Journal: The Journal of Experimental Medicine
Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations
Figure Lengend Snippet: TSP1 replacement does not suppress the rise in KLF2 and KLF4 after loss of KRIT1. (A and B) Analysis of TSP1, ZO-1, KLF2, and KLF4 mRNA levels by RT-qPCR in freshly isolated microvasculature from mice at P5 and P7 as indicated. Krit1 fl/fl littermate controls, at each developmental stage, were used to calculate percentage increase or decrease in Krit1 ECKO mice using the following formulas: % increase = 100 × ( X − F )/ F and % decrease = 100 × ABS[( F − X )/ F ], where X and F = mRNA abundance in Krit1 ECKO and Krit1 fl/fl BMECs, respectively (SEM, n = 4 or 6). (C) Representative confocal images of retinal vasculature stained for KLF4 (green), TSP1 (red), or isolectin B4 (turquoise). TSP1 is decreased and KLF4 is increased at areas of condensed vasculature ( n = 5 or 6 mice in each group). Bar, 25 µm. (D and E) Analysis of levels of KLF2 and KLF4 mRNA by RT-qPCR from Krit1 ECKO BMEC (D) or cerebellar tissue from Krit1 ECKO mice (E) treated with 3TSR, TSP1, or vehicle compared with Krit1 fl/fl BMEC or Krit1 fl/fl controls. Data are expressed as percentage increase or decrease in Krit1 ECKO using the same formulas as in A and B (SEM, n = 3 or 4 in each group). (F) HUVECs were transduced with lentivirus encoding shKrit1, KLF2, or KLF4, and the increase in KLF2 or KLF4 mRNA relative to cells transduced with lentivirus encoding GFP was measured by RT-qPCR (SEM, n = 4). (G) HUVECs were transduced with lentivirus encoding ShKrit1, KLF2, or KLF4 as described in F, and the decrease of TSP1 mRNA levels was measured relative to cells transduced with EGFP control lentivirus (SEM, n = 4 or 5). (H) Analysis of TSP1 protein levels in HUVECs transduced with lentivirus encoding KLF2 or KLF4 as assessed by Western blot analysis; lentivirus encoding GFP was used as a control (SEM, n = 4). White lines indicate intervening lanes have been spliced out. (I) Loss of endothelial KRIT1 increases expression of KLF2 and KLF4 transcription factors, contributing to CCM formation by downstream effects including suppressed TSP1 expression. 3TSR (TSP1 derivative) reduces CCM lesion formation by replacing functions of TSP1 such as blocking VEGF signaling. Loss of KRIT1 also leads to ROCK activation in a KLF2-dependent manner, and blocking ROCK can also ameliorate CCMs. Thus, blockade of these and other downstream targets of KLF2 and KLF4 may offer a general strategy to reduce CCM formation in humans. *, P
Article Snippet: RNA libraries were multiplexed and sequenced with 100-bp paired single-end reads (SR100) to a depth of ∼30 million reads per sample on an Illumina HiSeq2500.
Techniques: Quantitative RT-PCR, Isolation, Mouse Assay, Staining, Transduction, Western Blot, Expressing, Blocking Assay, Activation Assay