MALDI-tof/tof-MS of N-glysocylated naEFC membrane proteins using hydrazide-bead capture.

<p>The consensus motif for N-linked glycosylation is highlighted in bold.</p

Hematopoietic properties of naEFCs.

<p>naEFCs were seeded in MethoCult and growth factors GM-CSF, IL-3, SCF and EPO for 14 days prior to colony counting and staining with May Grunwald/Giemsa to assess cellular morphology. naEFCs formed blast-forming unit-erythroid (BFU-E), colony-forming units (CFU)-GEMM, -GM, -G and -M colonies in methylcellulose. Colony formation was photographed and quantified after 14 days and compared between naEFCs and freshly isolated CD133⁺ and CD133⁻ cells (mean ± sem, n = 3).</p

Fold change values of well-established markers in the naEFCs when compared with HUVEC.

↑<p>denotes significantly higher mRNA expression;</p>↓<p>denotes significantly lower mRNA expression.</p

naEFCs express mature EC markers and form perfused tubes in vivo.

<p>In (A), CFSE-labelled naEFCs mixed with Matrigel prior to injection into the flank of NOD/SCID mice, after 7 days the plugs were removed, processed and sections counterstained for nuclei with DAPI prior to imaging by confocal microscopy. The upper left image shows the cross section of a CFSE-naEFC generated tube-like structure (green) within which the nuclei of cells can be seen (blue) at 60× mag (arrows). The upper right image is the control plug in which no naEFCs were added. Images represent one experiment of n≥3. Similar sections were stained for CD144 and images captured by confocal microscopy with CFSE-naEFCs (green) exhibiting CD144 (red) as a cross section of a tube (lower left image) and CD144 staining in the junctions of the CFSE-naEFCs (lower right panel). Images are a representative of n≥3. In (B), similar experiments were executed and at day 7 post-implant the mice were injected i.v. with TRITC-lectin prior to exsanguinations, plugs removed, processed and sections counterstained for nuclei with DAPI prior to imaging by confocal microscopy. The representative image shows the cross section of a CFSE-naEFC generated tube-like structure (green, upper left image), TRITIC-lectin (red, upper right image), DAPI counterstain (blue, lower left image) and the merged image (lower right). In (C), CFSE-naEFCs were digested from explanted Matrigel plugs using dispase and phenotyped for hematopoietic progenitor cell and endothelial cell markers by flow cytometry (right panel); cells from contra-lateral control Matrigel plugs were similarly examined for antigen expression (left panel). In the histograms, the light dotted lines represent unstained cells and the dark lines represent stained cells of a representative of repeated experiments.</p

Gene expression analysis of naEFCs versus HUVEC.

<p>In (A), a heat map illustrating the hierarchical clustering of Log2 relative gene expression in 3 separate HUVEC and naEFC samples. In (B), scatter data showing the average gene expression data in naEFCs and HUVEC. The dots represent the gene expression of UCB CD133+ 4 day cultured naEFCs versus HUVEC. The diagonal lines indicate the cut off value of 1.5 fold activation and genes coloured on the basis of expression level (yellow, evenly expressed genes; blue, naEFC upregulated genes; red, naEFC downregulated genes). In (C), ICAM-3 mRNA levels in naEFCs and HUVEC as determined by qPCR with relative gene expression normalised to CycA. Data are expressed as relative fold change (mean ± sem) normalised to HUVEC, n = 3,*<i>p</i><0.05 versus HUVEC. In (D–F), flow cytometric analysis of ICAM-3 on (D) naEFCs, (E) HUVEC and (F) freshly isolated peripheral blood CD133⁺CD117⁺ gated cells. Light dotted line represents the unstained control and the dark line represents cells stained for ICAM-3. One representative experiment is shown n≥3.</p

Surface expression profiling of freshly isolated CD133⁺ cells, naEFCs and HUVEC.

<p>In (A), freshly isolated CD133⁺ cells were phenotyped for hematopoietic progenitor cell and endothelial cell markers by flow cytometry. In the histograms, the light dotted lines represent unstained cells and the dark lines represent stained cells of one representative experiment from n≥3. In (B), CD133⁺ enriched cells at 4 days of culture (naEFCs) and HUVEC were more extensively assessed for surface antigen phenotype. The histograms show one representative experiment from n≥3 with the light and dark lines as above. In (C), the pan-leukocyte marker CD45 and the myeloid markers CD11b and CD14 were examined with the light dotted lines representing unstained cells and the dark lines representing stained cells of one representative experiment from n≥3.</p

ICAM-3 mediates rolling and adhesion of naEFCs.

<p>In (A), still images of Video S1 illustrate the interaction of naEFCs with untreated (left panel), TNFα treated (5 ng/ml for 5 hours, middle and right panels) where naEFCs were pre-treated with an isotype control antibody (middle panel) or an antibody to ICAM-3 (right panel) prior to perfusion over HUVEC at 2 dynes/cm². In (B and C), data of rolling and adherent naEFCs is represented as the mean ± sem per field of view (fov) for n = 3;*<i>p</i><0.05 versus untreated; #<i>p</i><0.05 versus iso ctl. In (D and E), data of rolling and adherent whole blood treated with an isotype control or antibody to ICAM-3 is represented as the mean ± sem per field of view (fov) for n = 4–5;*<i>p</i><0.05 versus untreated; #<i>p</i><0.05 versus iso ctl.</p

Efficacy and safety of sparsentan versus irbesartan in patients with IgA nephropathy (PROTECT): 2-year results from a randomised, active-controlled, phase 3 trial

Background Sparsentan, a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist, significantly reduced proteinuria versus irbesartan, an angiotensin II receptor blocker, at 36 weeks (primary endpoint) in patients with immunoglobulin A nephropathy in the phase 3 PROTECT trial's previously reported interim analysis. Here, we report kidney function and outcomes over 110 weeks from the double-blind final analysis. Methods PROTECT, a double-blind, randomised, active-controlled, phase 3 study, was done across 134 clinical practice sites in 18 countries throughout the Americas, Asia, and Europe. Patients aged 18 years or older with biopsy-proven primary IgA nephropathy and proteinuria of at least 1·0 g per day despite maximised renin–angiotensin system inhibition for at least 12 weeks were randomly assigned (1:1) to receive sparsentan (target dose 400 mg oral sparsentan once daily) or irbesartan (target dose 300 mg oral irbesartan once daily) based on a permuted-block randomisation method. The primary endpoint was proteinuria change between treatment groups at 36 weeks. Secondary endpoints included rate of change (slope) of the estimated glomerular filtration rate (eGFR), changes in proteinuria, a composite of kidney failure (confirmed 40% eGFR reduction, end-stage kidney disease, or all-cause mortality), and safety and tolerability up to 110 weeks from randomisation. Secondary efficacy outcomes were assessed in the full analysis set and safety was assessed in the safety set, both of which were defined as all patients who were randomly assigned and received at least one dose of randomly assigned study drug. This trial is registered with ClinicalTrials.gov, NCT03762850. Findings Between Dec 20, 2018, and May 26, 2021, 203 patients were randomly assigned to the sparsentan group and 203 to the irbesartan group. One patient from each group did not receive the study drug and was excluded from the efficacy and safety analyses (282 [70%] of 404 included patients were male and 272 [67%] were White) . Patients in the sparsentan group had a slower rate of eGFR decline than those in the irbesartan group. eGFR chronic 2-year slope (weeks 6–110) was −2·7 mL/min per 1·73 m2 per year versus −3·8 mL/min per 1·73 m2 per year (difference 1·1 mL/min per 1·73 m2 per year, 95% CI 0·1 to 2·1; p=0·037); total 2-year slope (day 1–week 110) was −2·9 mL/min per 1·73 m2 per year versus −3·9 mL/min per 1·73 m2 per year (difference 1·0 mL/min per 1·73 m2 per year, 95% CI −0·03 to 1·94; p=0·058). The significant reduction in proteinuria at 36 weeks with sparsentan was maintained throughout the study period; at 110 weeks, proteinuria, as determined by the change from baseline in urine protein-to-creatinine ratio, was 40% lower in the sparsentan group than in the irbesartan group (−42·8%, 95% CI −49·8 to −35·0, with sparsentan versus −4·4%, −15·8 to 8·7, with irbesartan; geometric least-squares mean ratio 0·60, 95% CI 0·50 to 0·72). The composite kidney failure endpoint was reached by 18 (9%) of 202 patients in the sparsentan group versus 26 (13%) of 202 patients in the irbesartan group (relative risk 0·7, 95% CI 0·4 to 1·2). Treatment-emergent adverse events were well balanced between sparsentan and irbesartan, with no new safety signals. Interpretation Over 110 weeks, treatment with sparsentan versus maximally titrated irbesartan in patients with IgA nephropathy resulted in significant reductions in proteinuria and preservation of kidney function.</p