CD34 affinity pheresis attenuates a surge among circulating progenitor cells following vascular injury Adriana Harbuzariu, MD, Justine Kim, BS, E. Michael.

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CD34 affinity pheresis attenuates a surge among circulating progenitor cells following vascular injury Adriana Harbuzariu, MD, Justine Kim, BS, E. Michael Meyer, BA, Albert D. Donnenberg, PhD, Bryan W. Tillman, MD, PhD Journal of Vascular Surgery Volume 59, Issue 6, Pages 1686-1694 (June 2014) DOI: 10.1016/j.jvs.2013.05.043 Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 1 Carotid angioplasty mediates endothelial injury in an ovine model. Compared with uninjured native carotid artery (A), acute balloon injury achieves endothelial injury (B) as shown in these scanning electron microscope images of the luminal surface of the vessel (×1200). By 4 months, the area of endothelial injury has re-endothelialized (C) in these representative images (n = 3). Journal of Vascular Surgery 2014 59, 1686-1694DOI: (10.1016/j.jvs.2013.05.043) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 2 Flow analysis of sheep for CD34-positive cells. Blood specimens drawn preoperatively, or from the first postoperative day after nonvascular or vascular surgery were compared. A total of 300,000 events from peripheral blood specimens were gated sequentially for singlet events, CD34 positivity, DAPI positivity, and finally backscattered to reveal final population of cells highly expressing CD34. Increased numbers of CD34-positive cells can be seen after vascular injury (bottom right). Journal of Vascular Surgery 2014 59, 1686-1694DOI: (10.1016/j.jvs.2013.05.043) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 3 Surge of total CD34 and CD34-positive, lineage negative cells is observed among animals undergoing vascular injury. Flow cytometry of sheep undergoing either control surgery without vascular injury (A and C) (n = 3) as compared with sheep undergoing vascular injury (B and D) (n = 3). Shown are percent CD34+ or CD34+/CD45- cells from all events. A and B reveal percent of CD34 cells and suggest a surge as high as sevenfold compared with baseline following vascular injury (P = .05 on postoperative day 1). C and D indicate the percent of CD34+/CD45- cells, again revealing a surge among lineage negative progenitors. Results are expressed as mean ± standard error. Journal of Vascular Surgery 2014 59, 1686-1694DOI: (10.1016/j.jvs.2013.05.043) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 4 CD34 affinity agarose beads prepared with N,N′-Disuccinimidyl carbonate (DSC) reveal specific depletion of CD34 cells from ovine blood specimens. Blood specimens were incubated with no beads, commercial N-hydroxysuccinimide (NHS) sepharose beads conjugated to CD34 antibody, plain agarose beads, isotype control antibody (MOPC) DSC beads, or CD34 conjugated DSC beads (n = 3 for all conditions; standard error bars are shown). CD34-affinity DSC beads deplete CD34-positive cells in a statistically significant manner relative to isotype control affinity beads (P = .01). Journal of Vascular Surgery 2014 59, 1686-1694DOI: (10.1016/j.jvs.2013.05.043) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 5 CD34 affinity agarose beads attenuate the surge of CD34 cells following vascular injury. Shown are percent CD34+ or CD34+/CD45- cells from all events. Following vascular injury, a surge of CD34+ cells (A) and CD34+/CD45- (C) is seen despite pheresis with an isotype affinity column (n = 4). In contrast, a CD34 affinity column (n = 3) attenuates the surge and mediates a relative state of depletion among both CD34+ (B) and CD34+/CD45- cells (D). A statistically significant difference is observed among total CD34 expressing cells between the two groups (P = .029 on postoperative day 1). Results are expressed as mean ± standard error. Journal of Vascular Surgery 2014 59, 1686-1694DOI: (10.1016/j.jvs.2013.05.043) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 6 CD34 affinity column recovers large numbers of CD34-positive cells. Cells eluted following pheresis of animals undergoing vascular injury were counted and examined by flow cytometry. Relative to the rare cells recovered by an isotype column (A), CD34 affinity column (B) recovered an average of 77 million CD34-positive cells. Journal of Vascular Surgery 2014 59, 1686-1694DOI: (10.1016/j.jvs.2013.05.043) Copyright © 2014 Society for Vascular Surgery Terms and Conditions