IGSF3 overexpression markedly reduced pseudo-vascular network formation and decreased cell viability in both air- and cigarette smoke-exposed human lung microvascular endothelial cells.
Does IGSF3 overexpression improve angiogenic function in human lung microvascular endothelial cells exposed to cigarette smoke?
IGSF3 overexpression impairs angiogenesis and reduces cell viability in lung microvascular endothelial cells, suggesting a hormetic window for its role in microvascular repair.
Abstract Rationale A key mechanism of the emphysema phenotype in chronic obstructive pulmonary disease (COPD) is a net loss of lung capillaries, due to an imbalance of increased injury and decreased repair of lung microvascular endothelial cells (LMVEC) attributed to chronic cigarette smoking (CS). Microvascular repair requires robust cell proliferation and angiogenesis. We have shown that the Immunoglobulin Superfamily Member 3 (IGSF3), a transmembrane protein that is decreased by CS, is required for endothelial scratch-wound repair1, however, its role in angiogenesis remains undefined. We hypothesize that overexpression of IGSF3 will improve the angiogenic function of lung microvascular endothelial cells at baseline and during CS exposure. Methods To overexpress IGSF3, transformed human LMVECs (HuLEC-5a) were transduced with a lentivirus expressing tagged human IGSF3, using GFP tagged HuLEC-5a as control and validated by immunofluorescence and immunoblotting. IGSF3 knockdown was performed by siRNA. Cells were exposed to cigarette smoke (CSE 2%; University of Kentucky, 3R4F) or air control (AC) extracts. Cell viability was measured by CCK-8 assay. Angiogenic function was measured by pseudo-vascular network formation (PVNF) using automated tube-formation analysis (Wimasis). Results Exposures of sublethal concentrations of CSE (2%, 5h) decreased the complexity of PVNF, measured by a reduction in number of tubes. Compared to cells expressing basal levels of IGSF3, IGSF3 overexpression markedly reduced PVNF and qualitatively enhanced cell-to-cell clumping (Figure 1) even in the absence of CS exposure. Overexpression of IGSF3 was sufficient to decrease cell viability in both AC-exposed and CS-exposed cells. Fig. 1 Effect of IGSF3 overexpression (B) on Pseudo-Vascular Network Formation imaged at 5h compared to WT control (A) with respective automated analysis (C,D); scale bar = 200 μm. Conclusion Given the requirement for IGSF3 in endothelial scratch-wound repair, the anti-angiogenic effect of IGSF3 overexpression suggests that IGSF3 supports repair at optimal levels but impairs angiogenesis when overexpressed, defining a hormetic window that is both mechanistically essential and therapeutically targetable to preserve lung microvascular integrity. References 1. Schweitzer KS et al., JCI Insight. 2020 This abstract is funded by: DoD W81XWH-22-1-0255
Jost et al. (Fri,) conducted a other in COPD / Emphysema (in vitro model). IGSF3 overexpression and cigarette smoke extract vs. GFP tagged HuLEC-5a and air control extracts was evaluated on Pseudo-vascular network formation (PVNF) and cell viability. IGSF3 overexpression markedly reduced pseudo-vascular network formation and decreased cell viability in both air- and cigarette smoke-exposed human lung microvascular endothelial cells.