Pharmacological perturbation of the RAS alters the growth of hematopoietic progenitors in vitro. (A) Inhibition of ACE activity by the addition of lisinopril does not alter the recruitment into division of primitive hematopoietic cells. Single CD34⁺CD38⁻ UCB cells were sorted using the ACDU of the cell sorter into 60-well Terasaki plates (Roskilde, Denmark), containing serum-free medium, SCF, TPO, and Flt3L, as described in the text, together with lisinopril at the stated doses. Two plates (up to 120 cells) were set up per lisinopril concentration. Plates were scored after 4 to 12 hours to verify the presence of single starting cells in each well and then at 24-hour intervals up to day 5 to assess the time to first division and the subsequent proliferation of recruited cells. Shown is the percentage of cells that had divided at least once plotted against days in culture. The experiment was repeated 3 times with identical results. (B,C) Lisinopril inhibits myeloid colony formation by primitive CD34⁺CD38⁻ UCB cells. Clonogenic assays were established using CD34⁺CD38⁻ and CD34⁺CD38⁺ UCB cells (500 cells/plate in triplicate). Colony formation was stimulated by the addition of a combination of rHu IL-1, IL-3, IL-6, G-CSF, GM-CSF, SCF, and Epo. Lisinopril was added over the dose range 0.01 to 100 μM at culture initiation. Colony formation by CD34⁺CD38⁺-derived granulocyte progenitors (CFU-Gs; □), granulocyte-macrophage-progenitors (CFU-GMs; △), or erythroid progenitors (BFU-Es; ◇) was unaffected by lisinopril. In contrast, lisinopril at 100 μM suppressed the growth of CFU-Gs derived from the more primitive CD34⁺CD38⁻ population (P = .047). This figure is a representative of 3 separate experiments for both CD34⁺CD38⁻ and CD34⁺CD38⁺ populations. (D) Angiotensin II inhibits myeloid (CFU-G; ) but not erythroid (BFU-E; ▧) or CFU-Mix () colony formation of UCB CD34⁺ cells. Clonogenic assays were established using UCB CD34⁺ cells (500 cells per plate in triplicate) under serum-free conditions (as described in “Hematopoietic progenitor cell clonogenic assays”) using the same combination of cytokines as described in panel C in the presence of the stated doses of angiotensin II. The asterisks indicate P < .05. Shown is one representative example of 5 replicate experiments. (E) The inhibitory effects of angiotensin II on myeloid progenitor growth in vitro is reversed by the Ang II receptor type 1 (AT1) antagonist losartan. Colony assays were established as described in panel D. Angiotensin II was used at a dose of 10 μM where indicated and losartan at 100 nM. The asterisks indicate P < .05. Shown is 1 representative experiment from a total of 3.