Session Type: ACR Concurrent Abstract Session
Session Time: 2:30PM-4:00PM
In SLE Apolipoprotein L1 (APOL1) risk variants (RV) associate with cardiovascular and end stage renal disease. APOL1 induction initially promotes cellular maintenance through autophagy; but when prolonged disrupts lipid bilayers causing ion flux, mitochondrial stress, and cell death. We hypothesized that cytokine-induced RV expression impairs autophagy favoring pore formation in endothelial cells (ECs) implicating endothelial dysfunction as a mechanism for broader organ damage.
Methods: ECs were isolated from umbilical cords of 11 healthy African American subjects and genotyped for APOL1 by PCR sequencing (G0/G0 n=4, RV/G0 n=4 RV/RV n=3). CD31+ ECs (confirmed by FACS) were treated with IFNɣ (100 units/mL) for 24 hours and subsequent assessments included: APOL1 and autophagy marker LC3B protein measurements by immunoblot, LC3B staining by immunofluorescence, angiogenesis capacity on Matrigel matrix, and bioenergetic profiling by the Seahorse mitochondrial stress test method.
EC were >98% CD31 positive by flow cytometry. Across the genotypes, exposure to IFNɣ increased APOL1 protein expression 13.3±9.2 fold (p=0.012). To assess the ability of APOL1 over-expression to initiate autophagy, LC3B positive autophagosomes were counted by immunofluorescence. As expected, in G0/G0 ECs autophagosomes increased 41% when APOL1 was over-expressed. By contrast, autophagosomes decreased 7.1% in RV/G0 and 35.5% RV/RV ECs suggesting an autophagy defect. This result was confirmed by immunoblot. Autophagy-dependent angiogenesis was evaluated by plating resting and IFNɣ treated ECs on Matrigel matrix. At baseline, RV/RV ECs showed impaired angiogenesis capacity forming fewer junctions (J) and tubules (T) than RV/G0 and G0/G0 ECs (RV/RV: J: 539.9, T: 484.8; RV/G0: J: 841.8, T: 727.7, G0/G0: J: 810.8 T: 719.0, p=0.07). Overexpressing APOL1 with IFNɣ, impaired angiogenesis in both the RV/RV and RV/G0 ECs but not G0/G0 ECs (RV/RV: J: 353.0 T: 319.8; RV/G0: J: 297.8, T: 296.0, G0/G0: J: 739.0 T: 657.8, p=0.001). As a proof of concept, autophagy inhibitor ABT-737 was added to G0/G0 ECs resulting in a 42% decrease in junctions (p=0.036) and 52% decrease in tubules (p=0.012). Mitochondrial oxygen consumption of resting and IFNɣ treated ECs, was next assessed by bioenergetic profiling. For baseline, there were genotype-dependent differences in spare capacity (SC) and coupling efficiency (CE) (G0/G0: SC: 41.0±22.0 pmol/min CE: 439.8%; RV/G0: SC: 22.75 pmol/min±0.18 CE:119.9%; RV/RV SC: 24.6±4.71 pmol/min CE:100.35%). Upon adding IFNɣ, Proton leak increased 1.89 pmol/min in the G0/G0 ECs, 1.49 pmol/min in the RA/G0 ECs, and 7.50 pmol/min in the RA/RA ECs. This pattern is consistent with genotype dependent increased cation pore formation with electron escape across the inner mitochondrial membrane.
IFNɣ increases both ancestral and variant APOL1 intracellular accumulation in cultured ECs across genotype. In RV carrying ECs, this increased expression results in aberrant autophagy, angiogenesis, and mitochondrial energy production, all of which likely contribute to endothelial dysfunction, representing an underpin of broader organ damage.
To cite this abstract in AMA style:Blazer A, Rasmussen S, Markham A, Mehta-Lee S, Buyon JP, Clancy RM. Interferon-Induced APOL1 over-Expression Causes Autophagic Dysfunction and Mitochondrial Stress in Risk Variant-Carrying Endothelial Cells [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/interferon-induced-apol1-over-expression-causes-autophagic-dysfunction-and-mitochondrial-stress-in-risk-variant-carrying-endothelial-cells/. Accessed October 19, 2021.
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