Key Takeaways & Executive Findings
- •• Successfully constructed a vascular endothelial growth factor receptor 2 (VEGFR2) gene knockdown rat model using adeno-associated virus (AAV) technology. • In situ injection of VEGFR2 knockdown AAV achieved a 60% knockdown efficiency in rat muscle tissue. • VEGFR2 knockdown in muscle tissue led to a significant reduction in muscle fiber area and microvessel number. • The study provides a valuable experimental basis for further research on VEGFR2's role in cervical spondylosis and potential therapeutic strategies.
Abstract
BACKGROUND: Vascular endothelial growth factor receptor 2 is mainly expressed in vascular endothelial cells and plays a crucial role in angiogenesis, tissue repair, and the occurrence and development of diseases. Adeno-associated virus, due to its unique advantages, has been widely applied in mechanism research, disease modeling, and gene therapy fields. OBJECTIVE: To construct an adeno-associated viral vector for vascular endothelial growth factor receptor 2 gene knockdown in rat muscle tissue, determine the knockdown efficiency of vascular endothelial growth factor receptor 2 gene-knockdown adeno-associated virus in the rat sternocleidomastoid muscle and assess its effects on muscle and blood vessels. METHODS: The vector was constructed, and packaged into adeno-associated virus. Following target screening experiments in Sprague-Dawley rats, immunofluorescence assays were conducted to assess viral infection efficiency and vascular endothelial growth factor receptor 2 protein expression. Quantitative real-time PCR was used to measure vascular endothelial growth factor receptor 2 mRNA expression. Ultimately, the optimal shRNA sequences were determined to be Y29478 and the control sequence Y9957. Twelve Sprague-Dawley rats were randomly divided into an adeno-associated virus group and a control adeno-associated virus group for functional verification. The adeno-associated virus was injected into the rat sternocleidomastoid muscle. After 20 weeks, quantitative real-time PCR and western blot were used to detect vascular endothelial growth factor receptor 2 mRNA and protein expression in cervical muscle, hematoxylin-eosin staining was used to detect muscle fiber area, and CD31 immunohistochemistry was used to detect the number of microvessels in cervical muscle. RESULTS AND CONCLUSION: (1) The shRNA sequence and dose that could knock down vascular endothelial growth factor receptor 2 expression were successfully screened, and the knockdown efficiency of vascular endothelial growth factor receptor 2 in rat muscle tissue after in situ injection of vascular endothelial growth factor receptor 2 gene knockdown adeno-associated virus reached 60%. (2) Compared with the control adeno-associated virus group, the mRNA and protein expression of vascular endothelial growth factor receptor 2 in the adeno-associated virus group decreased, the muscle fiber area decreased, and the number of microvessels decreased. (3) A rat model of vascular endothelial growth factor receptor 2 gene knockdown was successfully constructed, and knockdown of vascular endothelial growth factor receptor 2 in muscle tissue caused a reduction in muscle fiber area and a decrease in the number of microvessels.
1. Introduction
Cervical spondylosis is commonly caused by long-term low-head posture leading to cervical muscle strain, which in turn causes cervical dynamic and static imbalance, thereby inducing symptoms such as pain and numbness. Therefore, the repair of muscle injury is the focus of prevention and treatment of cervical spondylosis [1-3]. Vascular endothelial growth factor receptor 2 (VEGFR2), also known as KDR, is mainly expressed in vascular endothelial cells. As a signal transduction receptor on the cell membrane, it mediates the biological effects of vascular endothelial growth factor signaling [4], and plays a key role in angiogenesis and endothelial dysfunction [5], participating in the regulation of autophagy and apoptosis [6-7]. Our research group has shown that VEGFR2 is closely related to cervical spondylosis, and intervention of VEGFR2 may be an effective strategy for the treatment of cervical spondylosis [8].
Adeno-associated virus (AAV) technology is a technique developed in recent years. As a vector, it transfects plasmids into target cells. Due to its high transfection efficiency and low immunogenicity, it is widely used in the construction of gene expression vectors [9-10], mechanism research [11-13], and even gene therapy [14-16]. By using AAV technology to construct gene knockdown or overexpression vectors, targeted control of target gene expression has been achieved in many successful cases, but the construction of a model for VEGFR2 gene knockdown has not been reported. This study used AAV technology to construct a VEGFR2 gene knockdown rat model, explore the function of VEGFR2 in cervical muscle tissue, and provide experimental evidence for further research on its mechanism and clinical treatment of cervical spondylosis.
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Qian Jiaming, Li Yumei, Wang Xiaole, Fang Ting, Liu Fushui (2026). Construction and functional verification of vascular endothelial growth factor receptor 2 gene knockdown rats. Chinese Journal of Tissue Engineering Research. https://doi.org/10.12307/2026.21578
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Frequently Asked Questions
What is the main objective of this study?
The main objective was to construct a vascular endothelial growth factor receptor 2 (VEGFR2) gene knockdown rat model using adeno-associated virus (AAV) technology and to evaluate the knockdown efficiency and its effects on muscle and blood vessels.
How was the VEGFR2 gene knockdown achieved?
The VEGFR2 gene knockdown was achieved by constructing an AAV vector carrying a short hairpin RNA (shRNA) sequence targeting VEGFR2 mRNA, which was then injected into the rat sternocleidomastoid muscle.
What were the key findings of the study?
The study successfully constructed a VEGFR2 knockdown rat model with a 60% knockdown efficiency. VEGFR2 knockdown led to a significant reduction in muscle fiber area and microvessel number, indicating its crucial role in muscle maintenance and angiogenesis.
What is the significance of this research?
This research provides a valuable experimental model for studying the role of VEGFR2 in cervical spondylosis and other muscle-related diseases, and may contribute to the development of targeted therapies.
What are the potential clinical applications of this study?
The findings suggest that VEGFR2 could be a therapeutic target for conditions involving muscle atrophy and impaired angiogenesis, such as cervical spondylosis. The AAV-mediated gene knockdown approach may be adapted for gene therapy applications.
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