Figure 1.
Heme regulated genes in endothelial cells.
Hierarchical cluster analysis of differentially expressed genes in primary lung endothelial cells, PMVECs (A) and PAECs (B) successfully segregates cultures treated with vehicle from those treated with 5 µM hemin. The unpaired student's t-test was used on a gene-by-gene basis to test for differential expression between hemin and vehicle-treated cultures. (C) Venn diagram showing the number of genes differentially regulated by hemin in PMVECs and PAECs.
Figure 2.
Induction of Nrf-2 regulated genes by hemin.
(A) Central role of Nrf2 in the response of the endothelium to heme. Genes whose expression was altered by hemin in both PAECs and PMVECs are shown. ROS = reactive oxygen species. (B) Microarray data of differentially expressed genes regulated by Nrf2 in PAECs and PMVECs treated with hemin. Data shown is mean fold change in gene expression as arbitrary units relative to the level of expression by control cells (n = 5).
Table 1.
Genes differentially regulated by hemin in PMVECs.
Table 2.
Genes differentially regulated by hemin in PAECs.
Figure 3.
Validation of microarray data by qRT-PCR.
(A) Total RNA from PAECs and PMVECs treated with hemin (0–25 µM) was analyzed for the expression of HO-1 and NQO1. Data shown is mean fold change relative to the vehicle (0 µM hemin) +/− SD for three independent experiments each in triplicate (n = 9). (B) Low-density array data showing changes in expression of fourteen genes in PMVECs treated with a concentration range (0–25 µM) of hemin for 7 days. Note the concentration-dependent increase in the number of genes altered by hemin (n = 12).
Figure 4.
Concentration- and time-dependent induction of HO-1 and NQO1 by hemin in endothelial cells.
(A) Western blot analysis confirming concentration dependent induction of HO-1 and NQO1 in PMVECs and PAECs treated with hemin for 7 days. Blots were probed for EF-1αto control protein loading. (B, C) Quantification of HO-1 and NQO1 protein expression in PAECs, PMVECs, BMVECs and DMVECs treated with hemin and vehicle, assayed by western blot analysis. Data shown is mean fold change in protein level as arbitrary units relative to the EF-1α-normalized expression in vehicle treated cells (n = 9). (D) Quantification of western blot showing variable timing of HO-1 and NQO1 induction by hemin (5 µM) in PAECs (n = 9).
Figure 5.
Organ-specific induction of HO-1 and NQO1 in SS mice.
Total RNA was isolated from the indicated organs from transgenic mice of the Townes model, expressing normal human hemoglobin (Hb AA n = 4), or with sickle trait (Hb AS n = 7) or SCD (Hb SS, n = 6). Expression of HO-1 (A) and NQO1 (B) was determined by relative quantitative real-time PCR. Data shown are the mean ± SD. *p<0.05.
Figure 6.
Heterogeneity of enhanced HO-1 expression in SS mice.
(A) Western blot analysis, of snap-frozen organs isolated from Townes SS, AS and AA mice, for HO-1 expression. (B) Quantitative data of western blot experiments. Data shown are mean arbitrary units of β-actin-normalized HO-1 expression in the indicated organs and genotypes of Townes mice (n = 6). (C) Western blot analysis of HO-1 in whole lungs of Berkeley sickle mice and control hemizygotes. (D) Quantitative data of western blot experiments for HO-1 in the Berkeley mice (n = 4). **p<0.01.
Figure 7.
Enhanced expression of NQO1 in SS mice.
(A) Western blot analysis for NQO1 expression in snap-frozen organs from Townes SS, AS and AA mice. (B) Quantitative data of western blot experiments. Data shown are mean arbitrary units of β-actin-normalized NQO1 expression in the indicated organs and genotypes (n = 6). (C) Western blot analysis of NQO1 in whole lungs of Berkeley sickle mice and control hemizygotes. (D) Quantitative data for NQO1 protein in the Berkeley mice lungs showing arbitrary units of β-actin-normalized expression (n = 4). *p<0.05, **p<0.01.
Figure 8.
Expression of HO-1 and NQO1 in lung endothelium of SCD patients.
Representative histological images (A) showing expression of HO-1 (i-ii), NQO1 (iii-iv) and vWF (v-vi) in the endothelium of post-mortem lung tissues of SCD patients and normal control. Note comparable intensities of HO-1 staining in both SCD and control endothelium (solid arrow). Robust staining of NQO1 in the endothelium of the SCD tissue (iii; solid arrow) compared to a relatively weak staining in the normal tissue (iv; open arrow). vWF staining of consecutive sections of both tissues demonstrates an intact endothelium in the NQO1 negative control tissue. Histological scores of HO-1 (B) and NQO1 (C) staining (SCD patients n = 17, normal controls n = 9). **p<0.01 and ***p<0.001.