Fig 2: The expression of soluble fms-like tyrosine kinase 1 (sFLT1) in kidney biopsies is positively correlated with kidney inflammation (n = 52 biopsies). sFLT1 protein expression localized in kidney biopsies by immunohistochemistry. A 4-tier grading system was used to semiquantitatively measure sFLT1 and inflammation in the kidney biopsies.

Fig 3: Upper panels (interstitial nephritis case): representative images of immunofluorescence assay for soluble fms-like tyrosine kinase 1 (sFLT1; red) and CD34 (endothelial marker; green) in a renal biopsy. The areas to the right of the asterisks show reduced or absent CD34 staining. Bars = 100 µm. Lower panels (anti-neutrophilic cytoplasmic antibody case): representative images of immunofluorescence assay for sFLT1 (red), and CD68 (macrophage marker; green) in a renal biopsy. Yellow immunofluorescence demonstrates colocalization of the 2 antigens, which demonstrates that macrophages express sFLT1 in kidney interstitium. Bars = 20 µm.

Fig 4: HBMSCs plated and cultured at high density differentiated toward functional ECs. To determine whether HBMSCs plated and cultured at high density differentiated toward EC over time, their morphology, EC functional properties, and expression of EC markers were determined. (A) Representative phase contrast images of (I) HBMSCs under standard culture conditions (MSC-St) for 48 h, (II) HBMSCs cultured at low density (HBMSC-L) for 14 days, or (III) HBMSCs cultured at high density (HBMSC-H) for 14 days. (IV) HUVECs under standard culture conditions were used as a positive cellular control. For culture conditions, see Materials and methods. Images were obtained using an Olympus (CK X41) microscope (4x objective). Inserts represent an enlarged region of each image. Scale bars = 200 µm. (B) (I–III) Immunofluorescence analysis showing DiI-Ac-LDL uptake in (I) HUVECs, (II) HBMSCs under standard culture conditions (HBMSC-St) for 48 h, and (III) HBMSCs cultured at high density (HBMSC-H) for 28 days. DiI-Ac-LDL uptake (red), DAPI (blue). Images obtained using an Olympus upright widefield fluorescence (BX51) microscope (20x objective). Scale bars = 20 µm. (IV–VI) Immunofluorescence analysis of VCAM-1 in cells stimulated with 10 µg/ml TNFa for 24 h. (IV) HUVECs under standard culture conditions for 48 h, (V) HBMSCs under standard culture conditions (HBMSC-St) for 48 h, and (VI) HBMSCs at high density (HBMSC-H) for 28 days. VCAM-1 (green), DAPI (blue). Images were obtained using a Nikon C1 upright confocal microscope (60x objective). Scale bars = 20 µm. (C,D) Immunofluorescence analysis of VE-cadherin, PECAM-1, vWF, and VEGFR1 expression in (I, IV) HUVECs under standard culture conditions for 48 h, (II, V) HBMSCs under standard culture conditions for 48 h, or (III, VI) HBMSCs cultured at high density (HBMSC-H) for (C) 28 days or (D) 14 days. VE-cadherin, PECAM-1, vWF, and VEGFR1 (green), phosphorylated VEGFR1 (red), DAPI (blue). Images were obtained using a Nikon C1 upright confocal microscope (60x objective) (C, I–VI; and D, IV–VI) or using an Olympus IX71 Deltavision microscope (40x objective) (D, I–III). Scale bars = 10 µm. White arrows highlight specific regions of localised immunoreactivity. Representative images from two independent experiments.

Fig 5: Neutralizing VEGF receptor antibodies and Akt inhibition upregulates RASSF1A and downregulates FoxM1 in mCRC. T84 and Colo 205 cells were treated with VEGF receptor 1 and VEGFR2 as indicated before (see Section 2.3). Protein expression from cell lysates was determined by immunoblotting for RASSF1A ((A), upper panel) and FoxM1 ((A), middle panel). Densitometry analysis was performed and normalyzed with GAPDH expression to demonstrate significant upregulation for RASSF1A (B) and downregulation of FoxM1 in the presence of neutralized (NT) VEGFR1 or VEGFR2 (C). (D,E) T84 and Colo 205 cells were treated with Akt inhibitor (wortmannin) with different doses (0–1 µM) for 24 h. Cell lysates were analyzed for pAkt (1st lane), total Akt (2nd lane), RASSF1A (3rd lane) expression by immunoblot analysis and quantified by densitometry (F,G). The results are from three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001).