Endothelial Cells Stably Infected with Recombinant Kaposi’s Sarcoma-Associated Herpesvirus Display Distinct Viscoelastic and Morphological Properties
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus that primarily infects endothelial cells and leads to the development of Kaposi’s sarcoma (KS), especially in people living with HIV. Kaposi’s sarcoma is a malignant tumor characterized by endothelial cell proliferation, typically presenting as cutaneous lesions. After infecting endothelial cells, KSHV induces significant changes in cell morphology and mechanical properties, which may serve as early diagnostic and therapeutic markers. However, quantitative studies on these changes remain limited, particularly in the field of cellular mechanics. Therefore, this study aims to explore the potential of these changes as diagnostic and therapeutic targets by quantitatively analyzing the morphological and mechanical changes in endothelial cells following KSHV infection.
Source of the Paper
This paper was co-authored by Majahonkhe M. Shabangu, Melissa J. Blumenthal, Danielle T. Sass, Dirk M. Lang, Georgia Schafer, and Thomas Franz. The authors are affiliated with multiple research institutions, including the Biomedical Engineering Research Centre at the University of Cape Town and the International Centre for Genetic Engineering and Biotechnology. The paper was published on April 5, 2025, in the journal Cellular and Molecular Bioengineering, with the DOI 10.1007/s12195-025-00848-z.
Research Process
1. Cell Culture and Viral Infection
The study utilized two endothelial cell lines: vascular endothelial cells (HUVEC-derived HuARLT2 cells) and lymphatic endothelial cells (LEC). These cells were infected with recombinant KSHV (rKSHV) via spinoculation, establishing stable infection models (HuARLT2-rKSHV and LEC-rKSHV). After infection, the cells were cultured in selection media containing puromycin for at least two weeks to ensure stable viral infection.
2. Mitochondria-Tracking Microrheology (MTM)
To investigate the mechanical properties of infected cells, the study employed mitochondria-tracking microrheology. After staining, live-cell imaging was performed using a confocal microscope to record the trajectories of mitochondria. By analyzing the mean squared displacement (MSD) of mitochondria and its power law exponent (α), the researchers assessed the viscoelastic properties of the intracellular environment.
3. Cell Morphology Analysis
The study captured images of infected and uninfected cells using phase-contrast and fluorescence microscopy and processed the images using Fiji software. By measuring the aspect ratio, roundness, and circularity of the cells, the researchers quantitatively analyzed the morphological changes following infection.
4. Data Analysis
The study used Python and GraphPad Prism for statistical analysis to evaluate the differences in morphology and mechanical properties between infected and uninfected cells. The normality of the data was assessed using the Shapiro-Wilk test, and group comparisons were performed using Welch’s t-test or the Mann-Whitney U test.
Key Findings
1. Changes in Cell Morphology
The study found that KSHV infection significantly increased the aspect ratio of HuARLT2 and LEC cells while reducing their roundness and circularity. Specifically, the aspect ratio of HuARLT2-rKSHV cells increased by 29.7%, while roundness decreased by 26.1% and circularity decreased by 25.7%. LEC-rKSHV cells also exhibited similar morphological changes, although the magnitude of the changes was smaller.
2. Changes in Cell Mechanical Properties
Mitochondria-tracking microrheology analysis revealed that KSHV infection significantly increased cellular deformability, as indicated by higher MSD values at short lag times (τ = 0.19 s). The MSD of HuARLT2-rKSHV and LEC-rKSHV cells was 49.4% and 42.2% higher, respectively, compared to uninfected cells. Additionally, the MSD power law exponent (α) of infected cells was significantly lower, indicating increased cytoplasmic viscosity.
Conclusion
This study demonstrates that KSHV infection of endothelial cells induces significant changes in morphology and mechanical properties, which may serve as biomarkers for the early diagnosis of Kaposi’s sarcoma. In particular, the increased cellular deformability and changes in cytoplasmic viscosity may provide important clues for developing new therapeutic strategies. Furthermore, the study found that compared to vascular endothelial cells, lymphatic endothelial cells exhibited smaller morphological changes after infection, but their mechanical properties changed more significantly, suggesting that intracellular mechanical properties may be more sensitive indicators of KSHV infection than morphological changes.
Research Highlights
- Quantitative Analysis of Morphological and Mechanical Changes After KSHV Infection: This study is the first to systematically analyze the morphological and mechanical changes in endothelial cells following KSHV infection using quantitative methods, providing new insights for the early diagnosis of Kaposi’s sarcoma.
- Application of Mitochondria-Tracking Microrheology: The study employed innovative mitochondria-tracking microrheology to precisely measure the viscoelastic properties of cells, offering a new tool for studying the cellular mechanics of viral infections.
- Potential of Cell Mechanical Properties as Diagnostic Markers: The study found that changes in cell mechanical properties were more significant than morphological changes after KSHV infection, providing a theoretical basis for developing diagnostic methods based on cell mechanics.
Research Value
This study not only deepens the understanding of the mechanisms of KSHV infection but also provides new directions for the early diagnosis and treatment of Kaposi’s sarcoma. In particular, changes in cell mechanical properties may become important targets for the development of non-invasive diagnostic tools in the future. Additionally, the high-throughput microrheology techniques used in this study offer new methodological references for research on other viral infections and cancers.
Additional Valuable Information
The research data and analysis code have been publicly released on the ZivaHub data repository of the University of Cape Town for reference and use by other researchers. Furthermore, the study received funding from multiple institutions, including the National Research Foundation of South Africa, ensuring the smooth progress of the research.
Through this study, scientists have not only revealed the biomechanical mechanisms of KSHV infection in endothelial cells but also laid the foundation for future diagnostic and therapeutic strategies based on cell mechanical properties.