The HUVEC cell line combined with CRISPR-Cas9 gene editing works so well!

Check out the perfect harmony between HUVEC and CRISPR-Cas9 gene editing

Abstract: Opening the doors to the research fields of Vascular Biology | Cardiovascular Diseases, etc

Human Umbilical Vein Endothelial Cells (HUVEC) were first successfully isolated and applied in the field of vascular biology and related research in the 1970s^[1]. Since then, the HUVEC cell line has become an indispensable cell model in research fields such as cardiovascular diseases, vascular biology, and human scaffolds. With the development of gene-editing technology, particularly CRISPR-Cas9 technology, more and more researchers are using this technology to deeply explore the molecular mechanisms of cells, disease pathogenesis, and new treatment methods. However, gene-editing technology is difficult to directly apply to primary cells because primary cells have a limited lifespan, low transfection efficiency, and difficulty forming single-cell clones^[2]. Therefore, researchers often choose to use immortalized cell lines in many cell gene editing experiments. So, what kind of research can be done with the combination of the immortalized HUVEC cell line and gene editing technology? Let's take a look!

Figure 1. HUVEC under microscope (40X)^[3]

PECAM-1 Cytoplasmic Structure Function Research

Platelet endothelial cell adhesion molecule (PECAM-1) is a type I transmembrane glycoprotein, mainly concentrated at the junctions between endothelial cells, which can promote leukocyte transendothelial migration, perceive shear and flow changes and maintain vascular barrier permeability. The homophilic interactions of PECAM-1's extracellular structural domains can help it perform these functions, but little is known about the role of its cytoplasmic structural domain in these processes. Liao et al^[4] used CRISPR/Cas9 gene-editing technology on the immortalized HUVEC cell line and successfully obtained a HUVEC cell line that only knocked out the PECAM-1 cytoplasmic structural domain (ΔCD-PECAM-1). By monitoring minor changes in the endothelial cell barrier using an Electric Cell-substrate Impedance Sensing (EICS), they observed that ΔCD-PECAM-1 normally accumulates at the junctions of endothelial cells. After disrupting the cell barrier with thrombin, these cells were able to restore barrier function more quickly. The molecular migration rate was assessed using fluorescence recovery after photobleaching (FRAP) analysis, and the results showed that ΔCD-PECAM-1 exhibits a higher migration rate within the plasma membrane, accelerating re-aggregation at the endothelial cell boundary to regulate vascular permeability. The above results reveal that the free movement of PECAM-1 within the plasma membrane is regulated by its cytoplasmic structural domain, thus affecting the efficiency of endothelial cells in establishing and maintaining the vascular permeability barrier. This finding provides important clues for further understanding the functions of endothelial cells and the regulation of vascular permeability.

Figure 2. Loss of PECAM-1 cytoplasmic structural domain enhances barrier recovery function^[4]

CCM Pathogenic Gene Regulation of Fibronectin Research

Cerebral cavernous malformations (CCM) are a kind of cerebrovascular lesion characterized by irregular capillary tangles. The disease is caused by mutations in the KRIT1 (CCM1), CCM2, and PDCD10 (CCM3) genes located on chromosomes 7q, 7p, and 3p. These gene mutations disrupt the connections between endothelial cells and lead to changes in small blood vessel permeability, thus causing vascular malformations. Schwefel and others^[5] used CRISPR-Cas9 technology to knock out the CCM1, CCM2, and CCM3 genes in immortalized HUVEC cells. The authors found that when HUVEC cells lack the CCM1, CCM2, or CCM3 genes, these cells reduce the expression of fibronectin. Then, they observed the effects on these gene-deficient cells by adding exogenous fibronectin. The results showed that the addition of fibronectin can rescue the abnormal phenotype of endothelial cells lacking CCM1, CCM2, or CCM3 genes, allowing the cells to partially or fully recover to a normal phenotype. It also showed that the CCM1, CCM2, and CCM3 genes have overlapping regulatory effects on the expression of fibronectin in endothelial cells.

Figure 3. The addition of fibronectin can rescue the abnormal phenotype of cells lacking the CCM1 or CCM2 gene^[5]

Development of Real-Time Monitoring Tools for E-Selectin Expression

E-selectin is a glycoprotein expressed on endothelial cells and plays an important role in many pathological processes such as inflammation. Ogrodzinski Lydia and the team^[6] developed a tool using CRISPR-Cas9 technology and NanoBiT technology that can monitor the expression of E-selectin on the cell surface in real time, thereby sensitively detecting the effects of different inflammatory cell factors or ligands on E-selectin expression. In this study, the authors used CRISPR-Cas9 technology to knock the HiBiT tag protein into the N-terminal of E-selectin in the immortalized HUVEC cell line, while externally adding LgBiT protein that cannot pass through the cell membrane. When LgBiT protein is fused with the HiBiT tag, it will produce a light signal, thereby achieving fluorescence detection of E-selectin. The results showed that after induction with inflammatory factors or ligands such as TNFα, IL-1α, IL-1β, LPS, VEGF 165a, and histamine, the expression of E-selectin in primary cells and immortalized cells was similar. In addition, the authors also evaluated the performance of these cells in the formation of blood vessels and angiogenesis. They found that primary HUVEC, immortalized HUVEC, and HiBiT-E-selectin-TERT2-HUVE can all form microvessels in 3D culture medium. However, because primary cells cannot form single-cell clones, each experiment requires re-transfection to obtain HUVEC cells expressing HiBiT tag-E-selectin. Moreover, the low gene- editing efficiency will reduce the light output of primary cells and make it more susceptible to other factors. But immortalized cells can make the light output more consistent in each experiment, so they can achieve more detailed real-time monitoring of the dynamics of E-selectin in living cells, which will become a powerful tool for future inflammation drug development.

Figure 4. Real-Time Monitoring Tool for E-Selectin Expression^[6]

 From the above cases, it can be seen that immortalized HUVEC and primary HUVEC have similarities in many research fields. In terms of gene-editing, the use of immortalized HUVEC can avoid some problems existing in primary cell models, such as low efficiency of CRISPR-Cas9 gene-editing, unstable expression of target genes, and research within a limited number of generations. The use of immortalized HUVEC cell lines not only improves the efficiency and repeatability of experiments, but also provides a more stable, lasting, and controllable cell model. Now, Ubigene has successfully modified the immortalized HUVEC cell line, and the feasibility of gene-editing has been confirmed to ensure its editing quality! If you are planning to get gene-editing cell models using the HUVEC, please come and consult with Ubigene, let us provide you with the one-stop customizatin service!

References

[1] Jaffe, Eric A., et al. "Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria." The Journal of clinical investigation 52.11 (1973): 2745-2756.

[2] Brandt, Camilla Blunk, et al. "HIF1A Knockout by Biallelic and selection-free CRISPR gene editing in human primary endothelial cells with ribonucleoprotein complexes." Biomolecules 13.1 (2022): 23.

[3] Medina-Leyte, Diana J., et al. "Use of human umbilical vein endothelial cells (HUVEC) as a model to study cardiovascular disease: A review." Applied Sciences 10.3 (2020): 938.

[4] Liao, Danying, et al. "CRISPR-mediated deletion of the PECAM-1 cytoplasmic domain increases receptor lateral mobility and strengthens endothelial cell junctional integrity." Life sciences 193 (2018): 186-193.

[5] Schwefel, Konrad, et al. "Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3." The FASEB Journal 34.7 (2020): 9018-9033.

[6] Ogrodzinski, Lydia, et al. "Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells." Iscience 26.7 (2023).