericytes that were untreated. A two-tailed student’s t-test was Aortic Aneurysms are a debilitating group of diseases that used in order to determine whether or not there was a are responsible for over 17,000 deaths a year in the United statistically significant difference between the cell lineages States alone [1]. Thoracic aortic aneurysms are a subset of (p < 0.05). aortic aneurysms that occur when there is a ballooning of the aorta as the artery passes through the chest. If left untreated METHODS this ballooning can lead to a tear in the aortic wall (dissection) The study was designed to differentiate pericyte cells into or a rupture, which lead to patient death [1]. Recent studies one of two blood vessel cell lineages, either endothelial cells or have shown that one of the major underlying causes of thoracic SMCs. The study was performed twice on two separate cell aortic aneurysms is tissue degeneration of the aorta caused by lines taken from bicuspid aneurysmal patients. The patients inefficient repair of the artery [2]. In order to better understand had undergone surgery to repair dissected thoracic aortic thoracic aortic aneurysms it is necessary to understand the aneurysms, and agreed to have their tissue harvested for study. underlying causes of this tissue degeneration. Upon reception, the harvested aortic tissue was separated and Progenitor cells have been shown to play an important role adventitia cells were grown in culture. Upon reaching in tissue repair throughout the human body including vessel confluency, the adventitia cells underwent FACS in order to repair [2]. Through progenitor cell’s ability to differentiate into isolate pericytes (CD146+) associated with the vasa vasorum. functionally relevant blood vessel lineages, tissue repair of The isolated pericyte cells were then subjected to one of three damaged aortas is possible. In previous studies, isolated possible differentiation treatments. All treatments lasted 14 pericyte cells, located in small blood vessels have been shown days and were run concurrently. to possess progenitor cell characteristics [3,4]. Through the In one treatment, the pericytes were grown in non-specific introduction of growth factors, recent studies have shown the basal growth media (Dulbecco’s Modified Eagle media, 10% ability of investigators to coax pericyte cells into specific Fetal Bovine Serum, 1% penicillin streptomycin) for 14 days linages of differentiation [5]. The vasa vasorum (vessel of with a change in growth media on days 0, 2, 4, 8, and 10. No vessels), the micro vessels located in the adventitia of larger growth factors were introduced to this population. The vessels, supplies blood and nutrients to larger vessels such as population was phase imaged at 0, 2, 4, 8, 10, and 14 days of the aorta and have been shown to be analogous to the small treatment. This population served as the negative control blood vessels previously described [3]. The vasa vasorum has group. been shown to possess pericyte cells and thus been The second treatment attempted to differentiate pericyte hypothesized as a progenitor cell niche, harboring cells that cells into aortic endothelial cells. The pericyte cells were display the potential to differentiate into functionally relevant grown in commericial endothelial cell basal growth media (Cell blood vessel cell types [6]. Applications Inc. San Diego, CA) for 14 days with a change in media on days 0, 2, 4, 8, and 10. At each change in media the OBJECTIVE cell population was also treated with vascular endothelial This study attempted to accomplish three objectives. First growth factor (VEGF 50 ng/mL) introduced into the media. was to determine if pericyte cells from aortic adventitia isolated The population was phase imaged at 0, 2, 4, 8, 10, and 14 days for progenitor cell markers (CD146+) through fluorescence of treatment. At the end of the 14 days of treatment, the phase activated cell sorting (FACS) had the ability to upregulate images of the cells pushed towards endothelial differentiation differentiation into endothelial cells and smooth muscle cells were qualitativealy compared to the negative control group. (SMCs) upon introduction to specific growth factors. Secondly The final treatment group attempted to differentiate the was to observe these changes qualitatively through imaging pericytes into aortic SMCs. The pericyte cells were grown in before and after differentiation treatment. The final objective commericial SMC basal growth media (Cell Applications Inc. was to quantify these changes in cell lineage through changes San Diego, CA) for 14 days with a change in media on days 0, in gene expression through the use of quantitative polymerase 2, 4, 8, and 10. At each change in media the pericyte cells were chain reaction (qPCR). also treated with transforming growth factor-beta 1 (TGF-B1 2 ng/mL) and platelet derived growth factor BB (PDGF-BB HYPOTHESIS AND STATISTICAL ANALYSIS 50 ng/mL) introduced into the media. The population was The study attempted to confirm the hypothesis that pericyte phase imaged at 0, 2, 4, 8, 10, and 14 days of treatment. At the cells isolated from aortic adventitia have the ability to end of the 14 days of treatment, the phase images of the cells differentiate into endothelial cells and SMCs. The study was pushed towards SMC differentiation were qualitativealy considered a success if gene expression levels of the pericytes compared to the negative control group. treated for differentiation were statistically different than the 1 After the 14 days of treatment, RNA isolation was performed on all cell populations. qPCR was then performed to compare the gene expression of the negative control to the endothelial differentiated population and the negative control to * the SMC population respectively. For gene expression comparison of the negative control to the endothelial differentiated cells the change in fold of endothelial specific markers was utilized. The markers that were used for the comparison were, Von Willabrand’s Factor (VWF), an adhesion protein found in endothelial cells, and CD31, a vessel growth protein found in endothelial cells. For gene expression Figure 2. Change in fold of CD31 expression of endothelial comparison of the negative control to the SMC differentiated treated cells compared to untreated pericytes. Standard error cells the change in fold of endothelial specific markers was bars are presented. Endothelial differentiated pericytes showed utilized. The markers that were used for the comparison were, a significant increase in CD31 expression (p < 0.01) actin, a contractile protein specific to SMC, and calponin, a calcium binding protein found in SMC. A two-tailed student t- DISCUSSION test was then performed to see if there was a statitcally The endothelial differentiated cells showed no significant significant difference in gene expression for each marker (p < phenotypic changes when compared to the untreated pericyte 0.05). cells, and did not look similar to confirmed endothelial cells. This can be explained to a large amount of variability in RESULTS endothelial cells appearance from cell line to cell line. The lack Figure 1 shows images of pericyte cells untreated, in change of expression of VWF can be explained as studies endothelial differentiation treated, and SMC differentiation suggest [7] that VWF is not universally strongly expressed in treated, along with confirmed SMC and endothelial cells for endothelial cells. The significant increase in CD31 expression comparison. Only the SMC differentiated cell lineage showed strongly supports the hypothesis that aortic pericyte cells any qualitative change towards the desired phenotype upon demonstrate the ability to differentiate into functionally completion of treatment. relevant blood vessel cell lineages. CD31 is universally expressed in endothelial cells and is considered the classic marker for whether a cell is considered endothelial or not. The SMC differentiated cells showed a more spindle like phenotypic appearance upon completion of treatment, similar to confirmed SMCs. The SMC differentiated cells also showed a trend of increase in actin and calponin expression. The phenotypic changes and trend change in gene expression support the hypothesis that aortic pericyte cells are capable of Figure 1. (top differentiating into functionally relevant blood vessel cell left) Untreated pericytes after 14 days of treatment, (top lineages such as SMCs. middle) Endothelial differentiation treated pericytes after 14 This study had several limitations. Only aneurysmal cell days of treatment, (top right) SMC differentiation treated lines, a small subset of the human population were tested. pericytes after 14 days of treatment, (bottom left) confirmed Additionally only two cell lines were tested. endothelial cells, (bottom right) confirmed SMCs. ACKNOWLEDGMENTS For endothelial gene comparison the treated pericytes The authors would like to acknowledge the patients for showed no change in VWF expression upon completion of their tissue donation, the UPMC Shadyside Cardiac Surgery treatment. However, as seen in Figure 2 the pericytes treated Department for tissue harvesting, Mary Kotlarczyk, Jen Hill, for endothelial differentiation showed an increase in CD31 and Marie Billaud for help in the lab, Vera Donnenberg for her expression upon completion of the 14 days of treatment assistance in FACS, and our funding sources, UPMC Health (p < 0.01). For SMC gene expression comparison the treated System and the National Heart, Lung, and Blood Institute who pericytes showed a non-significant trend of increase in both made this study possible. actin and calponin expression. REFERENCES 1. Center for Disease Control. (2013) 2. Shen (2012) doi: 10.1016/j.athoracsur2012.01.063 3. Corselli (2013) doi: 10.1002/cyto.a.22313 4. 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