3.4. Tubulogenesis and Angiogenesis Assays of VHL versus Control BOECsUpon isolation and expansion of BOECs, it was visually clear that BOECs derived from VHL patients appeared morphologically different from healthy controls (Figure 5A). While controls appear as elongated and in closer contact, VHL BOECs are less elongated, rather rounded, and sparsely distributed. Angiogenesis can be measured in vitro by two functional assays for ECs: tubulogenesis and wound healing. VHL patient-derived BOECs showed differences compared to the control group. In the tubulogenesis assay, VHL cells displayed a significantly reduced number of closed tubules (Figure 5B). On the other hand, they were slower on average when migrating to cover the discontinuity inflicted in the confluent monolayers or wound (Figure 5C).Open in a separate windowFigure 5Morphological and functional differences between ECs derived from either healthy subjects or VHL patients. (A) Control and VHL ECs photographed under the microscope once confluence levels were reached. (B) Tubulogenesis assay performed on ECs from control vs. VHL groups, photographed 3 h after cell seeding on Matrigel-matrix-coated wells. (C) Wound healing assay performed on ECs from control vs. VHL groups, photographed 6.5 h after wound infliction on confluent wells. Error bars denote ± SEM. Student’s t-test: ** p < 0.01; and *** p < 0.001.3.5. VEGF Signaling PathwayThe VEGF signaling pathway appeared upregulated in VHL BOECs, according to the GSEA analysis (Figure 6A). Western blotting was used to check the phosphorylation of regulatory kinases belonging to the VEGFA-VEGFR2 pathway (AKT and ERK1/2) (Figure 6B).Open in a separate windowFigure 6(A) VEGF signaling pathway appeared upregulated, according to the GSEA analysis. (B) ERK1/2 and AKT phosphorylation assessment by Western Blot. Densitometric quantification of phosphorylated/total protein ratios are shown for control subject (n = 2) or VHL patients (n = 2). Error bars denote ± SEM. Student’s t-test: * p < 0.05 and *** p < 0.001.3.6. VHL ECs Have Higher Expression of ROSFigure 7A shows a table collecting genes involved in the ROS metabolism, which are downregulated in VHL ECs. Accordingly, a measure of ROS was made from in vitro cultures of control and VHL-derived ECs. In Figure 7B, a representative histogram of the results is depicted. As expected, and supporting the RNAseq results, we found that VHL ECs have on average almost double the amount of ROS than their non-VHL counterpart.Open in a separate windowFigure 7(A) ROS metabolism modulation in RNAseq analysis in VHL ECs. (B) ROS levels in control and VHL cells, showing a significant increase in VHL samples. Error bars denote ± SEM. Student’s t-test: ** p < 0.01.3.7. VHL Gene Expression in VHL Patients’ ECsNo differences in VHL gene expression were found between the patients and the control group, despite the heterozygosis of the patients in the VHL gene. This result could be explained by the type of mutation these patients present: four out of five present missense mutations (Table 1, Figure 3). Only in sample 4, where the mutation affects the splicing site of the DNA, the RNA levels show a lower tendency, although not significant (Table S1). In order to visualize the translational effect of the patients’ mutations, we mapped them on the 3D structure of pVHL, highlighting the changes in amino acids (Figure 8A,B). The altered amino acids fall within areas of close contact between the different subunits, which could affect its proteolytic activity and the assembly of a functional proteasomal complex. Proper folding of the protein, an altered interaction with the substrates, or assembly of the E3 ligase complex, which most likely lead to its loss of function. Further computational analysis should be performed to elucidate the definite cause of how those mutations impair protein function.Open in a separate windowFigure 8(A) 3D complex structure (PDB ID: 4AJY) of the von Hippel–Lindau protein (yellow), Elongin B (green), and Elongin C (purple), bound to Hif1- alpha peptide (depicted in dark blue), highlighting the heterodimerization interface of this complex in a gray box and the position of the R64P mutation. (B) Inset of the VHL-ELOb/c interface with their respective involved amino acids are represented as sticks, while the relevant mutation positions are represented with spheres and labelled in bold. (C) Confocal staining of sample V1 (p.R167Q) shows the distribution of pVHL in the cell cytoplasm, distributed in the perinuclear region in VHL cells and throughout the cell in control ones.A misfolding of the protein could explain the differential distribution of pVHL in sample V1 (p.R167Q). Confocal staining of ECs shows that pVHL accumulates in the perinuclear region. This result suggests a likely aggregation of pVHL in endoplasmic reticulum cysts. Conversely, control ECs show a more heterogeneous distribution of pVHL throughout the cytoplasm (Figure 8C). We believe that these differences in cellular pVHL distribution are caused by the mutations in pVHL present in this patient.

1 15 c.525C>A p.Tyr175X x x Likely de novo Father and siblings -