Prostacyclin analog iloprost can reverse vascular dysfunction in scleroderma, according to study results published in Arthritis & Rheumatology. Researchers noted that iloprost stabilized adherens junctions, which are responsible for regulating endothelial barrier function and tubulogenesis, and reduced endothelial to mesenchymal transition (Endo-MT) in patients with systemic sclerosis (SSc).
Despite its short half-life, iloprost has been shown to improve symptoms of Raynaud phenomenon and promote the healing of digital ulcers in SSc. In the current study, the researchers sought to assess the cellular mechanisms of iloprost in improving vascular function in scleroderma.
Patients with SSc were recruited in the study from the University of Michigan Scleroderma program and matched by age, sex, and race/ethnicity with healthy control participants from the same region. Two 4 mm punch biopsies were obtained from the distal forearm of all participants. Dermal endothelial cells were isolated from all biopsies and treated with 150 nM of iloprost at various intervals. After treatment, immunofluorescence was used to visualize vascular endothelial (VE)-cadherin, β-catenin, and F-actin in the endothelial cells. Permeability was tested by measuring horseradish peroxidase (HRP) movement through endothelial cell monolayers. Matrigel tube formation assays were performed to test endothelial cell angiogenesis. Finally, quantitative polymerase chain reaction (PCR) was used to study gene expression after iloprost exposure.
The study cohort included 10 patients with SSc (mean age, 54.9±4.9 years) and 13 healthy control participants (mean age, 50.8±3.8 years). All patients had diffuse cutaneous SSc with Raynaud phenomenon at the time of biopsy. Before treatment with iloprost, adherens junctions were significantly disrupted in SSc cells compared with healthy endothelial cells. Junctional levels of VE-cadherin were significantly lower in the SSc vs healthy endothelial cells (P <.05). Iloprost had demonstrable effects on adherens junctions in both the SSc and control samples. In SSc endothelial cells, iloprost caused a sustained increase in clustering of β-catenin and VE-cadherin at cell junctions, which peaked at 10 minutes and then returned to baseline by 60 minutes of exposure. The increase in VE-cadherin clustering remained significantly elevated after 120 minutes.
Researchers suggested that compared with control endothelial cells, the SSc endothelial cells may show barrier dysfunction in vitro; HRP permeation was much higher in the SSc samples compared with the control samples. Iloprost was found to significantly decrease the permeability of SSc endothelial cells at 2 and 3 hours. In Matrigel assays, pretreatment of SSc endothelial cells with iloprost significantly enhanced tubulogenesis. Finally, iloprost also appeared to inhibit Endo-MT. Iloprost blocked Endo-MT in the endothelial cells that were treated with transforming growth factor beta (TGFβ).
To test the effect of the function-blocking antibody to VE-cadherin BV9, normal endothelial cells were treated with BV9 before the addition of iloprost. Effects of iloprost on permeability, tubulogenesis, and Endo-MT were found to be significantly reduced. Researchers hypothesized that the protective effects of iloprost are “dependent on [the] increased clustering of VE-cadherin at endothelial junctions.” Further study is necessary to better understand the mechanistic effects of iloprost, particularly as they relate to VE-cadherin.
Study limitations included the lack of inclusion of patients with limited cutaneous SSc and the variability in disease activity and medications between the patient and control group.
“The prolonged clinical endothelial protective effect of PGI2 [analogs] may…stem from their ability to stabilize [endothelial cell] adherens junctions resulting in vasculoprotection, including improved barrier function, normalization of dysregulated angiogenesis, and inhibition of Endo-MT,” the researchers concluded.
Tsou PS, Palisoc PJ, Flavahan NA, Khanna D. Dissecting the cellular mechanism of prostacyclin analogue iloprost in reversing vascular dysfunction in scleroderma. Arthritis Rheumatol. Published online October 1, 2020. doi:10.1002/art.41536