Background/Purpose: Systemic lupus erythematosus (SLE) induces endothelial cell dysfunction (ECD) that can manifest as glomerulonephritis or atherosclerosis. Lupus-prone mice lacking endothelial nitric oxide synthase (eNOS, a critical enzyme in endothelial cell (EC) function), have more severe proliferative nephritis. We hypothesized that SLE serum would induce ECD in vitro by reducing eNOS expression and increasing uncoupling of eNOS monomers, rendering the enzyme unable to produce protective nitric oxide (NO).

Methods: To address this hypothesis, we determined the extent to which serum from SLE patients induced ECD in vitro, whether serum-induced ECD was associated with reduced eNOS mRNA expression, and whether serum-induced ECD was reversed by addition of L-sepiapterin, a tetrahydrobiopterin donor that induces coupling of eNOS monomers to form functional, NO-producing homodimers. Human umbilical vein endothelial cells (HUVEC) were cultured with 20-50% serum from SLE patients (met ≥4 ACR criteria, mean SLE Disease Activity Index score = 4.6, n = 25) and connective tissue disease-free controls (n = 14) for 24 hours. Endpoints measured were: 1) EC NO production measured by staining with DAF-FM diacetate (an NO fluorescent probe) and performing flow cytometry; 2) EC eNOS mRNA measured via quantitative reverse transcription quantitative polymerase chain reaction (RTqPCR); and 3) Changes in HUVEC eNOS monomer and dimer formation in the presence of patient serum were determined by Western blot. The aformentioned endpoints were reassessed after co-culture of EC with serum and sepiapterin (5 μM). Data were reported as mean ± standard deviation and compared using a Student’s t-test or Kruskal-Wallis test.

Results: HUVECs cultured in 50% SLE serum displayed significantly reduced median fluorescence intensity (MFI) indicative of diminished NO production compared to EBM-2 media cultured cells ( 0.28 ± 0.38 vs. 1.08 ± 0.92 fold change respectively, p < 0.05). This reduction was reversed when cells were co-cultured in L-sepiapterin (5μM) and healthy control (n=5) or SLE (n=12) serum (1.13 ± 0.60 and 1.28 ± 2.8, respectively). Conversely, 20% SLE serum increased relative ratios of NOS3 mRNA expression vs. control as measured by reverse transcriptase quantitative PCR (RTqPCR) (1.9 ± 1.0 vs. 1.0 ± 1.2, p < 0.05). Western blot analysis revealed increased monomerization in cell cultured using SLE serum. However, preliminary studies suggest that the addition of L-sepiapterin restores dimerization of eNOS homodimers.

Conclusion: These results suggest that SLE serum alone can induce ECD through reduction of functional NO production in EC. The reversal of this in vitro ECD with sepiapterin suggests that SLE serum induces uncoupling of eNOS homodimers, a process that increases eNOS superoxide and reduces eNOS NO production. Both of these changes induce ECD. The observation that SLE serum increased (rather than decreased) eNOS mRNA expression suggests a feedback loop in enzyme expression in response to reduced NO production; although, this hypothesis is not addressed here. Determining the mechanism and molecular pathway through which SLE serum induces ECD could lead to targets for pharmacologic intervention.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/sle-serum-impairs-no-production-in-huvecs-through-induction-of-enos-uncoupling/