Figure 11.

Pericyte conditioned medium activates the Tie2/Akt signaling axis in HUVECs. Knockdown in human brain pericytes was performed using short hairpin RNA transduction particles against the insulin receptor (shIR) or green-fluorescent protein (shCtrl). (A) Knockdown efficiency with a multiplicity of infection of 15 results in a minor reduction of insulin receptor expression on protein level in shIR. (B) Secretion of Angpt1 into cell culture supernatant is reduced in shIR compared to shCtrl after 24 hours, upon stimulation with 100 nM insulin. (C) HUVECs were treated with serum-free cell-starvation medium (SFM), basal medium (BM) used for conditioning, or shCtrl or shIR conditioned medium (CM), either collected under untreated or 100 nM insulin-stimulated conditions, for 15 minutes to assess activation of the Tie2/Akt signaling axis. (D) Tie2 activation in HUVECs treated with conditioned medium from 100nM insulin-stimulated shIR is reduced compared to shCtrl. Data presented as mean ± SEM, paired t-test (used because within each experimental replicate, shIR or shCtrl were transduced in pericytes derived from a single donor), *P < .05, n = 9, 9 (A, D) and n = 5, 5 (B).