Abstract Body: Background:
Atherosclerosis develops in regions of blood vessels exposed to disturbed flow, such as bifurcations or curves, where endothelial cells (ECs) exhibit altered gene expression and cuboidal morphology. Suppression of Kruppel-like factor 2 (KLF2), a transcription factor critical for EC physiology, is a hallmark of these regions. Endothelial cell size, which influences vessel diameter, perfusion, and vascular responses, is also affected by flow conditions. Although KLF2’s role in regulating EC size has been demonstrated in zebrafish, its effects on human EC size and morphology, as well as its downstream targets, remain poorly understood.

Methods:
Human umbilical vein endothelial cells (HUVECs) were transduced with murine KLF2 (mKLF2) or GFP-only control vectors under static conditions. Epifluorescent imaging methodologies were employed: Cellpose was used for accurate segmentation and quantification of cell area, and PolarityJam facilitated analysis of morphological features—including eccentricity, axis ratio, and orientation. In parallel, NOS3 expression was quantified across four experimental groups: Ad-GFP control, Ad-mKLF2, Ad-mKLF2 with L-NAME (an eNOS inhibitor), and Ad-GFP with L-NAME.


Results:
Following transduction, Ad-mKLF2 cells exhibited a 22% increase in surface area compared to controls (95% CI: 15%–26%) and adopted a more elongated morphology. This elongated shape is important as it reflects improved alignment to shear stress. While control cells proliferated over 48 hours, increasing by 29% (95% CI: 9%–48%), mKLF2 cells did not divide, prioritizing growth without changes in nuclear size, thereby ruling out senescence or DNA damage. A positive correlation between GFP fluorescence intensity and cell area in individual mKLF2-expressing cells supported a direct role for KLF2 in modulating cell size and shape. Moreover, no significant size difference was observed between Ad-mKLF2 cells with or without L-NAME, indicating that eNOS is not a key determinant in this context. Expanding our analysis, we are now examining hundreds of downstream genes to further elucidate the regulatory network.

Conclusions:
These findings establish KLF2 as a key regulator of EC size and morphology, independent of eNOS, and lay the groundwork for future studies to define additional KLF2-regulated pathways. The insights gained have promising implications for developing therapeutic strategies to restore vascular homeostasis and combat atherosclerosis.