American Society of Hematology

Figure 6

$Figure 6. The endogenous vasoactive peptide apelin is strongly down-regulated through CD154 and other proinflammatory stimuli in ECs. (A) Schematic representation of the APLN full transcript (GenBank accession no. NM 017413) showing the coding sequence and the relative positions of the 2 polyadenylation sites within the 3′ noncoding sequence of the gene. The cDNA fragments (F3-R3 probe and F4-R4 probe) used to distinguish the alternative transcripts are also shown. (B) Relative APLN mRNA levels from msiRNA-2–treated (black columns) and siRNA-2–treated (white columns) HUVECs stimulated with Jurkat D1.1 (CD154+) cells for 4, 10, and 16 hours were assessed by real-time RT-qPCR using the housekeeping cyclophilin A gene (CYPA) as a control. Results are mean values plus or minus SEM from 2 independent experiments performed in triplicate. (C) Northern blot analysis of total RNA isolated from HUVECs either unstimulated, or stimulated with Jurkat D1.1 (CD154+) cells or with TNFα (100 U/mL) plus IFNγ (1000 U/mL) for 4 hours (left panels). Alternatively, HUVECs were treated with siRNA-2 or msiRNA-2 and further stimulated with Jurkat D1.1 (CD154+) T cells (right panels). All Northern blots were hybridized with the APLN-specific F3-R3 or F4-R4 probes as indicated, and a G3PDH probe was used for normalization. (D) At the protein level, cell extracts (40 μg of total protein/sample) from HUVECs either untransfected and unstimulated (lane 4), transfected with msiRNA-2, transfected with siRNA-2, or untransfected, and further stimulated for 4 hours with Jurkat D1.1 (CD154+) T cells (lanes 1, 2, and 3, respectively) were fractionated by 10% SDS-PAGE and probed with a 77–amino acid preproapelin-specific antibody. The housekeeping β-actin protein levels were assessed for lane normalization. (E) Relative APLN mRNA levels from HCtAECs either unstimulated, or stimulated with Jurkat D1.1 (CD154+) cells or with TNFα (100 U/mL) plus IFNγ (1000 U/mL) for 4 hours, were assessed by real-time RT-qPCR using the housekeeping cyclophilin A gene (CYPA) as a control. Results are mean values plus or minus SEM from 2 independent experiments performed in triplicate. (F) Apelin-36 immunostaining in human carotid arteries. A representative section from the carotid bifurcation showing the internal (ICtA) and external (ECtA) carotid arteries (central image: ×20). In the ICtA with advanced atheroma (type V lesion) there was almost absent apelin-36 immunostaining in the intraluminal ECs (). In the normal-looking ECtA, strong positive intraluminal EC immunostaining for apelin-36 was evident (). Both ICtA and ECtA presented significant endothelial CD40 immunoreactivity (). Peripheral images, ×400. The sections were computerized as color-encoded digitized images using a Nikon Eclipse 80i microscope equipped with a CFI Plan Fluor 4×/0.13 NA and 40×/0.75 NA air objectives, a Nikon DS-2Mv camera, and the DS-U2 image processing unit and analyzing software NIKON NIS-Elements version 3.21 (all Nikon, Melville, NY).$

The endogenous vasoactive peptide apelin is strongly down-regulated through CD154 and other proinflammatory stimuli in ECs. (A) Schematic representation of the APLN full transcript (GenBank accession no. NM 017413) showing the coding sequence and the relative positions of the 2 polyadenylation sites within the 3′ noncoding sequence of the gene. The cDNA fragments (F3-R3 probe and F4-R4 probe) used to distinguish the alternative transcripts are also shown. (B) Relative APLN mRNA levels from msiRNA-2–treated (black columns) and siRNA-2–treated (white columns) HUVECs stimulated with Jurkat D1.1 (CD154⁺) cells for 4, 10, and 16 hours were assessed by real-time RT-qPCR using the housekeeping cyclophilin A gene (CYPA) as a control. Results are mean values plus or minus SEM from 2 independent experiments performed in triplicate. (C) Northern blot analysis of total RNA isolated from HUVECs either unstimulated, or stimulated with Jurkat D1.1 (CD154⁺) cells or with TNFα (100 U/mL) plus IFNγ (1000 U/mL) for 4 hours (left panels). Alternatively, HUVECs were treated with siRNA-2 or msiRNA-2 and further stimulated with Jurkat D1.1 (CD154⁺) T cells (right panels). All Northern blots were hybridized with the APLN-specific F3-R3 or F4-R4 probes as indicated, and a G3PDH probe was used for normalization. (D) At the protein level, cell extracts (40 μg of total protein/sample) from HUVECs either untransfected and unstimulated (lane 4), transfected with msiRNA-2, transfected with siRNA-2, or untransfected, and further stimulated for 4 hours with Jurkat D1.1 (CD154⁺) T cells (lanes 1, 2, and 3, respectively) were fractionated by 10% SDS-PAGE and probed with a 77–amino acid preproapelin-specific antibody. The housekeeping β-actin protein levels were assessed for lane normalization. (E) Relative APLN mRNA levels from HCtAECs either unstimulated, or stimulated with Jurkat D1.1 (CD154⁺) cells or with TNFα (100 U/mL) plus IFNγ (1000 U/mL) for 4 hours, were assessed by real-time RT-qPCR using the housekeeping cyclophilin A gene (CYPA) as a control. Results are mean values plus or minus SEM from 2 independent experiments performed in triplicate. (F) Apelin-36 immunostaining in human carotid arteries. A representative section from the carotid bifurcation showing the internal (ICtA) and external (ECtA) carotid arteries (central image: ×20). In the ICtA with advanced atheroma (type V lesion) there was almost absent apelin-36 immunostaining in the intraluminal ECs (). In the normal-looking ECtA, strong positive intraluminal EC immunostaining for apelin-36 was evident (). Both ICtA and ECtA presented significant endothelial CD40 immunoreactivity (). Peripheral images, ×400. The sections were computerized as color-encoded digitized images using a Nikon Eclipse 80i microscope equipped with a CFI Plan Fluor 4×/0.13 NA and 40×/0.75 NA air objectives, a Nikon DS-2Mv camera, and the DS-U2 image processing unit and analyzing software NIKON NIS-Elements version 3.21 (all Nikon, Melville, NY).

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