VEGF165: A Key Factor in Angiogenesis Regulation and Disease Treatment

Vascular endothelial growth factor 165 (VEGF165), as the most biologically active subtype in the VEGF-A splice variant family, plays a central regulatory role in both physiological vascular formation and pathological angiogenesis. As a homodimeric glycoprotein with a molecular weight of approximately 45 kDa, VEGF165 can simultaneously bind to the tyrosine kinase receptors VEGFR-1 and VEGFR-2 on the surface of vascular endothelial cells, as well as the neuropilin (NRP) co-receptor, through its unique structural features, activating a complex downstream signaling network. This article comprehensively explores the molecular characteristics, expression regulation mechanisms, physiological and pathological functions of VEGF165, and its clinical application prospects in areas such as ischemic diseases, tumor therapy, and retinopathy, with a particular focus on the latest advancements in the therapeutic application of recombinant VEGF165 protein and the development of VEGF165-based gene therapy strategies. By systematically reviewing basic research and clinical translation achievements, we can gain a deeper understanding of the central role of this multifunctional growth factor in vascular biology and provide new insights for the diagnosis and treatment of related diseases.

Molecular Characteristics and Expression Regulation of VEGF165

The structural features of VEGF165 form the molecular basis of its biological activity. As the main subtype produced by alternative splicing of the VEGF-A gene, VEGF165 consists of an N-terminal region of 121 amino acid residues and a C-terminal exon 7-encoded region of 44 amino acid residues, forming a homodimeric glycoprotein with a molecular weight of approximately 45 kDa. Compared to VEGF121, VEGF165 retains the heparin-binding domain, enabling it to bind to heparan sulfate proteoglycans (HSPGs) in the extracellular matrix; compared to VEGF189, VEGF165 has moderate solubility, allowing effective diffusion in tissues. This unique structural balance makes VEGF165 the most widely distributed and biologically active VEGF subtype in vivo. X-ray crystallographic analysis reveals that the VEGF165 dimer exhibits a typical "cysteine knot" growth factor folding pattern, with each monomer containing two receptor-binding sites capable of simultaneously binding two VEGFR molecules, inducing receptor dimerization and activation. Notably, the C-terminal exon 7-encoded region of VEGF165 contains a neuropilin (NRP) binding motif, a feature absent in other VEGF subtypes, enabling VEGF165 to enhance its pro-angiogenic signals through NRP.

The expression regulation of VEGF165 involves a multi-level complex mechanism. At the transcriptional level, VEGF gene expression is directly regulated by hypoxia-inducible factor-1α (HIF-1α). Under hypoxic conditions, HIF-1α accumulates and binds to the hypoxia response element (HRE) in the VEGF gene promoter region, significantly enhancing transcriptional activity. Additionally, various growth factors (e.g., EGF, PDGF), cytokines (e.g., IL-1β, IL-6), and hormones (e.g., estrogen) can regulate VEGF expression through their respective signaling pathways. At the post-transcriptional level, the stability of VEGF mRNA is finely regulated by RNA-binding proteins (e.g., HuR) and microRNAs (e.g., miR-126, miR-200 family). Notably, the alternative splicing process of VEGF165 is strictly regulated by members of the serine/arginine-rich (SR) protein family, and the differential expression of these splicing factors in different tissues determines the proportional changes between VEGF165 and other subtypes. At the post-translational level, glycosylation modifications of VEGF165 affect its secretion efficiency and biological activity, while proteases (e.g., plasmin) can cleave it into smaller active fragments, regulating its functional duration.

The secretion and distribution characteristics of VEGF165 determine the spatiotemporal specificity of its biological effects. Unlike the fully soluble VEGF121, VEGF165, due to its heparin-binding domain, binds immediately to HSPGs on the cell surface or in the extracellular matrix after secretion, forming a local reservoir; the remaining portion diffuses into surrounding tissues in a soluble form. This partially soluble, partially matrix-bound property allows VEGF165 to establish a concentration gradient in tissues, guiding the directional migration of vascular endothelial cells. Under pathological conditions, such as in the tumor microenvironment or ischemic tissues, the activation of matrix metalloproteinases (MMPs) can release matrix-bound VEGF165, enhancing local angiogenesis. Notably, VEGF165 can also be packaged into extracellular vesicles like exosomes, enabling long-distance signal transmission, a mechanism that may play an important role in the formation of pre-metastatic niches in tumors. These complex secretion and distribution patterns provide new perspectives for understanding the role of VEGF165 in physiological and pathological processes.

The receptor system of VEGF165 constitutes the structural basis of its signal transduction. VEGF165 can simultaneously bind three types of cell surface receptors: VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and neuropilin (NRP-1/2). Among these, VEGFR-2 is the primary receptor mediating pro-angiogenic effects, and its activation triggers multiple downstream pathways, including PLCγ-PKC-MAPK, PI3K-Akt, and FAK-paxillin, promoting endothelial cell proliferation, migration, and survival. Although VEGFR-1 has a higher affinity than VEGFR-2, its tyrosine kinase activity is weaker, primarily serving a regulatory and buffering role. NRP, as a co-receptor, lacks catalytic activity but can enhance the binding of VEGF165 to VEGFR-2 and the efficiency of signal transduction. After receptor activation, the complex enters the cell through clathrin-mediated endocytosis, a process that regulates signal strength and affects receptor recycling. Notably, the binding affinity of VEGF165 to different receptors is influenced by the local microenvironment, such as the composition of the extracellular matrix and pH changes, which can modulate its receptor-binding properties, forming a complex signal regulation network.

Protein engineering of VEGF165 has made it possible to develop improved therapeutic molecules. Through genetic engineering techniques, researchers have successfully developed recombinant human VEGF165 (rhVEGF165), whose biological activity is comparable to that of natural VEGF165. Further structure-function analysis has revealed that the N-terminal region of VEGF165 is responsible for receptor binding, while the C-terminal heparin-binding domain affects its distribution and stability. Based on this knowledge, scientists have designed various VEGF165 variants: variants with enhanced heparin-binding capacity have a longer tissue retention time, making them suitable for treating chronic ischemic diseases; variants with reduced heparin-binding have greater diffusion capacity, suitable for extensive vascular regeneration; variants fused with specific antibody Fc segments have significantly extended half-lives. Additionally, site-directed mutagenesis has produced selective VEGF165 variants that specifically activate VEGFR-2 with minimal binding to VEGFR-1, thereby enhancing pro-angiogenic effects while reducing side effects. These protein engineering techniques not only deepen the understanding of the relationship between VEGF165 structure and function but also lay the foundation for developing safer and more effective therapeutic VEGF165.

Physiological Functions and Pathological Roles of VEGF165

The central role of VEGF165 in vascular development has been fully demonstrated through gene knockout experiments. In embryonic development studies, complete knockout of the Vegf gene led to embryonic lethality in mice, manifesting as a complete absence of the vascular system; whereas knock-in mice expressing only VEGF120 (equivalent to human VEGF121) survived but exhibited severe cardiovascular defects, including ventricular septal defects and vascular maturation disorders. These phenotypic differences indicate that VEGF subtypes containing heparin-binding domains (especially VEGF164, equivalent to human VEGF165 in mice) are indispensable for normal vascular development. During vascular formation, VEGF165 guides the directional migration (chemotaxis) of endothelial precursor cells through concentration gradients, promoting the formation of primary vascular plexuses; subsequently, it participates in vascular maturation and stabilization by regulating the recruitment of vascular smooth muscle cells and pericytes. Notably, VEGF165 plays a key role in organ-specific vascular patterning, such as the development of glomerular capillary loops in the kidneys and hepatic sinusoids in the liver, which rely on the precise spatiotemporal expression of VEGF165. These findings establish the central role of VEGF165 in angiogenesis and patterning.

The role of VEGF165 in tissue repair provides important insights for regenerative medicine. In adult tissues, VEGF165 expression is typically maintained at low levels but rapidly upregulated after tissue injury, initiating reparative angiogenesis. Ischemia, trauma, or inflammatory stimuli induce VEGF165 expression through HIF-1α-dependent and independent pathways, promoting neovascularization and improving tissue perfusion and oxygen supply. Preclinical studies have shown that local administration of exogenous VEGF165 can accelerate the repair of skin wounds, fractures, and myocardial ischemia. Notably, the reparative effects of VEGF165 are not limited to pro-angiogenesis but also include the mobilization of endothelial progenitor cells (EPCs) from the bone marrow to the injury site and the activation of tissue stem cells' regenerative potential through paracrine mechanisms. For example, in myocardial infarction models, VEGF165 treatment not only increases myocardial vascular density but also reduces scar formation and improves cardiac function, effects partly attributed to the activation of cardiac stem cells. These multifaceted repair functions make VEGF165 a molecule of great interest in tissue engineering and regenerative medicine.

The key role of VEGF165 in tumor angiogenesis has made it an important target for anti-cancer therapy. Most solid tumors exhibit upregulated VEGF165 expression, a result of tumor cells adapting to the hypoxic microenvironment caused by rapid proliferation. Unlike physiological angiogenesis, tumor-induced angiogenesis is markedly disordered: overexpression of VEGF165 leads to abnormal vascular morphology, increased permeability, and incomplete basement membranes, changes that support tumor growth and promote metastasis. At the molecular level, tumor-derived VEGF165 activates endothelial cell VEGFR-2 through autocrine and paracrine actions, promoting the formation and remodeling of tumor vascular networks. Clinical studies have shown that VEGF165 expression levels are significantly correlated with the malignancy, metastatic risk, and poor prognosis of various tumors (e.g., breast cancer, lung cancer, colorectal cancer). Notably, immune cells such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) can also secrete VEGF165, creating an immunosuppressive microenvironment that helps tumors evade immune surveillance. These findings provide a theoretical basis for the application of anti-VEGF165 therapy in oncology.

The dual role of VEGF165 in ocular diseases reflects the complexity of its biological effects. Under physiological conditions, intraocular VEGF165 expression is tightly regulated, maintaining corneal avascularity and retinal vascular homeostasis. Under pathological conditions, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), retinal ischemia leads to excessive VEGF165 expression, causing pathological angiogenesis and vascular leakage, threatening vision. Intravitreal injection of anti-VEGF165 drugs (e.g., bevacizumab, ranibizumab) has become the first-line treatment for these diseases. However, long-term complete inhibition of VEGF165 may impair the physiological functions of retinal ganglion cells and choroidal vessels, leading to retinal atrophy. This therapeutic paradox suggests that ideal anti-VEGF therapy should achieve precise regulation rather than complete inhibition. Recent studies are exploring treatment strategies based on the dose-dependent effects of VEGF165, such as low-dose VEGF165 promoting vascular normalization rather than complete inhibition of angiogenesis, which may yield better long-term visual outcomes.

The non-vascular roles of VEGF165 in the nervous system expand the traditional understanding of its functions. In addition to its pro-angiogenic activity, VEGF165 also acts as a neurotrophic factor directly affecting neurons and glial cells. Neurons, astrocytes, and microglia in the central nervous system can express VEGF165 and its receptors, forming an autocrine/paracrine regulatory network. In ischemic stroke models, VEGF165 promotes neuronal survival and axonal regeneration by activating the VEGFR-2/NRP-1 complex; in Alzheimer's disease, moderate increases in VEGF165 expression can improve cerebral blood flow and clear β-amyloid; in amyotrophic lateral sclerosis (ALS), the neuroprotective effects of VEGF165 may delay motor neuron degeneration. These non-vascular effects are primarily mediated through survival signaling pathways such as PI3K-Akt and MEK-ERK, distinct from the classic pro-angiogenic pathways. Notably, the effect of VEGF165 on blood-brain barrier integrity is concentration-dependent: physiological concentrations maintain barrier function, while high concentrations increase permeability. This characteristic is particularly important in brain tumor therapy, as anti-VEGF drugs may improve chemotherapy drug delivery by "normalizing" tumor vasculature.

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